WO1993000357A1 - Therapeutic polypeptides based on von willebrand factor - Google Patents

Abstract

A polypeptide patterned on a fragment of wild type mature von Willebrand factor (vWF) subunit having one or more binding sites of predetermined affinity for one or more of the ligands selected from the group consisting of collagen, glycosaminoglycans, proteoglycans, platelet glycoprotein Ibα, platelet glycoprotein IIb/IIIa, or coagulation factor VIII, said polypeptide having a modified amino acid sequence relative to that of said fragment and an increased binding affinity, relative to said predetermined affinity, for one or more of said ligands, including also such a polypeptide prepared by mutagenesis of a DNA sequence and patterned on wild type mature vWF subunit, and also a polypeptide in purified form patterned upon a parent polypeptide which comprises the wild type amino acid sequence of mature von Willebrand factor subunit, or a fragment thereof, and including also purified DNA sequences encoding such polypeptides, expression plasmids and viral expression vectors containing the DNA sequences, and therapeutic compositions comprising such polypeptides effective in the treatment of thrombosis, and methods for the use thereof, and also preparation of such polypeptides by mutagenesis of an encoding DNA sequence or covalent modification of wild type vWF.

Description

THERAPEUTIC POLYPEPTIDES BASED ON VON WILLEBRAND FACTOR Cross-Reference to Related Applications

This is a continuation-in-part of application Serial No. 07/675,529, filed March 27, 1991, which is a continuation-in-part of application Serial No. 07/613,004, filed November 13, 1990, which is a continuation-in-part of Serial No.

07/600,183, filed October 17, 1990, which is a continuation-in-part of Serial No. 07/519,606, filed May 7, 1990, which is a continuation-in-part of Serial No. 07/270,488, filed

November 4, 1988, now abandoned, which is a continuation of Serial No. 869,188, filed May 30, 1986, now abandoned.

Field of the Invention

This invention relates to polypeptides which are useful in the treatment of vascular disorders such as thrombosis. More particularly, this invention relates to therapeutic polypeptides based on von Willebrand factor (vWF), including, for example, polypeptides produced by recombinant DNA-directed methods.

The term "hemostasis" refers to those processes which comprise the defense mechanisms of the body against loss of circulating blood caused by vascular injury. Processes which are normal as a physiologic response to vascular injury may lead in pathologic circumstances, such as in a patient afflicted with atherosclerotic vascular disease or chronic congestive heart failure, to the formation of undesired thrombi (clots) with resultant vascular occlusion.

Impairment of blood flow to organs under such circumstances may lead to severe pathologic states, including myocardial infarction, a leading cause of mortality in developed

countries.

The restriction or termination of the flow of blood within the circulatory system in response to a wound or as a result of a vascular disease state involves a complex series of reactions which can be divided into two processes, primary and secondary hemostasis. Primary hemostasis refers to the process of platelet plug or soft clot formation. The platelets are non-nucleated discoid structures approximately 2-5 microns in diameter derived from megakaryocytic cells. Effective primary hemostasis is accomplished by platelet adhesion, the interaction of platelets with the surface of damaged vascular endothelium on which are exposed underlying collagen fibers and/or other adhesive macromolecules such as proteoglycans and glycosaminoglycans to which platelets bind.

Secondary hemostasis involves the reinforcement or crosslinking of the soft platelet clot. This secondary process is initiated by proteins circulating in the plasma (coagulation factors) which are activated during primary hemostasis, either in response to a wound or a vascular disease state. The activation of these factors results ultimately in the production of a polymeric matrix of the protein fibrinogen (then called "fibrin") which reinforces the soft clot.

Therapeutic drugs for controlling thrombosis have been classified according to the stage of hemostasis which is affected by the administration thereof. Such prior art compositions are typically classified as anticoagulants, thrombolytics and platelet inhibitors.

The anticoagulant therapeutics typically represent a class of drugs which intervene in secondary hemostasis.

Anticoagulants typically have no direct effect on an

established thrombus, nor do they reverse tissue damage.

Associated with the use of existing anticoagulants is the hazard of hemorrhage, which may under some conditions be greater than the clinical benefits otherwise provided by the use thereof. As a result, anticoagulant therapy must be closely monitored. Certain anticoagulants act by inhibiting the synthesis of vitamin K-dependent coagulation factors resulting in the sequential depression of, for example, factors II, VII, IX, and X. Representative anticoagulants which are used

clinically include coumarin, dicoumarol, phenindione, and phenprocoumon.

Thrombolytics act by lysing thrombi after they have been formed. Thrombolytics such as streptokinase and urokinase have been indicated for the management of acute myocardial infarctions and have been used successfully to remove

intravascular clots if administered soon after thrombosis occurs. However, the lysis effected thereby may be

incomplete and nonspecific, i.e., useful plasma fibrinogen, in addition to fibrin polymers within clots, is affected. As a result, a common adverse reaction associated with the use of such therapeutics is hemorrhage.

A third classification, antiplatelet drugs, includes drugs which suppress primary hemostasis by altering platelets or their interaction with other circulatory system

components. The present invention relates to this

classification of antiplatelet drugs.

Reported Developments

Specific antiplatelet drugs operate by one or several mechanisms. A first example involves reducing the availability of ionized calcium within the platelet cytoplasm thereby

impairing activation of the platelet and resultant

aggregation. Pharmaceuticals representative of this strategy include prostacyclin, and also Persatine^® (dipyridamole) which may affect calcium concentrations by affecting the concentration of cyclic AMP. Numerous side effects related to the administration of these compounds have been reported. An additional class of antiplatelet drugs acts by inhibiting the synthesis of thromboxane A₂ within the platelet, reducing the platelet activation response. Non- steroidal anti-inflammatory agents, such as ibuprofen, phenolbutazone and napthroxane may produce a similar effect by competitive inhibition of a particular cyclooxygenase enzyme, which catalyzes the synthesis of a precursor of thromboxane A₂. A similar therapeutic effect may be derived through the administration of aspirin which has been

demonstrated to irreversably acetylate a cyclooxygenase enzyme necessary to generate thromboxane A₂.

A third anti-platelet mechanism has involved the platelet membrane so as to interfere with surface receptor function. One such drug is dextran, a large branched polysaccharide, which is believed to impair the interaction of fibrinogen with platelet receptors that are exposed during aggregation. Dextran is contraindicated for patients with a history of renal problems or with cardiac impairment. The therapeutic ticlopidine is stated to inhibit platelet adhesion and aggregation by suppressing the binding of von Willebrand factor and/or fibrinogen to their respective receptors on the platelet surface. However, it has been found that ticlopidene possesses insufficient specificity to eliminate the necessity of administering large doses which, in turn, may be associated with clinical side effects.

The aforementioned pharmaceuticals are foreign to the body and may cause numerous adverse clinical side effects, there being no way to prevent such compounds from

participating in other aspects of a patient's physiology or biochemistry, particularly if high doses are required. It would be desirable to provide for pharmaceuticals having such specificity for certain of the reactions of hemostasis, that they could be administered to patients at low doses, such doses being much less likely to produce adverse effects in patients. An example of a pharmaceutical which is representative of a therapeutic that is derived from natural components of the hemostatic process is described in EPO Publication No. 317278. This publication discloses a method for inhibiting thrombosis in a patient by administering to the patient a therapeutic polypeptide comprised of the amino-terminal region of the α chain of platelet membrane glycoprotein lb, or a subfragment thereof.

The present invention is directed to the provision of antithrombotic polypeptides based on von Willebrand factor, one of the proteins of the hemostatic mechanism.

Summary of the Present Invention

In accordance with the present invention, there is provided: (A) a polypeptide comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain and having a predetermined affinity for the GPIbα receptor of platelets; and (B) an anionic material having affinity for said amino acid residue domain 509-695 or subfragment thereof and which, in the presence of both mature vWF and said polypeptide, has a greater affinity for said polypeptide than said mature vWF.

In preferred form, the present invention encompasses a derivatized polypeptide comprising: (A) a polypeptide portion comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain and having a predetermined affinity for the GPIbα receptor of platelets; and (B) an anionic portion having affinity for said amino acid residue domain 509-695 or subfragment thereof; wherein said portions are linked by one or more bonds; and wherein said derivatized polypeptide has an increased binding

affinity for GPIbα receptor relative to said predetermined affinity of said subunit or fragment. Another aspect of the present invention encompasses the provision of a process for preparing a derivatized

polypeptide comprising: (A) providing a polypeptide

comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain, and having a predetermined affinity for the GPIb receptor of platelets, the polypeptide including also one or more reactive groups; (B) providing an anionic material having affinity for said amino acid residue domain 509 to 695 or subfragment thereof of said polypeptide and having also one or more groups which are reactive with the reactive group (s) of said polypeptide; and (C) reacting said polypeptide and said anionic material under conditions such that the reactive groups of the polypeptide and of the anionic material form one or more covalent bonds, to thereby form a derivatized polypeptide that has an increased binding affinity for the GPIb receptor of platelets relative to the predetermined affinity of said polypeptide. Other aspects of the invention are set forth

hereinbelow.

Polypeptides of the present invention are useful as antithrombotic agents and include, for example, biologically active polypeptides which are effective in preventing adhesion of platelets to surfaces, in inhibiting activation or aggregation of platelets, and in inhibiting thrombosis. By way of explanation, it is noted that von Willebrand factor, that is, the protein on which the polypeptides of the present invention are based, exists in humans as a series of high molecular weight multimers of up to 30 glycosylated subunits per multimer. The subunits are believed to be identical, with each having an approximate molecular weight of 270,000 (270 kDa) . Each circulating "mature" human subunit consists of 2,050 amino acid residues. In the formation of a clot, an initial monolayer of platelets covering injured endothelial surfaces is believed to involve a bridging function in which surface bound multimeric vWF binds on the one side to components of the subendothelium, such as collagen or proteoglycans, and on the other side to the GPIb-IX receptor of a platelet membrane. It is believed that the interaction of multimeric vWF with glycoprotein Ib- IX complex (at GPIbα) results in platelet activation and facilitates the recruitment of additional platelets which function to form a growing thrombus. The rapidly

accumulating platelets are also crosslinked by the binding of fibrinogen. Of particular importance in this process is the multimeric and multivalent character of circulating vWF, which enables the macromolecule to effectively carry out its binding and bridging functions.

It is believed that compositions to which the present invention relates in effect compete with vWF factor for GPIbα receptors and inactivates the receptors so they are not available for interaction with vWF factor, the result being that the formation of clots is inhibited.

Polypeptides of the present invention possess high specificity for target binding domains on other

macromolecules, including platelet receptors involved in the hemostatic mechanism. Generally speaking, polypeptides of the present invention are believed to function by preventing platelet adhesion, activation and aggregation, and are expected to be effective at concentrations which are not associated with clinically disadvantageous side effects.

Brief Description of the Drawings

Figure 1 is a table which shows the previously reported amino acid and DNA sequence for the mature von Willebrand factor subunit (human) between residue 431 and residue 750 thereof (see also SEQ ID NO: 1).

Figure 2 is a drawing of the disulfide dependent

association of two 52/48 kDa vWF fragments to form a 116 kDa homodimer. Figure 3 is a graph which shows the effect of two Type IIB mutations on the ability of bacterially expressed vWF fragments to bind to platelets.

Figure 4 is a graph which shows the effect of a single Type IIB mutation on the ability of bacterially-expressed vWF fragments to bind platelets at two different concentrations of a monoclonal antibody which competes with vWF fragments for platelet GPIbα receptor.

Figure 5 is a map of the pCDM8 plasmid. Figure 6 is a graph which shows the effect of the Trp⁵⁵⁰→ Cys⁵⁵⁰ mutation on the affinity of the reduced and alkylated 36 kDa vWF fragment for platelet GPIbα receptor.

Figure 7 is a graph which shows the effect of the Trp⁵⁵⁰→ Cys⁵⁵⁰ mutation on the affinity of the 116 kDa homodimeric vWF fragment for platelet GPIbα receptor.

Figure 8 is a drawing of the preferred anionic oligomer 4-[4-[4-[4-[4-[4-[4-(2-Carboxyethyl-5-hydroxybenzyl)CMB]CMB]CMB]CMB]CMB]CMB]-2-carboxyethyl-5-methoxytoluene, wherein CMB represents 2-carboxyethyl-5-methoxybenzyl, useful in the formation of derivatized vWF polypeptides.

Figure 9 contains a graph (middle panel) showing the ability of certain fragments of vWF to inhibit binding of botrocetin to multimeric vWF. The upper panel of the figure shows the letter-number designations for peptides tested, and the lower panel shows efficacy of the certain active

peptides.

Figure 10 is a graph showing the effect of ATA-related compound on the binding of multimeric vWF to platelets. Figure 11 is a graph showing that the ATA-related

compound competes with botrocetin for binding sites on vWF. Definitions

Unless indicated otherwise herein, the following terms have the indicated meanings.

Pre-pro-vWF - von Willebrand factor is subject to extensive posttranslational processing. "Pre-pro-vWF" contains (from the N to the C terminus) a signal peptide comprised of approximately 22 amino acid residues, a propeptide of

approximately 741 amino acids, and then the approximate 2,050 residues of circulating vWF. Pro-vWF - The signal peptide has been removed from pre-pro-vWF.

Wild Type Amino Acid Sequence - refers to the amino acid sequence of mature vWF subunit, or of a fragment thereof, which is present in the large majority of humans, and refers also to any mutant amino acid sequence as isolated from vWF of a particular person if no detectable functional

differences in the vWF with respect to its interaction with GPIbα result therefrom in humans.

Peptide - for the purposes of this invention, the terms

"peptide" and "polypeptide" are interchangable.

Monomeric - when used with respect to polypeptides,

"monomeric" refers to a polypeptide which is not covalently linked to another polypeptide. "Dimeric" refers to a

covalent association of two monomers. Table 1 shows the standard three letter designations for amino acids as used in the application. TABLE I

Alanine Ala

Cysteine Cys

Aspartic Acid Asp

Glutamic Acid Glu

Phenylalanine Phe

Glycine Gly

Histidine His

Isoleucine He

Lysine Lys

Leucine Leu

Methionine Met

Asparagine Asn

Proline Pro

Glutamine Gln

Arginine Arg

Serine Ser

Threonine Thr

Valine Val

Tryptophan Trp

Tyrosine Tyr

Background Information

A "Sequence Listing" pursuant to 37 CFR §1.821(c) for nucleotide and amino acid sequences disclosed or referred to herein is appended and made part of this application.

As set forth above, the present invention encompasses the use of polypeptides which are based, for example, upon subunits or fragments of the natural occurring protein von Willebrand factor. For informational purposes, there is set forth hereafter information concerning this protein and its role in hemostasis and thrombosis.

Description of the Role of vWF in Hemostasis and Thrombosis vWF performs an essential role in normal hemostasis during vascular injury and is also of central importance in the pathogenesis of acute thrombotic occlusions in diseased blood vessels. Both of these roles involve the interaction of vWF with platelets which are induced to bind at the affected site and are then crosslinked. It is believed that single platelets first adhere to a thrombogenic surface after which they become activated, a process involving major metabolic changes and significant morphological changes within the platelet. Activation is evidenced by the discharge of platelet storage granules containing adhesive substances such as von Willebrand factor (an adhesive protein), and the expression on the surface of the platelet of additional functional adhesive sites, once activated, and as a part of normal hemostasis, platelet cells become aggregated, a process which involves extensive crosslinking of the platelet cells with additional types of adhesive proteins. As stated above, these processes are normal as a

physiologic response to vascular injury. However, they may lead in pathologic circumstances, such as in diseased

vessels, to formation of undesired platelet thrombi with resultant vascular occlusion. Other circumstances in which it is desirable to prevent deposition of platelets in blood vessels include the

prevention and treatment of stroke, and to prevent occlusion of arterial grafts. Platelet thrombus formation during surgical procedures may also interfere with attempts to relieve preexisting vessel obstructions.

The adhesion of platelets to damaged or diseased vessels occurs through mechanisms that involve specific platelet membrane receptors which interact with specialized adhesive molecules. One such platelet receptor is the glycoprotein Ib-IX complex which consists of a noncovalent association of two integral membrane proteins, glycoprotein lb (GPIb) and glycoprotein IX (GPIX). The adhesive ligand of the GPIb-IX complex is the protein von Willebrand factor which is found as a component of the subendothelial matrix, as a component of the α-granules secreted by activated platelets, and also as a circulating blood plasma protein. The actual binding site of the vWF to the GPIb-IX receptor has been localized on the amino terminal region of the α chain of glycoprotein lb which is represented by GPIb(α). As mentioned above, von Willebrand factor exists as a series of high molecular weight multimers of up to 30 glycosylated subunits per multimer in which the subunits are believed to be identical, with each having an approximate molecular weight of 270,000 (270 kDa). It is believed that the interaction of multimeric vWF with glycoprotein Ib-IX complex (at GPIb(α)) results in platelet activation and facilitates the recruitment of additional platelets to a now growing thrombus. The rapidly accumulating platelets are also crosslinked (aggregated) by the binding of fibrinogen at platelet glycoprotein IIb-IIIa receptor sites, and possibly also by vWF at these sites, and/or at additional glycoprotein Ib-IX receptor sites. In addition, the glycoprotein Ilb/IIIa receptor may also be involved in the formation of the initial monolayer of platelets. Of particular importance in this process is the multimeric and multivalent character of circulating vWF, which enables the macromolecule to

effectively carry out its binding and bridging functions.

Inactivation of the GPIbα or GPIIb/IIIa receptors on the platelets of a patient or inactivation of the binding sites for vWF located in the subendothelium of a patient's vascular system, thereby inhibiting the bridging ability of vWF, is considered important for treating or inhibiting thrombosis. It is believed that the composition of the present invention is effective in accomplishing the foregoing.

As mentioned above, the circulating "mature" human subunit of vWF consists of 2050 amino acid residues. The domain of the vWF subunit which binds to the platelet

membrane glycoprotein Ib-IX receptor (GPIb(α)) has been identified within a fragment of vWF. The fragment may be generated by trypsin digestion, followed by disulfide

reduction, and extends from approximately residue 449

(valine) of the circulating subunit to approximately residue 728 (lysine) thereof. This fragment has an apparent

molecular weight of approximately 52,000. The GPIbα binding domain of vWF comprises residues contained in two

discontinuous sequences cys⁴⁷⁴-Pro⁴⁸⁸ and Leu⁶⁹⁴-Pro⁷⁰⁸ within the fragment. Mohri, H. et al., J. Biol. Chem.. 263(34), 17901-17904 (1988).

Typically, the 52,000 molecular weight fragment is referred to as a "52/48" fragment reflecting the fact that human enzyme systems glycosylate the fragment contributing to its molecular weight. The amount of glycosylation varies from molecule to molecule, with two weights, 52,000 and

48,000, being most common. As expressed from recombinant bacterial host cells, the fragment lacks the

posttranslational glycosylation associated with expression thereof in mammalian cells. Without the additional weight contributed by glycosylation, the polypeptide has a molecular weight of approximately 33,000.

Current evidence indicates that the fragment also contains between residues 509 and 695 thereof binding domains for components of the subendothelium, such as collagen and proteoglycans, although other regions of the mature vWF subunit may be more important in recognizing these substances (an additional proteoglycan or heparin binding site is located in residues 1-272 of the mature subunit and an additional collagen binding site within residues 910-1110 thereof). The tetrapeptide Arg· Gly· Asp· Ser (SEQ ID NO: 2) (residues 1744 to 1747), a sequence which vWF shares with many other adhesive proteins, is believed to represent the platelet glycoprotein IIb-IIIa binding site. The primary and tertiary structure of von Willebrand factor and the location of functional domains thereof is reviewed by Titani, K. et al., "Primary Structure of Human von Willebrand Factor" in Coagulation and Bleeding Disorders: The Role of Factor VIII and von Willebrand Factor. T. Zimmerman and Z.M. Ruggeri, eds., Marcel Dekker, New York, 1989.

Figure 1 shows the previously reported amino acid and DNA sequence for the mature von Willebrand factor subunit (human) between residue 431 and residue 750. The 52/48 kDa fragment produced by tryptic digestion has an amino terminus at residue 449 (valine) and extends approximately to residue 728 (lysine). Amino acids are shown by standard three letter designations. The DNA sequence is represented by the coding strand (non-transcribed strand). Very little polymorphism has been reported in the 52/48 human sequence with one significant exception - histidine/aspartic acid at position 709, see Mancuso, D.J. et al. J. Biol. Chem., 264(33), 19514- 19527, Table V, (1989). (See also SEQ ID No: 1).

Brief Reference to Developments of Parent Applications

Parent applications of the present application, as identified hereinabove, disclose therapeutic polypeptides which have antithrombotic properties and which are based on mature vWF subunit or a fragment thereof or on a mutated form thereof, including recombinant forms of the aforementioned.

Thus, parent application Serial No. 07/519,606 discloses that a polypeptide derived from circulating (mature) vWF subunit (approximately residue 449 to approximately residue 728) or a fragment thereof is considered useful as an

antithrombotic pharmaceutical when added to blood in an amount sufficient to compete successfully with multimeric vWF for platelet GPlb(α) receptor sites. The pharmaceutical functions to prevent monolayer formation by, or crosslinking of, platelets in circumstances where thrombus formation is undesirable, such as in the treatment of vascular disorders. The '606 application identifies numerous publications which relate to the structure, function and molecular genetics of von Willebrand factor, such publications being incorporated herein by reference.

Parent Application Serial No. 07/600,183 discloses antithrombotic polypeptides which are based on the

aforementioned residue 449-728 region of vWF subunit and which are generated from recombinant bacterial host cells. Particular polypeptides which are described in the

application have improved stability owing to replacement by mutagenesis of cysteine residues therein, except those

involved in stabilizing the residue 509-695 loop region of the fragment. Parent application Serial No. 07/613,004 discloses a similar vWF fragment, but one which is generated from

recombinant mammalian host cells and which is further improved relative to the polypeptide of the '183 application in that it retains the normal tertiary structure of the fragment as determined from the comparable sequence of the blood-derived polypeptide, is properly glycosylated, and remains in monomeric and therapeutically useful form owing to replacement by mutagenesis of cysteine residues responsible for the association of vWF subunits.

The development of the present invention provides still further improvements in the antithrombotic field.

Detailed Description of the Invention

The Polypeptide

The polypeptide which comprises the composition of the present invention or which is the source of the polypeptide portion of the derivatized polypeptide of the present

invention comprises mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain, the subunit comprising 2,050 residues. This polypeptide, which, for convenience, is referred to herein on occasions as "vWF-based polypeptide", can be derived from natural sources, such as the blood, or by expression from recombinant cells,

including, for example, mammalian and bacterial cells and can comprise a structure which corresponds to the structure of the natural form thereof or a mutated form of the natural-occurring structure. Any mutated form of the vWF-based polypeptide can be used provided that the mutations do not substantially interfere adversely with the desired biological activity of the polypeptide.

Examples of effective mutated forms of the vWF-based polypeptide which can be used in the practice of the present invention are the subject of parent applications identified hereinabove and summarized below. vWF-Based Polypeptides of Parent

Application Serial No. 07/600,183

The vWF-based polypeptides of the '183 application are derived from the residue 449-728 region of the mature von Willebrand factor subunit and have antithrombotic properties.

In one embodiment, the polypeptides are patterned upon a parent polypeptide and comprise the amino acid sequence of that fragment of mature von Willebrand factor subunit which begins approximately at residue 441 (arginine) and ends at approximately residue 733 (valine), or any subset thereof, and have one or more of the cysteine residues normally present in the parent polypeptide, or subset thereof, deleted and/or replaced by one or more other amino acids. Such polypeptides have less tendency than the parent polypeptide, or subset thereof, to form intra or interchain disulfide bonds in aqueous media at a physiological pH.

In another embodiment of the '183 application, the polypeptide comprises the amino acid sequence from

approximately residue 441 (arginine) to approximately residue 733 (valine) of mature von Willebrand factor subunit, or any subset of said sequence which contains residues 509

(cysteine) and 695 (cysteine), and in which one or more of cysteine residues 459, 462, 464, 471, and 474 are deleted or replaced by one or more other amino acids. vWF-based polypeptides of the aforementioned embodiments can be made from DNA which encodes that fragment of mature von Willebrand factor subunit comprising essentially the amino acid sequence from approximately residue 441 (arginine) to approximately residue 733 (valine), or which encodes any subset of said amino acid sequence, a mutant polypeptide fragment, or subset thereof, which contains fewer cysteine residues than that of the comparable wild-type amino acid sequence. Preparation of the molecules comprises culturing a host organism transformed with a biologically functional expression plasmid which contains a mutant DNA sequence encoding a portion of said von Willebrand factor subunit under conditions which effect expression of the mutant von Willebrand factor fragment, or a subset thereof, by the host organism and recovering said fragment therefrom. The preferred means for effecting mutagenesis of cysteine codons in a vWF DNA to codons encoding amino acids incapable of disulfide bonding is based upon the site

directed mutagenesis procedure of Kunkel, T.A., Proc. Natl. Acad. Sci. U.S.A.. 82, 488-492 (1985). An important aspect of the development described in the '183 application is the provision of compositions of vWF-based polypeptides which are less prone to aggregation and denaturation caused by undesired disulfide bonding within the inclusion bodies of host expression cells (or resultant from inclusion body solubilization procedures) than previous preparations. The development employs mutagenesis to limit the number of cysteine residues present within said

polypeptides.

More specifically, the development encompasses the preparation of a mutant polypeptide fragment which

corresponds to that fragment of mature von Willebrand subunit having an amino terminus at residue 441 (arginine) and a carboxy terminus at residue 733 (valine), but which differs therefrom in that each of the cysteine residues thereof is replaced by a glycine residue. The development also

recognizes that the retention of a certain disulfide bond within polypeptides corresponding to the 449-728 vWF subunit region is particularly important for the design of

therapeutic molecules derived therefrom. To accomplish this, a cDNA clone encoding the von

Willebrand factor gene (for the pre-propeptide) is utilized. The cDNA is then subjected to enzymatic amplification in a polymerase chain reaction using oligonucleotides which flank the indicated region. The first oligonucleotide representing coding strand DNA contains an EcoRI site 5' to the codon for residue 441 (arginine) and extends to the codon for residue 446 (glycine). The second oligonucleotide, corresponding to non-coding strand DNA, encodes amino acids 725 to 733 and encodes 3' to codon 733 a HindIII restriction sequence. The resultant double stranded von Willebrand factor cDNA

corresponding to the amino acid sequence from residue 441 to residue 733 (of the mature subunit) is then inserted, using EcoRI and HindIII restriction enzymes, into the double stranded replicative form of bacteriophage M13mp18 which contains a multiple cloning site having compatible EcoRI and HindIII sequences. Following the procedure of Kunkel, T.A., Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985), site directed mutagenesis is performed using hybridizing oligonucleotides suitable for replacing all of the cysteine codons (residue positions 459, 462, 464, 471, 474, 509 and 695) with

individual glycine codons (see Example 1) or 5 of the

cysteine codons (residue positions 459, 462, 464, 471 and 474) with individual glycine codons (see Example 4). Mutant double stranded vWF cDNA fragments derived from the procedure are removed from M13mp18 phage by treatment with EcoRI and HindIII restriction endonucleases, after which the ends of the vWF cDNA fragments are modified with BamHI linkers.

The two types of mutant vWF cDNA, containing either 5 or 7 Cys to Gly mutations, are then separately cloned into the pET-3A expression vector (see Rosenberg, A.H. et al., Gene. 56, 125-136 (1987)) for expression from E.coli strain

BL21(DE3), Novagen Co., Madison, WI. pET-3A vehicle

containing cDNA for the vWF subunit fragment with 7 cysteine to glycine mutations is referred to as "p7E", and as "p5E" when the contained vWF cDNA fragment encoded the 5 above specified cysteine to glycine mutations. Mutant von

Willebrand factor polypeptides produced by bacterial cultures containing expression plasmid p5E were compared with those expressed from cultures containing p7E plasmids. The p5E molecule is capable of forming a disulfide bond between cysteine residue 509 and 695 whereas the p7E molecule cannot. The behavior of p5E and p7E extracts was examined using immunological methods (see Example 5). vWF-specific murine monoclonal antibodies RG-46 and NMC-4 were used as probes. RG-46 has been demonstrated to recognize as its epitope a linear sequence of amino acids, comprising residues 694 to 708 within the mature von Willebrand factor subunit. The binding of this antibody to its determinant is essentially conformation independent. Mohri, H. et al., J. Biol. Chem.. 263(34), 17901-17904 (1988). NMC-4 however, has as its epitope the domain of the von Willebrand factor subunit which contains the glycoprotein lb binding site. Mapping of the epitope has demonstrated that it is contained within two discontinuous domains (comprising approximately mature vWF subunit residues 474 to 488 and also approximately residues 694 to 708) brought into disulfide-dependent association (Mohri, H. et al., supra), although it was not determined whether the disulfide bond conferring this tertiary conformation in the native vWF molecule was

intrachain or interchain. Id. at 17903. Accordingly, 1.5 μg samples (of protein) were first run on 10% SDS-polyacrylamide gels so that the antigenic behavior of particular bands

(under reducing and nonreducing conditions) could be compared with results obtained by Coomassie blue staining.

Immunoblotting ("Western Blotting") according to a standard procedure, Burnette, A. Anal. Biochem. , 112, 195-203 (1981), was then performed to compare p5E and p7E extracts.

Briefly, it was found that, under nonreducing

conditions, the single chain p5E polypeptide fragment

(representing the sequence from residue 441 to residue 733) displays an approximate 120 fold increase in binding affinity for NMC-4 compared to the comparable cysteine-free species isolated from p7E. After electrophoresis under reducing conditions (utilizing 100 mM DTT), the single chain p5E species shows a remarkably decreased affinity for NMC-4, which was then very similar to that of the cysteine-free p7E species under either reduced or nonreduced conditions. NMC-4 also failed, under reducing or non-reducing conditions, to recognize as an epitope disulfide-linked dimers from the p5E extract.

