WO1995011691A1

WO1995011691A1 - Antithrombotic proteins

Info

Publication number: WO1995011691A1
Application number: PCT/US1994/009358
Authority: WO
Inventors: Thomas M. Connolly; Jerzy Karczewski; Richard G. Endris
Original assignee: Merck & Co., Inc.
Priority date: 1993-10-29
Filing date: 1994-08-15
Publication date: 1995-05-04
Also published as: AU7868394A

Abstract

Disagregin is a unique fibrinogen receptor antagonist isolated from the tick Ornithodoros moubata. Disagregin inhibits fibrinogen binding to platelets, and thus platelet aggregation, by binding to subunits of the fibrinogen receptor, glycoproteins IIb and IIIa. It is a 60 amino acid peptide which lacks both the arg-gly-asp(RGD) and lys-gly-asp sequences and has no homology to other regions of fibrinogen. It is useful in inhibiting the formation of the initial hemostatic plug which forms at the site of an injury.

Description

TITLE OF THE INVENTION ANTITHROMBOTIC PROTEINS

BACKGROUND OF THE INVENTION

Platelets play an important role in hemostasis as an integral component of the initial hemostatic plug which forms at the site of injury. Colman, R.W., and Walsh, P.N., Hemostasis and Thrombosis: Basic Principles and Clinical Practice, pp. 594-605 (1987). In order for this process to occur, platelets must first be activated after which they become competent to bind fibrinogen and form an aggregate via the heterodimeric complex of glycoproteins lib and Iϋa. Platelet membrane glycoprotein Ilb/IIIa is a member of the integrin family and contains binding sites for fibrinogen, von Willebrand factor and fibronectin. The binding of fibrinogen to glycoprotein Ilb/IIIa is a critical step in platelet aggregation and is thought to be mediated by two distinct sites on fibrinogen, the arg-gly-asp sequence on the alpha-chain, and residues 400-411 at the carboxyl terminus of the gamma-chain. Kloczewiak et al., "Platelet Receptor Recognition Site on Human Fibrinogen", Biochemistry 23, 1767-1774 (1984). The interaction of fibrinogen with its platelet receptor and the ensuing platelet aggregation can be inhibited by short synthetic peptides which contain the sequence arg-gly-asp. Plow et al., "Arginyl-Glycyl-Aspartic Acid Sequences and Fibrinogen Binding to Platelets", Proc. Natl. Acad. Sci. U.S.A. 82, 8057-8061 (1985). D'Souza et al, "Arginyl-Glycyl-Aspartic Acid (RGD): A Cell Adhesion Motif, Trends Biochem. Sci.. 16, 246-250 (1991). This sequence was identified as the cell recognition site on fibronectin and is present in a variety of proteins including fibrinogen, vWF, thrombospondin, collagen, vitronectin and osteopontin and is thought to mediate their adhesive functions. Ruoslahti and Pierschacher, "New Perspectives in Cell Adhesion: RGD and Integrins", Science 238, 491-497 (1987).

Both short synthetic peptides, and small, naturally occurring proteins containing the arg-gly-asp sequence bind to platelet glycoprotein Ilb/IIIa and inhibit fibrinogen dependent aggregation. These proteins, called disintegrins, found in snake venom and in leeches are 1,000-10,000 fold more potent as inhibitors of platelet aggregation (IC50 = 3xl0^-8 - 3xl0-⁷ M) than the short synthetic peptides. It is not known if their increased potency is due to the other regions of the larger proteins providing secondary binding sites for the fibrinogen receptor or if the other regions of these proteins promote a more favorable conformation of the arg-gly-asp region itself. In the case of the short synthetic peptides, the arg-gly-asp sequence is required for their inhibitory activity. The only exception is an lys for agr substitution. Barbourin, containing lys-gly-asp sequence, and synthetic peptides containing this sequence are as effective antagonists of the fibrinogen receptor, as the arg-gly-asp containing proteins and peptides. The present invention discloses a unique fibrinogen receptor antagonist isolated from the tick Ornithodoros moubata. which lacks both the sequence arg-gly-asp and lys-gly-asp and has no homology to other regions of fibrinogen.

