WO1991005566A1

WO1991005566A1 - Protease nexin i/dextran sulfate anticoagulant

Info

Publication number: WO1991005566A1
Application number: PCT/US1990/005936
Authority: WO
Inventors: Randal W. Scott
Original assignee: Invitron Corporation
Priority date: 1989-10-16
Filing date: 1990-10-16
Publication date: 1991-05-02
Also published as: AU6879091A

Abstract

Compositions and methods to prevent blood clotting by taking advantage of the ability of dextran sulfate to activate protease nexin I (PN-I) are disclosed. PN-I and dextran sulfate are utilized in combination and administered simultaneously or in tandem.

Description

PROTEASE. NEXIN I/DEXTRAN SULFATE ANTICOAGULANT

Technical Field

The invention relates to methods to regulate blood coagulation using protease nexin (PN-I). More specifically, the invention takes advantage of the activa¬ tion of PN-I anti-clotting activity by dextran sulfate.

Background Art

The clotting or coagulation of blood is known to be the result of the successive activation of a number of proteolytic enzymes^' in a cascade which results in the production of fibrin to provide physical support for the resulting clot. While such clotting is^', of course, a necessary and desirable protective mechanism to prevent blood loss and hemorrhaging internally and externally, there are circumstances in which blood clotting is clearly undesirable. Notable among these are the thromboses which are seen in heart, attack victims, and conditions wherein the coagulation proteases are systemically activated—a condition known as disseminated intravascular coagulation (DIC). In this latter case, rather than a single defined clot, the propensity for unwanted clotting is spread throughout the vascular system.

The DIC condition is characterized by a systemic depletion of antithrombin III (AT-III). DIC seems to occur frequently in leukemia patients, and anti-thrombin III has been included in some therapies for this condition. AT-III is the major inhibitor of coagulation proteases in the blood and maintains the hemostatic balance by irreversibly binding and inhibiting thrombin, Factor Xa, and several other procoagulant enzymes. Thus, AT-III is consumed when the blood coagulation cascade is driven in the procoagulant direction. In the condition DIC, the levels of AT-III in the blood drop dramatically, and uncontrolled diminution of the AT-III level can be fatal. Supplementation of AT-III^' is a known mode of treatment for this disease.

A serine protease inhibitor which is similar to AT-III in that it inhibits the coagulation cascade and is stimulated to do so by the addition of heparin is protease nexin I (PN-I). This protein was purified from fibroblasts as described by Scott, R.W. , et al. , J Biol Chem (1985) 260:7029-7034; and has been produced recombinantly in mammalian cells (McGrogan, M. , et al. , Biotechnology (1988) 6_^:172-177, incorporated herein by reference) . Human PN-I has been shown to exist in two closely related alpha and beta forms which differ slightly at a splice junction. Cloning and expression of the genes encoding the alpha and beta forms are also described in U.S. Ser. No. 07/025,450 filed 13 March 1987, assigned to the same assignee and incorporated herein by reference. While PN-I and AT-III are electrophoretically and i - munologically distinct, both exhibit anticoagulant activ¬ ity which is stimulated in the presence of heparin. Although AT-III is found at high levels in plasma, PN-I is evidently released into the plasma from platelets after their activation by thrombin (Gromke, R.S., et al. J Biol Chem (1987) 262:3030-3036) ■ PN-I is also found in fibroblasts and glial cells.

It has now been found that the anticoagulant activity of PN-I can be stimulated by dextran sulfate, at doses that are not similarly stimulatory with respect to the anticoagulant effect of AT-III. Thus, selective means to regulate the coagulation cascade independent of heparin are now made available.

Disclosure of the Invention The invention provides methods and compositions which are useful in regulating the coagulation cascade in human blood. These methods and compositions are based on the discovery that protease nexin I (PN-I) is stimulated in its inhibition of this cascade by dextran sulfate, a sulfated polysaccharide related to, but different from, heparin, which is a known stimulant for the anticoagulant activities of both PN-I and antithrombin III (AT-III).