The nitrocellulose filters used to produce autoradiographs based on NMC-4 were rescreened with RG-46 by

subtracting the initial NMC-4 exposure response, which was kept low through a combination of low antibody titer and short exposure time. The binding of RG-46 to the 36,000 kDa p7E polypeptide on the filters was the same whether reducing or non-reducing conditions were chosen, consistent with the replacement of all cysteines by glycine in the expressed polypeptide.

A large molecular weight vWF antigen (reactive to RG-46) was present in the p5E polypeptide extract under nonreducing conditions. These p5E vWF aggregates (reflecting interchain disulfide bonds) migrated under reducing conditions in the same position as the p7E polypeptide indicating disruption of their disulfide contacts. However, the large p5E interchain disulfide aggregates which are readily recognized under nonreducing conditions by RG-46 were not recognized by NMC-4 under either reducing or nonreducing conditions. It was thus demonstrated that the disulfide bond between residues 509 and 695 in native multimeric vWF subunits represents an

intrachain contact.

The disulfide bond between residues 471 and 474 of the mature vWF subunit has previously been shown to be an

intrachain contact, thus the aforementioned embodiment is able to suggest that interchain disulfide bond(s) in

multisubunit mature vWF would be formed using one or more of cysteine residues 459, 462 or 464. The 52/48 tryptic fragment of the mature vWF subunit has been established to comprise the amino acid sequence between residues 449 and 728. Contained within that sequence is a subfragment consisting approximately of residues 500 to 700 known as the A₁ domain. This domain has substantial amino acid sequence homology to the A₂ and A₃ domains of the 2,050 residue subunit and which are located at approximately residue positions 710-910 (A₂) and 910-1110 (A₃). See Titani, K. et al "Primary Structure of Human von Willebrand Factor" in Coagulation and Bleeding Disorders, Marcel Dekker, New York, 1989.

It has been discovered that these "A" domains also share substantial amino acid sequence homology (at minimum

approximately 15-20%) with similar domains in numerous other adhesive proteins. Twenty percent sequence homology is generally recognized as being far to great to appear by chance. As recognized by Mancuso, D.J. et al J. Biol Chem. 264(33) 19514-19527 (1989), these homologies clearly suggest that the vWF subunit is the product of a mosaic gene which contains subregions shared by many other proteins. These homologies probably arose from repeated gene segment

duplication and exon shuffling.

Pharmacologically active collagen binding polypeptides can be derived from the "A₃" domain. The A₃ domain contains also a pair of cysteine residues which are believed to form, in vivo, a loop analogous to the residue 509-695 A, loop structure. The potential utility of this new mutant vWF fragment as an inhibitor of the binding of multimeric vWF to collagen can be demonstrated following the procedures of Pareti, F.I. et al., J. Biol. Chem., 262(28), 13835-13841 (1987) and Mohri, H. et al., J. Biol. Chem., 264(29), 17361-17367 (1989). vWF-Based Polypeptides of Parent

Application Serial No. 07/613 , 004

The development described in the '004 application includes the recognition of certain of the roles that are performed by cysteine residues present in the residue 449-728 primary sequence fragment of the mature vWF subunit. In this connection, work associated with the development confirms that the cysteine 509-695 disulfide bond is an intrachain bond and provides for effective therapeutics incorporating the 509-695 bond for the purpose of treating thrombosis.

The antithrombotic polypeptides of the '004 application are based upon that amino acid sequence domain which

comprises approximately residues 449 to 728 of the mature von Willebrand factor subunit and which, if fully glycosylated, would be equivalent in weight to the 52/48 kDa vWF subunit fragment. In practice it is difficult to derive

therapeutically useful quantities of such polypeptides from blood plasma. Difficulties include effective separation of 116 kDa and 52/48 kDa fragments from other components of tryptic digests and effective sterilization of blood-derived components from human viruses such as hepatitis and HIV. In addition, methods reported in the literature to generate the 52/48 kDa monomer from the 116 kDa dimer have utilized complete disulfide reduction with resultant loss of tertiary structure. Certain important manipulations of the 52/48 fragment, such as replacement of selective cysteine residues to improve product utility and stability, can only be

accomplished in a practical sense by recombinant DNA

technology.

However, the production by recombinant DNA-directed means of therapeutic vWF polypeptides analogous to the 52/48 tryptic fragment has met with certain limitations. It is desirable that the polypeptide not only be made by the host cells but that it be correctly folded for maximum therapeutic utility. It is believed that the principal factor which has to date prevented the expression of the most therapeutically active forms of the 52/48 fragment is the incorrect folding of the molecule caused by the linking up of cysteine residues to form incorrect disulfide contacts. In addition, such polypeptides appear to exhibit hydrophobic properties or solubility problems which would not be encountered if they were to be contained within the entirety of the natural vWF subunit, or were properly glycosylated. Of critical importance, therefore, to the synthesis of vWF-derived therapeutic polypeptides is the selection of conditions which minimize the formation of improper disulfide contacts. Prior expression of such polypeptides from

recombinant DNA in host bacterial cells has certain

disadvantages. With reference to the embodiments of the aforementioned '183 application, newly produced vWF

polypeptides are unable to escape from the host cells, causing them to be accumulated within insoluble aggregates therein (inclusion bodies) where the effective concentration of cysteine residues was extremely high. Under these

circumstances, disulfide bonds not characteristic of the vWF molecule as it naturally exists in the plasma are encouraged to, and do, form either within the inclusion bodies or during attempts to solubilize the polypeptide therefrom.

The development of the '004 application provides a solution to these difficulties by causing the vWF-derived polypeptides to be expressed in mammalian cells using a DNA sequence which encodes the polypeptide and which also encodes for a signal peptide, the presence of which causes the vWF polypeptide to be secreted from the host cells. Incorrect disulfide bond formation is minimized by limiting the

accumulation of high local concentrations of the polypeptide as in inclusion bodies. In addition, enzymes present in the host eucaryotic cells, unlike bacteria, are able to glycosylate (add

carbohydrate chains to) the vWF-derived polypeptides

resulting in therapeutic molecules which more closely

resemble domains of vWF molecules derived from human plasma. The recombinant 116 kDa polypeptide generated according to the development of the '004 application is demonstrated to represent a dimer of the subunit fragment consisting of residues 441-730 and possesses an amount of glycosylation equivalent to that found in the comparable region of plasma-derived vWF. There follows hereafter a description of the types of therapeutic vWF-derived polypeptides which have or may be generated according to the effective recombinant procedures of the '004 application. Stated broadly, the polypeptides of the '004 application include any fragment of mature von Willebrand subunit comprising that sequence of amino acids between approximately residue 449 and approximately residue 728, or a subfragment thereof, from which at least one of cysteine residues 459, 462 and 464 thereof is removed. Such removal reduces the tendency of the fragment to form undesired interchain

disulfide bonds (and resultant dimers) with the result that therapeutic utility as an antithrombotic is improved.

A further aspect of the embodiment encompasses a

glycosylated form of the above defined polypeptides.

In the design of antithrombotic polypeptides derived from the aforementioned region of vWF, it is preferred that cysteine residues be retained at positions 509 and 695 so that the tertiary structure of the GPIb(α) binding domain of the mature vWF subunit fragment is preserved.

Also preferred is a glycosylated polypeptide derived from the aforementioned region of vWF in which cysteine residues are retained at positions 509 and 695 and in which each of cysteine residues 459, 462 and 464 is deleted or replaced by residues of other amino acids.

Additionally preferred is a glycosylated polypeptide derived from the aforementioned region of vWF in which cysteine residues are retained at positions 509 and 695 and in which any one of cysteine residues 459, 462 and 464 is deleted or replaced by a single residue of another amino acid. Important factors involved in the design of, or further modification to, the preferred mutant polypeptides

(antithrombotics) of the invention are described hereafter.

Potential binding sites for collagens and

glycosaminoglycans (or proteoglycans) exist in the 449-728 tryptic fragment in the loop region between cysteine residues 509 and 695. In the event that binding at these sites by such macromolecules impairs the antithrombotic therapeutic utility of any of the recombinant polypeptides by, for example, also providing bridging to collagen, the polypeptide can be redesigned (for example, by proteolysis, covalent labelling or mutagenesis) to delete or alter the loop region, or a subdomain thereof.

It is known that both platelets and von Willebrand factor molecules contain large numbers of negative charges such as, for example, those contributed by sialic acid. Such charges can facilitate desirable mutual repulsion of the molecules under non-injury conditions. The addition of one or more positively charged residues of lysine and/or of arginine extending, for example, from the amino and/or from the carboxy terminus of the 52/48 tryptic fragment or

recombinant equivalents thereof can overcome electrical repulsions with respect to the GPIb(α) receptor, thus

facilitating use of the fragment as an antithrombotic

therapeutic.

In addition, and with respect to polypeptides patterned upon the 449-728 vWF subunit fragment, it is within the scope of the invention to remove certain cysteine residues by site directed mutagenesis and to thereafter inactivate any

remaining cysteine residues by chemical inactivation thereof, such as, for example, by S-carboxymethylation.

There follows hereafter a discussion of means by which polypeptides can be prepared and, in particular, by which such polypeptides can be effectively secreted from host cells in proper folded form and possessing preferably only those disulfide bonds whose presence is consistent with therapeutic utility.

Essential elements necessary for the practice of the embodiment are: (A) a DNA sequence which encodes the residue 449-728 domain of the mature vWF subunit; (B) an expression plasmid or viral expression vector capable of directing in a eucaryotic cell the expression therein of the aforementioned residue 449-728 domain; and (C) a eucaryotic host cell in which said expression may be effected. The expression of the DNA sequence of the von Willebrand factor subunit fragment is facilitated by placing a

eucaryotic consensus translation initiation sequence and a methionine initiation codon upstream (5') to the residue 449- 728 encoding DNA. The vWF DNA sequence may be a cDNA

sequence, or a genomic sequence such as, for example, may be produced by enzymatic amplification from a genomic clone in a polymerase chain reation. Expression of the residue 449-728 encoding sequence is further facilitated by placing

downstream therefrom a translation initiation codon such as TGA. The vWF-polypeptide so expressed typically remains within the host cells because of the lack of attachment to the nascent vWF polypeptide of a signal peptide. In such a situation, purification of proteins expressed therein and the extraction of pharmacologically useful quantities thereof are more difficult to accomplish than if the polypeptide were secreted into the culture medium of the host cells. Such expression systems are nonetheless useful for diagnostic assay purposes such as, for example, testing the proper function of platelet GPIb-IX receptor complexes in a patient. In the preferred practice of the invention in which the polypeptide is secreted from the host cell, there is provided a vWF-encoding DNA sequence for insertion into a suitable host cell in which there is also inserted upstream from the residue 449-728 encoding sequence thereof a DNA sequence encoding the vWF signal peptide (see Example 7). Other vWF-encoding DNA sequences corresponding to different regions of the mature vWF subunit, or corresponding to the propeptide, or to combinations of any of such regions, may be similarly expressed by similarly placing them downstream from a vWF signal peptide sequence in a suitable encoding DNA. When attached to the amino terminal end of the residue 449-728 fragment of the vWF subunit, the signal peptide causes the fragment to be recognized by cellular structures as a polypeptide of the kind to be processed for ultimate

secretion from the cell, with concomitant cleavage of the signal polypeptide from the 449-728 fragment.

With respect to the construction of a eucaryotic expression system and the expression therein of the tryptic 52/48 kDa domain of mature subunit vWF (the residue 449-728 fragment), it has been found (see Example 7) to be conveneint to manipulate a slightly larger fragment represented by residues 441 (arginine) to 730 (asparagine). Other similar fragments containing small regions of additional amino acids (besides the 449-728 residue sequence), which additional amino acids do not significantly affect the function of said fragment, may also be expressed.

Similarly, functional fragments may be expressed from which, when compared to the 449-728 fragment, several residues adjacent to the amino and carboxy terminals have been removed as long as the GPIb(α) binding sequences are not compromised.

It has also been found to be effective, with respect to the construction of a suitable DNA sequence for encoding and expressing the residue 441-730 fragment, to cause to be inserted between the DNA encoding the carboxy terminus of the signal peptide and the codon for residue 441 codons for the first three amino acids of the vWF propeptide (alanineglutamic acid-glycine) said codons being naturally found directly downstream (3') to the signal sequence in the human vWF gene. A wide variety of expression plasmids or viral

expression vectors are suitable for the expression of the residue 441-730 mature vWF subunit fragment or similar vWF fragments. One factor of importance in selecting an

expression system is the provision in the plasmid or vector of a high efficiency transcription promoter which is directly adjacent to the cloned vWF insert.

Another factor of importance in the selection of an expression plasmid or viral expression vector is the

provision in the plasmid or vector of an antibiotic

resistance gene marker so that, for example, continuous selection for stable transformant eucaryotic host cells can be applied.

Examples of plasmids suitable for use in the practice of the invention include pCDM8, pCDM8^nco, pcDNA1, pcDNA1^nco, pMAM^nco and Rc/CMV. Preferred plasmids include pCDM8^nco, pcDNA1^nco, pMAM^nco and Rc/CMV.

Examples of viral expression vector systems suitable for the practice of the invention include those based upon retroviruses and those based upon baculovirus Autographa californica nuclear polyhedrosis virus.

Representative host cells comprising permanent cell lines suitable for use in the practice of the invention include CHO-K1 Chinese hamster ovary cells, ATCC-CCL·61; COS-1 cells, SV-40 transformed African Green monkey kidney, ATCC-CRL 1650; ATT 20 murine pituitary cells; RIN-5F rat

pancreatic β cells; cultured insect cells, Spodoptera

frugiperda; or yeast (Sarcomyces).

Example 7 contains a detailed explanation of preferred procedures used to express and secrete the 441-730 sequence. In that Example, the fragment is secreted as a homodimer held together by one or more disulfide bonds involving cysteine residues 459, 462 and 464. Expression of monomeric fragments useful as antithrombotics necessitates control be made of the disulfide bonding abilities of the monomers which is achieved most preferably by mutagenesis procedures as described below.

A variety of molecular biological techniques are

available which can be used to change cysteine codons for those of other amino acids. Suitable techniques include mutagenesis using a polymerase chain reaction, gapped-duplex mutagenesis, and differential hybridization of an

oligonucleotide to DNA molecules differing at a single nucleotide position. For a review of suitable codon altering techniques, see Kraik, C. "Use of Oligonucleotides for Site Specific Mutagenesis", Biotechniques. Jan/Feb 1985 at page 12.

It preferred to use the site-directed or site-specific mutagenesis procedure of Kunkel, T.A., Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985). This procedure takes advantage of a series of steps which first produces, and then selects against, a uracil-containing DNA template. Example 1

explains in detail the mutagenesis techniques used to create mutant vWF cDNA. Other publications which disclose site-directed

mutagenesis procedures are: Giese, N.A. et al., Science.

236, 1315 (1987); U.S. Patent No. 4,518,584; and U.S. Patent No. 4,959,314.

It is also preferred to cause to be substituted for one or more of the cysteine codons of the wild type DNA sequence codons for one or more of the following amino acids: alanine, threonine, serine, glycine, and asparagine. Replacement with alanine and glycine codons is most preferred. The selection of a replacement for any particular codon is generally independent of the selection of a suitable replacement at any other position.

The following are representative examples of the types of codon substitutions which can be made, using as an example cysteine residue 459: (A) the codon for cysteine 459 could be replaced by a codon for glycine; or

(B) the codon for cysteine 459 could be replaced by two or more codons such as one for serine and one for glycine, such replacement resulting in a new amino acid sequence: His⁴⁵⁸-Ser^459(a)-Gly^459(b)-Gln⁴⁶⁰; or

(C) the codon for cysteine 459 could be deleted from the cDNA, such deletion resulting in a shortened amino acid sequence represented by - - - His⁴⁵⁸- Gln⁴⁶⁰- - -; or

(D) one or more codons for residues adjacent to

cysteine residue 459 could be deleted along with codon 459 as represented by - - Glu⁴⁵⁷-Gln⁴⁶⁰- -.

It is contemplated that codons for amino acids other than alanine, threonine, serine, glycine or asparagine will also be useful in the practice of the invention depending on the particular primary, secondary, tertiary and quaternary environment of the target cysteine residue.

It is considered desirable to provide as a replacement for any particular cysteine residue of the 449-728 tryptic vWF subunit fragment an amino acid which can be accommodated at the cysteine position with minimal perturbation of the secondary structure (such as α-helical or β-sheet) of the wild type amino acid sequence subsegment within which the cysteine position is located. Alanine, threonine, serine, glycine and asparagine will generally be satisfactory because they are, like cysteine, neutrally charged and have side chains which are small or relatively small in size.

Substantial research has been conducted on the subject of predicting within which types of structural domains of proteins (α-helix, β-sheet, or random coil) one is most likely to find particular species of amino acids. Serine is a preferred amino acid for use in the practice of this invention because it most closely approximates the size and polarity of cysteine and is believed not to disrupt α-helical and β-sheet domains. Reference, for example, to Chou, P.Y. et al.,

Biochemistry, 13(2), 211-222 (1974) and Chou, P.Y. et al., "Prediction of Protein Conformation," Biochemistry. 13(2), 222-244 (1974) provides further information useful in the selection of replacement amino acids. Chou, P.Y. et al.

predicted the secondary structure of specified polypeptide sequence segments based on rules for determining which species of amino acids therein are likely to be found in the center of, for example, an alpha helical region, and which residues thereof would be likely to terminate propagation of a helical zone, thus becoming a boundary residues or helix breakers. According to Chou, P.Y. et al., supra, at 223, cysteine and the group of threonine, serine, and asparagine are found to be indifferent to α-helical structure, as opposed to being breakers or formers of such regions. Thus, threonine, serine and asparagine are likely to leave

unperturbed an α-helical region in which a potential target cysteine might be located. Similarly, glycine, alanine and serine were found to be more or less indifferent to the formation of β-regions. It is noted that serine, threonine and asparagine residues represent possible new sites of glycosylation making them potentially unsuitable replacement residues at certain positions in secretory proteins subject to glycosylation. Generally, the primary consideration which should be taken into account in connection with selecting suitable amino acid replacements is whether the contemplated

substitution will have an adverse effect on the tertiary structure of the fragment. Thus, other amino acids may be suitable as acceptable substitutes for particular cysteine residues as long as the new residues do not introduce undesired changes in the tertiary structure of the 449-728 fragment. Reactivity with NMC-4 antibody is recommended as a test of whether a mutant polypeptide has the desired

therapeutic properties.

The specific protocol used to generate the mutant vWF residue 441-730 fragment containing cysteine to glycine substitutions at each of residue positions 459, 462 and 464 is described in Example 9. The expression plasmid used therein was designated pAD4/Δ3C.

The specific protocol, adapted from that of Example 9, and which was used to generate the three mutant residue 441- 730 fragments, each of which contains a different single Cys → Gly mutation (at positions 459, 462 or 464) is described in Example 14. The respective expression plasmids used therein were designated pAD4/G⁴⁵⁹, PAD4/G⁴⁶² and pAD/G⁴⁶⁴ (collectively "the pAD4/ΔlC plasmids").

An important aspect of the development of the '004 application is the provision of glycosylated 52/48 kDa monomeric fragments of the vWF subunit having substantial elements of normal tertiary structure. Such fragments have a reduced tendency to form dimers which tend to be unsuitable for use as antithrombotic therapeutics.

Following the above described procedures for site directed mutagenesis, residue 441-730 vWF fragments were produced in which one or more of cysteine residues 459, 462 and 464 were replaced with glycine residues. Examples 9, 10 and 11 below explain the mutagenesis and cell culture

conditions necessary to create COS-1 cell transformants expressing these mutant vWF polypeptides. Examples 6 to 8 describe the properties of the molecules so derived in comparison with the recombinant 116 kDa polypeptide produced from pAD4/WT transformed COS-1 cells.

Preferred polypeptides for use in the practice of the present invention are described in the aforementioned '004 application and represent the glycosylated residue 441 to 730 subunit sequence, expressed from mammalian cells, in which one or more of cysteine residues 459, 462 and/or 464 thereof are deleted or replaced by other amino acids, thereby

avoiding disulfide induced dimerization of the fragment (see Examples 9-14). Preferred compositions of the invention comprising vWF-based polypeptides and an anionic material may be formulated in solution, as mixtures or admixtures, as lyophilizied powders as frozen samples, in gels, or in other pharmacologically suitable forms.

The affinity for GPIbα of the polypeptide portions of the invention which are derived from vWF may be measured by any of the assay procedures provided in the Examples of the invention or disclosed in references cited or taught in this application.

It has also been mentioned that the derivatized

polypeptides resultant from the practice of the invention have increased binding affinity for platelet GPIbα receptor relative to the affinity therefor of the component vWF-based polypeptide portion. This means that affinity is increased by approximately 10% or that approximately 10% less

derivatized polypeptide than nonderivatized polypeptide is needed to attain a particular level of binding to platelets.

Amino Acid Subdomains of vWF Subunit which

Facilitate Binding of the Anionic Material

As mentioned above, it is well established that

circulating vWF does not bind to platelets in the blood absent some stimulus associated with vascular injury or a vascular disease state that triggers vWF's participation by vWF in clot formation. It is believed that such a stimulus, for example, binding of vWF to negatively charged

proteoglycans or to collagen fibers at a damaged site in the vascular subendothelium triggers a change in multimeric vWF, converting it from an inert substance to an adhesive molecule involved in platelet adhesion, and primary hemostasis.

Botrocetin, a highly negatively charged protein

extracted from the venom of the snake Bothrops jararaca (see Example 23) is believed to introduce, in vitro,

conformational changes which have the effect of changes resultant in vivo from the binding of vWF to the above-mentioned components of the vascular subendothelium, or other related stimuli, indicative of vascular damage. When so bound vWF is believed to exhibit a "switched on"

conformation.

It is known (see Example 3) that botrocetin binds to vWF within the fragment thereof containing amino acid sequence positions 441-733 and thus the GPIb binding domain. Certain amino acid subdomains of the loop region (residues 509-695) of the aforementioned fragment are crucial to the binding of botrocetin.

Derivatized polypeptides of the present invention have built into their structures those features which provide the "switched on" conformation, or structure having a particular distribution of electric charges characteristic of activated vWF.

Parent application Serial No. 07/675,529 discloses that such a conformation or distribution of charge may be induced in the antithrombotic polypeptide by copying into the amino acid sequence thereof certain mutations associated with Type lib von Willebrand disease.

An additional approach to such active conformations is provided in Example 31 below which describes the mapping of the botrocetin binding site in the vWF subunit. The binding site (or more precisely subdomains of polypeptide sequence crucial for binding) is identified as within the residue 509-695 loop region of the subunit. Accordingly, provision of vWF subunits, or fragments thereof, having increased binding affinity for the GPIbα receptors of platelets, and hence increased utility in the prevention and treatment of

thrombosis, may be accomplished by manipulation of the loop regions, and in particular of the net positively charged subdomains therein.

Described in conceptual form, the invention recognizes the importance of enhancing the binding affinity of

antithrombotic vWF-based polypeptides for platelet GPIbα receptors by manipulation of the cysteine 509 to cysteine 695 loop region thereof. This is accomplished generally by allowing an anionic material to become linked to the vWF-based polypeptide portion, the anionic material having had also an affinity for all or part of said loop region. There follows hereafter a description of peptide subdomains which are important in the binding of anionic material to the vWF subunit and fragments thereof.

With respect to the subdomains of said domain needed to define a binding site for the anionic material, it is preferred that there be contained in the polypeptide portion one or more, and most preferably all of the 3 subdomains identified in Example 31 as having the highest botrocetin binding activity, specifically

F8 (residues 539-553) (SEQ ID NO: 22)

Asp Met Met Glu Arg Leu Arg He Ser Gln Lys Trp Val Arg Val

F4 (residues 569-583) (SEQ ID NO: 23)

Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg He Ala Ser Gln

E4-3 (residues 629-643) (SEQ ID NO: 24)

Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys [positions 644, 645 are also lysine]

Also preferred is the use of those subdomains identified in Example 31 as overlapping with the most active (preferred) subdomains shown above.

In connection with the selection of preferred and most preferred subdomains within the loop region, it must be noted that, for example, not all peptides are equally soluble.

Peptide F8-7a could not be tested at the higher of two concentrations (Figure 9, middle panel) and may otherwise have been the most active peptide. Preferred also are other subdomains of the residue 509-695 loop not mentioned above but in which are identified type IIb von Willebrand disease mutations. In this regard preferred is the peptide sequence surrounding residue 511 (arginine).

The Anionic Material

Generally speaking, anionic material which comprises the composition of the invention or which can be the source of the anionic portion of the derivatized polypeptide of the present invention functions by inducing a change in the structure of the vWF-based polypeptide and/or in its

functional properties. The induced change may be an

alteration in the conformation of the vWF-based polypeptide, neutralization of certain electric charge thereof, or another effect capable of inducing in the polypeptide an enhanced ability to bind to platelet GPIbα receptors. Accordingly, anionic material useful in the practice of the invention is capable of facilitating the interaction of multimeric vWF or of fragments thereof with GPIbα receptors, the interaction with fragments thereof being used to inhibit the thrombotic activity of the multimers.

Various types of compounds are useful as the anionic material. Such materials may be organic or inorganic in nature. It is particularly noted that such materials, may be overall, of cationic character, but, nonetheless, possess functional subdomains of anionic character capable of

interacting with multimeric vWF or fragments thereof. The functional result of the effects of the anionic material is demonstrated, for example, in Example 32 below using 4-[4-[4-[4-[4-[4-[4-(2-Carboxyethyl-5-hydroxybenzyl)CMB]CMB]CMB]CMB]CMB]CMB]-2-carboxyethyl-5-methoxytoluene, wherein "CMB" represents 2-carboxyethyl-5-methoxybenzyl, as an anionic material. Figure 10

demonstrates a well known behavior of multimeric vWF,

relevant to evidencing the utility of anions, that is, binding of the protein to platelets does not occur absent the presence of a stimulus which initiates binding. According to the practice of the invention, anionic materials are used to initiate or to enhance binding of vWF-based polypeptides to platelets, the polypeptides being incapable of causing platelet adhesion to damaged vascular subendothelium but, by occupying platelet receptors, capable of preventing such binding by multimeric vWF. The anionic material referred to in Example 32 was also demonstrated (see Example 33) to compete with negatively charged botrocetin for positively charged binding sites on multimeric vWF. Additional assays useful in confirming the functional behavior of such "anionic materials" are provided elsewhere in the application.

The structure of the anionic species preferred presently in the practice of the invention is shown in Figure 8. Such charged compounds are referred to herein as "anionic

oligomers". Functional comparisons with similar compounds have demonstrated that modification of the terminal rings is least likely to perturb the desired polypeptide binding properties. Accordingly, additional effective compounds may be prepared by substituting into the terminal rings groups such as, for example, hydrogen, halogen, alkyl, aryl, O-alkyl, O-aryl or phenol. Such compounds can be made by any suitable method. A preferred method of preparation is the subject of U.S. patent application Serial No. 07/703,061, the text of which is incorporated by reference. See also Example 35 for a representative method of synthesizing a preferred anionic material.

With respect to the design of other effective compounds, preservation of at least some of the 6 carboxyl groups of the 6 internal rings of the preferred anion (Figure 8), or alternately substitution therefor by other negatively charged groups, such as, for example, sulfate, nitrate or phosphate, acetate or propionate, is recommended. It is expected that increasing the size of the anionic oligomer by insertion of additional monomeric units will result in effective

compounds.

In preferred form, the anionic material should remain permanently in contact with the vWF subunit or fragment, unless by binding therewith temporarily, it imparts a permanent conformational change in the polypeptide which remains after dissociation, instances of such behavior being known in other types of applications.

As has been mentioned, the anionic material has affinity for the amino acid domain of the vWF subunit comprising residues 509-695 or a subfragment thereof. It is believed that the affinity is due to electrostatic attraction between the negative groups of the anionic material and postive groups of the polypeptide. Such affinity may manifest itself in the formation of bonds, for example, hydrophobic,

electrostatic, hydrogen, and covalent bonds, between the anionic material and the vWF-based polypeptide. It is noted that these bonds may or may not involve the subregions of either portion whereby original affinity was conferred. The affinity of the anionic material for the amino acid residue 509-695 domain of the vWF-based polypeptide portion need not be based solely or primarily on electrostatic interactions. For example, electrostatic interactions may cause initial attraction of the two portions, the complex being further stabilized by other forces such as hydrophobic interactions and hydrogen bonds. Once associated, such complexes may later be covalently linked.

Linking may be maintained by one or more covalent or noncovalent bonds. In the case of noncovalent bonding, utility as an antithrombotic is dependent on the preferential affinity of the anion for the subunit or fragment in relation to native multimeric vWF. Once dissociated from the subunit or fragment, if the anion is bound to multimeric vWF, there is potentially triggered thrombotic bridging activity.

Accordingly, if noncovalently linked derivatized polypeptides are used in the practice of the invention, the dissociation constant of the anion-polypeptide complex should be at least one and preferably several orders of magnitude lower than that of the comparable dissociation constant of a complex of anion and multimeric vWF. There follows a discussion of methods which are useful in the preparation of covalently derivatized polypeptides of von Willebrand factor subunit or fragments thereof. To form a covalent link or bond between a polypeptide portion and the anionic portion of the derivatized polypeptide, both the polypeptide and the anionic material must possess one or more reactive groups which enable the coupling thereof. There follows a discussion of suitable representative reactive groups. Such reactive groups may preexist in the original structure of the polypeptide or anionic material or be covalently added after the isolation or production of the polypeptide and anionic material.

In connection with the utilization or preparation of the reactive groups, certain general requirements should be met. Firstly, it is desired that the polypeptide and anionic material, upon mixing, be bound in a reproducible orientation or set of orientations. In this regard, it is believed that regions of the anionic material having appropriately spaced negative charge are attracted to appropriately spaced

positive charges such as within subdomains F8, F4 and E4-3 of the polypeptide (see Example 31). The resulting complex may then be stabilized by additional noncovalent bonds.

Secondly, it is desired that suitable reactive groups of both the polypeptide and anionic material be positioned

sufficiently close to permit reaction. As described below, photoaffinity labels which insert into common bonds such as C-C or C-H insure a local coupling, as almost every group in the polypeptide can receive the reacting ligand.