SUMMARY OF THE INVENTION

A platelet aggregation inhibitor was identified in the salivary gland of the tick Ornithodoros moubata. The purified inhibitor, a 6 kDa protein named "disagregin", inhibits ADP-stimulated platelet aggregation in plasma. Disagregin also inhibits platelet aggregation induced by other agonists, including collagen, epinephrine, PAF, ristocetin, thrombin, and the thrombin receptor peptide, TRP (as described in Scarborough et aL, J. Biol. Chem. 267, 13146-13149). It does not, however, effect platelet shape change induced by these agonists or thrombin-induced dense granule release. Disagregin inhibits platelet aggregation by binding to both subunits of the platelet fibrinogen receptor, glycoproteins lib and Ula. Unlabeled disagregin and the snake venom protein disintegrin, echistatin, both compete for this binding.

Disagregin also completely blocks platelet adhesion to fibrinogen and partially inhibits platelet adhesion to collagen and to fibronectin. However, unlike echistatin, disagregin has no effect on the adhesion of human umbilical cord vein endothelial cells to fibrinogen or vitronectin. This result is consistent with the ability of disagregin to bind selectively to platelet glycoproteins lib and Hla. Sequence analysis of disagregin revealed 60 residues comprising a unique protein. Unlike other fibrinogen antagonists it does not contain the arg-gly-asp cell recognition sequence, or a conservative substitution, and it has no structural homology with the arg-gly-asp-containing snake venom disintegrins. Thus, disagregin is unique both in structure and function and may serve as a useful tool for the design of therapeutical ly useful antithrombotic agents.

DETAILED DESCRIPTION OF THE INVENTION

PREPARATION OF GLAND EXTRACT

Salivary glands from 60 Ornithodoros moubata ticks were dissected and placed in ice-cold extraction buffer, containing 20 mM Bis-Tris, pH 7.0, 0.1 M NaCl, 2 mM EDTA, 50 μM chymostatin, 5 μM pepstatin, 50 μM leupeptin, 10 μM E-64 and 50 μM PMSF (TEB). The glands were homogenized in three glass homogenizers in 3 x 1 ml extraction buffer. Homogenate was centrifuged in an Eppendorf microfuge for 5 minutes after which the supernatant was collected (Sup. I). The pellets were resuspended in 3 x 0.5 ml extraction buffer, homogenized and again centrifuged to form a supernatant (Sup. II) which was added to Sup. I. The pellets were suspended in 3 x 0.5 ml extraction buffer containing 50 mM octyl-β-glucoside and centrifuged as before. The obtained supernatant (Sup. UJ) was added to pooled Sup. I and II, resulting in 6 mis of soluble gland homogenate.

PURIFICATION OF DISAGREGIN

The 6 mis of soluble salivary gland homogenate was extracted by mixing it with 4 mis of 100% ACN/0.1 % TFA, keeping it on ice for 10 minutes with constant stirring and then centrifuging it for 10 minutes at 4000 rpm in a Beckman centrifuge. The supernatant was dried down in 10 x 1 ml aliquots. The resulting pellet was washed in 1 ml of extraction buffer, centrifuged and the supernatant vacuum-dried. The dried, ACN extracted material (40 mg) was dissolved in 800 μl H2θ, centrifuged, and the supernatant collected (Sup. I). Pellet was washed in 1 ml H2O, centrifuged and supernatant collected (wash I). Pellet was washed one more time and supernatant again collected (wash II). Wash I and wash II were speed-vac concentrated to a small volume and added to Sup. I to give a total volume of 850 μls. Eight hundred and ten (810) μls was filtered through a 0.45 mm filter and applied to a Bio-Sil SEC-125 size exclusion HPLC column previously equilibrated in 20 mM sodium acetate, pH 5.0, containing 0.5 M NaCl and 0.1 % PEG at a flow rate of 0.5 ml/min. Fractions were collected every 30 seconds and tested for their inhibitory effect on ADP-induced platelet aggregation at 1 :100 dilution. A single peak of aggregation inhibitory activity eluted at approximately 6000 Da. The peak inhibitory fractions were pooled and applied to a C8 reverse phase HPLC column. The bound proteins were eluted by a 20-30% gradient of acetonitrile over 40 min. The aggregation inhibitory activity eluted at approximately 26% acetonitrile/0.1 % TFA, as shown in Fig. 2B. Collected fractions were vacuum dried, dissolved in 100 μl H2O, diluted 1 :150 and assayed for aggregation inhibitory activity. The final purification was achieved by re-chromatographing the peak inhibitory fractions on the same C8 column with an isocratic 26% acetonitrile/0.1 % TFA gradient.