Thus, in one aspect, the invention is directed to a method to mitigate unwanted blood coagulation in animals which comprises administering a composition which will activate the coagulation inhibition by the PN-I/ dextran sulfate complex. PN-I and dextran sulfate are co- administered in effective amounts, preferably^' simultaneously. In other aspects, the invention is directed to compositions useful in the method of the invention which contain both PN-I and dextran sulfate, and to improved formulations of PN-I. In still other aspects, the invention -is directed to anticoagulant compositions for use in contacting whole blood to prevent clotting and to methods to prevent clotting of blood by use of these compositions.

Brief Description of the Drawings Figure 1 shows the amino acid sequences of PN-I alpha (Fig 1A) and PN-I beta (Fig IB) as deduced from the cloned human genes. The sequence differences at the splice junctions are overlined.

Figure 2 shows the effect of various polysaccharides and glycosaminoglycans on thrombin inhibi¬ tion by PN-I. Figure 3 shows the effect of dextran sulfate on thrombin inhibition by PN-I and AT-III.

Figure 4 shows the effect of pH on PN-I stability.

Modes of Carrying out the Invention

As used herein, protease nexin I (PN-I) refers to a protein which is active in the standard assay for PN- I described below and which has an amino acid sequence substantially similar to that of Figure 1. This assay depends on the ability of the PN-I protein to complex to thrombin, and the acceleration of this complexation by heparin. Two forms of native PN-I are shown in Figure 1 herein; it is, of course, understood that inocuous modifications can be made to some portions of this amino acid sequence without destroying the PN-I activity. Accordingly, PN-I as herein defined includes proteins with amino acid sequences modified from and thus "referenced to" and substantially similar to the sequences shown in Figure 1 and which retain the essential activity of PN-I. One can set arbitrary levels of homology required—i.e., PN-I may be defined as proteins having an amino acid sequence at least 80% homologous to that shown in Figure 1, preferably 90% homologous and more preferably 95% homologous while retaining the characteristic PN-I activity. It is .understood that not all sequence modifications will permit the resulting protein to retain activity; however, the resultant of these modifications is readily tested in the assay described below. Furthermore, it is understood that fragments of the amino acid sequence may constitute proteins which retain activit —again, the criterion for falling within the definition is readily evaluated with respect to any particular fragment.

Further, with respect to defining PN-I, it is understood that while the amino acid sequence of the protein is referenced to those in Figure 1 with respect to sequence encoded by the gene, post--translational modifica¬ tions may alter the status of the side chains of particular residues, and covalent bonding to additional residues, such as, most commonly, sugar residues, is to be expected when the protein is produced in eucaryotic cells. It is also recognized that the status of amino acid sequences is pH-dependent and, accordingly, acid addition salts or basic salts of the protein are also included in the definition so long as activity is retained. "Dextran sulfate" is an art-recognized term for a sulfated polysaccharide. It is generally not found natively, but is prepared by treating dextran with sulf ric acid and neutralizing the product. Dextran itself is a polymer of alpha-D-glucose units which is isolated from a number of bacterial sources. Differences in compositions from various bacteria are generally due to the degree of branching. Dextran sulfate is a known anticoagulant (Walton, K.W. , Proc Roy Soc Med (1951) 4:563) but at doses about 10X higher than heparin. Also, dextran sulfate may be prepared in a range of molecular weights; the higher molecular weight forms — i.e. > about 20 kd, appear to have enhanced toxicity.

PN-I Assay To assess PN-I activity, samples to be tested are diluted in a suitable buffer of pH 7-8, such as "assay buffer" (3 itiM sodium phosphate, pH 7.4, 137 mM NaCl, 1% polyethylene glycol, 0A% Tween 20, 1 mg/ml aprotinin, plus or minus 2 units/ml heparin (170 USP unit/mg, Sigma Chemical Company, St. Louis, Missouri)) to 100 ul sample volumes. Samples are combined with 100 ul human alpha thrombin at 0.25 NIH units/ml in assay buffer, and incubated for 5-30 min at room temperature or 37°C. The resultant thrombin activity is measured in a standard colorimetric assay using substrate S.2238 (Kabi) at a final concentration of 0.2 mM. One unit of PN-I activity is defined as that amount of PN-I required to inhibit 1 NIH unit of thrombin. In the alternative, 100 M Tris HC1, pH 8.1 with 160 mM NaCl, 1% polyethylene glycol, 0.1% Tween 20, 1 mg/ml aprotinin plus or minus 2 units/ml heparin may be used as assay buffer.