The types of modifications which can be applied are well known in the art. The methodology is reviewed, for example, in Jakoby, W.B. and Wilcheck, M. eds., Methods in Enzvmology. 46, (1977). It is preferred that the labels be applied to the anionic material, for reaction with reactive groups on a polypeptide. Suitable labelling groups as shown below include I, N₃-,CH₂Br, CH₂Cl and structures (a) representing α, 6 unsaturated aldehyde or ketone; structures (b) aryl azides linked via alkyl or alkoxy groups; structures (c) diazoketone wherein "A" can be trifluoromethyl or carboxy or H, or structures (d) aryl ketones.

Highly preferred labelling groups include photolabile reagents, such as (a)-(d) above, anchored in the anionic oligomer by a covalent bond. Photolysis of the complex then leads to the generation of a highly reactive species that reacts immediately by insertion rapidly with directly

proximal bonds in the polypeptide. By inserting into a wide variety of bonds, including C-C and C-H, reproducible

geometry of orientation into the polypeptide may be obtained.

There follows hereafter a representative method for coupling a photolabile benzophenone, a reactive group useful in the practice of the invention to an anionic oligomer.

Methods for the preparation of compound (1) above are described in aforementioned Patent Application Serial No.

07/703,061. The pathway for conversion of an aryl iodide to an aryl trimethylstannane follows the procedure of A.M.

Echavarren and J.K. Stille, J. Amer. Chem. Soc., 109, 5478 (1987) and references therein. See also H. Azizian, C.E.

Eaborn, A. Pidcock, J. Organomet. Chem.. 215, 49 (1981). The coupling of an aryl stannane with an acid chloride is also a known process. (J.K. Stille, Angew, Chem. Int. Eng., 25, (1986), 508 and references therein.).

A recommended protocol for the production of the

photoaffinity labelling agent would be as follows:

Step 1

To a solution of 3.44 g of (1) in 10 ml of THF is added 0.116 g of tetrakis (triphenylphosphine) palladium followed by 0.8 ml of hexamethylditin. The resulting solution is warmed to reflux and stirred until the reaction mixture burns black, the solution is cooled, concentrated under reduced pressure and the residue purified by flash chromatography to yield the product.

Step 2

To a solution of 1.49 g of stannane (2) in 5 ml of HMPA is added 0.055 g of bis (triphenylphophine) benzyl palladium chloride followed by 0.3 ml of benzoyl chloride. The resulting solution is warmed to 65°C and stirred until the solution becomes black. The reaction mixture is cooled, diluted with ether and washed thoroughly with water, and then brine. The organic extract is dried over magnesium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography to yield the product.

Step 3

To a solution of 0.25 g of (3) in 2 ml of THF, 2 ml of methanol is added 2 ml of 15% sodium hydroxide. The

resulting mixture is stirred at room temperature for 30 hours. Concentrated hydrochloric acid is added until the solution has a pH less than 2. The mixture is then extracted with a mixture of methylene chloride/methanol/ethyl acetate. The organic extract is washed with brine, dried over

magnesium sulfate and concentrated to yield the product.

Antibodies with Therapeutic Activity

Antibodies, and particularly conformation dependent antibodies, are powerful tools for analyzing the structure and function of macromolecules. By blocking macromolecular interactions, antibodies can also have important therapeutic utility.

Accordingly, this invention includes within its scope an antibody which is specific for a vWF-based and derivatized polypeptide which is made by a process which involves

immunizing animals with a derivatized polypeptide patterned upon the mature vWF subunit or a fragment thereof.

Procedures useful in immunizing animals with vWF-based derivatized polypeptides are well known in the art.

Therapeutic Compositions One or more of the derivatized polypeptides of the present invention can be formulated into parmaceutical preparations for therapeutic, diagnostic, or other uses. To prepare them for intravenous administration, the compositions are dissolved in water containing physiologically compatible substances such as sodium chloride (e.g. at 0.35-2.0 M), glycine, and the like and having a buffered pH compatible with physiological conditions, which water and

physiologically compatible substances comprise a

pharmaceutically acceptable carrier.

The amount to administer for the prevention or

inhibition of thrombosis will depend on the severity with which the patient is subject to thrombosis, but can be determined readily for any particular patient.

Examples

The following Examples are representative of the practice of the invention. The Example section of

aforementioned U.S. applications Serial No. 07/600,183, 07/613,004 and 07/675,529 are incorporated herein and are useful in the practice of the present invention.

I. Construction of vWF Polypeptides

for the Invention

Example 1 - Expression of a mutant cysteine-free mature von

Willebrand factor subunit fragment having an amino terminus at residue 441 (arginine) and a carboxy terminus at residue 733 (valine)

Preparation of a cDNA Clone from

pre-pro-von Willebrand Factor mRNA A cDNA clone encoding the entire von Willebrand factor gene (for the pre-propeptide) was provided by Dr. Dennis Lynch, Dana-Farber Cancer Institute, Boston, MA and was prepared as described in Lynch, D.C. et al., Cell, 41, 49-56 (1985). It had been deemed probable that the size of vWF mRNA would likely exceed that of human 28S type rRNA.

Accordingly, total RNA from endothelial cells (the major source of plasma vWF) was sedimented in sucrose gradients, with RNA larger than 28S being selected for construction of a cDNA library.

This enriched fraction was further purified using two separate cycles of poly(u)-Sephadex^® chromatography to select for RNA species (mRNA) having 3' polyadenylated ends. Lynch et al., supra, estimated the prevalence of vWF mRNA in this fraction at about 1 in 500, which fraction was used to generate a cDNA library of approximately 60,000 independent recombinants. To generate the cDNA library, standard techniques were used. The mRNA population was primed using an oligo (dT) primer, and then transcribed with a reverse transcriptase. The RNA strands were then removed by alkaline hydrolysis, leaving cDNA anticoding strands (equivalent to transcribed strands) which were primed by hairpin looping for second strand synthesis using DNA polymerase I. The hairpin loop was removed with S, nuclease and rough ends were repaired with DNA polymerase I.

GC tailing, Maniatis, T. et al., Molecular Cloning. 2nd ed., v.l, p.5.56 (1987), was then used to anneal the cDNA into plasmid vector pBR322. Oligo(dC) tails were added to the cDNA fragments with terminal transferase and were

annealed to oligo(dG) tailed pBR322. The plasmids were transformed into ampicillin sensitive E.coli. strain HB101 for propagation. Suitable clones were identified after screening with ³²P-labelled cDNA prepared as reverse

transcriptase product of immunopurified vWF polysomes.

Positive clones were subcloned into pSP64 (Promega Co.,

Madison, WI). Primer Directed Amplification of cDNA cDNA representing the full length pre-pro-vWF gene from pSP64 was subjected to enzymatic amplification in a

polymerase chain reaction. Based upon the established nucleotide sequence of the pre pro-vWF gene, Bonthron, D. et al. Nucl. Acids Res.. 14(17), 7125-7127 (1986); Mancuso, D. et al., J. of Biological Chemistry, v.264 (33), 19514-19527 (1989) oligonucleotides flanking the region of interest (designated (1), SEQ ID NO: 3, and (2), SEQ ID NO: 4) were prepared. All oligonucleotides used herein were synthesized by the phosphoramidite method , Sinha, et al., Tetrahedron

Letters, 24, 5843 (1983), using a model 380B automated system, Applied Biosystems, Foster City, CA.

Oligonucleotide (l) (SEQ ID NO: 3)

5'ACGAATTC CGG CGT TTT GCC TCA GGA3 '

EcoRI Αrg₄₄₁ Gly₄₄₆

Oligonucleotide (2) (SEQ ID NO: 4)

3'GG GAC CCC GGG TTC TCC TTG AGG TAC CAT TCGAAG5' 5'cc ctg ggg ccc aag agg aac tec atg gta agcttc3' Leu₇₂₅ Met₇₃₂Val₇₃₃HindIII

The oligonucleotides overlap the ends of the coding region for that fragment of the mature vWF subunit which can be produced by digestion with trypsin and which begins with residue 449 (valine) and ends with residue 728 (lysine).

Oligonucleotide (1) corresponds to coding strand DNA

(analogous with mRNA) for amino acid positions 441 to 446 and adds an EcoRI restriction site 5' to the codon for amino acid 441. Oligonucleotide (2) corresponds to the non-coding strand (transcribed strand) of mature vWF DNA for amino acids positions 725-733 and adds a HindIII restriction site 3' to the codon for amino acid 733. The coding strand

complementary to (2) is shown in lower case letters.

Using the above oligonucleotides with the full length cDNA as template, a cDNA fragment corresponding to mature vWF residues Nos. 441-733, and containing EcoRI and Hind III linkers, was then synthesized in a polymerase chain reaction following the method of Saiki, R.K. et al. Science. 239, 487-491 (1988). The procedure utilizes a segment of double-stranded vWF cDNA, a subsegment of which is to be amplified, and two single-stranded oligonucleotide primers (in this case

oligonucleotides (1), (2)) which flank the ends of the subsegment. The primer oligonucleotides (in the presence of a DNA polymerase and deoxyribonucleotide triphosphates) were added in much higher concentrations than the DNA to be amplified.

Specifically, PCR reactions were performed with a DNA thermal cycler (Perkin Elmer Co., Norwalk, CT/Cetus

Corporation, Berkeley, CA) using Taq polymerase (Thermus aquaticus). The reactions were run in 100 μℓ volumes

containing 1.0 μg of pre-pro-vWF cDNA, 1.0 μg of each

synthetic oligonucleotide primer, and buffer consisting of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.1% gelatin (BioRad Co., Richmond, CA) and 200 mM of each dNTP. PCR conditions were 35 cycles of 30 seconds at 94 °C, 30 seconds at 52°C and 1 minute at 72°C. Amplified fragments were then purified and isolated by electrophoresis through a 2% agarose gel, Maniatis et al., Molecular Cloning. A Laboratory Manual. 164-170, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1982).

The vast majority of polynucleotides which accumulate after numerous rounds of denaturation, oligonucleotide annealing, and synthesis, represent the desired double-stranded cDNA subsegment suitable for further amplification by cloning.

For some experiments, cDNA corresponding to the mature vWF fragment beginning at amino acid sequence position 441 and ending at position 733 was prepared and amplified

directly from platelet mRNA following the procedure of

Newman, P.J. et al. J. Clin. Invest., 82, 739-743 (1988).

Primer nucleotides No. 440 and 733 were utilized as before with the resulting cDNA containing EcoRI and HindIII linkers. Insertion of cDNA into M13mpl8 Cloning Vehicle The resultant double stranded von Willebrand factor cDNA corresponding to the amino acid sequence from residue 441 to 733 was then inserted, using EcoRI and HindIII restriction enzymes, into the double stranded replicative form of bacteriophage M13mp18 which contains a multiple cloning site having compatible EcoRI and HindIII sequences.

M13 series filamentous phages infect male (F factor containing) E.coli strains. The infecting form of the virus is represented by single stranded DNA, the (⁺) strand, which is converted by host enzymes into a double stranded circular form, containing also the minus (-) strand, which double stranded structure is referred to as the replicative form (RF) . The ability to isolate a stable single stranded (⁺) form of the virus is particularly useful to verify the integrity of any cloned sequences therein. See Messing, J., Meth. Enzymology. 101, 20-78 (1983); Yanish-Perron, C. et al., Gene. 33, 103-109 (1985).

Accordingly, the vWF cDNA insert was completely

sequenced using single-stranded dideoxy methodology (Sanger, F. et al. Proc. Natl. Acad. Sci USA. 74, 5463-5467 (1977)), utilizing the single-stranded (⁺) form of M13mp18, to confirm that the vWF cDNA fragment contained the correct coding sequence for mature vWF subunit residues 441-733.

Site-Directed Mutagenesis to Replace Cysteine Residues Cysteine residues 459, 462, 464, 471, 474, 509, and 695, within the mature vWF fragment corresponding to amino acids 441 to 733, were replaced with glycine residues by

substitution of glycine codons for cysteine codons in the corresponding cDNA. In order to accomplish this,

oligonucleotides (see Sequence Listing ID NOS: 5-8)

encompassing the region of each cysteine codon of the vWF cDNA were prepared as non-coding strand (transcribed strand) with the corresponding base substitutions needed to

substitute glycine for cysteine. The oligonucleotides used were as follows: Oligonucleotide (3) (SEQ ID NO: 5)

3'GGA CTC GTG CCG GTC TAA CCG GTG CAA CTA CAA CAG5 ' 5'cct gag gac ggc cag att ggc cac ggt gat gtt gtc3' Pro Glu His Gly Gln Ile Gly His Gly Asp Val Val

459 462 464

(simultaneously replacing cysteines 459, 462, 464).

Oligonucleotide (4) (SEQ ID NO: 6)

3'TTG GAG TGG CCA CTT CGG CCG GTC CTC GGC5'

5'aac etc acc ggt gaa gcc ggc cag gag ccg3'

Asn Leu Thr Gly Glu Ala Gly Gln Glu Pro

471 474

(simultaneously replacing cysteines 471, 474)

Oligonucleotide (5) (SEQ ID NO: 7)

3 'CTA AAG ATG CCG TCG TCC G5'

5'gat ttc tac ggc agc agg c3'

Asp Phe Tyr Gly Ser Arg

509

(replacing cysteine 509)

Oligonucleotide (6) (SEQ ID NO: 8)

3 'TCG ATG GAG CCA CTG GAA CGG5'

5'age tac ctc ggt gac ctt gcc3'

Ser Tyr Leu Gly Asp Leu Ala

695

replacing cysteine 695) Hybridizing oligonucleotides are shown in capital letters and are equivalent to the transcribed strand (non-coding DNA). The equivalent coding strand is shown in lower case letters with the corresponding amino acids shown by standard three letter designation, (for designations see Table 1)

As elaborated below, cysteines 459, 462 and 464 were replaced simultaneously using oligonucleotide (3). Cysteine residues 471 and 474 were then replaced simultaneously using oligonucleotide (4). Cysteine residues 509 and 695 were then replaced individually using oligonucleotides (5) and (6) respectively.

The cysteine to glycine cDNA substitutions were

accomplished following the procedure of Kunkel, T.A., Proc. Natl. Acad. Sci. USA, 82,488-492 (1985) which procedure repeats a series of steps for each oligonucleotide and takes advantage of conditions which select against a uracil containing DNA template:

(A) M13mp18 phage, containing wild type vWF cDNA corresponding to amino acid

positions 441 to 733, is grown in an

E.coli CJ236 mutant dut^_ung^_strain in a uracil rich medium. Since this E.coli strain is deficient in deoxyuridine triphosphatase (dut^_), an intracellular pool of dUTP accumulates which competes with dTTP for incorporation into DNA. (see Shlomai, J. et al. J. Biol. Chem., 253(9), 3305-3312 (1978). Viral DNA synthesized under these conditions includes several uracil insertions per viral genome and is stable only in an E.coli strain which is incapable of removing uracil, such as

(ung^_) strains which lack uracil

glycosylase. Uracil-containing

nucleotides are lethal in single stranded (⁺) M13mp18 DNA in ung⁺ strains due to the creation of abasic sites by uracil glycosylase.

(B) Single-stranded (⁺) viral DNA is isolated from culture media in which phage were grown in E.coli strain CJ236 dufung^". The single stranded (⁺) form of the virus contains the specified vWF cDNA at its multiple cloning site which cDNA is equivalent to the nontranscribed vWF DNA strand. (C) Oligonucleotide (3), which contains codon alterations necessary to substitute glycines for cysteines at positions 459, 462 and 464, is then annealed in vitro to single stranded (⁺) phage DNA.

Generally, a wide range of

oligonucleotide concentrations is

suitable in this procedure. Typically 40 ng of oligonucleotide was annealed to 0.5-1.0 jug M13mp18 phage (⁺) DNA.

(D) All missing sequence of the M13mp18(^") strand is then completed in vitro using T₇ DNA polymerase and T₄ DNA ligase in a dTTP rich environment thereby generating a transcribable vWF cDNA sequence

corresponding to amino acid positions 441 to 733 of the mature vWF subunit.

(E) The double stranded M13mp18 phage, now containing a thymine normal (-) strand and a (⁺) strand with several uracil substitutions, is transformed into a wild type E.coli XL-1 Blue (Stratagene, La Jolla, CA) strain which contains normal levels of uracil glycosylase and

deoxyuridine triphosphatase.

(F) Uracil glycosylase and other enzymes

present in the new host initiate

destruction of the uracil-containing (⁺) strand of the double-strand phages, leading after replication in the host of remaining phage (-) strand DNA to the presence of stable thymine-normal double stranded (RF) DNA which reflects the glycine mutations induced by the

oligonucleotide.

(G) Steps (A) to (F) of the above process are then repeated for each of

oligonucleotides (4), (5) and (6) until each successive cysteine codon of the vWF sequence within the M13mp18 phage has been replaced by a glycine codon.

(H) Upon completion of mutagenesis procedures the sequence of the vWF cDNA insert was reconfirmed using the single stranded DNA dideoxy method. (Sanger, F. et al., supra)

Construction of Expression Plasmids. The double stranded vWF cDNA fragment containing 7 sitespecific cysteine to glycine mutations is then removed from M13mp18 phage by treatment with EcoRI and HindIII restriction endonucleases, after which the ends of the fragment are modified with BamHI linkers (Roberts, R.J. et al. Nature.

265, 82-84 (1977)) for cloning into a high efficiency E.coli expression vector. The particular expression vector chosen is plasmid pET-3A, developed by Rosenberg, A.H. et al. Gene, v.56, 125-135, (1987) and which is a pBR322 derivative containing a high efficiency (ølO) T7 transcription promoter directly adjacent to the BamHI linker site. When containing the above-specified fragment of mutant vWF cDNA, the pET-3A vehicle is refered to as "p7E" or p7E expression plasmid.

A second pET-3A-derived expression plasmid (designated p7D) was constructed containing the identical vWF coding sequence cloned into the plasmid in the opposite orientation. p7D should be unable to express the vWF polypeptide fragment.

A third expression plasmid (pJD18) contains wild type 52/48 tryptic vWF fragment cDNA encoding the vWF amino acid sequence between residues 441 and 733, (with 7 cysteines) in the same pET-3A vector.

The p7E (or p7D and pJD18) expression plasmids were then cloned into an ampicillin sensitive E.coli strain, BL21(DE3), Novagen Co., Madison WI, according to a well established protocol Hanahan, D., J. Mol. Biol., 166, 557-580 (1983). Strain BL21(DE3) is engineered to contain a gene for T7 RNA polymerase so that the vWF insert can be transcribed with high efficiency.

Expression of Mutant vWF Polypeptides Three separate samples of E.coli strain BL21(DE3) containing respectively p7E, p7D or pJD18 expression plasmids were innoculated into 5-6 ml of 2X-YT growth medium

containing 200 μg/ml of ampicillin, and grown overnight at 37°C to create fully grown cultures. 2X-YT growth medium contains, per liter of water, 10 gm Bacto-tryptone, 10 gm yeast extract and 5 gm NaCl. Five ml of each overnight culture was then innoculated into 500 ml of 2X-YT medium, again containing 200 μg/ml of ampicillin and grown for 2 hours at 37°C with shaking. After the 2 hour incubation period, the cultures were induced for protein expression by addition of isopropyl-betad-thiogalactopyranoside to a concentration of 5 mM. The incubation was then continued for 3 hours at 37°C.

A high level of expression of vWF polypeptide was obtained with p7E and pJD18 resulting in the generation of cytoplasmic granules or "inclusion bodies" which contain high concentrations of vWF polypeptide in essentially insoluble form. Solubilization of vWF polypeptide was accomplished according to the following procedure. As explained in

Example 2, p7E and pJD18 extracts responded very differently to solubilization procedures. See Maniatis, T. et al.,

Molecular Cloning. 2nd ed., vol. 3, Sec. 17.37, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, for a general discussion of the properties of, and successful manipulation strategies for, inclusion bodies.

The cells were harvested by centrifugation at 4000 g for 15 minutes in a JA-14 rotor at 4°C. The pelleted cells were washed in 50 ml of ice cold buffer (0.1 M NaCl, 10 mM Tris pH 9.0, 1 mM EDTA) and repelleted by centrifugation at 4000 g at 4°C.

The cell pellets from p7E, p7D and pJD18 cultures were each redissolved in 5 ml of lysing buffer and kept ice-cold for 30 minutes. The lysing buffer comprises a solution of sucrose 25%(w/v), 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM ethylene diaminetetraacetic acid (EDTA), 2 mg/ml lysozyme and 50 mM Tris hydrochloride, adjusted to pH 8.0.

After the 30 minute incubation, aliquots of 1.0 Molar MgCl₂ and MnCl₂ were added to make the lysing solution 10 mM in each cation. Sixty μg of DNAseI (Boehringer-Mannheim) was then added and the incubation was continued at room

temperature for 30 minutes.

Twenty ml of buffer No. 1 (0.2 M NaCl, 2 mM EDTA, and 1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1- propanesulfonate (CHAPS), 1% (w/v) Non-idet 40, and 20 mM Tris hydrochloride, pH 7.5) was then added to the incubation mixture. The insoluble material was pelleted by

centrifugation at 14,000 g (12,000 rpm in a JA-20 rotor) for 30 minutes at 4°C.

The relatively insoluble pelleted material derived from each culture (which contains the desired polypeptides except in the case of p7D) was washed at 25°C in 10 ml of buffer No. 2 (0.5% (w/v) Triton X-100 surfactant, 2 mM EDTA, 0.02 M Tris hydrochloride, pH 7.5) and vortexed extensively. The

suspension was centrifuged at 14,000 g for 30 minutes at 4°C and the supernatant was then discarded. The process of resuspension of the pelleted material in buffer No. 2, vortexing and centrifugation was repeated twice. Each pellet was then washed in 5 ml of buffer No. 3 (0.02 M Tris hydrochloride, pH 7.5, and 2 mM EDTA) at 25°C and vortexed extensively. The suspension was then

centrifuged at 4°C for 30 minutes at 14,000 g after which the supernatant was discarded leaving a pellet of inclusion body derived material (the "wet pellet") with a clay-like

consistency (With respect to the following final steps, and in replacement therefor, see also Example 20 which presents an additional improved procedure). The insoluble pellet was slowly redissolved in an 8 Molar urea solution held at room temperature for 2 hours, after which solubilization was continued overnight at 4°C. The urea-soluble material was extensively dialyzed against a solution of 0.15 M NaCl containing 20 mM Hepes (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) (pH 7.4) ("Hepes-buffered saline") at 4°C.

The solublized peptide extracts were assayed for purity (Example 2), used in vWF binding inhibition assays (Example 3) or subject to further purification. Further purification steps should not be delayed and the samples should remain cold.

The cysteine-free vWF polypeptide (comprising subunit positions 441 to 733) constitutes more than 75% of the material solubilized from the inclusion bodies according to the above procedure. Further purification of the cysteine-free mutant vWF polypeptide was accomplished by redialyzing the partially purified peptide extract against 6 M

guanidine-HCl, 50 mM Tris-HCl, pH 8.8 followed by dialysis against 6 M urea, 25 mM Tris-HCl, 20 mM KCl, 0.1 mM EDTA, pH 8.0. The extract was then subjected to high performance liquid chromatography using Q-Sepharose^® Fast Flow

(Pharmacia, Uppsala, Sweden) for anion exchange. The column was preequilibrated with 6 M urea, 25 mM Tris-HCl, 20 mM KCl, 0.1 mM EDTA pH 8.0. Elution of the vWF polypeptide utilized the same buffer except that the concentration of KCl was raised to 250 mM. Polypeptide samples used for further assays were redialyzed against 0.15 M NaCl, 20 mM Hepes, pH 7.4. However, long term storage was best achieved in urea buffer (6 M urea, 25 mM Tris-HCl, 20 mM KCl, 0.1 mM EDTA pH 8.0. Final p7E-vWF polypeptide percent amino acid

compositions (by acid hydrolysis) compared closely with values predicted from published sequence information

(Bonthron, D. et al. and also Mancuso, D. et al. in Example 1, supra; see also Figure 1).

Example 2 - Characterization of the cysteine-free

mutant von Willebrand factor fragment

produced by expression plasmid P7E

Urea-solubilized and dialyzed polypeptides extracted from inclusion bodies of cultures containing expression plasmids p7E, p7D and pJD18 were analyzed using

polyacrylamide gel electrophoresis (PAGE) and immunoblotting.

Characterization by SDS-Polyacrylamide Gel Electrophoresis

The purity and nature of the expression plasmid

extracts, which had been urea-solubilized and then

extensively dialyzed, were first analyzed using the

denaturing sodium dodecylsulfate-polyacrylamide gel

electrophoresis procedure of Weber, K. et al. J. Biol. Chem.. 244, 4406-4412 (1969), as modified by Laemli, U.K. Nature, 227, 680-685 (1970) using an acrylamide concentration of 10%. The resultant gels were stained with Coomassie blue and compared.

The extract from expression plasmid p7E contains as the major component, the mutant von Willebrand factor polypeptide which migrates with an apparent molecular weight of

approximately 36,000 Daltons. The polypeptide appears as a single band under both reducing conditions (addition of between 10 and 100 mM dithiothreitol "DTT" to the sample for 5 min at 100°C prior to running the gel in a buffer also containing the same DTT concentration) and nonreducing conditions, which result is consistent with the substitution of glycine residues for all of the cysteine residues therein. No vWF polypeptide could be extracted from host cells

containing p7D expression plasmids as expected from the opposite orientation of the vWF cDNA insert. The cysteine-containing vWF polypeptide expressed by host cells containing pJD18 plasmids, and which contains the wild type amino acid sequence of the 52/48 fragment, (herein represented by a residue 441 to 733 cloned fragment) behaved differently under reducing and nonreducing conditions of electrophoresis. The wild-type sequence expressed from pJD18 forms intermolecular disulfide bridges resulting in large molecular weight aggregates which are unable to enter the 10% acrylamide gels. After reduction (incubation with 100 mM DTT for 5 min at 100°C), the vWF peptide migrates as a single band with a molecular weight of approximately 38,000.

Characterization by Immunoblotting

Polypeptides expressed from p7E, p7D and pJD18 were further characterized by immunoblotting ("Western blotting") according to a standard procedure Burnett et al., A. Anal. Biochem.. 112, 195-203, (1981) and as recommended by reagent suppliers. Samples containing approximately 10 μg of protein from the urea-solubilized and dialyzed inclusion body

extracts of host cells (containing p7E, p7D and pJD18

plasmids) were subjected to electrophoresis on 10%

polyacrylamide gels, Laemli, U.K. Nature. 227, 660-665

(1970), in the presence of 2% concentration of sodium dodecyl sulfate.

The proteins were blotted and immobilized onto a

nitrocellulose sheet (Schleicher and Schuell, Keene, NH) and the pattern was then visualized using immunoreactivity.

The von Willebrand factor-specific monoclonal antibodies (from mice) used to identify the polypeptides were RG-46 (see Fugimura, Y. et al. J. Biol. Chem.. 261(1), 38l-385 (1986), Fulcher, CA. et al. Proc. Natl. Acad. Sci. USA. 79, 1648-1652 (1982)), and NMC-4 (Shima, M. et al. J. Nara Med.

Assoc.. 36, 662-669 (1985)), both of which have epitopes within the expressed vWF polypeptide of this invention. The secondary antibody (¹²⁵I-rabbit anti-mouse IgG), labelled by the method of Fraker, P.J. et al. Biochem.

Biophvs. Res. Commun., 80, 849-857 (1978)), was incubated for 60 minutes at 25°C on the nitrocellulose sheet. After rinsing, the sheet was developed by autoradiography.

Peptide extracts from host cells containing p7E and pJD18 expression plasmids display strong immunoreactivity for RG-46 antibody and a weaker but definite affinity for NMC-4 antibody. As expected, peptide extracts from p7D plasmids show no immunoreactivity with either RG-46 or NMC-4.

Example 3 - Inhibition of botrocetin-induced binding

of vWF to platelets by the cysteine-free

mutant polypeptide expressed by p7E

It has been demonstrated that botrocetin, extracted from the venom of Bothrops jararaca modulates the in vitro binding of multimeric von Willebrand factor to platelets (Read, et al. Proc. Natl. Acad. Sci.. 75, 4514-4518 (1978)) and that botrocetin binds to vWF within the region thereof containing amino acid sequence positions 441-733 (of the mature

subunit), and thus the GPIb binding domain. (Andrews, R.K. et al., Biochemistry. 28, 8317-8326 (1989)).

The urea-solubilized and dialyzed polypeptide extracts, obtained (according to the method of Example 1) from cultures containing expression plasmids p7E, p7D and pJD18, were tested without further purification for their ability to inhibit botrocetin-induced vWF binding to formalin-fixed platelets on a dose dependent basis.

Formalin-fixed platelets, prepared according to the method of MacFarlane, D. et al., Thromb. Diath. Haemorrh. 34, 306-308 (1975), were pre-incubated at room temperature for 15 minutes with specified dilutions of peptide extracts obtained from cultures containing pJD18, p7D, and p7E plasmids.

Botrocetin, (Sigma, St. Louis, MO) to a final concentration of 0.4 μg/ml, and ¹²⁵I-labelled multimeric vWF (isolated from human plasma cryoprecipitate according to the method of

Fulcher, CA. et al. Proc. Natl. Acad. Sci. USA. 79, 1648- 1652 (1962), and labelled according to the method of Fraker, P.J. et al. Biochem. Biophys. Res. Commun.. 80, 649-657

(1978)) were then added to the incubation mixture, and the amount of ¹²⁵I- vWF bound to the platelets was determined.

¹²⁵I-vWF binding to the platelets was referenced against 100% binding which was defined as the amount of ¹²⁵I- vWF bound in the absence of added peptide extracts. Peptide extracts from expression plasmids p7D, and also pJD18 (unreduced and unalkylated) could not compete with plasma-derived vWF for platelet GPIb receptor binding sites. The peptide extract from plasmid p7E was effective in a dose dependent manner (using a range of 0 to 100 μg extract/ml) in inhibiting vWF binding. The concentration of ureasolubilized polypeptide extract (μg/ml) in the incubation mixture reflects the total protein concentration from the extract. Addition of peptide extracts to the reaction mixture causes certain nonspecific effects which raise apparent initial binding to 110% of the value found in the absence of the added peptide extracts. The ¹²⁵-IvWF

concentration used was 2μg/ml.