The aggregation inhibitory activity eluted as a single peak, which coeluted with the peak of absorbance at 215 nm. Salivary glands dissected from 60 ticks yielded -40 mg soluble, crude extract and -90 μg of purified aggregation inhibitor. The isolated inhibitor migrated on 20% SDS-PAGE as a single band at approximately 6 kDa. Reduction of disulfides by mercaptoethanol and boiling did not alter the mobility of the protein isoelectric focusing revealed a pi of 7.35.

RADIOLABELING OF DISAGREGIN

Purified disagregin was radiolabeled with ^^[I]Na by 7 min. incubation with 1 mCi 125[rjNa in the presence of 1 iodobead (Pierce) at room temperature. Unreacted reagents were removed by passing the reaction mixture through a Cl 8 SEP-PAC cartridge (Waters), and eluting the 125 [i] -disagregin with 50% acetonitrile/0.1 % TFA. The specific activity of the radiolabeled disagregin was 169 mCi/mg, based on amino acid composition analysis of the radiolabeled protein. Iodination of disagregin did not impair its ability to inhibit platelet aggregation.

AMINO ACID ANALYSIS

The amino acid composition of disagregin was determined after a 20 h acid hydrolysis. This analysis suggested a protein of molecular weight of approximately 6987 as shown in Table 1. The sequencing of native disagregin yielded 60 amino acid residues of comparable composition. Reduced and carboxymethylated disagregin was also sequenced. The identity of the first 40 amino acid residues was confirmed and the presence of four cysteins revealed. Disagregin was digested with trypsin or chymotrypsin and the resulting peptides resolved and sequenced. Sequence analysis of one of the resulting peptide fragments confirmed the identity of the next 10 amino acid residues, 42_arg-51g . Sequence analysis of another resulting peptide fragment revealed the sequence of the final 12 carboxyl terminal amino acids residues, 49_aι_a_60_aι_a? which overlap the sequence obtained from the native protein. The complete primary sequence of disagregin and the carboxyl terminal peptide are shown in Sequence ID listing 1. The 60 residues and the molecular weight of 6987 calculated from the sequence data are in good agreement with the values obtained from the amino acid composition analysis and PAGE, as is the molecular weight obtained from FAB-MS analysis (7113+1.4). Disagregin does not contain the arg-gly-asp sequence common to other disintegrins and it has no significant homology with any known protein, as determined by homology search of PIR and SwissProt databases using FASTA software. BINDING OF DISAGREGIN TO PLATELETS

In order to investigate the mechanism of disagregin interaction with platelets, the protein was radiolabeled with ^ [j] _ancj its ability to bind to platelets assessed. Washed platelets at 3x10° platelets/ml were incubated with -^ -^^ [I] -disagregin for 30 min. in PB at room temperature; the free ^ ^^ [I] -disagregin was separated from that bound to the platelets by centrifugation through 20% sucrose in thin tip tubes (Sarstedt, Princeton, NJ). The bound ^-^^[I]-disagregin was monitored by counting the radioactivity in the amputated tip of the tube. Radiolabeled disagregin specifically bound to platelets; the binding was saturable and dependent on divalent cations. Schatchard analysis revealed 27,990±2070 binding sites per platelet and K >=55±10 nM. Stimulation of platelets with ADP did not significantly effect the affinity (KD=50±10 nM), or the number of binding sites per platelet (28,040±2030 ). Unlabeled disagregin and the snake venom disintegrin echistatin, both competed for this binding with comparable potencies.