For determination of PN-I-specific activity, protein concentrations were obtained from absorbance readings at 280 n using an extinction coefficient of 6.1 for a 1% solution. A purified protein is considered to have PN-I activity if its specific activity in.this assay as determined on the basis of the pure protein is at least 25% of that shown by PN-I alpha or PN-I beta measured in this assay. (It is recognized that proteins other than PN-I, such as antithrombin III, have PN-I activity.) Of course, mixtures of active purified PN-I proteins with other non-interferring and nontoxic proteins can be used in the method of the invention

Preparation of PN-I PN-I can be prepared by isolation of native material from fibroblasts or glial cells, for example as described by Scott, R.W., et al., J Biol Chem (1985) 260:7029-7034 (supra) or by using recombinant techniques as described by McGrogan, M. , et al., Biotechnology (1988) :172-177 (supra) . The recombinant form of PN-I is conveniently produced in Chinese hamster ovary (CHO) cells, though other recombinant systems such as the insect-based bacculovirus system, yeast systems or even bacterial systems could also be used. A convenient control system for CHO cells employs an SV40 promoter, and the construction of a convenient expression vector is described in the illustrative examples below. •

The PN-I synthesized is purified from the culture medium or native sources using techniques analogous described by Scott (supra) . Utility and Administration

The PN-I/dextran sulfate compositions of the invention are prepared according to standard pharmaceuti¬ cal formulation procedures, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, latest edition. In general, when administered to an animal subject, the compositions can be administered by injection, subcutaneously, intra¬ muscularly, or intravenously. The injectables can be prepared in conventional forms as liquid solutions or suspensions, solid forms suitable for solution or suspen¬ sion in liquid prior to injection or as emulsions. Suit¬ able excipients are, for example, water, saline, dextrose, glycerol, various buffers such as Hank's solution or Ringer's solution, etc. The compositions may contain nontoxic auxiliary substances such as wetting or emulsify¬ ing agents, pH buffering agents, and so forth such as sodium acetate, sorbitan monolaurate, and so forth.

Systemic or parenteral administration can also be made through transdermal or transmucosal formulations. Generally such formulations contain penetrants such as bile salts or fusidic acids. Transdermal or transmucosal administration can be through suppositories or sprays. For example, nasal sprays are commonly formulated as aerosols containing the active ingredient in finely divided form along with a surfactant and propellant. Typical nontoxic surfactants include the partial esters of fatty acids such as lauric or palmitic acid with an alaphatic polyhydric alcohol s.uch as glycerol or erythritol. Suitable propellants include alkanes of up to 5 carbons arid fluorinated or fluorochlorinated alkanes.

In addition, topical formulations for localized treatment to effect wound healing or other localized effects can be applied. These are generally in the form of gels, ointments, lotions, pastes, salves, and the like. While oral compositions are more difficult to formulate, administration via an oral route is also envisioned. In addition, various slow-release forms of drug dispensation have been devised which may be appropriate for administration of the compositions of the invention. The active ingredients are administered in amounts effective in the desired degree of mitigation of coagulation. When both PN-I and dextran sulfate are administered, the levels of dextran sulfate can be lowered considerably from those at which dextran sulfate has been used alone. Suitable dosage levels for these active ingredients in combination are in the range of 1-40 g/kg PN-I and 1-10 mg/kg of dextran sulfate with the goal of achieving 10-100 ug/ml blood concentrations of PN-I and 1-10 ug/ml blood concentrations of dextran sulfate. Administration of the two active components may be made simultaneously, or in any order within an effective time proximity, for example within 30-40 min of each other, preferably 1-5 min of each other.