Example 4 - Expression of a mutant vWF fragment of

reduced cysteine content containing a

disulfide-dependant conformation

Utilizing the procedures of Example 1, except as

modified below, a mutant vWF polypeptide fragment

(corresponding to the mature vWF subunit sequence from residue 441 to residue 733) was prepared in which the

cysteines at positions 459, 462, 464, 471 and 474 were each replaced by a glycine residue. Cysteine residues were retained at positions 509 and 695, and allowed to form an intrachain disulfide bond. Site directed mutagenesis was performed only with oligonucleotides No. 459 and 471, thereby substituting glycine codons only at positions 459, 462, 464, 471 and 474. Upon completion of mutagenesis procedures, the sequence of the mutant vWF cDNA was confirmed using the single-stranded dideoxy method.

The double-stranded form of the vWF cDNA insert

(containing 5 cysteine to glycine mutations) was then removed from M13mp18 phage by treatment with EcoRI and HindIII restriction endonucleases, modified as in Example 1 with

BamHI linkers, and cloned into pET-3A. The pET-3A vehicle so formed is referred to as "p5E" or p5E expression plasmid.

The p5E expression plasmids were then cloned into ampicillin sensitive E.coli strain BL21(DE3), Novagen Co., Madison, WI, according to the procedure of Hanahan, D., J. Mol. Biol.. 166, 557-580 (1983). The p5E mutant polypeptide was expressed from cultures of E.coli BL21(DE3) following the procedure of Example 1 except that solubilization of

inclusion body pellet material in the presence of 8 Molar urea need not be continued beyond the initial 2 hour period at room temperature, at which point redissolved material had reached a concentration of 200 μg/ml. Oxidation of cysteine residues 509 and 695 to form a disulfide bond was

accomplished by dialysis overnight against Hepes-buffered saline. Formation of intrachain rather than interchain disulfide bonds is favored by allowing thiol oxidation to proceed at a low protein concentration such as 50-100 μg/ml.

As in Example 1 pertaining to the p7E extracts, final purification of urea-solubilized inclusion body preparations was accomplished by dialysis against the 6 M guanidine and 6 M urea buffers followed by anion exchange chromatography. Example 5 - Characterization of the mutant vWF

fragment produced by expression plasmid p5E

The mutant von Willebrand factor polypeptides produced by cultures containing expression plasmid p5E were

characterized utilizing the procedures of Example 2, and in particular compared with the vWF fragment expressed by plasmid p7E.

Urea-solubilized and dialyzed polypeptides extracted from inclusion bodies (according to the procedure of Example 4) were compared with similar extracts from p7E plasmid cultures produced as in Example 1.

Characterization by SDS-Polyacrylamide Gel Electrophoresis

The denaturing sodium dodecylsulfate gel procedure of Example 2 was used to compare the p5E vWF fragments, which can form disulfide bonds using cysteine residues 509 and 695, with the p7E fragment which has no cysteine residues.

Electrophoresis was conducted using 7.5 μg of protein extract per lane on 10% acrylamide gels under reducing (100 mM dithiothreitol) and non-reducing conditions. Under reducing conditions, and after staining with

Coomassie blue, extracts from p7E and p5E have identical electrophoretic mobilities.

Electrophoresis under nonreducing conditions, however, demonstrates the effects of disulfide bonds involving

residues 509 and 695. A substantial amount of the p5E extract appears as a high molecular weight complex (resulting from interchain disulfide bonds) which enters the gel only slightly. Densitometric scanning of the gels of initial preparations indicates that approximately 25% of the p5E polypeptide material found on nonreducing gels is represented by monomers of the 441-733 fragment having an apparent molecular weight of approximately 38,000. The percent of monomer present in p5E extracts can be improved significantly by conducting urea solubilization, dialysis, and thiol oxidation at a more dilute protein concentration, such as 50- 100 μg/ml, to favor intrachain rather than interchain

disulfide bond formation. This p5E monomeric species has a slightly higher

mobility during electrophoresis under nonreducing conditions than the comparable p7E product species which has no cysteine residues. The mobilities of these p5E and p7E monomeric 38 kDa species appear identical under reducing conditions. The slightly accelerated mobility of a polypeptide which retains tertiary structure in the presence of SDS under nonreducing conditions, when compared to the mobility of the homologous polypeptide which the anionic detergent converts completely into a negatively charged fully rigid rod under said

conditions, is generally considered suggestive of the

presence of an intrachain disulfide bond.

Characterization by Immunoblotting

The behavior of p5E and p7E extracts were also examined using immunological methods. As in Example 2, vWF-specific murine monoclonal

antibodies RG-46 and NMC-4 were used as probes. RG-46 has been demonstrated to recognize as its epitope a linear sequence of amino acids, comprising residues 694 to 708, within the mature von Willebrand factor subunit. The binding of this antibody to its determinant is essentially

conformation independent. Mohri, H. et al., J. Biol. Chem., 263(34), 17901-17904 (1988).

NMC-4 however, has as its epitope the domain of the von Willebrand factor subunit which contains the glycoprotein lb binding site. Mapping of the epitope has demonstrated that it is contained within two discontinuous domains (comprising approximately mature vWF subunit residues 474 to 488 and also approximately residues 694 to 708) brought into disulfide-dependent association, Mohri, H. et al., supra, although it was unknown whether the disulfide bond conferring this tertiary conformation in the native vWF molecule was

intrachain or interchain. Id. at 17903.

7.5 μg samples (of protein) were first run on 10% SDS polyacrylamide gels so that the antigenic behavior of particular bands (under reducing and nonreducing conditions) could be compared with results obtained above by Coomassie blue staining. Immunoblotting was performed as in Example 2 to compare p5E and p7E extracts. Application of antibody to the nitrocellulose sheets was usually accomplished with antibody solutions prepared as follows. Mice were injected with B-lymphocyte hybridomas producing NMC-4 or RG-46. Ascites fluid from peritoneal tumors was collected and typically contained approximately 5 mg/ml of monoclonal antibody. The ascites fluid was mixed (1 part per 1000) into blocking fluid (PBS containing 5% (w/v) non-fat dry milk, Carnation) to minimize non-specific

background binding. The antibody-containing blocking fluid was then applied to the nitrocellulose. Under nonreducing conditions, the single chain p5E polypeptide fragment (representing the sequence from residue 441 to residue 733) displayed an approximate 120 fold

increase in binding affinity for NMC-4 compared to the comparable cystein-free species isolated from p7E also representing the primary sequence from residue 441 to 733. After electrophoresis under reducing conditions (utilizing 100 mM DTT) , the single chain p5E species showed a remarkably decreased affinity for NMC-4, which was then very similar to that of the cysteine-free p7E species under either reduced or nonreduced conditions. NMC-4 also fails, under reducing or non-reducing conditions, to recognize as an epitope

disulfide-1inked dimers from the p5E extract.

The nitrocellulose filters used to produce autoradiographs based on NMC-4 were rescreened with RG-46 by

subtracting the initial NMC-4 exposure response, which was kept low through a combination of low antibody titer and short exposure time. The binding of RG-46 to the p7E 36,000 kDa polypeptide on the filters is the same whether reducing or non-reducing conditions were chosen, consistent with the replacement of all cysteines by glycine in the expressed polypeptide.

A large molecular weight vWF antigen (reactive to RG-46) is present in the p5E polypeptide extract under nonreducing conditions. These p5E vWF aggregates (reflecting interchain disulfide bonds) migrate under reducing conditions in the same position as the p7E polypeptide indicating disruption of their disulfide contacts. However, the large p5E interchain disulfide aggregates which are readily recognized under nonreducing conditions by RG-46 are not recognized by NMC-4 under either reducing or nonreducing conditions. It is thus demonstrated that the disulfide bond between residues 509 and 695 in native multimeric vWF subunits represents an

intrachain contact.

Example 6 - Inhibition of the binding of an anti-GPIb

monoclonal antibody by p5E polypeptide

Monoclonal antibody LJ-Ib1 is known to completely inhibit von Willebrand factor-platelet glycoprotein lb interaction. Handa, M. et al., J. Biol. Chem.. 261(27), 12579-12565 (1986). It reacts specifically with the amino terminal 45 kDa domain of GPIbα which contains the vWF binding site. Vicente, V. et al., J. Biol. Chem.. 265, 274-280 (1990).

To assess the inhibitory activity of p5E extracts on antibody binding, a concentration of LJ-Ib1 was first

selected which would, in the absence of p5E extracts, provide half-maximal binding. LJ-Ib1 was iodinated by the procedure of Fraker, D.J. et al., Biochem. Biophys. Res. Commun.. 80, 849-657 (1978) using I¹²⁵ from Amersham, Arlington Heights, IL and Iodogen (Pierce Chemical Co., Rockford, IL). Washed platelets were prepared by the albumin density gradient technique of Walsh, et al., Br. J. Haematol.. 36, 281-298 (1977), and used at a count of 1 × 10⁸/ml. Half-maximal binding of antibody to platelets was observed at 10 μg/ml LJ-Ib1 concentration, which

concentration was selected for p5E polypeptide inhibition studies.

The p5E polypeptide extract was purified according to the procedure of Example 4 including final purification of the urea-solubilized inclusion body preparation by dialysis against 6.0 M guanidine and urea solutions followed by Q-Sepharose^® chromatography.

To evaluate binding, platelets were incubated for 30 minutes at 22-25°C with LJ-Ib1 (10 μg/ml) and concentrations of purified p5E polypeptide (.002-10.0 μMolar). At the end of the incubation platelets with bound radioactivity were separated from free antibody by centrifugation at 12000 g through a 20% sucrose layer, in 0.15 M NaCl, 20 mM Hepes, pH 7.4, hereinafter "Hepes-buffered saline" buffer in a

microcentrifuge tube. Inhibition of LJ-Ib1 binding was plotted in the presence of 2 μg/ml botrocetin (Sigma Chemical Co., St. Louis, MO) and in the absence of botrocetin.

Less than 5 percent of the ¹²⁵I label bound to the

platelets was contributed by labelled substances other than LJ-Ib1 as determined by binding competition experiments in the presence of a 100 fold excess of unlabelled LJ-Ib1.

Background labelling was subtracted from data points. Binding of ¹²⁵I LJ-Ib1 was expressed as a percentage of a control assay lacking recombinant polypeptides. Fifty percent inhibition of ¹²⁵I LJ-Ib1 binding to platelets was achieved at 10 μM of p5E polypeptide without botrocetin whereas in the presence of botrocetin (2 μg/ml), 50% inhibition may be achieved at less than 0.1 μM. It is known that botrocetin induces in

circulating multisubunit von Willebrand factor and single subunits thereof a conformational change which enhances or permits binding to the GPIbα receptor. This example demonstrates that the p5E polypeptide (containing an

intrachain cysteine 509-695 bond) behaves very much like native circulating von Willebrand factor with respect to how its activity is modulated by botrocetin. Structural

similarity is therefore indicated.

Example 7 - Expression of homodimeric 116 kDa

von Willebrand factor fragment in

stable mammalian transformants

This example is illustrative of conditions under which a DNA sequence encoding the mature vWF subunit fragment having an amino terminus at residue 441 (arginine) and a carboxy terminus at residue 730 (asparagine) may be expressed, and of the secretion from cultured mammalian host cells of a

glycosylated homodimeric form of the 441-730 vWF fragment having native tertiary structure.

Expression of the 116 kDa homodimer is achieved using a DNA construct in which the following structural elements are assembled in a 5' to 3' direction (referring to the coding or nontranscribed strand):

(A) a eucaryotic consensus translation initiation

sequence, CCACC; and

(B) the initiating vWF methionine codon followed by the remaining 21 amino acids of the vWF signal peptide; and

(C) the coding sequence corresponding to the first

three amino acids from the amino terminus region of the vWF propeptide; and

(D) the coding sequence for vWF amino acid residues

441-730; and

(E) the "TGA" translation termination codon.

Preparation of a cDNA Clone from

Pre-pro-von Willebrand Factor mRNA

The cDNA clone, pvWF, encoding the entire pre-pro-vWF gene was obtained from Dr. Dennis Lynch, Dana-Farber Cancer Institute, Boston, MA and was prepared as described in Lynch, D.C et al., Cell. 41, 49-56 (1985). Preparation of pvWF was described in Example 1.

Primer Directed Amplification of cDNA - Phase I

The cDNA representing the full length pre-pre-vWF gene from pSP64 was subjected to enzymatic amplification in a polymerase chain reaction according to the method of Saiki, R.K. et al. Science. 239, 487-491 (1988), as described in Example 1.

For PCR amplification, the following oligonucleotides were synthesized by the phosphoramidite method, Sinha, et al., Tetrahedron Letters. 24, 5843 (1983), using a model 380B automated system, Applied Biosystems, Foster City, CA.

Oligonucleotide (7) - see SEQ ID NO: 9

5' - GTCGACGCCACCATGATTCCTGCCAGA - 3'

Sail Met

Oligonucleotide (8) - see SEQ ID NO: 10

5' - TCAGTTTCTAGATACAGCCC - 3'

XbaI

In designing the oligonucleotides used herein, reference was made to the established nucleotide sequence of the pre pro-vWF gene, Bonthron, D. et al., Nucl. Acids Res.. 14(17), 7125-7127 (1986); Mancuso, D. et al., J. Biol. Chem..

264(33), 19514-19527 (1989).

Oligonucleotide (7) was used to create a Sail

restriction site fused 5' to a eucaryotic consensus

translation initiation sequence (CCACC) preceding the

initiating methionine codon of the vWF cDNA. See Kozak, M. Cell. 44, 183-292 (1986).

Oligonucleotide (8) hybridizes with the non-transcribed strand (coding strand) of the vWF cDNA and overlaps with nucleotides which are approximately 360 base pairs from the initiating methionine in the pre-pro-vWF cDNA, thus spanning (at residues 120 and 121 within the pre-pro-vWF cDNA

sequence) an XbaI restriction site.

The polymerase chain reaction therefore synthesized a cDNA fragment, containing (reading from 5' to 3' on the coding strand) a SalI site, a consensus initiation sequence, an initiating methionine codon, the codon sequence for the signal peptide, and approximately, the first 100 codons of the propeptide, followed by an XbaI site.

Insertion of cDNA into M13mp18 Cloning Vehicle The amplified cDNA fragment was then inserted, using SalI and XbaI restriction enzymes, into the double stranded replicative form of bacteriophage M13mp18 which contains a multiple cloning site having compatible SalI and XbaI sequences. The resulting clone is known as pADl. See

Arrand, J.R. et al. J. Mol. Biol., 118, 127-135 (1978) and Zain, S.S. et al. J. Mol. Biol., 115, 249-255 (1977) for the properties of SalI and XbaI restriction enzymes respectively. The vWF cDNA insert was completely sequenced using single- stranded dideoxy methodology (Sanger, F. et al. Proc. Natl. Acad. Sci. USA. 74, 5463-5467 (1977)) to confirm that the VWF cDNA fragment contained the correct vWF coding sequence.

Primer Directed Amplification of cDNA - Phase II cDNA corresponding to mature vWF amino acid residues 441 to 732 was then amplified in a polymerase chain reaction.

For amplification, the pvWF clone encoding the entire pre-pro-vWF gene was used. Alternatively, a cDNA corresponding to mature subunit residues 441 to 732 may be prepared and then amplified directly from platelet mRNA following the procedure of Newman, P.J. et al. J. Clin. Invest.. 82, 739-743 (1988).

Suitable flanking oligonucleotides were synthesized as follows: Oligonucleotide (9) - see SEQ ID NO: 11

Oligonucleotide (10) - see SEQ ID NO: 12

5' - G- AAGCTT- AC- CAT- GGA- GTT- CCT- CTT- GGG - 3'

HindIII Met Ser Asn Arg Lys Pro

732 731 730 729 728 727

-or- 3' - GGG- TTC- TCC- TTG- AGG- TAC- CA- TTCGAA- G - 5'

Pro Lys Arg Asn Ser Met HindIII

727 728 729 730 731 732

(equivalent to anticoding strand)

The ends of the double stranded vWF cDNA fragment product were then modified with BamHI linkers (Roberts, R.J. et al. Nature, 265, 82-84 (1977)), digested with BamHI, and inserted into the BamHI site of pAD1, which site is directly downstream(3') from the XbaI site. The resultant plasmid was designated pAD2.

Loopout Mutagenesis of pAD2. Site-directed (loopout) mutagenesis was then performed to synchronize the reading frames of the first insert with the second insert simultaneously deleting all propeptide codon sequence (except that encoding the first 3 amino terminal residues of the propeptide), and the remaining bases between the XbaI and BamHI sites.

As a loopout primer, the following oligonucleotide was utilized which encodes the four carboxy-terminal amino acid residues of the signal peptide, the three amino-terminal residues of the propeptide, and amino acid residues 441 to 446 of the mature vWF subunit sequence.

Oligonucleotide (11) - see SEQ ID NO: 13

5' - GGGACCCTTTGTGCAGAAGGACGGCGTTTTGCCTCAGGA - 3'

Arg₄₄₁ Gly₄₄₆

The loopout of undesired nucleotide sequence was

accomplished following the procedure of Kunkel, T.A., Proc. Natl. Acad. Sci. USA, 82, 488-492 (1985). This procedure involves the performance of a series of steps to take advantage of conditions which select against a uracil

containing DNA template:

(A) M13mp18 phage (containing cDNA corresponding to the consensus translation initiation sequence, the signal peptide, approximately the first 121 amino acids of the propeptide, residual intervening

M13mp18 polylinker sequence, and codons

corresponding to mature subunit sequence residues 441 to 732) is grown in an E.coli CJ236 mutant dut-ung- strain in a uridine rich medium. Since this E.coli strain is deficient in deoxyuridine triphosphatase (dut-), an intracellular pool of dUTP accumulates which competes with dTTP for incorporation into DNA. (see Shlomai, J. et al. J. Biol. Chem.. 253(9), 3305-3312 (1978). Viral DNA synthesized under these conditions includes several uracil insertions per viral genome and is stable only in an E.coli strain which is incapable of removing uracil, such as (ung-) strains which lack uracil glycosylase. Uracil-containing nucleotides are lethal in single stranded (⁺) M13mp18 DNA in ung⁺ strains due to the creation of abasic sites by uracil glycosylase.

(B) Single-stranded (⁺) viral DNA is isolated from

culture media in which phage were grown in E.coli strain CJ236 dufung^". The single stranded (⁺) form of the virus contains the specified vWF cDNA at its multiple cloning site. This cDNA is equivalent to the transcribed vWF cDNA strand. (C) Oligonucleotide (11) is then annealed in vitro to single stranded (⁺) phage DNA, thereby looping out the undesired sequence. Generally, a wide range of oligonucleotide concentrations is suitable in this procedure. Typically 40 ng of oligonucleotide was annealed to 0.5-1.0 μg M13mp18 phage (⁺) DNA. (D) All missing sequence of the M13mp18(-) strand is then completed in vitro using T₇ DNA polymerase and T₄ DNA ligase in an environment containing dTTP, dGTP, dATP and dCTP, thereby generating a chimeric vWF cDNA sequence without the undesired

intermediate sequence.

(E) The double stranded M13mp18 phage, now containing a thymine normal (-) strand and a (⁺) strand with several uracil substitutions, is transformed into a wild type E.coli XL-1 Blue (Stratagene, La Jolla,

CA) strain which contains normal levels of uracil glycosylase and deoxyuridine triphosphatase.

(F) Uracil glycosylase and other enzymes present in the new host initiate destruction of the uracilcontaining (⁺) strand of the double stranded phages, leading after replication in the host of remaining phage (-) strand DNA to the presence of stable thymine-normal double stranded (RF) DNA which reflects the desired deletion. Upon completion of mutagenesis procedures, the sequence of the vWF cDNA insert was confirmed using the single stranded DNA dideoxy method. (Sanger, F. et al., supra).

A second mutagenesis procedure, following steps (A) to (F) above, was performed to add to the cDNA insert a

translation termination codon (TGA), and an XbaI restriction site (TCTAGA) . The oligonucleotide, again synthesized by the phosphoramadite method and containing also sequence homology at its 3' end with the M13mp18 vehicle sequence, was as follows. The stop codon was added after residue 730.

Oligonucleotide (12) - see SEQ ID NO: 14

5'- GGGCCCAAG· AGG· AAC· TGA· TCTAGA· AAGCTTGGCACTGGC -3'

Arg₇₂₉Asn₇₃₀ XbaI The final M13mp18 recombinant containing the desired construct as a SalI - XbaI insert was designated pAD3-l. In addition to the XbaI site created 3' to the termination codon, an XbaI site exists in the polylinker region of

M13mp18 directly 5' to the SalI site. The vWF insert was again sequenced by the dideoxy method to verify organization and integrity of the components.

Cloning of the SaIl - XbaI Fragment of

PAD3-1 Into the pBluescript II KS(-) Vector The SalI-XbaI fragment was then removed from pAD3-l (as contained within the XbaI-XbaI fragment) and inserted into pBluescript II KS(-) vector (Stratagene, La Jolla, CA) which had been previously digested with XbaI. pBluescript II KS(-) contains an XhoI restriction site which is 5' to the XbaI insert and a NotI site which is directly 3' to the XbaI insert. A resultant plasmid selected as having the proper insert orientation was designated pAD3-2. Reference to the restriction map for pBluescript II KS(-) shows that an EcoRI site is present in the polylinker region thereof between the XhoI restriction site and the XbaI site, and is therefore useful (see Example 21 below) for inserting vWF gene

sequences containing Type IIB mutations into pCDM8 vectors so that stable mutant transformants can be generated.

Construction of Plasmids for Integration into Mammalian Cells A selection procedure, based on aminoglycosidic

antibiotic resistance, was then employed to select

continuously for transformants which retained the vWF

expression plasmid. pCDM8 vector (developed by B. Seed et al. Nature. 329, 840-842 (1987) and available from Invitrogen, San Diego, CA) was modified by Dr. Timothy O'Toole, Scripps Clinic and

Research Foundation, La Jolla, CA to include a neomycin resistance gene (phosphotransferase II) that was cloned into the BamHI restriction site of pCDM8 as a part of a 2000 base pair BamHI fragment. The site of the BamHI insert is

indicated by an arrow in Figure 5. The protein produced by the neomycin(neo) gene also confers resistance against other aminoglycoside antibiotics such as Geneticin^® G418 sulfate (Gibco/Life Technologies, Inc., Gaithersburg, MD). The neo gene is provided by the Tn5 transposable element and is widely distributed in procaryots. Lewin, J., Genes, 3rd ed., p.596, Wiley & Sons (1987). The final construct places the neo gene under the control of an SV40 early promoter. Several other suitable expression vectors containing neomycin resistance markers are commercially available:

pcDNA 1*° (Invitrogen, San Diego, CA), Rc/CMV (Invitrogen, San Diego, CA) and pMAM⁰⁰⁰ (Clontech, Palo Alto, CA). If

necessary, the vWF fragment may be differently restricted or modified for expression capability in these other expression plasmids.

The XhoI-Notl fragment of pAD3-2 was therefore inserted into pCDM8^nco which had been restricted with XhoI and NotI. Ampicillin sensitive E.coli strain XS-127 cells (Invitrogen, San Diego, CA) were transformed with the resultant ligated DNA mixture following the method of Hanahan, D., J. Mol.

Biol.. 166, 557-580 (1983).

Plasmids from resultant colonies were characterized by restriction mapping and DNA sequencing to identify colonies which contained the intended insert. One such appropriate plasmid (designated pAD5/WT) was maintained in E.coli strain XS-127, and was selected for mammalian cell transformation procedures.

Prior to use in transforming mammalian cells,

supercoiled plasmids (pAD5/WT) were recovered from host

E.coli by an alkaline cell lysis procedure, Birnboim, H.C and Doly, J., Nucleic Acids Research. 7,1513 (1979), followed by purification by CsCl/ethidium bromide equilibrium

centrifugation according to Maniatis, T. et al., Molecular Cloning, 2nd ed., p. 1.42, Cold Spring Harbor Laboratory Press (1987).

Transformation of Chinese Hamster Ovary Cells pAD5/WT was introduced into CHO-K1 Chinese hamster ovary cells (ATCC-CCL- 61) by a standard calcium phosphate-mediated transfection procedure. Chen, C et al. Mol. Cell. Biol., 7(8), 2745-2752 (1987).

CHO-K1 cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Gibco/Life Technologies, Inc.,

Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS), 0.5 mM of each nonessential amino acid (from NEAA supplement, Whittaker, Walkersville, MD) and 2 mM L-glutamine under a 5% CO₂ atmosphere, and then

subcultured 24 hours prior to transformation at a density of 1.5 × 10⁵ cells per 60 mm tissue culture dish (approximately 25% of confluence). CHO-K1 cells have a doubling time in DMEM/10%FCS of approximately 16 hours under these conditions.

To accomplish transformation, pAD5/WT plasmids were recovered from cultures of E.coli strain XS-127, according to the method of Birnboim, H.C and Doly, J., Nucleic Acids

Research. 7, 1513 (1979). Ten μg of plasmids were applied to the cells of each 60 mm dish in a calcium phosphate solution according to the method of Chen et al., supra. After

inoculation with plasmid, the cells were maintained in

DMEM/10% FCS for 8 hours at 37°C in a 5% CO₂ atmosphere.

The growth medium was then replaced with a solution of phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HP0₄-7H₂0/1.4 mM KH₂PO₄, pH 7.4, hereinafter "PBS",

containing also 10% (v/v) glycerol. The cultures were then maintained in glycerol-PBS for 2 minutes to increase the efficiency of transformation (see Ausukel, et al., eds.

Current Protocols in Molecular Biology, p.9.1.3, Wiley & Sons (1987) . After 2 minutes the glycerol-PBS solution was

replaced with DMEM/10% FCS. After approximately 24 hours of growth at 37°C in a 5% CO₂ atmosphere, the cells were trypsinized as follows. Growth medium for each dish was replaced by 3 ml of 0.25% trypsin in PBS. Trypsinization was conducted for 3 minutes. The

trypsin-containing medium was removed and the dishes were then placed in the incubator for a further 15 minutes after which the cells were resuspended in DMEM containing 10% fetal calf serum. The cells from each dish were then split 20 fold, and plated at a density of 3 × 10⁴ cells/60 mm dish (approximately 5% of confluence).

Production of stable transformants, which have

integrated the plasmid DNA, was then accomplished by adding Geneticin^® G418 sulfate to the 60 mm dishes to a

concentration of 0.8 mg/ml. Growth was continued for 10-14 days at 37°C in a 5% CO₂ atmosphere. Surviving independent colonies were transferred to 12- well plates using cloning rings and then grown for another seven days in DMEM/10% FCS supplemented with 0.8 mg/ml of Geneticin^®. Under these conditions, 3 to 7 surviving colonies per plate were apparent after 10-14 days. Approximately 100 stable transformants can be isolated from each original 60 mM dish originally

containing approximately

5 × 10⁵ cells at a plate density of 50-70% of confluence.

Fifty to seventy percent of G418-resistant cell lines produce the 441-730 mature vWF subunit fragment. The

specific geometry of integration of each clone presumably prevents expression in all cases. Stable transformants were then cultured and maintained at all times in medium

containing Geneticin^® G418 sulfate (.8 mg/ml) to apply continuous selection.

Colonies expressing the recombinant 441-730 vWF

polypeptide were detected by dot-blot analysis on nitrocellulose after lysis in disruption buffer (see Cullen,

Methods in Enzvmology. 152, 684-704 (1987)) comprising 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, 10 mM benzamidine, 1 mM PMSF, 1% (w/v) Non-idet 40 (an octylphenol-ethylene oxide condensate containing an average of 9 moles of ethylene oxide/mole phenol), Sigma, St. Louis, MO.

RG-46 (see Fugimura, Y. et al. J. Biol. Chem.. 261(1), 381-385 (1986) and Fulcher, CA. et al. Proc. Natl. Acad. Sci. USA, 79, 1648-1652 (1982)) was used as the primary antibody. The secondary antibody (¹²⁵I-rabbit anti-mouse IgG) which had been labelled by the method of *Fraker, P.J. et al. Biochem. Biophys. Res. Commun. , 80, 849-857 (1978) was incubated for 60 minutes at 25°C on the nitrocellulose sheet. After rinsing, the nitrocellulose was developed by

autoradiography to identify those colonies expressing the vWF fragment.

Secretion of the von Willebrand Factor Fragment

Secretion of the 441-730 mature vWF subunit fragment into the culture medium by CHO-K1 cells was confirmed by immunoprecipitation and immunoaffinity chromatography of culture medium.

Confluent transformed CHO-K1 cells were rinsed three times with PBS to remove bovine vWF and then incubated in DMEM without FCS for 16 hours at 37°C in a 5% CO₂ atmosphere. To a 5 ml volume of the culture medium was added a 1/10 volume (0.5 ml) of lOx immunoprecipitation buffer (10×IPB) which comprises 100 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 10 mM EDTA, and 10% (w/v) Non-idet 40. It has been established that bovine vWF-derived polypeptides present in fetal calf serum do not react with NMC-4.

The mixture was then incubated for 16 hours at 4°C with approximately 0.05 mg of NMC-4 or 0.05 mg of RG-46 murine monoclonal anti-vWF antibody (or 0.1 mg of both) allowing formation of IgG-vWF complexes. Immune complexes were precipitated by taking advantage of the affinity of protein A (isolated from the cell wall of Staphylococcus aureus) for constant regions of heavy-chain antibody polypeptides

following generally the method of Cullen, B. et al., Meth. Enzymology. 152, 684-704 (1987). See also Harlow, E. et al. eds, Antibodies. A Laboratory Manual. Chapters 14-15, Cold Spring Harbor Laboratory Press (1988) .

Protein A-Sepharose^® beads were purchased from Sigma, St. Louis, MO. Immune complexes were then precipitated with the beads in the presence of 3 M NaCl/1.5 M glycine (pH 8.9), and washed twice with lx IPB and then once with 1x IPB without Non-idet 40.

Immunoprecipitated proteins were then electrophoresed in polyacrylamide gels containing sodium docecyl sulfate (SDS-PAGE) following the method of Weber, K. et al., J. Biol.