CROSSLINKING OF DISAGREGIN TO PLATELETS

In order to localize the disagregin binding site on the surface of platelets, radiolabeled disagregin was incubated with washed platelets and then crosslinked with BS3. Platelets were washed as described above for the adhesion studies and resuspended in PB without BSA. 125[i]__£ji_sagregin at 7.6 nM was incubated with the platelets at room temperature for 15 min., followed by the addition of 0.5 mM BS3 crosslinker. After 10 min. the crosslinking reaction was terminated by the addition of 100 mM Tris. The platelets were washed 3 times in 50 mM Tris, pH 7.4, 2 mM EDTA and 0.14 M NaCl (TBS/EDTA), then dissolved in TBS/EDTA, containing 1% Nonidet P40, 100 μM PMSF, 10 μM E-64, 50 μM leupeptin and 5 μM pepstatin. The Nonidet P40 soluble fraction was subjected to SDS-PAGE on 8% polyacrylamide gels and the presence of crosslinked disagregin visualized by autoradio- graphy. The identity of the disagregin-platelet surface glycoprotein complexes was analyzed by immunoprecipitation studies with antibodies against known platelet glycoproteins. -^^^[I]-disagregin formed complexes with two major surface glycoproteins, migrating under reduced conditions at 135 and 120 kDa and at 150 and 110 kDa when not reduced. Formation of the complexes was dependent on the presence of the chemical crosslinker (no complexes formed in the absence of crosslinker), and was competed for by a 100 fold excess of unlabeled disagregin, thus demonstrating the specificity of the complex formation. The monoclonal antibodies, P2 and SZ21 , directed against platelet glycoproteins Hb HIa and Hla, respectively, immunoprecipi- tated both complexes formed with disagregin. Nonimmune IgG and SAM1 , an IgG which recognizes the fibronectin receptor, VLA-5 complex, precipitated no platelet-disagregin complexes.

PLATELET FUNCTIONAL ACTIVITY ASSAYS

Blood was collected from healthy human volunteers free of drugs for at least 10 days and the platelets or platelet rich plasma isolated as previously described. Connolly et al, J. Biol. Chem. 267, 6893-6898. An aggregometer (Chronolog Corp., Havertown, PA) was used to monitor aggregation inhibitory activity of samples compared to control responses. The rate and extent of aggregation in platelet rich plasma were calculated by the software program Agglink provided by Chronolog. Adhesion of platelets was measured in 96-well microtiter plates (Costar, Cambridge, MA) coated with 50 μl of protein solution at 40 μg/ml (collagen in 5 mM acetic acid, fibrinogen or fibronectin in PBS) for 1 hour followed by blocking of non specific sites by incubation with heat denatured BSA as previously described. Keller et al., J. Biol. Chem. 267, 6899-6904. The washed platelets were suspended at 3xl0°/ml in a modified Tyrode's buffer containing 5 mM HEPES, pH 7.35, 134 mM NaCl, 3 mM KCl, 0.3 mM NaH2P04, 2 mM MgCl2, 5 mM glucose, 12 mM NaHCθ3 and 3.5 mg/mJ bovine serum albumin, platelet buffer (PB), and incubated with the coated wells at room temperature for 45 min. followed by removal of non adherent platelets, washing and measurement of the adhered platelets by BCA protein assay (Pierce). The soluble gland extract fraction (SGE) was found to inhibit aggregation in a concentration dependent manner, with an IC50 = 15 μg/ml. At 50 μg/ml, the SGE also completely abolished platelet aggregation induced by other platelet agonists including collagen, platelet activating factor, epinephrine, ristocetin and the thrombin receptor peptide. Purified disagregin inhibited ADP-induced platelet aggregation in PRP in a concentration-dependent manner. It inhibited both the final amplitude and the slope and of aggregation with the same potency, IC50120120 nM. At the highest concentration tested disagregin completely inhibited aggregation, but had no effect on platelet shape change. The aggregation of platelets in PRP induced by other common platelet agonists including collagen, platelet activating factor, ristocetin, epinephrine and the thrombin receptor peptide, as well as aggregation of washed platelets induced by thrombin was completely blocked in the presence of 770 nM disagregin. In contrast, disagregin had no effect on aggregation independent platelet dense granules release induced by thrombin. Disagregin also completely inhibited platelet adhesion to fibrinogen in a concentration dependent manner with an IC50 = 60 nM. It also partially blocked platelet adhesion to fibronectin and to collagen.