In addition to therapeutic use, the compositions of the invention may also be used in vitro to prevent clotting in whole blood samples. In this application, the composition is added to whole blood in ^'an amount of ap¬ proximately 10-100 ug/ml PN-I and 1-10 ug/ml dextran sulfate. The following examples are intended to il¬ lustrate but not to limit the invention.

Preparation A: Production of Recombinant PN-I

A.l Construction of the Host Vector pSTH-MDH pSVoriHBV3' contains the origin of replication and early and late promoters of SV40 upstream of the 3' termination sequences from the hepatitis B surface antigen gene with insertion sites for a foreign gene between them. pSVoriHBV3' is constructed from pML, SV40, and HBV. pML is digested with EcoRI, blunted with Klenow, and then digested with Hindlll. The vector fragment containing the E. coli origin of replication and the ampicillin resist¬ ance gene is isolated and ligated to the isolated 540 bp fragment containing the early and late promoters and origin of replication of SV40, obtained by digestion of SV40 DNA by Hindlll and Hindi. The resulting vector, designated pSVori, is then digested with BamHI for accept¬ ance of a 585 bp fragment isolated from a BamHI/Bglll digest of HBV DNA which contains the 3 ' termination sequences of the surface, antigen gene. Correct orienta¬ tion is confirmed by restriction analysis - digestion with Hindlll and BamHI yields a 350 bp fragment from the cor¬ rect vector. The resulting ligated vector, pSVoriHBV3 ' , thus contains the SV40 promoter and origin sequences upstream of the HBV terminator and permits a coding sequence to be inserted conveniently between them.

Also prepared was ptPA-BALl7, which contains the tailored upstream portion of the tPA gene in a bacterial replication vector. The tPA cDNA is furnished by the vec- tor pMON-1068, which is a bacterial vector containing an insert of the entire cDNA sequence obtained for tPA as described in Pennica, D., et al., Nature (1983) 301:214- 221. Of course, any bacterial replication vector contain¬ ing this coding sequence could just as well have been used, and the restriction sites designated below fall within the disclosed sequence of the tPA cDNA set forth in the Nature reference. pMON-1068 is first digested with BamHI to excise the tPA encoding cDNA and then with BAL-31 to chew back at each end of the gene. Digestion with BAL- 31 was continued until analysis of the lengths and sequence of linear fragments indicated that the 5 ' end of the fragment was within 17 bp of the ATG start codon. The precise distance of chew-back is not critical so long as it is within sufficiently short distance to permit the ATG to be placed an operable distance from the promoter in the expression cassette. A separation in this fragment of the 5' terminus from the ATG of about 10 bp is, in fact, preferred. The selected linear fragment was then digested with Sad, which cuts inside the coding sequence of the tPA gene, and the resulting blunt/Sad fragment was isolated. This contains the suitably tailored 5' end of the gene and was ligated into SacI/HincII-digested pUC13 to give the intermediate plasmid ptPA-BAL17. pUC-DHFR was used as a cloning vector for the DHFR-encoding sequences, absent their associated control sequences. pUC-DHFR was constructed by digesting pDHFR-11 (Simonsen, C.C., et al. , Proc Natl Acad Sci USA (1983) :2495-2499) with Fnu4HI, blunting with Klenow and then digesting with Bglll to isolate the 660 bp fragment as there described, and ligating this fragment into pUC13 which had been digested with Hindi and BamHI. Thus, pUC- DHFR represents a straightforward cloning vector for DHFR analogous to the ptPA-BAL17 vector described for the 5 ' portion of the tPA gene above.