Chem.. 244, 4406-4412 (1969), or as modified by Laemli, U.K., Nature. 227, 680-685 (1970), using an acrylamide

concentration of 10%. Samples of immune-complexed vWF protein were dissociated prior to electrophoresis by heating at 100°C for 5 minutes in non-reducing and 2% SDS-containing acrylamide gel sample buffer to disrupt non-covalent bonds. The protein A-Sepharose^®4B beads were spun down and

discarded. Visualization was accomplished with Coomassie blue staining which revealed the dominant vWF-derived

polypeptide species to have an apparent molecular weight, based on molecular weight markers, of about 116,000 daltons.

Protein bands in duplicate gels were blotted and

immobilized onto nitrocellulose sheets (Schleicher & Schuell Co. , Keene, NH) and the pattern was then visualized using immunoreactivity according to the highly sensitive "Western blot" technique. Burnette, et al., A. Anal. Biochem.. 112, 195-203 (1981).

The von Willebrand factor-specific monoclonal antibodies (from mice) used to identify the polypeptides were RG-46 (see Fugimura, Y. et al. J. Biol. Chem.. 261(1), 381-385 (1986), Fulcher, CA. et al., Proc. Natl. Acad. Sci. USA. 79, 1648-1652 (1982)), and NMC-4 (Shima, M. et al., J. Nara Med.

Assoc.. 36, 662-669 (1985)), both of which have epitopes within the expressed vWF polypeptide of this invention. The secondary antibody (¹²⁵I-rabbit anti-mouse IgG), labelled by the method of Fraker, P.J. et al., Biochem.

Biophys. Res. Commun., 80, 849-857 (1978)), was incubated for 60 minutes at 25°C on the nitrocellulose sheet. After rinsing, the sheet was developed by autoradiography.

Growth medium from non-transformed CHO-K1 cells shows no immunoreactivity with RG-46 and NMC-4 anti-vWF monoclonal antibodies under identical conditions.

The 116 kDa fragment may also be isolated from the culture medium of CHO-K1 cells using immunoaffinity

chromatography. Approximately 300μg of the 116 kDa fragment can be recovered from 500 ml of culture medium derived from transformed CHO-K1 culture plates using NMC-4 antibodies coupled to particles of Sepharose^®4B. Example 8 - Induction of platelet aggregation by

the homodimeric 116 kDa von Willebrand factor fragment derived from the

culture medium of stable CHO-K1 transformants

The tryptic 116 kDa fragment has been previously

characterized as a dimer consisting of two identical

disulfide-linked subunits each corresponding to the tryptic 52/48 kDa fragment of vWF and containing the mature subunit sequence from residue 449 to residue 728. Owing to its bivalent character, the dimeric 116 kDa fragment can support ristocetin-induced platelet aggregation whereas the

constituent 52/48 kDa subunit cannot (see Mohri, H. et al., J. Biol. Chem.. 264(29), 17361-17367 (1989)).

Stable pAD5/WT CHO-K1 transformants, and untransformed CHO-K1 cells as controls, were each grown to 90% of

confluence in DMEM/10% FCS, at 37°C in a 5% CO₂ atmosphere. The 60 mm plates were then rinsed twice with PBS and the incubation was continued in DMEM (without FCS) for 24 hours. The resultant serum-free culture medium was collected and concentrated (at 18°C) 300 fold in a centrifugation filtration apparatus, Centricon 30, Amicon Co., Lexington,

MA.

Dose-dependent platelet aggregation curves were obtained by the addition of concentrated culture medium from pAD5/WT transformed cells to platelets. No aggregation was seen in the presence of control culture medium derived from

untransformed CHO-K1 cells. Platelets for the assay were prepared using albumin density gradients according to the procedure of Walsh, et al. British J. of Hematology. 36, 281- 298 (1977). Aggregation was monitored in siliconized glass cuvettes maintained at 37 °C with constant stirring (1200 rpm) in a Lumi-aggregometer (Chrono-Log Corp., Havertown, PA) .

Aggregation experiments followed generally the procedure of Mohri, H. et al., J. Biol. Chem.. 264(29), 17361-17367

(1989). Two to ten μl quantities of 300-fold concentrated FCS-free DMEM from cultures of pAD5/WT-transformed and control untransformed CHO-K1 cells (CM) were brought up to 100 μl by dilution with "Hepes" buffered saline, comprising 20 mM Hepes, N-[2-hydroxyethyl]piperazine-N'[2-ethanesulfonic acid], (pH 7.4), and 0.15 M NaCl. The 100 μl samples were then mixed with 200 μl of platelet suspension (4 × 10⁸/ml) and then incubated with stirring in the aggregometer for 5 minutes. Ristocetin was then added to a final

concentration of lmg/ml at the injection timepoints (time zero). Aggregation was monitored by recording changes in light transmittance. Platelet aggregation can be observed with as little as 100 μl of unconcentrated serum-free medium from pAD5/WT-transformed cell lines. Serum-free medium from control untransformed cultures concentrated up to 300 fold, and assayed at up to 10 μl concentrated medium/100 μl sample did not induce platelet aggregation.

Preincubation with Monoclonal Antibodies

As a further control to confirm the specificity of the ristocetin-induced 116 kDa vWF fragment-platelet interaction, platelets were preincubated with anti-platelet glycoprotein lb monoclonal antibody LJ-Ib1 which has been specifically demonstrated to block vWF-platelet GPIb-IX receptor

interaction (Handa, et al., J. Biol. Chem. , 261, 12579-12585 (1986)).

Platelets subjected to this preincubation did not exhibit an aggregation response whereas platelets similarly preincubated with monoclonal antibody LJ-CP3 (TrapaniLombardo et al., J. Clin. Invest.. 76, 1950-1958 (1985) gave an effective aggregation response. LJ-CP3 has been

demonstrated to block platelet GPIIb/IIIa receptor sites and not vWF-specific GPIb-IX receptors. To perform the assays antibody LJ-Ib1 or antibody LJ-CP3 was added, at a

concentration of 100 μg/ml, to the platelet/serum mixture while the mixture was being stirred in the aggregometer, and at a timepoint one minute prior to the point when ristocetin (to 1 mg/ml) was added.

Example 9 -Construction of a mammalian transformant for

the expression of the monomeric 441-730 mature von Willebrand factor subunit fragment with cysteine- to-glycine mutations at residues 459, 462 and 464 This example is illustrative of conditions under which a DNA sequence encoding a mature vWF subunit fragment, which has an amino terminus at residue 441 (arginine) and a carboxy terminus at residue 730 (asparagine) and which further contains glycine residues substituted for cysteine residues at positions 459, 462 and 464 thereof, can be constructed and transfected into mammalian cells.

The SalI-XbaI insert of pAD3-2 (see Example 7) was removed by restriction and then cloned into pcDNA1 vector (Invitrogen, San Diego, CA) which had been previously

digested with XhoI and XbaI restriction enzymes. Since XhoI and Sail restriction sites contain identical internal

sequences -TCGA- / -AGCT- , a Sail restricted fragment may be annealed into an XhoI site. The fragments were ligated with T₄ DNA ligase; however the integrity of the XhoI site was not restored. This plasmid construct was designated pAD4/WT. Site-directed mutagenesis using M13mp18 pAD4/WT was restricted with EcoRI and SmaI enzymes.

pcDNAl vector contains an EcoRI site within its polylinker region which is upstream from the XhoI ("SalI") site but contains no SmaI site. As shown in Figure 1 (SEQ ID NO: 1), a unique SmaI site (CCCGGG) is contained within the vWF cDNA insert, spanning mature subunit residues 716 (glycine) to residue 718 (glycine).

Accordingly, an approximate 950 base pair EcoRI-SmaI fragment of pAD4/WT was subcloned into the EcoRI-SmaI site within the polylinker region of M13mp18 phage. The vWF sequence in M13mp18 was then mutagenized and reinserted into the previously restricted pAD4/WT construct leading to reassembly of the intact residue 441-730 vWF sequence. The mutagenesis followed the procedure of Example 1 and Kunkel, T.A. , supra, and utilized the following

oligonucleotide.

Oligonucleotide (13) - see SEQ ID NO: 15

3' - GGACTCGTGCCGGTCTAACCGGTGCCACTACAACAG - 5' 5' - cctgagcacggccagattggccacggtgatgttgtc - 3'

Gly₄₅₉ Gly₄₆₂ Gly₄₆₀

The hybridizing oligonucleotide is shown (3' → 5') in capital letters and is equivalent to transcribed strand (noncoding strand DNA). Underlined letters indicate the single base mutations for the mutant codons. The equivalent coding strand is shown in lower case letters with the corresponding glycine substitutions identified by three letter designation.

The mutant 950 base pair EcoRI-SmaI fragment was then re-inserted into the EcoRI-SmaI site of the previously restricted pAD4/WT plasmid. The mutant construct was

designated pAD4/Δ3C To facilitate long-term storage and propagation, pAD4/Δ3C was transformed into ampicillin

sensitive E.coli strain XS-127 according to the method of Hanahan, D., J. Mol. Biol.. 166, 557-580 (1983). Consistent with the procedures of Example 1, the sequence of the mutant cDNA was confirmed by the dideoxy method and the plasmid was purified by CsCl/ethidium bromide equilibrium centrifugation. Transformation of COS-1 cells pAD4/Δ3C was introduced into COS-1 cells (SV 40

transformed African Green monkey kidney cells, ATCC - CRL 1650) by a standard calcium phosphate-mediated transfection procedure. Chen, C et al., Mol. Cell. Biol.. 7(8), 2745- 2752 (1987).

COS-1 cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Gibco/Life Technologies, Inc.,

Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) under a 5% CO₂ atmosphere, and then subcultured 24 hours prior to transformation at a density of 1.5 × 10⁵ cells/60 mm tissue culture dish (approximately 25% of confluence). COS-1 cells have a doubling time in DMEM/10% FCS of approximately 20 hours under these conditions.

To accomplish transformation, pAD4/Δ3C plasmids were recovered from cultures of E.coli strain XS-127 according to the method of Birnboim, H.C and Doly, J., Nucleic Acids

Research, 7, 1513 (1979). Ten μg of plasmids were applied to the cells of each 60 mm dish in a calcium phosphate solution according to the method of Chen et al., supra. After

inoculation with plasmid, the cells were maintained in

DMEM/10% FCS for 8 hours at 37°C in a 5% CO₂ atmosphere.

The growth medium was then replaced with a solution of phosphate-buffered saline/10% (v/v) glycerol. The cultures were then maintained in glycerol-PBS for 2 minutes to

facilitate the production of transformants (Ausukel, et al. eds, Current Protocols in Molecular Biology, p.9.1.3, Wiley & Sons (1987)). After 2 minutes, the glycerol-PBS solution was replaced with DMEM/10% FCS. Antibiotic resistance was not used to select for stable transformants. The cells were then maintained at 37°C in DMEM/10% FCS in a 5% CO₂ atmosphere.

Example 10 -Transformation of COS-1 cells by pAD4/WT plasmids

COS-1 cells were also transformed successfully with pAD4/WT plasmids. Although antibiotic resistance was not used to select for stable transformants, transient expression of the 116 kDa fragment therefrom was particularly useful for the purpose of comparing the properties of the 116 kDa mutagenized polypeptide produced by pAD4/Δ3C plasmids to those of the pAD4/WT 116 kDa homodimer.

Following the procedures of Example 9, pAD4/WT plasmids were recovered from storage cultures of E.coli strain XS-127. Transformation of COS-1 cells with pAD4/WT was then

accomplished using the procedures of Example 9. The cells were then maintained at 37°C in DMEM/10% FCS in a 5% CO₂ atmosphere.

Example 11 - Construction of mammalian transformants

which express mutant 441-730 mature von

Willebrand factor subunit fragments

wherein each mutant contains a single

cysteine-to-glycine substitution

Following the procedures of Example 9, and using

suitable oligonucleotides for site-directed mutagenesis, three plasmids (pAD4/G⁴⁵⁹, pAD4/G⁴⁶² and PAD4/G⁴⁶⁴, collectively referred to as "pAD4/ΔlC plasmids") were constructed. Such plasmids are identical to pAD4/WT except that each contains a single base pair mutation which corresponds to a single cysteine to glycine substitution at mature vWF subunit residue positions 459, 462 and 464 respectively. The

oligonucleotides used are identical to oligonucleotide (13) used to prepare pAD4/Δ3C except that each contains only one of the three mutant codons of that oligonucleotide, the other two codons being represented by the wild type coding

sequence. To facilitate long-term storage and propagation, samples of pAD4/G⁴⁵⁹, pAD4/G⁴⁶², and pAD4/G⁴⁶⁴ were each cloned into ampicillin sensitive E.coli strain XS-127 following the method of Example 9.

Consistent with the procedures of Example 9, the sequences of the mutant cDNAs were confirmed by the dideoxy method and the plasmids were purified by CsCl/ethidium bromide equilibrium centrifugation.

Transformation of COS-1 cells with either pAD4/G⁴⁵⁹, PAD4/G⁴⁶² or PAD4/G⁴⁶⁴ plasmids was accomplished according to the protocol of Example 9. Antibiotic resistance was not used to select for stable transformants. The cells were then maintained at 37°C in DMEM/10% FCS in a 5% CO₂ atmosphere.

Example 12 - Expression and characterization

of von Willebrand factor subunit fragments by COS-1 cells transformed

with pAD4/WT and pAD4/Δ3C plasmids

COS-1 cells which had been transformed with pAD4/Δ3C or pAD4/WT plasmids according to the procedures of Examples 9 and 10 respectively were cultured to express the encoded vWF DNA as explained below. COS-1 cells similarly transformed with pcDNAl plasmid vector (not containing a vWF cDNA insert) were used as controls.

COS-1 cells at a density of 4-5 × 10⁵/60 mm dish were transformed by adding, at time zero, 10 μg of pAD4/WT, pAD4/Δ3C or pcDNAl plasmid. Following the procedure of

Examples 9 and 10, the cells were glycerol-shocked after a period of 8 hours. The cells were then covered with DMEM/10% FCS at 37°C in a 5% CO₂ atmosphere for 32 hours.

The cells for each culture were then rinsed three times with PBS and the incubation was continued with DMEM (without FCS) which was supplemented with ³⁵S-methionine (Amersham Co., Arlington Heights, IL) having a specific activity of 1000 Ci/mmol to a final concentration of 100 μCi/ml. The cells were returned to the incubator for 16 hours, after which time the respective culture media were harvested for purification by immunoprecipitation of secreted vWF polypeptides.

Immunoprecipitation followed generally the procedure of Example 7. Five ml volumes of culture media were incubated with 0.5 ml of 10X immunoprecipitation buffer, 0.05 mg of NMC-4 antibody and 0.05 mg of RG-46 antibody for 16 hours.

Treatment with protein A-Sepharose^®4B was performed according to Example 7. Samples of IgG-complexed vWF protein were dissociated prior to SDS-PAGE in SDS-containing sample buffer.

For analysis of the vWF polypeptides under reducing conditions, the sample buffer was modified to contain 100 mM dithiothreitol (DTT).

Results The gels run under reducing and non-reducing conditions were dried and subject to autoradiography to develop the ³⁵S label. No ³⁵S-labelled protein was detected as an

immunoprecipitate derived from control cultures of COS-1 cells (transformed by unmodified pcDNAl vehicle) under either reducing or non-reducing conditions.

COS-1 cells transformed with pAD4/WT plasmids produce, under non-reducing conditions, a prominent ³⁵S-labelled band of an approximate apparent molecular weight of 116,000. This value is consistent with proper mammalian glycosylation of the 441-730 fragment. When run under reducing conditions, no 116 kDa material is apparent, consistent with the reduction of the disulfide bonds which stabilize the 116 kDa homodimer. Under reducing conditions, a prominent ³⁵S-labelled band is visualized of approximately 52,000 apparent molecular weight. The apparent 52 kDa value is again consistent with proper glycosylation of the reduced monomeric 441-730 fragment. The gel lanes corresponding to transformation with pAD4/Δ3C show no apparent 116 kDa material. Instead a band is apparent, under reducing and non-reducing conditions, at an apparent molecular weight of approximately 52,000. Thus, mutagenesis to replace cysteine residues 459, 462 and 464 within the 441-730 vWF fragment with glycine residues results in the successful expression of a non-dimerizing polypeptide presumably having only intrachain (471 to 474 and 509 to 695) disulfide bonds. Interaction with NMC-4 (see also Example 7) is known to require an intact 509 to 695 intrachain disulfide bond, thereby demonstrating the presence of native wild type tertiary structure in the polypeptide produced by pAD4/Δ3C

The presence in the gels of low molecular weight ³⁵S- labelled material (under reducing and non-reducing

conditions) probably indicates that not all vWF polypeptides produced by pAD4/WT constructs successfully dimerize and that proteolysis and/or incomplete glycosylation of the

polypeptide may prevent higher yields. Proteolysis and/or incomplete glycosylation also presumably affect the yield of the monomeric vWF polypeptide produced by the pAD4/Δ3C transformants. Some high molecular weight aggregate material (essentially not entering the gels) is present in non-reduced samples from pAD4/WT and pAD4/Δ3C Example 13 -Use of NMC-4 monoclonal antibody to

immunoprecipitate vWF polypeptides secreted by PAD4/WT and PAD4/Δ3C transformed COS-1 cells

The NMC-4 monoclonal antibody has as its epitope the domain of the von Willebrand factor subunit which contains the glycoprotein lb binding site. Mapping of the epitope has demonstrated that it is contained within two discontinuous domains (comprising approximately mature vWF subunit residues 474 to 488 and also approximately residues 694 to 708) brought into disulfide-dependent association by an intrachain (residues 509 to 695) disulfide bond. Thus, reactivity with NMC-4 is important evidence of whether a particular recombinant 441-730 mature vWF subunit fragment has assumed the tertiary structure of the analogous wild type residue 441-730 domain. Accordingly, the procedure of Example 12 was followed to characterize vWF polypeptides secreted by pAD4/WT and pAD4/Δ3C transformed COS-1 cells, with the modification that immunoprecipitation of the culture media was effected solely with NMC-4 antibody (0.05 mg NMC-4 per 5 ml of culture media to which 0.5 ml of 10X immunoprecipitation buffer had been added).

Samples were run under reducing and non-reducing

conditions. Consistent with the results of Example 12, the major component isolated from pAD4/WT culture medium has an apparent molecular weight of 116 kDa under non-reducing conditions and 52 kDa under reducing conditions.

Although only a small fraction of the total pAD4/Δ3C derived vWF polypeptide material binds to NMC-4 (compared to conformation independent RG-46), a band of apparent molecular weight of 52 kDa is visible under reducing and non-reducing conditions in gels of NMC-4 immunoprecipitates.

Example 14 - Expression and characterization of

von Willebrand factor subunit fragments produced by COS-1 cells transformed with

PAD4/G⁴⁵⁹. PAD4/G⁴⁶² or PAD4/G⁴⁶⁴ plasmids

Transformation of COS-1 cells by either pAD4/G⁴⁵⁹,

PAD4/G⁴⁶² or pAD4/G⁴⁶⁴ plasmid (collectively the "pAD4/ΔlC plasmids") was accomplished according to the procedure of Example 11. Culture media were analyzed for secreted vWF polypeptide according to the procedure of Example 7, using only NMC-4 for immunoprecipitation.

³⁵S-labelled proteins, prepared according to Example 12, were immunoprecipitated by NMC-4 and run in SDS-polyacrylamide gels under reducing and non-reducing conditions and compared with vWF antigen produced by pAD4/WT and pAD4/Δ3C transformants.

Substitution of any one of the 3 cysteines (459, 462, 464) believed responsible for interchain disulfide contacts in native mature subunits prevents the formation of the homodimeric 116 kDA polypeptide characteristic of pAD4/WT transformed COS-1 cells. These three vWF antigens with a single glycine substitution appear predominantly as monomeric polypeptides of an apparent molecular weight of 52,000 under reducing or non-reducing conditions. That the predominant material has an apparent molecular weight of 52 kDa is strongly suggestive of correct glycosylation by the COS-1 cell transformants duplicating glycosylation seen in the human 52/48 kDa tryptic vWF fragment. Some proteolyzed and/or inadequately glycosylated vWF antigen (molecular weight less than 52 kDa) is also apparent in the gels. The relatively small fraction of pAD4/Δ3C vWF polypeptide which is successfully folded and secreted, thereby presenting an NMC-4 epitope, was shown by the low intensity of the pAD4/Δ3C transformant autoradiograph band of apparent 52,000 molecular weight.

II. Introduction of Type IIB Mutations into vWF Polypeptides

Example 15 - Genetic characterization of patients

with Type IIB von Willebrand disease This example demonstrates the procedure used to identify the mutation(s) in the mature von Willebrand factor subunit responsible for Type IIB von Willebrand disease in particular patients. Patients selected for screening were previously determined to fulfill all of the criteria for a diagnosis of Type IIB von Willebrand disease. See Ruggeri, Z.M. et al., N. Engl. J. Med.. 302, 1047-1051 (1980).

The propositus determined to have a vWF gene with a Trp⁵⁵⁰ → Cys⁵⁵⁰ mutation is identified as patient No. 7 in the study reported in Kyrle, P.A. et al., Br. J. Hemat., 69, 55-59 (1988). The propositus determined to have a vWF gene with an Arg⁵¹¹ → Trp⁵¹¹ mutation is identified as patient No. 8 in the same study. Samples of blood were drawn from patients after obtaining informed consent according to the Declaration of Helsinki and institutional guidelines.

Platelets were collected from 50 ml of blood drawn into a 5 ml volume of 3.2% trisodium citrate as anticoagulant. The residual total platelet RNA was then isolated by

ultracentrifugation through a cesium chloride cushion

following the procedure of Newman, P.J. et al., J. Clin.

Invest.. 82, 739-743 (1988).

To generate double stranded cDNA, standard techniques were used. Total platelet RNA was primed for first-strand cDNA synthesis with a vWF-specific oligonucleotide

corresponding to the non-coding strand (transcribed strand) for mature vWF subunit residues 899-908.

Oligonucleotide (14) - see SEQ ID NO: 16

3' GGA CTG GAC CAC- GAC- GTC- TCC- ACG- ACG- AGG- TTCGAA 5' Pro Ser HindIII

899 908

The primed vWF mRNA population was then used as template for reverse transcriptase (from Moloney murine leukemia virus, Gibco/Bethesda Research Laboratories, Gaithersburg, MD) according to the procedure of Maniatis, T. et al.,

Molecular Cloning. 2 ed., Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY (1989). The RNA strands were then removed by alkaline hydrolysis and the first strand cDNA was primed for second strand synthesis using DNA polymerase I and then amplified in a polymerase chain reaction ("PCR") as described in Example 1 using oligonucleotide 14, and also oligonucleotide 15 (equivalent to coding strand, nontranscribed strand DNA, corresponding to amino acid residues 428-436). Oligonucleotide (15) - see SEQ ID NO: 17

5' GAATTC GTT GAC CCT GAA GAC TGT CCA GTG TGT 3'

EcoRI Val Cys

428 436 The product of a vWF pseudogene (said gene having an intron-exon arrangement similar to that of the functional gene within the region thereof corresponding to the mRNA region selected for amplification) was avoided in the PCR reaction by selecting priming oligonucleotides complementary to exons 23 and 24 (Mancuso, D.J. et al., J. Biol. Chem., 264, 19514-19527 (1989)) which are separated in the

functional gene by a 2000 base pair intron. Amplified DNA of the predicted length was therefore verified to be derived from platelet cDNA and not from genomic DNA corresponding to small quantities of leukocytes or other cells which may have contaminated the platelet preparation.

The amplified 1.4 kilobase cDNA fragment corresponding to mature subunit residues 428-908 was then subjected to further rounds of PCR amplification which split the fragment into two smaller overlapping cDNA regions (corresponding to amino acid residues 440-670 and 660-905) to facilitate sequence analysis.

Priming oligonucleotides therefor were synthesized

(according to the method of Example 1) to correspond

approximately to the first twenty nucleotides on the 5'

(upstream) and 3' (downstream) ends of each of the two overlapping fragments and contained also either an EcoRI (if 5') or HindIII (if 3') restriction sequence so that the amplified 440-670 or 660-905 sequences so prepared could be inserted into M13mp18 phage for sequencing. Resultant double stranded vWF cDNA corresponding to the residue 440-670 or 660-905 fragment was then inserted into the multiple cloning site of the double stranded replicative form of bacteriophage M13mp18 using EcoRI and Hind III restriction enzymes, and then sequenced by the single-stranded dideoxy method (Example 1, Sanger, F. supra) One patient was found to have a mutation at the codon corresponding to mature subunit residue 550 that specified a Trp to Cys mutation (^5' TGG^3' → ^5'TGC^3'). The transversion mutation destroys an Avail restriction site overlapping codons for residues 550-552 of the mature subunit. Absence of the restriction site was confirmed in the patient's genomic DNA. Another patient was found to have a mutation at the codon corresponding to mature subunit residue 511 specifying an Arg to Trp mutation (^5'AGG^3'→ ⁵TGG^3') . Both patients were found to be heterozygous for their particular amino acid substitutions, a finding consistent with the autosomal dominant mode of inheritance seen in most Type IIB patients. Ruggeri, Z.M. et al., N. Engl. J. Med. , 302, 1047-1051 (1980). In the event the mutation or mutations responsible for the altered properties of the mature vWF subunit from other particular Type IIB patients cannot be resolved into the above region of the mRNA, corresponding to residues 428-908, other regions may be selected for study using suitable oligonucleotides with reference to the published DNA sequence for the vWF gene.

Example 16 - Mutagenesis of peptide subdomains of

vWF to create additional vWF-derived

polypeptides having Type IIB-like properties It has been demonstrated that the GPIb(α) binding domain of vWF is formed primarily by residues contained in two discontinuous sequences, comprising approximately Cys⁴⁷⁴-Pro⁴⁸⁸ and approximately Leu⁶⁹⁴-Pro⁷⁰⁸ maintained in proper

conformation in native vWF by disulfide bonding. Mohri, H. et al., J. Biol. Chem.. 263(34), 17901-17904 (1988). It has also been demonstrated that an intrachain disulfide bond which is necessary to provide that conformation is formed by cysteine residues 509 and 695 (U.S. Application Serial No. 07/600,183, filed October 17, 1990 and Examples 1-6 reported herein) . The present development provides substantial evidence that the "loop" region of the mature vWF subunit (between residues 509 and 695) modulates the binding

properties toward GPIbα of the above mentioned primary sequence regions of vWF. The following methods are

representative of techniques which can be employed to (A) identify within the "loop" region of vWF further potentially important primary sequence subdomains or specific amino acids involved in modulating binding of vWF to GPIbα; and/or (B) create artificial vWF-derived polypeptide sequences with altered modulating or binding activity. Method 1 Random mutagenesis of the "loop"

to generate antithrombotic or

antihemorrhagic therapeutic polypeptides

Using vWF DNA from plasmid p5E (which encodes the amino acid sequence comprising mature subunit residues 441 to 733 in which the cysteine residues at positions 459, 462, 464, 471 and 474 thereof are replaced by glycine residues) , and random mutant oligonucleotides which will sequentially span the entire 187 amino acid "loop", novel variant DNA sequences can be constructed which encode variant vWF-derived

polypeptides. Resultant potential therapeutic polypeptides can be screened for relative binding affinity (1) in direct binding assays for affinity to GPIbα, or (2) in botrocetin or ristocetin induced binding assays, or (3) to conformation dependent vWF-specific antibodies. Random mutagenesis experiments can also be performed using vWF DNA constructs suitable for expression in mammalian cells such as those of Example 7.

Preparation of Oligonucleotides

Mutant oligonucleotides suitable for site directed mutagenesis protocols and spanning sequential 10 amino acid subdomains of the loop (for example corresponding to amino acids 510 - 519, 520 - 529, 530 - 539) can be generated using a procedure designed to yield a randomly mutagenized

oligonucleotide population. Hutchison, CA. et al., Proc. Natl. Acad. Sci.. USA. 83, 710-714 (1986). The randomized vWF oligonucleotide is then hybridized, for example, to

M13mp18 to copy the mutation into a residue 441-733 encoding DNA sequence.

The method of Hutchison, CA. et al. relies on automated synthesis of the oligonucleotide from the 3' end. In the Hutchison procedure, a random oligonucleotide population suitable for causing permutation of the residues between positions 504 and 524 of the mature vWF subunit would be constructed as follows. The oligonucleotide corresponds to transcribed strand DNA. As the chain is then built stepwise by the nonenzymatic 3'→5' addition of subsequent bases

(comprising the part of the vWF loop region to be surveyed), each of the four nucleoside phosphoramidite reservoirs

(A,T,G,C) for oligonucleotide synthesis would be "doped" with a small amount of each of the other three bases.

Incorporation of one of the "doping" nucleotides would result in a mutant oligonucleotide. The amount of doping can be adjusted to control results. The resultant randomized population of mutant oligonucleotides is then used in the standard site directed mutagenesis protocol (Example 1) to construct a pool of mutagenized vWF "loop" DNA sequences in M13mp18 corresponding to the mature vWF subunit residue 441733 fragment and suitable for subcloning into a bacterial expression system. It is possible to control the number of mutations per molecule by controlling the composition of the base mixtures. For example, it is possible to select for only single base pair substitutions or to select for molecules which have 2, 3, 4, or more mutations. The procedure developed by

Hutchison, supra typically employed solutions of each of the four bases in which approximately 1.5% impurity of each of the other three bases contaminates the original base

solutions. Mutagenesis using this particular doped mixture resulted in roughly 41% of clones with no base substitutions, 40% with one, 15% with two, 3% with three and 0.7% with four (for target nucleotide sequences corresponding to 10 amino acids). The resultant mutant M13mp18 populations are then subject to restriction (Example 1) , and the mutagenized DNA sequences are inserted into vectors or plasmids such as pET- 3A for expression in host bacterial cells. Large scale screening of mammalian clones is generally much more

difficult than for bacterial clones. However, promising mutations identified in bacterial constructs may later be inserted into mammalian or other eucaryotic host cells for further testing or for commercial-scale polypeptide

production.