CELL ADHESION ASSAY

The specificity of the inhibitory effect of disagregin was determined by examining its ability to inhibit the adhesion of human endothelial cells, which lack the glycoprotein πb/HJa complex, Phillips et al., Blood 71 , 831 -843, to fibrinogen and vitronectin. Human umbilical cord endothelial cells were grown as described, Cines et al., J. Clin. Invest. 69, 123-128. Briefly, cells were grown to confluence on rat type I collagen matrix, then harvested by brief trypsinization, washed twice and suspended at 2x10^ cells/ml in PB, containing 2 mM CaC)2. The adhesion assay was as described for platelets above except the cells were incubated at 37°C for 90 min. Disagregin at a concentration 20-fold greater than its IC50 to inhibit platelet adhesion to fibrinogen (2 μM) had no inhibitory effect, whereas 2 μM echistatin, a concentration only 8-fold greater than its IC50 to inhibit platelet adhesion to fibrinogen, completely abolished cell adhesion to both adhesive proteins.

TABLE 1

AMINO ACID COMPOSITION OF DISAGREGIN

Amino Acid Residues/mol acid hydrolysis³)

Aspartic Acid + asparagine 8 Serine 6

Proline 2

Alanine 2

Valine 1

Tyrosine 3 Histidine 3

Arginine 4

Threonine 2

Glutamic Acid + glutamine 7

Glycine 6

Cysteine ₂b)

Methionine 1

Phenylalanine 1

Lysine 3

Tryptophan n/d

^a) -Amino acid analysis after hydrolysis with 6M HCl at

100°C for 20 hours b) Cysteine was detected in non-reduced disagregin

n/d - not determined TABLE 2

EFFECT OF DISAGREGIN ON PLATELET AGGREGATION

Agonist Aggregation

Final cone. Control Disagregin³ Amplitude Slope Amplitude Slope

ADP 20 μM 75 105 0 5

Collagen 5 μg/ml 71 110 2 27

PAF 5 μg/ml 62 94 0 7

Epinephrine 1 μM 70 42 0 6

TRP 10 μM 90 186 10 15

Thrombinb I nM 75 110 6 15

LEGEND

Platelet activation was induced in platelet rich plasma after 2 min. preincubation of platelets with disagregin. Aggregation was monitored in the aggregometer as increase in light transmittance as described in Methods. a = 770 nM disagregin b = washed platelets

TRP = thrombin receptor peptide (Scarborough et al, (1992) J. Biol Chem. 267. 13146-13149)

Therapeutic Utility

Disagregin may be administered in any situation where inhibition of human or mammalian platelet aggregation or adhesion is desired. Disagregin may be used in surgery on peripheral arteries (arterial grafts, carotid endarterectomy) and in cardiovascular surgery where manipulation of arteries and organs, and/or the interaction of platelets with artificial surfaces, leads to platelet aggregation and consumption. The aggregated platelets may form thrombi and thromboemboli. Disagregin may be administered to these surgical patients to prevent the formation of thrombi and thromboemboli.

Extracorporeal circulation is routinely used for cardiovascular surgery in order to oxygenate blood. Platelets adhere to surfaces of the extracorporeal circuit. Disagregin may be administered to prevent adhesion.

Other applications of disagregin include prevention of platelet thrombosis, thromboembohsm and reocclusion during and after thrombolytic therapy and prevention of platelet thrombosis, thromboembolism and reocclusion after angioplasty of coronary and other arteries and after coronary artery bypass procedures. Disagregin may also be used to prevent myocardial infarction.

Disagregin may be administered by any convenient means which will result in its delivery into the blood stream in substantial amount. It may be combined with thrombolytic agents such as plasminogen activators or streptokinase in order to inhibit platelet aggregation. It may also be combined with anticoagulants such as heparin, aspirin or warfarin. Intravenous administration is presently contemplated as the preferred administration route. Disagregin is soluble in water, and may therefore be effectively administered in solution.