Finally, a separate cloning vector for the termination sequences derived from the hepatitis B surface antigen gene, pUC-HBV3 ' , was constructed by digesting HBV DNA with BamHI and Bglll and isolating the 585 bp frag¬ ment, as described above, and ligating this fragment into BamHI-digested pUC13. pSV-tPA17, which contains the full-length tPA coding sequence under control of SV40 promoter and HBV terminating sequences was prepared as a three-way ligation of the vector fragment from pSVoriHBV3 ' digested with Hindlll and BamHI, which thus provides the promoter and terminator along with vector sequences; the 3' portion of tPA obtained by Sacl/Bglll digestion of pMON-1068; and the tailored 5^r portion of the tPA coding sequence, which was obtained as a Hindlll/Sacl digest of ptPA-BAL17. The resulting ligation mixture was transfected into E. coli, the transformants selected for ampicillin resistance, and plasmid DNA containing the desired pSV-tPA17 isolated. The counterpart vector for DHFR expression, designated pSV-DHFR, was also obtained in a three-way ligation. Again the vector fragment obtained from Hindlll/BamHI digestion of pSVoriHBV3 ' was used to provide the control sequences , and the 5 ' and 3 ' portions of the DHFR coding sequence were obtained by digestion of pUC- DHFR with Hindlll and Sad (partial) and with Bglll and Taql (partial), respectively. The ligation mixture was used to transform E. coli, ampicillin resistant transformants were selected, and plasmid-DNA, designated pSV-DHFR was isolated.

A single plasmid containing a weak expression system for the DHFR coding sequence was also prepared. This plasmid, pMDH, was obtained in a 3-way ligation using the 1 kb fragment obtained by EcoRI/Taql (partial) diges¬ tion of pDR34, the vector fragment from EcoRI/Sall- digested pML, and the 3' .end of the gene isolated from Sad (partial) /Sail digested pSV-DHFR. (The pDR34 vector is described by Gasser, C.S., et al . , Proc Natl Acad Sci USA (1982) 29:6522-6526, supra) and contains the mouse

DHFR gene linked to its own promoter.) The resulting vec¬ tor, pMDH, is analogous to pSV-DHFR, except that the DHFR gene is under control of the urine DHFR promoter. The weak expression cassette residing on pMDH and strong expression cassette residing on pSV-tPA17, when used in admixture to transfeet suitable DHFR-deficient cells, thus constitute one embodiment of the expression system of the invention.

Finally, pSTH-MDH, which contains the expression cassettes for tPA and for DHFR on a single vector, was constructed as a three-way ligation of the appropriate isolated fragments of ρSV-tPA17, pMDH, and pUC-HBV3 ' . ρSV-tPAl7 is digested with SacII and Sail, pMDH with EcoRI and Smal, and pUC-HBV3 ' with SacII and EcoRI. A.2 Construction of PN-I Expression Vectors

The coding sequences from the EcoRI cassettes of PN-33 or PN-18 shown in Figure 1 were each ligated into pSTH-MDH. In the resulting vectors, pSNalphaH-dhfr and pSNbetaH-dhfr, the amplifiable DHFR sequences are under control of the native promoter and followed by the termination sequences of the hepatitis surface antigen gene, and the PN-I sequences are under control of the SV40 early promoter and are also followed by hepatitis surface antigen termination sequences.

The DNAs encoding PN-I alpha and PN-I beta were inserted into the host vector pSTH-MDH, in place of the tPA expression cassette, using three-way ligations in which the 5' and 3' ends of the coding sequences and ap- propriate portions of the expression systems were inserted as separate fragments.

For construction of pSNalphaH-dhfr, pNexalpha- HBV3'R1, a vector containing the C-terminal encoding_,por¬ tion of the gene followed by the hepatitis termination sequences was digested with Bglll and EcoRI and the 3' end of the expression system isolated. The vector pSV- Nexalpha, which contains the 5' end of the expression system, was digested with Bglll and Sail and the vector portion containing the nexin 5' end isolated. These frag- ments were ligated with the DHFR selectable marker, excised from pSTH-MDH by ^"EcoRI/Sall digestion, in a three- way ligation and transformed into E_;_ coli for selection and amplification. Plasmid DNA representing the desired construction, pSNalphaH-dhfr, was isolated from the suc- cessful transformants.