The mutant clones can then be screened in GPIbα binding assays or in binding assays with vWF-specific monoclonal antibodies (as described below). Mutant clones having cell lysates which exhibit enhanced platelet binding or antibody response can be sequenced to determine the amino acid alteration(s) responsible for the mutant phenotype. In this way a very systematic analysis of the loop region of vWF can be performed and mutations which alter the binding of vWF to GPIbα can be identified. The mutagenesis techniques of Method 1 above is equally applicable to permuting the amino acid sequence regions of the mature subunit believed to represent the actual GPIbα binding site (Leu⁴⁶⁹-Asp⁴⁹⁸ and Glu⁶⁸⁹-Val⁷¹³) for the purpose of enhancing their GPIbα affinity. Method 2 Random mutation of targeted

subdomains to develop therapeutic polypeptides

To date, five residue positions within the residue 441-733 region of mature vWF subunit have been identified which when appropriately mutated result in potentiation of platelet aggregation response and with Type IIB disease. These sites are at amino acid positions 511 (Arg→Trp), 543 (Arg→Trp), 550 (Trp→Cys), 553 (Val→Met), and also possibly 561 (Gly→Asp). (The patient with a mutation at position 561 exhibits some Type IIB-like disease symptoms, namely enhanced platelet aggregation with low doses of ristocetin and was treated at the Greene Hospital of Scripps Clinic and Research

Foundation, La Jolla, CA). Since the known mutations indicate primary sequence subdomains wherein Type IIB

properties can be generated, random mutagenesis of the DNA corresponding to short peptide sequences directly adjacent to these particular sites would be emphasized. Randomly

mutagenized oligonucleotides, prepared and used as described above, and which span domains of approximately 10 amino acids adjacent to residues 510, 520, 530, 540, 550, 560, 570 and 580 can be utilized.

Method 3 Mutagenesis of specific target amino

acids to develop therapeutic polypeptides

Two of the known mutations which correlate with Type IIB von Willebrand disease result in replacement of a wild type codon, encoding a positively charged amino acid, with a codon corresponding to a neutral residue. It is probable that the electrical charge of particular subdomains in the "loop" must be maintained for proper in vivo function (i.e. preventing interaction of GPIbα with circulating multimeric plasma vWF until vascular injury triggers a sequence of events resulting in a conformational change in the vWF molecule or its GPIbα receptor). Positively charged amino acids that are proximal to arginine residues 511 and 543 can also be specifically mutated to code for amino acids which are neutral or possess, at physiological pH, negatively changed side chains. In addition, Type IIB disease-conferring neutral mutant codons such as Trp⁵⁴³, may be replaced by codons for other neutral or negatively charged amino acids. Representative further target amino acid sites predicted to yield mutant

polypeptides having Type IIB properties and resultant

therapeutic utility include the arginine residues at

positions 524, 545, 552, 571, 573, 578 and 579, the lysine residues at positions 534 and 549 and the histidine residues at positions 559 and 563. Method 4 Generation of additional mutant oligonucleotide constructs having therapeutic activity

There are numerous additional mutagenesis strategies which can be used to probe the specific structural features and amino acid sequence requirements therefor which confer upon the vWF loop region the ability to modulate GPIbα binding. Such strategies are also useful in constructing vWF-derived polypeptides containing, for example, mutant loop regions which are useful as therapeutics. Representative, additional mutagenesis strategies are hereafter described.

In the practice of this invention, effective

substitutions need not be made at the exact residue positions corresponding to the targeted wild type residues. For example, substitution of cysteine for Lys⁵⁴⁹ or Val⁵⁵¹ or for other nearby residues instead of for Trp⁵⁵⁰ may be performed, with the resultant polypeptides being subjected to screening for therapeutic utility.

Table 2 presents representative examples of potentially useful amino acid substitutions, deletions and additions which accomplish net reduction of positive charge at or adjacent to specific sites. Similar strategies can be employed at or adjacent to other specific residues of vWF to accomplish net reduction of negative charge or to break or form a hydrogen bond, salt bridge, or hydrophobic contact.

Table 2

with respect to the sequence: Ser- Arg- Leu -

510 512 a substitution of a neutral for Arg₅₁₁ → a neutral residue or negatively charged residue: such as Gly, Ser, Asn, Ala, or

Gln₅₁₁, or, for example, Asp an insertion of a negatively Arg₅₁₁- Asp- Leu₅₁₂

charged residue: a deletion of a positively Cys- Ser- Leu

charged residue: 510 512

Cloned vWF polypeptide constructs reflecting known Type IIB mutations may also be subject to the above mentioned random mutagenesis procedures and then screened for

restoration of normal binding function such as, for example, having a normal response in a modulator-induced GPIbα binding assay. For example, it may be demonstrated that a particular "reversion" mutation proximal to residue 511 would compensate for, and nullify the effect of the original Arg→Trp⁵¹¹ Type IIB mutation. The associated DNA sequence can then be determined to identify the relevant counteracting amino acid. Such procedures can be used to give important further evidence as to which other residue positions in the mature subunit vWF amino acid sequence are important modulators of GPIbα

binding. Screening of mutant vWF-derived

polypeptides for enhanced GPIbα binding activity

There is hereafter presented an effective method to screen randomly mutagenized mature vWF subunit polypeptide sequences for enhanced GPIbα binding activity, and resultant enhanced therapeutic utility.

To perform the assays, a device used for the enzyme-linked immunofiltration assay technique (ELIFA), Pierce Chemical Co., Rockford, IL, can be adapted in combination with immobilization of the mutant vWF-derived polypeptides to be tested. It is considered most efficient to initially test the effect of mutant codons on vWF polypeptides expressed from bacterial constructs and to then copy potentially useful mutations (using, for example, mutagenesis in M13mp18 vehicle and the procedure of Example 22) into a mammalian expression construct. High levels of mutant vWF polypeptides

corresponding to mutant DNA sequences can be expressed from pET-3A type bacterial expression plasmids such as p5E.

Mutant polypeptides constitute a major portion of host E.coli cell lysates and can be readily screened for GPIbα affinity.

Accordingly, site directed mutagenesis can be performed following the procedure of Example 1 using as template in M13mp18 the vWF fragment corresponding to p5E expression plasmid (Example 4) which because of the use of BamHI linkers in assembly of p5E is recovered therefrom and inserted into M13mp18 as an XbaI/HindIII fragment (see Example 17) . For the oligonucleotide pool, oligonucleotides each having randomly mutagenized residue 505 to 524 sequences are used.

The mutagenized population of M13mp18 constructs can be cloned into pET-3A plasmids after which the expression plasmids can be transformed into E.coli BL21 (DE3) following the procedure of Example 1. Preparation of mutant

polypeptide extracts from E.coli BL21(DE3) for screening follows the procedure of Example 1 with the final step being solubilization of extracted inclusion body material with 8 M urea at room temperature for 2 hours.

Resultant extracts of expressed mutant p5E-type vWF polypeptides are immobilized following the manufacturer's instructions onto a nitrocellulose membrane (0.45μ pore size) using 96-well sample application plates (Easy-Titer^® ELIFA System, Pierce Co., Rockford, IL) and a vacuum chamber.

Commercially available pump materials can be used. The apparatus is suitable for screening large series of clone lysates in an ELIFA or dot blot system and allows also quantitative transfer of sample fluids to underlying

microtiter wells without cross contamination.

Immobilization of the vWF polypeptides is accomplished by causing a suitable volume, such as 200 μl, of each resuspended inclusion body pellet material (in 8 M urea) to be vacuum-drawn through the individual wells to the

nitrocellulose membrane over a 5 minute period. Several 200 μl volumes of Hepes-buffered saline are then drawn through the membrane to remove urea. The protein binding capacity of the membrane is then saturated by passing through it three consecutive 200 μl aliquots of HEPES/BSA buffer herein comprising 20 mM Hepes, pH 7.4, 150 mM NaCl, and 1% w/v bovine serum albumin

(Calbiochem, La Jolla, CA) . After completion of the above procedure to minimize background caused by nonspecific interaction, a 50 μl volume of HEPES/BSA containing botrocetin (at approximately 0.5 μg/ml) or containing ristocetin ( at approximately 1 mg/ml) can be vacuum drawn through the nitrocellulose membrane again over a 5 minute period. The ristocetin-induced precipitation of bacterially-expressed vWF polypeptides observed under some test conditions is not expected to cause difficulty in this assay as the polypeptide is already immobilized.

GPIb(α) represented by its external domain,

glycocalicin, or the 45 kDa tryptic fragment thereof is next applied to the nitrocellulose using the vacuum system and the 96-well plate. The GPIbα fragments are purified and

1²⁵iodinated by standard procedures. Vicente, V. et al., J. Biol. Chem.. 265, 274-280 (1990). 50 μl aliquots of

HEPES/BSA containing ¹²⁵I-GPIbα fragments (0.25 μg/ml having a recommended specific activity of between approximately 5×10⁸ and approximately 5×10⁹ cpm/mg) can then be vacuum drawn through the nitrocellulose filter over 5 minutes. The membrane is then allowed to dry and discs

corresponding to the position of each application well are cut out and counted in a γ scintillation spectrometer to determine bound radioactivity. An autoradiograph of the membrane can also be obtained before cutting out the discs in order to ascertain that there was no leakage of radioactivity from one well to another. The counting process may be facilitated by scanning the developed autoradiogram in a densitometer to digitize the intensity of developed spots. As long as the autoradiogram is not excessively

overdeveloped, beyond the linear region of response, useful qualitative results are obtained.

An alternate procedure to derive from individual host E.coli clones an impure extract which can be screened in immunoblot or dotblot procedures is as follows. A large set of individual E.coli colonies carrying separate randomly mutagenized vWF inserts is picked and grown overnight as separate cultures. The cultures are then diluted 1:100 and grown to an OD₆₀₀ of 1.0. vWF fragment synthesis is induced by adding isopropyl-/.-d-thiogalactopyranoside (IPTG) , U.S.

Biochemicals, Cleveland, OH, to 5 mM and continuing growth for approximately 2.5 hours. The cells are harvested by centrifugation for 1 minute at 10,000 g and then washed and repelleted (at 10,000 g) 3 times with phosphate buffered saline (0.14 M NaCl, 0.1 M Na₂HPO₄ pH 7.0). The bacterial pellet is then solubilized by boiling for 10 minutes in a buffer comprising 0.01 M Na₂HPO₄, 10 mM Na₂EDTA, 1% (w/v) sodium dodecylsulfate, pH 7.0. The incubation is continued for 2 hours at 60°C in the presence also of 10 mM

dithiothreitol (DTT). Suitable volumes (such as 200 μl) of such extracts can be used directly in ELIFA apparatus or dot immunoblot analyses. Prior to adding ¹²⁵I-GPIbα to the plate several rinses of Hepes-buffered saline are washed through the wells. This extract preparation technique is applicable to the screening requirements posed by Examples 25-30. vWF derived polypeptides from colonies representing the most intense response are selected for confirmation of enhanced binding using methods such as subjecting purified or partially purified extracts therefrom as appropriate to (A) immunoblotting according to the procedure of Example 2 with conformation-dependent NMC-4 antibody; (B) assaying for ability to inhibit botrocetin-induced vWF binding to

formalin-fixed platelets on a dose dependent basis (Example 3); or (C) assayed for ability to inhibit the binding of anti GPIbα monoclonal antibodies to platelets (Example 6) . The procedure of Example 6 can be readily adapted as a

supplementary screening system for other target receptors besides GPIbα (see Examples 25-30).

Clones which confer enhanced positive responses in these systems are then subjected to standard DNA sequencing

procedures to identify the vWF gene mutations responsible for the mutant properties. The appropriate mutations may be copied, according to the procedure of Example 22, into a vWF DNA sequence within a plasmid (such as a pAD8/WT-pCDM8^neo expression plasmid) suitable for expression in CHO-K1 cells. Further characterization, such as enhanced potential for induction of platelet aggregation by 116 kDa homodimers thereof can then be performed.

Example 17 - Selection of oligonucleotides for

expression in E.coli of fragments

of mature von Willebrand factor subunit reflecting Type IIB disease mutations

This example demonstrates the mutagenesis strategy for expression in E.coli of the vWF subunit sequence encoded by p5E or p7E plasmid, said constructs containing also Trp⁵¹¹ or cys⁵⁵⁰ mutations. p7E and p5E expression plasmids (Examples 1 and 4) were recovered from cultures of E.coli strain BL21 (DE3) according to the alkaline lysis procedure of Birnboim, H.C and Doly, J. , Nucleic Acids Research. 7, 1513 (1979). The p5E and p7E constructs contain, in reference to the vWF BamHI insert, an upstream XbaI site and a downstream HindIII site. The BamHI site at which the vWF sequence is inserted is positioned directly between an upstream

initiating methionine codon of the parent plasmid and a downstream stop codon thereof. Rosenberg, A.H. et al., Gene, 56, 125 (1987). As a result of the structure of pET-3A and the position of the BamHI site therein, expressed p5E or p7E vWF polypeptides contain also a 17 residue amino terminal sequence extension derived from the gene 10 capsid protein of the vector.

Accordingly the XbaI/HindIII fragment was removed from p5E or p7E and inserted into M13mpl8 which had been

previously restricted with XbaI and HindIII. Site directed mutagenesis was then performed in M13mp18, according to the procedure of Examples 1 and 4 using the following oligonucleotides (for p7E) to insert either a Trp⁵¹¹ or Cys⁵⁵⁰ mutation.

Oligonucleotides are equivalent to non-coding strand (transcribed strand) DNA with the secreted single stranded (+) form of M13mp18 DNA containing coding strand vWF DNA. Similar manipulations were performed to insert either the Trp⁵¹¹ (using oligonucleotide 18) or Cys⁵⁵⁰ (again using oligonucleotide 17) mutation into a p5E construct. With respect to the Trp⁵¹¹ p5E insertion, the hybridizing oligo nucleotide reflected a Cys⁵⁰⁹ codon instead of the previously inserted glycine⁵⁰⁹ mutation as shown below.

Oligonucleotide (18) - see SEQ ID NO: 20

↓

3' G-ATG ACGTCGACCGATGACTT 5'

Tyr₅₀₈Cys₅₀₉ Trp₅₁₁

Example 18 Effect of recombinant von Willebrand factor p5E fragments reflecting Type IIB mutations on the binding of anti-glycoprotein lb monoclonal antibody LJ-Ibl to platelets

Following the procedure of Example 17, the von

Willebrand factor DNA sequence as contained within p5E

(Example 4) was mutagenized to contain either a tryptophan codon or a cysteine codon corresponding to residue positions 511 and 550 respectively.

The mutant polypeptides were expressed in E.coli strain BL21(DE3), and then solubilized from inclusion bodies, according to the procedure of Example 4.

Final purification of the monomeric p5E, p5E-Trp⁵¹¹ and p5E-Cys⁵⁵⁰ polypeptides was accomplished as in Example 4 by dialysis against 6 M guanidine and urea buffers followed by anion exchange chromatography.

The ability of p5E-Trp⁵¹¹, p5E-Cys⁵⁵⁰, and p5E polypeptides to compete with LJ-Ib1 for binding to platelets was

demonstrated over a polypeptide concentration of between about 0.03 and about 2.5 μM. LJ-Ib1 was prepared as

described in Handa, M. et al., J. Biol. Chem.. 261(27), 12579-12585 (1966), iodinated according to the procedure of Example 6 above, purified on protein-A Sepharose^® (Sigma, St. Louis, MO) according to the method of Ey, P.L. et al.,

Immunochemistry, 15, 429-436 (1978) and then used in the competition assays at a concentration of 20 μg/ml. Washed platelets (used at 1 × 10⁸/ml) were also prepared according to the procedure of Example 6.

The assay is based on the ability under certain

circumstances of native vWF to inhibit the binding to

platelets of the anti-glycoprotein lb monoclonal antibody LJ- Ib1. The antibody is also a potent inhibitor of vWF binding to platelets, indicating that the epitope of LJ-Ib1 must overlap with the vWF binding site in GPIbα.

Incubations were performed by mixing the purified fragments at specified concentrations (Figure 3) with washed platelets and ¹²⁵I-LJ-Ib1 for 30 minutes at 22-25°C After the incubation separation of platelet-bound from free antibody was achieved by centrifugation through a layer of 20% sucrose in Hepes-buffered saline at 12,000 g for 4 minutes. See Ruggeri, Z.M. et al., J. Clin. Invest.. 72, 1-12 (1983).

Residual antibody binding (Figure 3) is expressed as a percentage of binding determined from control incubation mixtures containing LJ-Ib1 (20 μg/ml) in 20 mM Hepes, 150 mM NaCl, pH 7.4 without vWF fragments. The figure demonstrates that both the Trp⁵¹¹ and the Cys⁵⁵⁰ Type IIB mutations increase the affinity of the purified polypeptide for GPIbα.

Example 19 - Effect of the recombinant p5E vWF

fragment containing a Cys⁵⁵⁰ mutation

on LJ-Ib1 binding to platelets at different monoclonal antibody concentrations

This example further demonstrates, at two different monoclonal antibody concentrations, the effect of the Trp⁵⁵⁰ to Cys⁵⁵⁰ mutation on the binding of the residue 441-733 vWF subunit fragment to platelet GPIbα. Following the procedure of Example 18, vWF polypeptide (p5E or p5E-Cys⁵⁵⁰) was incubated at various concentrations (Figure 4) with washed platelets (1 × 10⁸/ml) for 30 minutes at 22-25°C but in the presence of either 6 or 20 μg/ml of LJ-Ib1. The assay otherwise followed the procedure of Example 18. As demonstrated in Figure 4, the Cys⁵⁵⁰ polypeptide competes for platelet GPIbα receptor at both antibody concentrations with a higher affinity than the wild type p5E polypeptide. The affinity of the p5E-Cys⁵⁵⁰ molecule for platelet receptor is at least 5-fold greater than that of the "wild type" p5E molecule. An additional procedure to test the behavior of vWF polypeptides containing Type IIB

mutations is dependent on direct binding of ¹²⁵I-labelled polypeptides to platelets. It is noted also that most of the p5E-Cys⁵⁵⁰ molecules in the samples whose activity was

demonstrated in Figures 3 and 4 are dimers.

The results of Examples 18 and 19 are consistent with the hypothesis that amino acid substitutions in the cysteine 509-695 loop region of the mature vWF subunit are responsible for the molecular basis of Type IIB von Willebrand disease.

Example 20 - An improved procedure to solubilize

recombinant

bacterially-expressed and disulfide- stabilized residue 441-733 vWF fragments

An optimized procedure for the solubilization of "p5E-type" polypeptides with formation of disulfide bonds therein has been developed which is believed to comprise an improved method to practice this aspect of the invention. Following this new procedure, a sample of "wet pellet" (Example 1) weighing approximately 350 mg was dissolved in 10 ml of a buffer composed of 6 M guanidine-HCl, 50 mM Tris, pH 8.8. Dithiothreitol (DTT) was then added to a final concentration of 10 mM, and the mixture was allowed to set at 37°C for two hours. The protein concentration of the solution was

determined by a bicinchoninic acid (BCA) titration kit

(Pierce Chemical Co., Rockford, IL). A typical final result is 2 mg/ml.

Seven ml of the above solution was then diluted to 140 ml with 3 M guanidine-HCl, 50 mM Tris, pH 8.8 and then subjected to dialysis for approximately 24 hours against Hepes-buffered saline using several reservoir changes. Oxidation of thiol groups occurs during dialysis as the DTT is removed.

The desired oxidized p5E-type molecule can be purified by reverse phase HPLC on a 1x30 cm C8 column (Vydac Co., Hesperia, CA) using an acetonitrile gradient. The remaining components of the eluting solvent are a constant amount of n- propanol (3%) and a constant amount of trifluoroacetic acid (0.1%), with the balance being spectrograde purity water.

The recommended acetonitrile gradient profile is 30% for 5 minutes, increasing linearly to 56% in 35 minutes, to 70% in 5 minutes, and maintained at 70% for an additional 10 minutes. The column is operated at a constant flow rate of 2.5 ml/min. The oxidized monomer of p5E itself (Example 4) corresponds to the most hydrophilic major peak, eluting at approximately 40 minutes.

Example 21 - Expression in stable mammalian transformants of the homodimeric 116 kDa von Willebrand factor fragment containing a Trp⁵⁵⁰ to Cys⁵⁵⁰ mutation

This example is illustrative of conditions under which a DNA sequence encoding the mature vWF subunit fragment having an amino terminus at residue 441 (arginine) and a carboxy terminus at residue 730 (asparagine), and containing also a

Type IIB mutation may be expressed in a stable mammalian cell transformant with secretion therefrom of the polypeptide.

The mutation strategy of Example 9 was adopted, with

modifications, to insert the Cys⁵⁵⁰ codon mutation into a pCDM8^neo construct.

Following the procedure of Birnboim, H.C and Doly, J., Nucleic Acids Research, 7, 1513 (1979), pAD4/WT plasmids were recovered from storage cultures of E.coli strain XS127. The approximate 950 base pair EcoRI-SmaI fragment of pAD4/WT (Example 9) was subcloned into the EcoRI-SmaI site within the polylinker region of M13mp18 phage. The vWF sequence in M13mp18 was then mutagenized according to the site directed mutagenesis protocol of Example 1. Oligonucleotide 17 (see Example 17) was used to insert the Trp⁵⁵⁰→Cys⁵⁵⁰ mutation.

The oligonucleotide is equivalent to non-coding strand (transcribed strand) DNA with the secreted single stranded (+) form of M13mp18 DNA containing coding strand vWF DNA.

The mutagenized DNA sequence was recovered as the EcoRI-SmaI fragment and subcloned into pAD5/WT (Example 7) which had been previously digested with EcoRI and SmaI.

Review of the cloning strategy of Example 7 discloses that the XhoI-Notl fragment of pAD3-2 (containing the

expression construct for wild type vWF residues 441-730) contains the following sequence of elements

5 '-Xhol EcoRI XbaI-SalI-vWF expression construct-XbaI-NotI-3' wherein the EcoRI site is acquired by cloning the XbaI-restricted vWF insert into pBluescript II KS(-). A SmaI site is defined within the vWF coding sequence corresponding to amino acid residues 716-718. Reference to Figure 5 shows that the XhoI-NotI fragment can be appropriately inserted into pCDM8-type vectors for expression. Consequently, insertion of the mutagenized EcoRI-SmaI fragment as a

replacement for the equivalent EcoRI-SmaI fragment of pAD5/WT creates a construct from which the Type IIB-mutated 441-730 vWF sequence can be expressed.

No SmaI restriction sites are contained in the parent pCDM8 plasmid. (The complete nucleotide sequence of pCMD8 is available from Invitrogen, San Diego, CA) . In addition to the SmaI site at vWF residues 716-718, an additional SmaI site is contributed to the pAD5/WT construct as part of the pBluescript II KS(-) polylinker (upstream from the vWF "XbaI-XbaI" insert and downstream from the EcoRI site). A further SmaI site arises in the 2000 base pair neomycin resistance gene fragment cloned into the BamHI site of pCDM8 to create pCDM8^neo. A strategy of (1) partial digestion with SmaI, and (2) agarose gel purification of the appropriately restricted vehicle fragment was used to assure reassembly of the proper expression vector. Five μg of pAD5/WT plasmid were incubated with 5 units of EcoRI for 60 minutes at 37°C resulting in complete digestion of the site and a homogenous population of linear fragments. A partial digest with SmaI was then accomplished using 5 μg of linearized plasmid as substrate for 0.1 unit of SmaI (at 37 °C for 15 minutes). Plasmid fragments were purified on an agarose sizing gel and a population of linearized plasmid of approximately 7.3 kb having been cleaved at the vWF residue 716-718 site was selected for insertion of the mutagenized EcoRI-SmaI

fragment. The Arg⁵¹¹ to Trp⁵¹¹ mutation may be similarly expressed in a 116 kDa homodimer using oligonucleotide (18) in the

mutagenesis protocol. Alternatively, using two or more complete cycles of mutagenesis in M13mpl8, the Trp⁵¹¹, Cys⁵⁵⁰ and further Type IIB mutations may be expressed in a single polypeptide.

Experimental procedures for effecting stable

transformation of Chinese hamster ovary cells and for

immunopurification of secreted 116 kDa vWF polypeptide were described in Example 7. For the purpose of purifying vWF polypeptides containing IIB mutations according to the present example, however, an immunoaffinity column procedure was used.

Twenty mg of purified NMC-4 antibody were coupled to CNBr-activated Sepharose^® 4B beads (Pharmacia, Uppsala,

Sweden). The column was preequilibrated with 0.5 M LiCl, 50 mM Tris-HCl, pH 7.4, containing 0.05% (w/v) NaN₃.

Culture plates containing confluent CHO-K1 cells were covered with DMEM/10% FCS and incubated for 24 hours. The medium was then collected. In a typical experiment, 500 ml of resultant culture medium containing secreted polypeptides were then applied to the immunoaffinity column. The column was then extensively washed with 15 bed volumes of

equilibration buffer. vWF antigens were then eluted using a solution of equilibration buffer containing also 3 M NaSCN. The eluted vWF polypeptides were concentrated by

ultracentrifugation and then dialyzed against Hepes-saline buffer (150 mM NaCl, 20 mM Hepes, pH 7.4). Protein

concentrations were determined using the bicinchoninic acid titration method (Pierce Chemical Co., Rockford, IL).

An alternate strategy for the transfer of Type IIB mutation codons to pAD5/WT expression constructs is to transform pAD3-2 into E.coli CJ236 and select for bacterial colonies resistant to ampicillin (conferred by plasmid) and chloramphenicol (conferred by host CJ236). An individual colony is grown in 2X-YT culture medium to late log phase and diluted 1:100 in fresh medium in the presence of VCS-M13 (helper filamentous phage available from Strategene, La

Jolla, CA) see Maniatis, T. et al., eds. Molecular Cloning. 2nd ed., Cold Spring Harbor Laboratory Press, 1989. After another overnight incubation, single-stranded uracil-containing DNA is isolated from secreted filamentous phage and the DNA is subjected to the standard extension reaction associated with mutagenesis using mutant oligonucleotides that are identical to the coding strand of vWF except for the intended mutation(s). After the extension reaction, the DNA is transformed into E.coli XL-1 Blue cells, (Stratagene, La Jolla, CA), selected with ampicillin and tetracycline and the resultant colonies characterized for the presence of mutant plasmids. The vWF inserts within the mutant plasmids are sequenced completely to confirm the absence of any additional mutagenic errors. The vWF insert is cloned into pcDM8^neo as an XhoI/NotI fragment as described above for the generation of PAD5/WT.

An additional strategy is to transform pAD5/WT into CJ236 and to select on plates containing chloramphenicol and kanamycin (Kan^r is conferred by the neomycin gene). A single resistant colony is picked and grown as described above for preparation of single-stranded DNA. An extension reaction using a coding strand oligonucleotide followed by

transformation into XS-127 results in colonies with

mutations, the frequencies ranging from 20-100%, depending upon the oligo and purity of the single-stranded DNA used in the mutagenesis reaction. Mutant colonies are sequenced to verify the targeted mutation, and the lack of any unexpected mutation. The mutant plasmids are ready for transformation into CHO-K1 cells for the establishment of stable cell lines.

Example 22 - Construction of a stable mammalian

transformant for the expression of the

monomeric 441-730 mature von Willebrand factor subunit fragment with cysteine-to- glycine mutations at residues 459, 462

and 464 and containing also one or

more mutations reflective of Type IIB disease

Following the procedure of Examples 9 and 21, an EcoRI- SmaI fragment may be removed from pAD4/WT plasmid and

subjected to two or more successive rounds of site directed mutagenesis in M13mp18 to (A) replace one or more of cysteine residues 459, 462 and 464 with, for example, glycine or alanine codons and (B) substitute one or more mutant codons identified from Type IIB patients or one or more codons which confer on the resulting polypeptide properties reflective of Type IIB von Willebrand disease.

Oligonucleotide 13 can be used to substitute glycine codons for each of the above specified cysteine codons thereby preventing formation of the 116 kDa homodimer and leading to the expression of 52/48 kDa monomers with wild type tertiary structure. A second round of mutagenesis using, for example, oligonucleotide 17 or 18 is used to insert Type IIB point mutations, in this case Cys⁵⁵⁰ or Trp⁵¹¹.

Similarly, monomeric residue 441-730 fragments

reflecting Type IIB codon mutations and only one or two of glycine substitutions at positions 459, 462 and 464 may be made following the above procedure and using the

oligonucleotides of Example 11. Alternate or additional strategies for the transfer of Type IIB mutant codons to pAD5 constructs according to this Example, and from which can be generated 52/48 kDa monomeric fragments, are provided in Example 21 above. Example 23 - Effect of reduced and alkylated recombinant von Willebrand factor fragment reflecting the Cys⁵⁵⁰ Type IIB mutation on the

binding of anti-glycoprotein Ib monoclonal antibody LJ-Ib1 to platelets

This example demonstrates that residue position 550 in the mature vWF subunit has no direct effect on binding to GPIbα but is important in the context of modulating the structure of the vWF subunit and hence activity of the GPIbα binding region (residues 474-488 and 694-708). p5E polypeptide was expressed and purified according to an improved procedure which modifies the method of Example 1. The corresponding p5E-Cys⁵⁵⁰ polypeptide was similarly

expressed and purified. The inclusion body solubilization method of Example 1 was followed up to the solubilization step which utilized 6 M guanidine HCl, 50 mM Tris, pH 8.8, said solution now containing 10 mM dithiothreitol.

Incubation in this solution, according to the new procedure, continued for 60 minutes at 37°C p5E and p5E-Cys⁵⁵⁰

polypeptides were then S-carboxymethylated with iodoacetamide according to the procedure of Fujimura, Y. et al., J. Biol. Chem., 262, 1734-1739 (1987) . The extract was then subjected to high performance liquid chromatography first using Q-Sepharose^® Fast Flow (Pharmacia, Uppsala, Sweden) for anion exchange followed by cation exchange on a Protein-Pack SP 8HR column (Waters Co., Bedford, MA) .