In one exemplary application, a suitable amount of disagregin is intravenously administered to a heart attack victim undergoing angioplasty. Administration occurs during or several minutes prior to angioplasty, and is in an amount sufficient to inhibit platelet aggregation, e.g. an amount which achieves a steady state plasma concentration of between about 0.05 - 2 μM. The dose required to achieve the target steady state plasma concentration depends on characteristics of the patient including age, weight, condition, etc., but can be readily determined by an ordinarily skilled physician. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: CONNOLLY, THOMAS M. KARCZEWSKI, JERZY ENDRIS, RICHARD

(ii) TITLE OF INVENTION: ANTITHROMBOTIC PROTEIN

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE .ADDRESS:

(A) ADDRESSEE: MERCK & CO. , INC.

(B) STREET: P.O. BOX 2000, 126 E. LINCOLN AVENUE

(C) CITY: RAHWAY

(D) STATE: NJ

(E) COUNTRY: USA

(F) ZIP: 07065

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: PARR, RICHARD S.

(B) REGISTRATION NUMBER: 32,586

(C) REFERENCE/DOCKET NUMBER: 18931Y

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (908)594-4958

(B) TELEFAX: (908)594-4720

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 amino acids

(B) TYPE: amino acid

(C) S1RANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Ser Asp Asp Lys Cys Gin Gly Arg Pro Met Tyr Gly Cys Arg Glu Asp 1 5 10 15

Asp Asp Ser Val Phe Gly Trp Thr Tyr Asp Ser Asn His Gly Gin Cys

20 25 30

Trp Lys Gly Ser Tyr Cys Lys His Arg Arg Gin Pro Ser Asn Tyr Phe

35 40 45

Ala Ser Gin Gin Glu Cys Arg Asn Thr Cys Gly Ala

50 55 60

Claims

WHAT IS CLAIMED IS:

1. A polypeptide in substantially purified form containing the amino acid sequence as described in SEQ ID NO:l.

2. A preparation for inhibiting fibrinogen mediated aggregation of human platelets comprising a polypeptide containing the amino acid sequence of Claim 1.

3. A preparation for inhibiting platelet adhesion to fibrinogen comprising the polypeptide containing the amino acid sequence of Claim 1.

4. A preparation for inhibiting platelet adhesion to collagen and fibronectin comprising the polypeptide containing the amino acid sequence of Claim 1.

5. A method of inhibiting fibrinogen mediated aggregation of human platelets, platelet adhesion to fibrinogen or platelet adhesion to collagen or fibronectin comprising incubating platelets or platelet rich plasma with the polypeptide of Claim 1.

6. A method of inhibiting fibrinogen mediated aggregation of human platelets, platelet adhesion to fibrinogen or platelet adhesion to collagen or fibronectin comprising incubating platelets or platelet rich plasma with the preparation of Claim 2.

7. A method of inhibiting fibrinogen mediated aggregation of platelets, platelet adhesion to fibrinogen, or platelet adhesion to collagen or fibronectin in humans comprising administering to an individual the polypeptide of Claim 1.

8. A method of inhibiting fibrinogen mediated aggregation of platelets, platelet adhesion to fibrinogen or platelet adhesion to collagen or fibronectin in humans comprising administering to an individual the preparation of Claim 2.

9. A process for purifying the polypeptide of Claim 1 comprising the steps of:

a) solubilizing and homogenizing Ornithodoros moubata salivary glands in an extraction buffer comprising a buffered salt having a fixed pH and protease inhibitors to produce the homogenate; b) centrifuging the homogenate to produce supernatant protein suspension fractions; c) combining and drying down the supematants, resuspending pellets and applying them to a size exclusion HPLC column; d) pooling peak inhibitory fractions and applying to a C8 RP- HPLC column; e) rechromatographing peak inhibitory fractions from the C8 column of (d) on same C8 column; and f) selecting fractions characterized by inhibitory activity.