In a precisely^' similar manner, pSNbetaH-dhfr was constructed, but using pNexbeta-HBV3'RI and pSV-Nexbeta in place of the corresponding PN-Ialpha-containing vectors. Common to these constructions is a vector containing the SV40 early promoter operably linked to the nexin 5' end which is common to both alpha and beta forms. This intermediate vector, pSV-NexBall, was constructed using PN-33, pUClδ, and pSVoriHBV3' . PN-33 was cut with EcoRI to excise the PN-I-containing inserts, cut back with Bal31 and then digested with Sad to obtain a tailored nexin insert containing the 5' end through the ATG start codon. This fragment was ligated into a HincII/SacI- digested pUC18 vector fragment to obtain pUCNex-Ball. pUCNex-Ball was cut with Hindlll and Sail to excise the nexin 5' end fragment common to the alpha and beta forms which was then ligated into the Hindlll/Sall-digested pSVori-HBV3' vector fragment (see below) to give the desired pSVNex-Ball.

An additional vector containing 3' sequences, pSVNex3'HBV, was constructed by digesting PN-33 with EcoRI and Hpal, isolating the 1650 bp fragment and ligating it into pUC18 digested with EcoRI and Smal to obtain pUC- Nex3' . pUC-Nex3' was supplied with hepatitis termination sequences by digesting with Hindlll and BamHI and ligating the isolated nexin 3' end into Hindlll/BamHI-digested pSVori-HV3' (see below) to obtain the desired pSVNex3'HBV. The two additional intermediate vectors, pSVNexalpha and pSVNexbeta, were obtained using the 540 bp internal fragment from PN-33 or PN-18, as appropriate, and the corresponding 5' and 3' ends from pSVNex-Ball and pSVNex3'HBV. In each case, pSVNex-Ball was digested with Bglll and Sail and pSVNex3'HBV with Hindlll and Sail and ligated in the three-way ligation with the Bglll/HindiII 540 bp internal fragment of PN-Ialpha from PN-18 or PN- Ibeta from PN-33 to obtain pSVNexalpha and pSVNexbeta, respectively. These were modified to place an EcoRI site at the extreme 3' end of the expression system by insert¬ ing the Bglll/SacII insert of pSVNexalpha or pSVNexbeta, as appropriate, into BamHI/SacII-digested pUC-HBV3'. The resulting vectors, pNexalphaHBV3'RI and pNexbetaHBV3'RI, were then used in the constructions described above. The resulting vectors can be generically named pSNH-dhfr. B. Expression of the PN-I Gene

The vectors were transfected into DHFR deficient CHO cells, which were then amplified in methotrexate and cultured for the production of PN-Ialpha or PN-Ibeta. The PN-I is secreted into the medium as the signal sequence is retained in the construct and is compatible with the host cells .

The media of the transformed cells are assayed for PN-I production using the thrombin binding assay described by Eaton, D.L., et al. , J Cell Physiol (1983)

117 : 175-185. Briefly, serum-free medium preincubated with confluent cell cultures for 72 hr was centrifuged to remove cell debris. Labeled thrombin ( 125I-Th) at 0.1 ug/ ml was incubated with this medium for 45 min at 37 C.

125I-Th-PN complexes were resolved by SDS-polyacrylamide gel electrophoresis using 7% gels, under conditions which do not dissociate the Th-PN complex, and quantitated in a gamma scintillation counter, assuming that PN and Th are present in equimolar amounts in Th-PN complexes . The complexes formed are confirmed to contain PN-I by immunoprecipitation with PN-I rabbit antiserum.

C. Purification of Recombinant PN-I

The PN-Ialpha or PN-Ibeta is purified by applying the conditioned media to cation exchange chromatography using S-Sepharose Fast Flow equilibrated in 20 mM citrate pH 6.0, 10 mM EDTA, 0.2 M NaCl. The column is then washed with the equilibration buffer and eluted with 20 mM citrate pH 6.0, 3.5 M NaCl. The fractions containing PN-I (as determined by

PN-I assay) are then desalted by gel filtration on a Sephadex G-25 column to reduce the ionic strength to the equivalent of 20 mM citrate pH 6.0, 0.3 M NaCl, and applied to a heparin agarose affinity column equilibrated in the same buffer. The PN-I is then eluted with gradient in 20 mM citrate pH 6.0 to 1.5 M NaCl. The fractions containing PN-I are then concentrated and further purified over size exclusion column containing Sephacryl HR 200 equilibrated in 20 mM citrate, 0.3 M NaCl. The resulting PN-I is homogeneous with respect to HPLC and SDS PAGE.