The resultant polypeptides contain glycine residues at positions 459, 462, 464, 471 and 474 and chemically

inactivated cysteines at positions 509 and 695, and in the case of p5E-Cys⁵⁵⁰, an additional chemically inactivated cysteine at position 550. Consistant with the lack of glycosylation arising in the bacterial expression system, the polypeptides have apparent molecular weights of approximately 36 kDa.

Binding inhibition assays were performed generally according to the procedure of Example 18 with 10 μg/ml of ¹²⁵I- LJ-Ib1 being used to evaluate the inhibitory effect of vWF polypeptides on antibody binding. Ten μg/ml is approximately the concentration of LJ-Ib1 at which, in these assays, half- maximal binding of antibody to platelets is acheived.

Various concentrations of vWF-derived polypeptide (Figure 6) were used with the constant amount of LJ-Ib1. Non-specific binding was determined in the presence of a 100 fold excess of unlabelled LJ-Ib1 and has been subtracted from all data points. Binding of the antibody was again expressed as a percentage of that measured for the control mixture lacking recombinant polypeptide.

Figure 6 demonstrates comparative antibody binding inhibition results for the reduced and alkylated p5E molecule (r36/Trp⁵⁵⁰) and for the mutant reduced and alkylated p5E molecule carrying also a reduced and alkylated cysteine at position 550 (r36/Cys⁵⁵⁰).

It can be seen (Figure 6) that, in the reduced and alkylated fragments (which have no stable tertiary

structure), substitution of Trp⁵⁵⁰ by cysteine does not effect binding to GPIbα, presumably because the GPIbα binding

sequences are already exposed. It is anticipated that

numerous other amino acid species could also occupy position 550 without effect under these assay conditions. It is likely that only when the polypeptide is assembled into a three dimensional structure having conformational domains mimicking those of the native subunit that the effects of mutations altering the activity of the loop region are

evident. The result of this Example is in contrast to that of Example 18 (Figure 3) where the Cys⁵⁵⁰ mutation

substantially enhanced the binding of bacterially-expressed polypeptide to platelets to the exclusion of LJ-Ib1 in the context of a p5E construct which polypeptide possessed the 509-695 loop.

It has also been discovered that addition of botrocetin at a concentration of approximately 0.4 μg/ml up to about 10 μg/ml or higher, to a suitable concentration of bacterially-expressed residue 441-733 vWF fragment (such as approximately 0.5 μ Molar) substantially enhances the ability of the vWF fragment to inhibit the binding of an anti-GPIbα monoclonal antibody to GPIbα, as measured by the concentration of the vWF fragment necessary to achieve half-maximal inhibition. Specific variants of the vWF fragment for which the effect can be demonstrated includes the p5E molecule containing an intrachain disulfide bond, reduced and alkylated p5E

polypeptide, the p7E polypeptide, and the fragment comprising residues 445-733 when reduced and alkylated. As was

previously noted, the expressed residue 441-733 fragments of the invention contain attached to the amino terminal residue (441) a 17 residue amino acid sequence derived from the gene 10 capsid protein of the pET-3A vector. It is very likely that complexes of the residue 441-730 mature vWF subunit fragment or subfragments thereof (and whether or not

glycosylated), formed with other appropriate molecules will enhance the affinity (and resultant antithrombotic utility) of the vWF fragment or subfragment for GPIbα, or for other known or potential receptors or ligands. In addition, the therapeutic effects of such complexes may be appropriately mimicked or duplicated by effecting within the residue 441-730 fragment appropriate amino acid sequence mutations.

Example 24 - Inhibition of antibody binding

to platelets by mutant and

non-mutant homodimeric 116 kDa

fragments expressed from CHO-K1 cells

For this example, measurement of binding inhibition was performed according to the procedure of Example 23 except that ristocetin (Sigma, St. Louis, MO) was added to a final concentration of 1 mg/ml at a point in time 30 minutes prior to centrifugation. Wild type recombinant 116 kDa homodimer (Trp⁵⁵⁰) was prepared as described in Example 7. The DNA corresponding to mutant 116 kDa homodimer (Cys⁵⁵⁰) was prepared according to the procedure of Example 21 with expression thereof following the procedure of Example 7, as modified in Example 21.

The inhibitory effects of mammalian-expressed vWF fragments on anti-GPIbα antibody binding to platelets were found to be different in certain respects than those of the fragments expressed from bacteria. The wild type recombinant 116 kDa homodimers performed similarly to native multimeric vWF in that they effectively inhibit antibody binding in the presence of ristocetin (and also botrocetin) but are

ineffective in its absence (Figure 7). However, in contrast to the results seen with the native sequence 116 kDa

homodimer (referred to as rll6/Trp⁵⁵⁰ in Figure 7), the

rll6/Cys⁵⁵⁰ homodimer effectively inhibits LJ-Ib1 binding without ristocetin, although the inhibitory effect is further enhanced when ristocetin is added thus reproducing the

classic functional abnormality of Type IIB von Willebrand factor. In the presence of ristocetin, the ability of the 116 kDa homodimer to inhibit antibody binding is increased approximately 10 fold as a result of the Trp⁵⁵⁰→ Cys⁵⁵⁰

mutation.

The combined results of Examples 23 and 24 strongly suggest that the two segments of the 52/48 kDa fragment believed to represent the actual GPIbα binding site (residues 474-488 and 694-708) may be prevented from effectively

interacting with the GPIbα receptor when the vWF subunits possess a native conformation such as presented by

circulating vWF. Disruption of tertiary structure (as in the case of reduced and alkylated E. coli-expressed polypeptides) or modulation thereof (as in circulating vWF of Type IIB patients, or in normal vWF molecules affected by a stimulus associated with a thrombotic or wound event) results in proper exposure of the binding sequences of vWF for GPIbα.

An additional procedure to test the behavior of homodimeric vWF polypeptides containing Type IIB mutations (or antithrombotic monomers patterned thereon and derived by mutation of cysteine residues at one or more of positions 459, 462 and 464) is dependent on direct binding of ¹²⁵I- labelled polypeptide to platelets. III. Development of Additional Antithrombotic Polypeptides

Example 25 - Screening of mutant antithrombotic

polypeptide fragments patterned on the residue 441-730 region of mature von Willebrand factor subunit

having enhanced affinity for collagen

Following the procedure of Method 1 of Example 16, random mutagenesis can be performed on a cDNA corresponding to the residue 441-730 vWF fragment, to target the collagen binding domain encoded therein. Mohri, H. et al., J. Biol. Chem.. 264(29), 17361-17367 (1989) have determined that the mature subunit residue sequence 512-673 is necessary (in the dimeric 116 kDa vWF fragment) to support binding to collagen. Binding to collagen was further reported therein to require intact disulfide bonds, an observation which was stated to have at least two possible explanations.

As stated by Mohri, H. et al., collagen may bind

elements within the residue sequence 512-673, or to the residue 597-621 subdomain thereof, when an appropriate disulfide-stabilized tertiary structure is present.

Alternatively, the actual binding regions in the 52/48 kDa fragment are outside the 512-673 region but require

stabilization of a functional conformation by said internal region. Roth, G.J. et al.. Biochemistry. 25, 8357-8361

(1986) have identified a Type III collagen-binding domain as within the residue 542-662 sequence of the fragment.

For the purpose of identifying vWF-derived

antithrombotic polypeptides with enhanced collagen binding ability, all or part of a cDNA encoding the 441-730 fragment can be subject to random mutagenesis. The population of resultant mutagenized vWF DNA sequences is then reinserted into pET-3A plasmid, as an XbaI-HindIII insert, for

transformation of host bacterial cells followed by expression therein and large scale screening for desired mutant

phenotypes. Mutations giving enhanced binding may then be inserted into mammalian or other eucaryotic host cell constructs for further testing.

A large scale screening assay suitable for detecting enhanced affinity of the mutant polypeptides for collagen can be patterned upon the screening assay for GPIbα binding in Example 16, with appropriate modifications.

Specifically, the mutagenized population of M13mp18 constructs is cloned into pET-3A plasmid followed by

transformation of E.coli BL21(DE3) . Partial purification of bacterial inclusion body lysates follows the procedure of Example 16. Contacting of the resultant vWF fragments with ristocetin or botrocetin is omitted. ¹²⁵I-monomeric type III collagen is applied to the nitrocellulose instead of ¹²⁵I-GPIbα as in Example 16. Monomeric type III collagen is prepared according to the procedure of Roth, G.J. et al.,

Biochemistry. 25, 6357-8361 (1986) and iodinated following the method of Bolton, A.E. and Hunter, W.M. , Biochem. J..

133, 529-539 (1973). Application of 50 μl aliquots Of monomeric collagen (0.25 μg/ml) having a specific activity of between approximately 5 × 10⁸ and approximately 5 x 10¹⁰ cpm/mg should result in an adequate bound signal in relation to nonspecific binding. Other suitable quantities and specific activities can be determined and substituted as necessary. The alternate assay of Example 16 based upon incubation with SDS and then DTT to prepare lysates is equally applicable. Additional screening strategies useful to confirm the properties of a much smaller number of bacterial clones which gave positive responses in the above assay (and using

labelled vWF fragments and unlabelled collagen) are provided by the binding assays of Pareti, F.I. et al., J. Biol. Chem.. 262(28), 13835-13841 (1987) and Mohri, H. et al., J. Biol. Chem., 264(29), 17361-17367 (1989). The ¹²⁵I labelling procedures described herein allow for specific activities varying over many orders of magnitude so that a wide range of receptor (ligand) and vWF fragment concentrations can be interacted. Example 26 - Screening of mutant antithrombotic polypeptide fragments patterned on the residue

441-730 region of mature von Willebrand factor subunit having enhanced affinity for glycosaminoglycans or proteoglycans The binding of heparin to vWF has been determined to involve one or more amino acid subsequences within the residue 512-673 domain. It is likely that this binding activity is conferred by limited linear subsequences within the above stated region since it has been demonstrated that both the intact disulfide-stabilized 116 kDa homodimer and the reduced and alkylated 52/48 kDa monomer are equally effective in inhibiting vWF-heparin interaction. Mohri, H. et al., J. Biol. Chem., 264(29), 17361-17367 (1989).

The mutagenesis strategy of Example 16, method 1 is used to create a randomized population of DNA sequences in

bacterial clones, with the screening of suitable colonies following the procedure of Example 25 except that

radiolabelled heparin is substituted for collagen as binding ligand. Labelling is accomplished by subjecting heparin sodium salt (porcine intestinal mucosa, grade II, Sigma, St. Louis, MO) or similar material to derivatization with

fluoresceinamine followed by iodination of the conjugate.

The ¹²⁵I labelling procedure allows for specific activities of heparin varying over many orders of magnitude so that a wide range of receptor (ligand) and vWF fragment concentrates can be interacted. Smith, J.W. and Knauer, D.J. Anal. Biochem., 160, 105-114 (1987).

As noted previously, it is possible that the collagen, and more importantly, the heparin binding domains of

antithrombotic polypeptides patterned upon 52/48 kDa vWF fragment will prevent the anti-GPIbα activity of the

molecule, such as by causing the polypeptide to be bound at nonspecific "heparin binding sites" throughout the vascular system. The random mutagenesis procedure of this Example could also be used to screen for a mutant binding subsequence having less affinity for glycosaminoglycans (or

proteoglycans) or collagen than present in the wild type sequence, thereby providing an additional alternate method of inactivating said binding activities. Additional screening strategies useful to confirm the desired properties expressed in a much smaller number of clones giving positive responses in the first assay (and using labelled vWF fragments and unlabelled heparin) are provided by Mohri, H. et al., J. Biol. Chem.. 264(29), 17361- 17367 (1989) and Fugimura, Y. et al., J. Biol. Chem.. 262(4), 1734-1739 (1987).

Example 27 - Screening of mutant antithrombotic

polypeptide fragments patterned on the A₃ domain of mature von

Willebrand factor subunit having enhanced affinity for collagen

Following the procedure of Example 1, a double stranded cDNA encoding the entire vWF protein (for the pre-propeptide) is amplified in a polymerase chain reaction using synthetic oligonucleotides selected to flank the A₃ domain encoding region, said oligonucleotides carrying also 5' or 3'

restriction sequences suitable for creating a vWF insert in the multiple cloning site of M13mp18. The strategy of

Examples 16 and 25 is again applied to generate, by random mutagenesis of subregions of the encoding cDNA, mutant vWF polypeptides with potential enhanced binding activity toward collagen. The collagen binding region of the A₃ domain is stated to comprise residues 948-998 thereof (Roth, G.J. et al., Biochemistry, 25, 8357-8361 (1986)) although it is anticipated that other subdomains of the domain may

participate in binding. Example 28 - Screening of mutant antithrombotic

polypeptide fragments patterned on

mature von Willebrand factor subunit

having enhanced affinity for the platelet glycoprotein IIb/IIIa receptor site

The screening assay of Example 16 for mutant vWF-derived polypeptides having enhanced platelet GPIbα binding activity is modified as described below to identify mutant vWF polypeptides having enhanced platelet GPIIb/IIIa receptor binding affinity.

The region of vWF cDNA selected for PCR amplification is recommended to encompass a region corresponding to

approximately 100 amino acid residues on either side of the Arg- Gly- Asp- Ser sequence (subunit residues 1744-1747).

Oligonucleotides for amplification are again designed to contain 5' and 3' terminal restriction sequences so that the cDNA may be inserted into M13mp18 phage for random

mutagenesis. Preparation of oligonucleotides for random mutagenesis of the target domain (focusing on the residues directly proximal to and including Arg- Gly- Asp- Ser) follows Method 1 of Example 16. With respect to the binding assay, neither botrocetin or ristocetin is applied to the nitrocellulose. ¹²⁵I-GPIIb/IIIa purified by the method of Fitzgerald, L.A. et al., Anal. Biochem.. 151, 169-177 (1965) or Newman, P.J. and Kahn, R.A., Anal. Biochem.. 132, 215-216 (1983) and labelled by the method of Bolton, A.E. and Hunter, W.M., Biochem. J.. 133,529-539 (1973) is substituted for ¹²⁵I-GPIbα. Applied to the 96-well plates are 50 μl aliquots of HEPES/BSA containing GPIIb/IIIa at a suitable concentration thereof, such as approximately 0.25 μg/ml or higher, with a specific activity of between approximately 5 × 10⁸ and approximately 5 × 10¹⁰ cpm/mg. The ¹²⁵I labelling procedure allows for specific activities varying over many orders of magnitude so that a wide range of receptor (ligand) and vWF fragment

concentrations can be interacted. An additional screening strategy useful to confirm the properties of a much smaller number of bacterial clones giving positive responses in the first assay (and using labelled vWF fragments and unlabelled GPIIb/IIIa is provided by Ruggeri, Z.M. et al., J. Clin. Invest.. 72, 1-12 (1983).

Example 29 - Screening of mutant antithrombotic polypeptide fragments patterned on the residue

1-272 region of mature von Willebrand

factor subunit having enhanced affinity for glycosaminoglvcans and proteoglycans

The strategy of Example 26 is applied using a suitable amplified cDNA to generate mutant polypeptides derived from the residue 1-272 domain of mature vWF subunit with potential enhanced binding activity toward glycosaminoglycans and proteoglycans.

Example 30 - Screening of mutant antithrombotic

polypeptide fragments patterned on

the residue 1-272 region of mature

von Willebrand factor subunit having

enhanced affinity for factor VIII

The general strategy of Examples 16 and 25 is applied to generate and detect mutant polypeptides patterned on vWF with enhanced binding activity toward factor VIII. Coagulation factor VIII, purified by the method of Fulcher, CA. and Zimmerman, T.S., Proc. Natl. Acad. Sci. USA. 79, 1648-1652 (1982) and labelled with ¹²⁵I to specific activities comparable to that of the other ligands in Examples 25-29 is used. The ¹²⁵I labelling procedure allows for specific activities varying over many orders of magnitude so that a wide range of

receptor (ligand) and vWF fragment concentrates can be interacted.

Example 31 - Inhibition of botrocetin-vWF

complex formation by synthetic vWF peptides This example demonstrates the identification of

subdomains of the residue 509-695 sequence of vWF subunit which form, or are essential components of, the binding site for botrocetin.

In order that these subdomains be identified, fifty-four synthetic overlapping peptides, each fifteen residues in length were constructed following the method of Houghten, et al., Proc. Natl. Acad. Sci.. USA, 82, 5131-5135 (1985) as adapted by Mohri, H. et al., J. Biol. Chem.. 263, 17901-17904 (1988). As shown in Figure 9, each peptide had 5 or 10 residues in common with the two preceeding or following peptides in the series, so that the entire length of the vWF residue 441-733 sequence could be probed.

Botrocetin for the assay was purified following

generally the procedure of Fujimura, Y. et al., Biochemistry. 30, 1957-1964 (1991). The resultant product was then

biotinylated according to the following procedure:

Purified botrocetin (100 μg) was dialyzed against 0.1 M NaHCO₃ for 18 hr at 4°C An equal amount (w/w) of sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LCbiotin, Pierce Co., Rockford, IL) dissolved in dimethyl sulfoxide was then added to the botrocetin solution and the resultant mixture incubated for 2 hours in the dark at 22-25°C The resultant biotinylated botrocetin was dialyzed against a buffer comprising 20 mM Hepes (N-[2- hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]), 0.15 M NaCl, pH 7.35 (hereinafter "Hepes-saline") for 18 hr at 4°C and stored at -70°C until used.

Inhibition of botrocetin-vWF complex formation by the synthetic vWF fragments was performed using commercially available 96 well ELISA-type polystyrene microtiter plates coated with (for each well) 100 μl of a solution of vWF

(purified by the method of Ruggeri, Z.M. et al., J. Clin.

Invest.. 72, 1-12 (1983), and DeMarco, L., J. Clin. Invest.. 68, 321-328 (1981)) at a concentration of 10 μg/ml. The coating solution also contained 0.05 M NaHCO₃, pH 9.6, and was applied for 2 hours at 22-25°C (An equally acceptable coating solution is 150 mM NaCl, 40 mM phosphate buffered at pH 8.0). The coating solution was removed and the plastic surface was saturated with 200 μl of 1% BSA using a 1 hour incubation at 22-25°C Wells so prepared were used after overnight storage at 4°C. When ready for use, the BSA solution was aspirated from the wells, which were then washed 3 times with 200 μl of "PBS/NP-40" buffer comprising 150 mM NaCl, 0.1% Nonidet^® P-40 (polyoxyethylene (9) p-tert-octylphenol), 40 mM sodium phosphate pH 7.4.

50 μl aliquots were then prepared containing the

following: biotinylated botrocetin at a fixed concentration of 0.2 μg/ml, the synthetic fragments at the indicated concentrations (see Figure 9, middle panel, open bars

indicate 0.5 mM synthetic peptide, filled bars indicate 0.1 mM synthetic peptide), and 5 mg/ml BSA in Hepes-saline.

Hepes-saline with BSA was used for the control mixture. The aliquots were then added to the wells. After a 30 minute incubation at 22-25°C, the wells were washed three times with 200 μl of PBS/NP-40 buffer. The bound botrocetin was

detected utilizing peroxidase-conjugated streptavidin and peroxidase substrate O-phenylenediamine.

The detection procedure was as follows. The washed wells were incubated for 20 minutes at 22-25°C with 50 μl of peroxidase-conjugated streptavidin solution (0.25 μg/ml in PBS/NP-40 containing also 1% BSA), Zymed Laboratories. The streptavidin solution was removed and the wells were again washed 3 times with PBS/NP-40. 50 μl of the peroxidase substrate, O-phenylenediamine (1 mg/ml) was added. After a 15 minute incubation, the reaction was blocked with 2 M H₂SO₄.

Inhibition of botrocetin-vWF complex formation by the synthetic vWF peptides was determined by measuring the intensity of developed color, at 490 nm, in an automated ELISA plate scanner. The intensity of color correlated with the amount of biotinylated botrocetin that had formed a complex with surface bound vWF. In the absence of synthetic peptide binding of biotinylated botrocetin was saturable at a concentration of 1.0 μg/ml.

Residual binding of botrocetin (lower panel, Figure 9) measured in the presence of synthetic fragments was also expressed as the percentage of the value for the control mixture, comprising Hepes-saline buffer. Data points thereof represent the mean and range of three separate experiments with the synthetic fragments. Three non-contiguous peptides, located in the residue 509-695 loop region of the 52/48 kDa fragment and designated F8 (residues 539-553), F4 (569-583) and E4-3 (residues 629-643) exhibited the ability to inhibit binding of botrocetin to immobilized multimeric vWF by approximately 60-75% (Figure 9, middle panel).

Peptides that overlap with the three non-contiguous peptides (F8, F4, and E4-3), such as F8-7a (residues 544-558), F3 (574-588), F6 (559-573), E3 (634-648) and E4 (624-638) were also among those that exhibited inhibitory

activity, although to a lesser degree. Peptide code

designations refer to peptides as presented in the upper panel of Figure 9. The IC₅₀ values (concentrations giving half maximal inhibition) are 150 μM (for peptides F8 and E4-3) and 400 μM for peptide F4. Example 32 - Effect of an anionic oligomer on

binding of multimeric vWF to platelets

The anionic oligomer shown in Figure 8 was prepared following the methods described in U.S. Patent Application

Serial No. 07/703,061, the text of which is incorporated herein by reference. The compound was dispensed from a stock solution (2.8 mg/ml in distilled water) for activity assays.

The pH of the stock solution was maintained at about 9 to improve compound solubility by addition of NH₄OH generating an ammonium salt. Additional reactants for the assay were prepared as follows. ¹²⁵I-labelled multimeric vWF (isolated from human plasma cryoprecipitate according to the method of Fulcher, CA. et al., Proc. Natl. Acad. Sci. USA, 79, 1648-1652 (1982) and labelled according to the method of Fraker, P.J. et al., Biochem. Biophys. Res. Commun., 80, 849-657 (1978) was prepared as a stock solution (200 μg/ml in Hepes-saline buffer). Formalin-fixed platelets were prepared according to the method of MacFarlane, D. et al., Thromb. Diath.

Haemorrh., 34, 306-306 (1975). The platelets were stored at 4°C in Hepes-saline.

To initiate the assay, i.e. inducing dose-dependent binding of vWF to the platelets, an appropriate aliquot of stock solution containing the anionic oligomer was used to achieve a final concentration thereof from 1 to 200 μg/ml in the reaction solution (Hepes-saline) containing normal multimeric ^I25I-vWF (at 2 μg/ml) and formalin-fixed platelets (at 1 × 10⁸/ml).

Figure 10 demonstrates the result achieved by the addition of increasing concentrations of anionic oligomer to the mixture containing ^I25I-vWF and fixed platelets. Binding of vWF to platelets was found to be dependent on the dose of anionic oligomer, with no bound ¹²⁵I counts detectable above background absent the use of the oligomer. The oligomer is thus demonstrated to be an inducer of vWF-platelet

interaction, said inducing activity being similar to the botrocetin-induced binding of vWF to platelets. Nonspecific binding was subtracted from all data points before

calculation of residual "specific" binding. Nonspecific binding of anionic oligomer to multimeric vWF was determined by adding NMC-4 antibody to bind to the active site of vWF (see text above) or LJ-P19 antibody to inhibit binding to GPIbα. General methods for providing conformation specific antibodies (like LJ-P19) which react with the N-terminal 45 kDa domain of GPIbα are provided in Handa, M. et al., J.

Biol. Chem., 261(27), 12579-12585 (1986). In a separate experiment, ¹²⁵I-vWF binding to formalin- fixed platelets was measured as a function of ¹²⁵I-vWF

concentration in the presence of a constant amount (100 μg/ml) of the anionic oligomer. Binding of vWF was

determined to be saturable providing a dissociation constant (K_D), by isotherm analysis, of approximately 3 × 10^-9 moles/L. The determined K_D was found to be remarkably similar to that for botrocetin binding to vWF.

Example 33 - Demonstration of competition

of anionic oligomer and

botrocetin for binding sites on vWF

This example demonstrates that the anionic oligomer inhibits formation of the botrocetin-vWF complex in a dose-dependent fashion. The experiment was performed following the procedure of Example 31 except that specific

concentrations of anionic oligomer replaced the series of synthetic peptides thereof.

As in Example 31, biotinylated-botrocetin was delivered in a 50 μl aliquot at a fixed concentration of 0.2 μg/ml. Contained also in a series of such 50 μl aliquots were different concentrations of anionic oligomer (about 1 to about 120 μg/ml). Bound botrocetin was determined as a function of the anionic oligomer concentration in the 50 μl aliquots. As demonstrated in Figure 11, the presence of oligomer markedly inpairs formation of the botrocetin-vWF complex. The maximum inhibition achieved (approximately 80%) was observed at the highest concentration of oligomer tested, 120 μg/ml. Competition of oligomer and botrocetin for binding sites on vWF was also demonstrated in a dot blot procedure.

Aliquots of multimeric vWF preincubated with different concentrations of oligomer were spotted onto nitrocellulose dots. The dots were then soaked for 2 hours at 22-25°C using blocking solution of 1% BSA, 0.05% Nonidet^® P-40 (Sigma

Chemical Co., St. Louis, MO) in Hepes-saline. It is thus demonstrated that once formed, the complex is very stable. The dots were then soaked for 1 hour in a solution of ¹²⁵I- botrocetin (40 ng/ml) in Hepes-saline containing also 1% BSA and 0.05% Nonidet^® P-40. The membrane dots were then washed three times, for 10 minute intervals, in the same solution without botrocetin followed by air drying and

autoradiography.

Example 34 - Random mutagenesis of vWF subunit

polypeptide subdomains 539-553,

569-583 and 629-643 to generate antithrombotic polypeptides with increased affinity for negatively charged modulators

Oligonucleotides corresponding to DNA sequences which code for the polypeptide subdomains 539-553, 559-583 and 629- 643 (or for portions thereof or for overlapping subdomains) but which contain random mutations can be used to make novel mutant DNA sequences which code for vWF polypeptides

containing amino acid mutations within, or proximal to, the above subdomains. vWF polypeptides comprising residue 441- 733 fragment, expressed from recombinant host cells, and containing the mutant subdomains can be screened for

increased binding affinity for negatively charged modulators such as botrocetin or anionic oligomer. Many such modified polypeptides represent polypeptide parts which when combined with anionic parts according to the practice of the invention form derivitized polypeptides of enhanced antithrombotic activity.

Randomly mutagenized oligonucleotides can be prepared for the regions of amino acids 539-553, 569-583 and 629-643 as in Example 16 above, following the procedure of Hutchison, CA. et al., Proc. Natl. Acad. Sci. USA, 83, 710-714 (1986).

The randomly mutagenized oligonucleotides prepared thereby can be used to replace wild type sequences

corresponding to the above polypeptide subdomains by cloning the oligonucleotides into M13mp18 phage containing vWF wild type polypeptide sequence (amino acids 441-733) for site-directed mutagenesis , again following the procedures of

Example 16. Following the site-directed mutagenesis procedures presented in Example 16, each subdomain can be separately mutagenized, or any 2 or all 3 subdomains thereof can be mutagenized in the same DNA polymer, for incorporation of encoded mutations into full length (residue 441-733) encoding DNA.

Expression of vWF polypeptide 441-733 containing random mutations in subdomains 539-553, 569-583 and 629-643 can be accomplished in bacterial cells as in Example 1 and in eucaryotic cells as in Example 7. Expression of resultant vWF polypeptides into the culture medium of host eucaryotic cells can be monitored as in Example 7.

Example 35

Synthesis of Anionic Oligomer for

Conversion to Photo Affinity Label

Preparation of 2-(2-methoxycarbonylethyl)-4-[4-[4-[4-[4-[4- [4-[[2-(2-methoxycarbonylethyl)-4-iodo-5-acetyl]benzyl]-[2- (2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-5-methoxytoluene

Step 1: Preparation of 2-bromo-5- trimethylacetyloxybenzaldehyde

To a suspension of 3-hydroxybenzaldehyde (36.0g) in chloroform (800 ml) is added a solution of bromine (15.8 ml) in chloroform (60 ml) over a period of about 2.5 minutes. The mixture is stirred for 5 minutes, washed with dilute sodium bicarbonate solution, then water, and the organic solution is dried over magnesium sulfate, filtered and concentrated in vacuo. Dry toluene is azeotroped from the residue and this residue is dissolved in methylene chloride (700 ml). To this solution is added triethylamine (55 ml), trimethylacetic anhydride (70 ml), and 4-dimethylaminopyridine (DMAP) (2.0g). The solution is stirred for 14 hours, diluted with ether, and washed with water, then brine. The organic solution is dried over magnesium sulfate, concentrated in vacuo and the residue purified by flash chromatography, eluting with 5% ether in hexanes, to give the desired product. Step 2: Preparation of 2-tert-butyldimethylsilyloxymethyl- 4-trimethylacetyloxybromobenzene

A solution of 2-bromo-5-trimethylacetyloxybenzaldehyde (52g) is tetrahydrofuran (THF) (400 ml) is cooled to -78°C and 0.5M sodiumborohydride in diglyme (182 ml) is added. The solution is stirred at room temperature for 20 minutes, quenched with 1M HCl (200 ml), and diluted with ether. The ether solution is washed with six portions of water, dried over magnesium sulfate, filtered, and concentrated in vacuo. Dry toluene is azeotroped from the residue and this residue is dissolved in anhydrous dimethylformamide (DMF) (400 ml) and imidazole (15 g), tert-butyldimethylchlorosilane (30 g) and DMAP (1.26g) are added. The solution is stirred for 30 minutes, diluted with ether and the ether solution washed with water, dried over magnesium sulfate, filtered and concentrated in vacuo. The residue is purified by flash chromatography, eluting with 2% ether in hexanes, to give the desired product.

Step 3: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4- trimethylacetyloxycinnamate

To a solution of 2-tert-butyldimethylsilyloxymethyl-4-trimethylacetyloxybromobenzene (48g) in dry dimethylformamide (460 ml) is added bis (triphenylphosphine)palladium dichloride (2.5g). The resulting solution is degassed and dry

triethylamine (67 ml) added, followed by methyl acrylate (42.6ml). The resulting solution is stirred at 135°C for about 1 hour, cooled, diluted with ether, washed with water, dried over magnesium sulfate, filtered, then concentrated under reduced pressure. The residue is purified by flash chromatography, eluting with 10% ether in hexanes, to give the desired product. Step 4: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4- trimethylacetyloxyhydrocinnamate

A solution of methyl 2-tert-butyldimethylsilyloxymethyl-4-trimethylacetyloxycinnamate (40.8g) and

tris (triphenylphosphine) rhodium chloride (2.5g) in dry benzene (500 ml) is degassed, then stirred under an

atomsphere of hydrogen at 40°C for 48 hours. The hydrogen atmosphere is replaced by nitrogen and the solution

concentrated in vacuo. The residue is diluted with ether and the mixture filtered, and the filtrate concentrated in vacuo to give the desired product. Step 5: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4-hydroxy-5- iodohydrocinnamate

A solution of methyl 2-tert-butyldimethylsilyloxymethyl- 4-trimethylacetyloxy hydrocinnamate (8.16g) and 25% sodium methoxide in methanol (3.9 ml) in methanol (40 ml) is stirred at room temperature for 30 minutes and diluted with ether. The ether solution is washed with 1M hydrochloric acid, brine, dried over magnesium sulfate, concentrated in vacuo and the residue dissolved in methylene chloride (145 ml). To this solution is added morpholine (4.25g), then iodine

(6.09g) and the resulting suspension stirred at room

temperature for 2 hours and diluted with ether. The ether solution is washed with 1M HCl (25 ml), then brine, dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue is purified by flash chromatography, eluting with 15% ether in hexanes, to give the desired product.

Step 6: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4-methoxy-5- iodohydrocinnamate

A suspension of 60% sodium hydride in mineral (1.2g) in THF (56 ml) is cooled to -20°C and of solution of methyl 2- tert-butyldimethylsilyloxymethyl-4-hydroxy-5- iodohydrocinnamate (I2.6g), iodomethane (5 ml) and 1,3- dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) (8 ml) in THF (20 ml) is added. The cold bath is removed, the mixture stirred for about 2 hours, and diluted with ether. The ether solution is washed with 0.1M HCl, brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The residue is purified by flash chromatography, eluting with 10% ether in hexanes, to give the desired product.

Step 7: Preparation of methyl 2-hydroxymethyl-4- trimethylacetyloxyhydrocinnamate

To a solution of methyl 2-tert-butyldimethylsilyloxymethyl-4-trimethylacetyloxyhydrocinnamate (32.6g) in acetonitrile (350 ml) is added 48% hydrofluoric acid (4.6 ml) and the solution stirred at room temperature for 40 minutes, the diluted with ether. The ether solution is washed with water, dilute sodium bicarbonate solution, brine, dired over magnesium sulfate, and concentrated in vacuo. To the residue is added triethylamine (5 ml) and this purified by flash

chromatography, eluting with 60% ether in hexanes, to give the desired product.

Step 8: Preparation of methyl 2-bromomethyl-4- trimethylacetyloxyhydrocinnamate

To a solution of 2-hydroxymethyl-4-trimethylacetyloxyhydrocinnamate (22.6g) in THF (800 ml) is added

triphenylphosphine (31.4g), then recrystallized N-bromosuccinimide (20g). The mixture is stirred for about 15 minutes, concentrated in vacuo, and the residue purified by flash chromatography, eluting with 30% ether in hexanes, to give the desired product.

Step 9: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl hydrocinnamate

To a suspension of zinc dust (3.64g) in THF (4 ml) is added freshly distilled 1,2-dibromoethane and the mixture is warmed to reflux, stirred for about 1 minute, then cooled to -5°C A solution of 2-bromomethyl-4-trimethylacetyloxyhydrocinnamate (8.28g) in THF (20 ml) is added over about 1.3 hours, and the mixture is stirred for and additional 30 minutes. Stirring is stopped and the solid allowed to settle. The supernatent liquid is transferred via cannula to a solution of methyl 2-tert-butyldimethylsilyloxymetHyl-4-methoxy-5-iodohydrocinnamate (9.26g) and

tetrakis (triphenylphosphine)palladium (1.17g) in THF (40 ml). The resulting solution is stirred at 60°C for about 2 hours, diluted with ether, washed with 5% aqueous ammonia, the brine. The ether solution is dried over magnesium sulfate, concentrated in vacuo, and the residue purified by flash chromatography, eluting with a gradient of 20% to 30% ether in hexanes to give the desired product. Step 10: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2- methoxycarbonylethyl)-5-hydroxy-4-iodo]benzyl hydrocinnamate

Using essentially the procedure of Example 35, Step 5, and purifying the crude product by flash chromatography, eluting with a gradient of 20% to 25% ether in hexanes, the desired product is prepared from methyl 2-tert- butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl

hydrocinnamate.

Step 11: Preparation of methyl 2-tert- butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2- methoxycarbonylethyl)-5-methoxy-4-iodo]benzyl hydrocinnamate

Using essentially the procedure of Example 35, Step 6, and purifying the crude product by flash chromatography, eluting with a gradient of 20% to 25% ether in hexanes, the desired product is prepared from methyl 2-tert-butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2-methoxycarbonylethyl)-5-hydroxy-4-iodo]benzyl hydrocinnamate.

Step 12: Preparation of methyl 2-hydroxymethyl-4-methoxy-5- [2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzylhydrocinnamate

Using essentially the procedure of Example 35, Step 7, and purifying the crude product by flash chromatography, eluting with a gradient of 60% to 80% ether in hexanes, the desired product is prepared from methyl 2-tert-butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzylhydrocinnamate. Step 13: Preparation of methyl 2-bromomethyl-4-methoxy-5-[2- (2-methoxycarbonylethyl)-5- trimethylacetyloxy]benzylhydrocinnamate Using essentially the procefure of Example 35, Step 8, and purifying the crude product by flash chromatography, eluting with 40% ether in hexanes, the desired product is prepared from methyl 2-hydroxymethyl-4-methoxy-5-[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl

hydrocinnamate. Step 14: Preparation of methyl 5-[4-[4-[[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]- [2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2- (2-methoxycarbonylethyl)-5-methoxy]benzyl]-4- methoxy-2-tert-butyldimethylsilyloxymethylhydrocinnamate

Using essentially the procedure of Example 35, Step 9, and purifying the crude product by flash chromatography, eluting with ether/methylene chloride/hexane (4:1:5), the desired product is prepared from 2-bromomethyl-4-methoxy-5- [2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl hydrocinnamate and methyl 2-tert-butyldimethylsilyloxymethyl-4-methoxy-5-[2-(2-methoxycarbonylethyl)-5-methoxy-4-iodo]benzyl hydrocinnamate.

A second product, 1,2-bis-[2-(2-methoxycarbonylethyl)-4-[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxybenzyl]-5-methoxyphenyl]ethane, is also isolated.

Step 15: Preparation of methyl 5-[4-[4-[[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]- [2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2- (2-methoxycarbonylethyl)-5-methoxy]benzyl]-4- methoxy-2-hydroxymethylhydrocinnamate

Using essentially the procedure of Example 35, Step 7, and purifying the crude product by flash chromatography, eluting with 90% ether in methylene chloride, the desired product is prepared from methyl 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-tert- butyldimethylsilyloxymethylhydrocinnamate.

Step 16: Preparation of methyl 5-[4-[4-[[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]- [2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2- (2-methoxycarbonylethyl)-5-methoxy]benzyl]-4- methoxy-2-bromomethylhydrocinnamate

Using essentially the procedure of Example 35, Step 8, and purifying the crude product by flash chromatography, eluting with ether/methylene choride/hexanes (4:1:5), the desired product is prepared from methyl 5-[4-[4-[[2-(2- methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-hydroxymethylhydrocinnamate.

Step 17: Preparation of methyl 5-[4-[4-[[2-(2- methoxycarbonylethyl)-5-hydroxy-4-iodo]benzyl]-[2- (2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy- 2-tert-butyldimethylsilyloxymethylhydrocinnamate Using essentially the procedure of Example 35, Step 5, and purifying the crude product by flash chromatography, eluting with ether/methylene chloride/hexanes (3:1:6), the desired product is prepared from methyl 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-tert-butyldimethylsilyloxymethylhydrocinnamate Step 18: Preparation of methyl 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-methoxy-4-iodo]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy- 2-tert-butyldimethylsilyloxymethylhydrocinnamate

Using essentially the procedure of Example 35, Step 6, and purifying the crude product by flash chromatography, eluting with ether/methylene chloride/hexanes (3:1:6), the desired product is prepared from methyl 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-hydroxy-4-iodo]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-tert-butyldimethylsilyloxymethylhydrocinnamate.

Step 19: Preparation of 2-(2-methoxycarbonylethyl)-4-[4-[4- [4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-5- trimethylacetyl]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-5- methoxybenzyloxy-2-tert-butyldimethylsilane

Using essentially the same procedure of Example 35, Step 9, and purifying the crude product by flash chromatography, eluting with ether/methylene chloride/hexanes (2:1:2), the desired product is prepared from 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-methoxy-4-iodo]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-tert-butyldimethylsilyloxymethylhydrocinnamate and methyl 5-[4-[4-[[2-(2-methoxycarbonylethyl)-5-trimethylacetyloxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-4-methoxy-2-bromomethylhydrocinnamate.

A second product, 1,2-bis-[4-[4-[4-[[2-(2-carboxyethyl)-5-trimethylacetyloxy]benzyl]-[2-(2-carboxyethyl)-5- methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2- carboxyethyl)-5-methoxy]phenyl]ethane, is also isolated.

Step 20: Preparation of 2-(2-methoxycarbonylethyl)-4-[4-[4- [4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-5- trimethylacetyl]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2- methoxycarbonylethyl)-5-methoxy]benzyl]-5- methoxytoluene

To a solution of the silylether product from Example 35, Step 19 (0.253g) and acetic acid (1 ml) in THF (4 ml) is added palladium hydroxide (0.15g) and the resulting

suspension stirred under an atmosphere of hydrogen at 40ºC for about 48 hours. The hydrogen atmosphere is replaced by nitrogen and the mixture diluted with methylene chloride.

The catalyst is removed by filtration and the filtrate concentrated in vacuo to give the desired product.

Step 21: Preparation of 2-(2-methoxycarbonylethyl)-4-[4-[4- [4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-4-iodo-5- hydroxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-5-methoxytoluene

Using essentially the same procedure of Example 35, Step 5, and purifying the crude product by flash chromatography, the desired product is prepared from the product of Step 20. Step 22: Preparation of 2-(2-methoxycarbonylethyl)-4-[4-[4- [4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-4-iodo-5- acetyl]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5- methoxy]benzyl]-5-methoxytoluene

To a solution of the product of step 21, in methylene chloride is added 2 equivalents of triethylamine followed by 2 equivalents of acetic anhydride. The mixture is stirred at room temperature for 30 min then diluted with ethyl acetate, washed with brine, dried with magnesium sulfate, concentrated and the crude product purified by flash chromatography.

NAMES FOR COMPOUNDS OF STEPS 1-3 OF COMPOUND BEFORE COUPLING PROCEDURES TO VWF

COMPOUND OF STEP 1:

2-(2-methoxycarbonylethyl)-4-[4-[4-[4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-4-trimethylstannyl-5-acetoxy]benzyl][2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-5-methoxytoluene

COMPOUND OF STEP 2:

2-(2-methoxycarbonylethyl)-4-[4-[4-[4-[4-[4-[4-[[2-(2-methoxycarbonylethyl)-4-benzoyl-5-acetoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-[2-(2-methoxycarbonylethyl)-5-methoxy]benzyl]-5-methoxytoluene COMPOUND OF STEP 3:

2-(2-carboxyethyl)-4-[4-[4-[4-[4-[4-[4-[[2-(2-carboxyethyl)-4-benzoyl-5-hydroxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-[2-(2-carboxyethyl)-5-methoxy]benzyl]-5-methoxytoluene

Screening of vWF polypeptides comprising residues 441-733 and containing mutations in peptide subdomains 539-553,

569-583 and 629-643 for enhanced ability to bind botrocetin

Screening of host cells (or colonies thereof) for the production of mutant residue 441-733 polypeptides having probable antithrombotic utility when used as polypeptide portions linked to anionic portions can be accomplished by a two-stage screening system. In the first stage, a large series of cell lysates is applied to nitrocellulose, followed by blocking with bovine serum albumin, using generally the procedure of Example 16 provided in the subsection thereof entitled, "Screening of mutant vWF-derived polypeptides for enhanced GPIbα binding activity" using relative binding of

¹²⁵I-botrocetin to detect the most useful polypeptides. Further screening of mutant polypeptides giving apparent positive responses in the first screening, to rule out false positives, can be accomplished using purified polypeptide (purified by the procedure of Example 18) in complex with botrocetin to inhibit binding of ¹²⁵I-LJ-IB1 antibody to GPIbα receptor of platelets following also the assay procedure of Example 18. Many polypeptides giving a positive response in both assays are expected, when linked to the particular anionic molecules provided by the invention, to have enhanced antithrombotic utility.

Deposit of Strains Useful in Practicing the Invention

Deposits of biologically pure cultures of the following strains were made under the Budapest Treaty with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. The accession numbers indicated were assigned after successful viability testing, and the requisite fees were paid.

Access to said cultures will be available during

pendency of the patent application to one determined by the Commissioner of the United States Patent and Trademark Office to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C §122, or if and when such access is required by the Budapest Treaty. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application and said cultures will remain permanently available for a term of at least five years after the most recent request for the furnishing of samples and in any case for a period of at least 30 years after the date of the deposits. Should the cultures become nonviable or be inadvertantly destroyed, they will be

replaced with viable culture(s) of the same taxonomic

description.

Strain/Plasmid ATCC No. Deposit Date

E.coli p5E BL21 (DE3) 96.3 ATCC 68406 9/19/90

E.coli XS127 96.4 ATCC 66407 9/19/90

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Ruggeri, Zaverio M. and

Ware, Jerry, inventors

on behalf of Scripps Clinic and Research Foundation

(i) APPLICANT: Chang, Michael N.

McGarry, Daniel G. and

Regan, John R., inventors

on behalf of Rhone-Poulenc Rorer Inc. (ii) TITLE OF INVENTION: Therapeutic Polypeptides Based on von

Willebrand Factor

(iii)NUMBER OF SEQUENCES: 24

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Scripps Clinic and Research

Foundation

(B) STREET: 10666 North Torrey Pines Road

(C) CITY: La Jolla

(D) STATE: California

(E) COUNTRY: United States

(F) ZIP: 92037

(A) ADDRESSEE: Rhone-Poulenc Rorer Inc.

(B) STREET: 660 Allendale Road

(C) CITY: King of Prussia

(D) STATE: Pennsylvania

(E) COUNTRY: United States

(F) ZIP: 19406

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 1.2 megabyte 5 1/4" floppy (B) COMPUTER: Proteus IBM PC comp. (80266) (C) OPERATING SYSTEM: MS DOS version 3.2

(D) SOFTWARE: WordPerfect 5.1 conv. to ASCII (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 28-Jun-1991

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: This appl. is a c-i-p of

(A) APPLICATION NUMBER: US 07/675,529

(B) FILING DATE: 27-Mar-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Barron, Alexis

(B) REGISTRATION NUMBER: 22,702

(C) REFERENCE/DOCKET NUMBER: P16,633-F

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 923-4466

(B) TELEFAX: (215) 923-2189

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 960

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAA GAC TGT CCA GTG TGT GAG GTG GCT GGC 30

Glu Asp Cys Pro Val Cys Glu Val Ala Gly

435 440

CGG CGT TTT GCC TCA GGA AAG AAA GTC ACC 60

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr

445 450

TTG AAT CCC AGT GAC CCT GAG CAC TGC CAG 90

Leu Asn Pro Ser Asp Pro Glu His Cys Gln

455 460

ATT TGC CAC TGT GAT GTT GTC AAC CTC ACC 120

Ile Cys His Cys Asp Val Val Asn Leu Thr

465 470

TGT GAA GCC TGC CAG GAG CCG GGA GGC CTG 150

Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu

475 480

' GTG GTG CCT CCC ACA GAT GCC CCG GTG AGC l8O

Val Val Pro Pro Thr Asp Ala Pro Val Ser

485 490

CCC ACC ACT CTG TAT GTG GAG GAC ATC TCG 210

Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser

495 500

GAA CCG CCG TTG CAC GAT TTC TAC TGC AGC 240

Glu Pro Pro Leu His Asp Phe Tyr Cys Ser

505 510

AGG CTA CTG GAC CTG GTC TTC CTG CTG GAT 270

Arg Leu Leu Asp Leu Val Phe Leu Leu Asp

515 520

GGC TCC TCC AGG CTG TCC GAG GCT GAG TTT 300

Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe

525 530

GAA GTG CTG AAG GCC TTT GTG GTG GAC ATG 330

Glu Val Leu Lys Ala Phe Val Val Asp Met

535 540

ATG GAG CGG CTG CGC ATC TCC CAG AAG TGG 360

Met Glu Arg Leu Arg Ile Ser Gln Lys Trp

545 550

GTC CGC GTG GCC GTG GTG GAG TAC CAC GAC 390

Val Arg Val Ala Val Val Glu Tyr His Asp

555 560

GGC TTC CAC GCC TAC ATC GGG CTC AAG GAC 420

Gly Ser His Ala Tyr Ile Gly Leu Lys Asp

565 570

CGG AAG CGA CCG TCA GAG CTG CGG CGC ATT 450

Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile

575 580

GCC AGC CAG GTG AAG TAT GCG GGC AGC CAG 480

Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln

585 590

GTG GCC TCC ACC AGC GAG GTC TTG AAA TAC 510

Val Ala Ser Thr Ser Glu Val Leu Lys Tyr

595 600

ACA CTG TTC CAA ATC TTC AGC AAG ATC GAC 540

Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp

605 610

CGC CCT GAA GCC TCC CGC ATC GCC CTG CTC 570

Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu

615 620 CTG ATG GCC AGC CAG GAG CCC CAA CGG ATG 600 Leu Met Ala Ser Gln Glu Pro Gln Arg Met

625 630

TCC CGG AAC TTT GTC CGC TAC GTC CAG GGC 630 Ser Arg Asn Phe Val Arg Tyr Val Gln Gly

635 640

CTG AAG AAG AAG AAG GTC ATT GTG ATC CCG 660 Leu Lys Lys Lys Lys Val Ile Val Ile Pro

645 650

GTG GGC ATT GGG CCC CAT GCC AAC CTC AAG 690 Val Gly Ile Gly Pro His Ala Asn Leu Lys

655 660

CAG ATC CGC CTC ATC GAG AAG CAG GCC CCT 720 Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro

665 670

GAG AAC AAG GCC TTC GTG CTG AGC AGT GTG 750 Glu Asn Lys Ala Phe Val Leu Ser Ser Val

675 680

GAT GAG CTG GAG CAG CAA AGG GAC GAG ATC 780 Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile

685 690

GTT AGC TAC CTC TGT GAC CTT GCC CCT GAA 810 Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu

695 700

GCC CCT CCT CCT ACT CTG CCC CCC CAC ATG 840 Ala Pro Pro Pro Thr Leu Pro Pro His Met

705 710

GCA CAA GTC ACT GTG GGC CCG GGG CTC TTG 870 Ala Gln Val Thr Val Gly Pro Gly Leu Leu

715 720

GGG GTT TCG ACC CTG GGG CCC AAG AGG AAC 900 Gly Val Ser Thr Leu Gly Pro Lys Arg Asn

725 730

TCC ATG GTT CTG GAT GTG GCG TTC GTC CTG 930 Ser Met Val Leu Asp Val Ala Phe Val Leu

735 740

GAA GGA TCG GAC AAA ATT GGT GAA GCC GAC 960 Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp

745 750 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Arg Gly Asp Ser

4

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ACGAATTC CGG CGT TTT GCC TCA GGA 26

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GAAGCT TAC CAT GGA GTT CCT CTT GGG CCC CAG GG 35

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GAC AAC ATC AAC GTG GCC AAT CTG GCC GTG CTC AGG 36 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CGG CTC CTG GCC GGC TTC ACC GGT GAG GTT 30

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

G CCT GCT GCC GTA GAA ATC 19

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GGC AAG GTC ACC GAG GTA GCT 21

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GT CGACGCCACC ATG ATT CCT GCC AGA 27 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TCAGTTTCTA GATACAGCCC 20

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ACGAATTC CGG CGT TTT GCC TCA GGA 26

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GAAGCTTAC CAT GGA GTT CCT CTT GGG 27

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GGG ACC CTT TGT GCA GAA GGA 21

CGG CGT TTT GCC TCA GGA 39 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GGG CCC AAG AGG AAC TGA 18

TCTAGAAAGC TTGGCACTGG C 39

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GAC AAC ATC ACC GTG GCC 18

AAT CTG GCC GTG CTC AGG 36

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

AAGCTT GGA GCA GCA CCT 18

CTG CAG CAC CAG GTC AGG 36

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GAATTC GTT GAC CCT GAA 18

GAC TGT CCA GTG TGT 33

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

TC CAG TAG CCA GCT GCC GTA G 21

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

C CAC GCG GAC GCA CTT CTG GG 21

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Single stranded

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

TT CAG TAG CCA GCT GCA GTA G 21

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 320

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Glu Asp Cys Pro Val Cys Glu Val Ala Gly

435 440

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr

445 450

Leu Asn Pro Ser Asp Pro Glu His Cys Gln

455 460 Ile Cys His Cys Asp Val Val Asn Leu Thr

465 470

Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu

475 480

Val Val Pro Pro Thr Asp Ala Pro Val Ser

485 490

Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser

495 500

Glu Pro Pro Leu His Asp Phe Tyr Cys Ser

505 510

Arg Leu Leu Asp Leu Val Phe Leu Leu Asp

515 520

Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe

525 530

Glu Val Leu Lys Ala Phe Val Val Asp Met

535 540

Met Glu Arg Leu Arg Ile Ser Gln Lys Trp

545 550

Val Arg Val Ala Val Val Glu Tyr His Asp

555 560

Gly Ser His Ala Tyr Ile Gly Leu Lys Asp

565 570

Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile

575 580

Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln

585 590

Val Ala Ser Thr Ser Glu Val Leu Lys Tyr

595 600

Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp

605 610 Arg Pro Glu Ala Ser Arg He Ala Leu Leu

615 620

Leu Met Ala Ser Gln Glu Pro Gln Arg Met

625 630

Ser Arg Asn Phe Val Arg Tyr Val Gln Gly

635 640

Leu Lys Lys Lys Lys Val He Val He Pro

645 650

Val Gly He Gly Pro His Ala Asn Leu Lys

655 660 Gln He Arg Leu He Glu Lys Gln Ala Pro

665 670

Glu Asn Lys Ala Phe Val Leu Ser Ser Val

675 680

Asp Glu Leu Glu Gln Gln Arg Asp Glu He

685 690

Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu

695 700

Ala Pro Pro Pro Thr Leu Pro Pro His Met

705 710

Ala Gln Val Thr Val Gly Pro Gly Leu Leu

715 720

Gly Val Ser Thr Leu Gly Pro Lys Arg Asn

725 730

Ser Met Val Leu Asp Val Ala Phe Val Leu

735 740

Glu Gly Ser Asp Lys He Gly Glu Ala Asp

745 750

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22

Asp Met Met Glu Arg Leu Arg Ile Ser Gln 540 545

Lys Trp Val Arg Val

550

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23

Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg 570 575

Arg He Ala Ser Gln

580

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15

(B) TYPE: Amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Arg Met Ser Arg Asn Phe Val Arg Tyr Val 630 635

Gln Gly Leu Lys Lys

640

Claims

We claim:

1. A composition comprising: (A) a polypeptide comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain and having a predetermined affinity for the GPIb receptor of

platelets; and (B) an anionic material having affinity for said amino acid residue domain 509 to 695 or

subfragment thereof and which, in the presence of both mature vWF and said polypeptide, has a greater affinity for said polypeptide than said mature vWF.

2. A derivatized polypeptide comprising: (A) a polypeptide portion comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain and having a predetermined affinity for the GPIbα receptor of platelets; and (B) an anionic portion having affinity for said amino acid residue domain 509-695 or subfragment thereof; wherein said portions are linked by one or more bonds; and wherein said derivatized

polypeptide has an increased binding affinity for GPIbα receptor relative to said predetermined affinity of said subunit or fragment.

3. A deriviatized polypeptide according to Claim 2 wherein said domain includes one or more of amino acid residue subdomains 539-553, 544-558, 559-573, 569-583, 574-598, 624-639, 629-643, and 634-648, or a subset thereof, and wherein the anionic portion has affinity for one or more of said subdomains or subsets theJeof.

4. A derivatized polypeptide according to Claim 3 wherein the polypeptide portion comprises the fragment of mature von Willebrand factor subunit from approximately residue 441 (arginine) to approximately 733 (valine).

5. A derivatized polypeptide according to Claim 4 wherein each of said portions has one or more reactive groups and wherein the anionic portion and the polypeptide portion are linked by one or more covalent bonds involving the reactive groups.

6. A derivatized polypeptide according to Claim 5 wherein the covalend bond(s) is formed by reaction of an azide group attached to the anionic portion and a group

attached to the polypeptide portion.

7. A derivatized polypeptide according to Claim 5 wherein the covalent bond(s) is an amide bond formed by reaction of a carboxyl group attached to the anionic portion and an amino group attached to the polypeptide portion.

8. A derivatized polypeptide according to Claim 5 wherein the covalent bond(s) is formed by reaction of an

bromoacetyl group attached to the anionic portion and a group attached to the polypeptide portion.

9. A derivatized polypeptide according to Claim 5 in which the anionic portion comprises an anionic oligomer.

10. A derivatized polypeptide according to Claim 5 in which the anionic oligomer is 4-[4-[4-[4-[4-[4-[4-(2- Carboxyethyl-5-hydroxybenzyl)CMB]CMB]CMB]CMB]CMB]CMB]-2- carboxyethyl-5-methoxytoluene, wherein CMB represents 2- carboxyethyl-5-methoxybenzyl.

11. A derivatized polypeptide according to Claim 2 in which the polypeptide portion and anionic portion are linked by noncovalent bonds.

12. A derivatized polypeptide according to Claim 2 wherein

the source of the polypeptide portion is a polypeptide expressed from a recombinant DNA molecule in a host cell.

13. A derivatized polypeptide according to Claim 3 wherein the source of the polypeptide portion is a polypeptide expressed from a recombinant DNA molecule in a host cell.

14. A derivatized polypeptide according to Claim 12 wherein the host cell is a mammalian cell.

15. A derivatized polypeptide according to Claim 13 wherein the host cell is a mammalian cell.

16. A derivatized polypeptide according to Claim 10 wherein the source of the polypeptide portion is a polypeptide expressed from a recombinant DNA molecule in a mammalian host cell.

17. A process for preparing a derivatized polypeptide

comprising:

(A) providing a polypeptide comprising mature vWF subunit or a fragment of the subunit, the subunit or fragment including amino acid residue domain 509 to 695 or a subfragment of the domain, and having a

predetermined affinity for the GPIb receptor of

platelets, the polypeptide including also one or more reactive groups;

(B) providing an anionic material having affinity for said amino acid residue domain 509 to 695 or subfragment thereof of said polypeptide and having also one or more groups which are reactive with the reactive group (s) of said polypeptide; and

(C) reacting said polypeptide and said anionic material under conditions such that the reactive groups of the polypeptide and of the anionic material form one or more covalent bonds, to thereby form a derivatized polypeptide that has an increased binding affinity for the GPIb receptor of platelets relative to the predetermined affinity of said polypeptide.

16. A process for producing a mutant polypeptide patterned upon a polypeptide which is the source of the polypeptide portion Claim 2 and having, relative to said polypeptide portion when contained in a derivatized polypeptide, increased utility as an antithrombotic, said process comprising the steps of:

(A) providing a population of oligonucleotides

corresponding to one or more of mature vWF subunit DNA sequences encoding mature subunit amino acid residue subdomains 539-553, 544-558, 559-573, 569- 583, 574-588, 624-638, 629-643, or 634-648, or subsets thereof, said oligonucleotides containing a population of random mutations;

(B) using the population of oligonucleotides in a

mutagenesis procedure with a vWF subunit or vWF fragment-encoding DNA sequence as template thereby creating a random population of mutagenized vWF subunit or vWF fragment-encoding sequences;

(C) inserting the random population of vWF resultant mutant DNA sequences into plasmids or vectors creating a population of expression plasmids or viral expression vectors;

(D) inserting the resultant population of expression plasmids or viral expression vectors into suitable host cells;

(E) screening individual colonies or cultures of

resultant host cells for expression of mutant vWF polypeptide portions having, relative to the unmutagenized polypeptide portion and in the presence of botrocetin, an increased activity to inhibit GPIbα-vWF interaction;

(F) having determined the mutant DNA sequence of a host cell from which is expressed mutant polypeptide portion having said increased activity;

(G) expressing the mutant DNA sequence, or an additional DNA sequence which is constructed to reflect the changes identified in the mutant sequence, in a host cell; and

(H) isolating the mutant polypeptide portion produced thereby.

19. A process according to Claim 18 in which the mutant polypeptide portion produced thereby is patterned upon the sequence of amino acids comprising essentially approximately residue 441 to approximately residue 733 of mature vWF subunit.

20. A mutant vWF-derived polypeptide portion produced by the process of Claim 18.

21. A purified DNA sequence encoding a mutant fragment of mature von Willebrand factor subunit produced by the process of Claim 18.

22. An expression plasmid or viral expression vector

containing a DNA sequence according to Claim 21.

23. A recombinant eucaryotic or procaryotic host cell

transformed with an expression plasmid or viral

expression vector according to Claim 22.

24. A therapeutic composition which is effective in treating or inhibiting thrombosis which comprises

(A) a pharmaceutically acceptable carrier; and

(B) a derivatized polypeptide according to Claim 2.

25. An antibody which is specific for a derivatized

polypeptide according to Claim 2, said antibody being made by a process of immunizing animals with a

derivatized polypeptide according to Claim 2 and then isolating the antibodies generated thereby.

26. A method of inhibiting aggregation of platelets which

comprises contacting platelets with an effective amount of one or more derivatized polypeptides according to Claim 2.

27. A method of inducing aggregation of platelets which

comprises contacting platelets with an effective amount of composition according to Claim 1.