D. Characterization of Recombinant PN-I

The heparin modulating effects of the re¬ combinant PN-I prepared as above were shown to be similar to those of PN-I purified from human fibroblasts. The native protein reacts with thrombin in the absence of heparin at a rate faster than AT-III; the second order rate constant is k = 6.0 x 10 5M-1 sec-1; the second order rate constant for inhibition by native PN-I in the presence of heparin is enhanced (k=1.2 x 10 8 M—1 sec—1). For the recombinant PN-I, similarly, heparin enhanced the rate of inhibition of thrombin, and the rate of inhibition of thrombin by PN-I was faster both in the presence and absence of heparin than the rate of inhibition of thrombin by AT-III.

Example 1 Activity Enhancement Profile of PN-I Thrombin inhibition was measured in the colorometric assay described in the general description section of the specification above. PN (0.25 Units/ml) and thrombin (0.25 NIH Units/ml) were coincubated in the absence of stimulator or in the presence of increasing concentrations of various polysaccharides, including dextran sulfate, heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid were added to the reaction mixtures. Figure 2 shows the results.

As shown in Figure 2, both heparin and dextran sulfate are able to enhance the ability of PN-I to inhibit thrombin, although the profiles are different in that a higher concentration of dextran sulfate appears necessary to achieve measurable but submaximal effects. Both polysaccharides are able to effect 100% inhibition at a concentration of less than or equal to 1 ug/ml.

A similar study was conducted using AT-III as the inhibitor of thrombin. While approximately 1 ug/ml dextran sulfate activated PN-I (both native and re¬ combinant) to obtain 100% thrombin inhibition, even 10 ug/ ml of dextran sulfate showed little enhancement effect on AT-III mediated inhibition; at 1 ug/ml only about 10% thrombin inhibition was obtained.

Example 2 Formulations for Injection Fifty mg of PN-I plus 150 mg of dextran sulfate are dissolved in 10 ml of 200 mM citrate, 1.5 M NaCl, pH 6.0 and then diluted to 100 ml with sterile water.

In an alternate composition, 100 mg of PN-I and 100 mg of dextran sulfate are dissolved in 15 ml of a 1% . dextrose solution containing 200 mM citrate, 1.5 M NaCl, pH 6.0 and the resultant diluted to 100 ml using Ringer's solution.

In another composition, 500 mg of PN-I is dis¬ solved in 10 ml 2% sorbitol containing 20 mM sodium succinate, 300 mM NaCl, pH 5.5. To this is added 200 mg dextran sulfate dissolved in 10 ml sterile water. The mixture is then diluted to 100 ml using isotonic phosphate buffer pH 7.4.

In an aerosol composition, 5 mg of PN-I and 15 mg of dextran sulfate are solubilized in 5 ml 20 mM citrate, pH 6.0, 150 mM NaCl- To this solution is added 25 ml of 50/50 mixture of water/ethanol containing 0.2 g ascorbic acid and 0.02 g acetyl pyridinium chloride as a preservative. The resulting solution is mixed with 10 ml of a 5% solution of sodium fusidate. The resulting mixture is packaged into spray squeeze bottle for inhalation. A suppository containing 100 mg each of PN-I and dextran sulfate is prepared by suspending these amounts in grated theobroma oil, and the active ingredients worked into the oil. The resulting paste is then molded into a suppository.

A suppository is prepared by mixing 50 mg each of PN-I and dextran sulfate in melted polyethylene glycol, molecular weight 4000. The melt is placed into a mold and cooled to a plastic consistency. A topical formulation is prepared by mixing 200 mg PN-I and 200 mg dextran sulfate with 2 grams each Span 60 and Tween 60, 5 g mineral oil, 10 g petrolatum, 100 mg each of methyl and propyl paraben and 10 mg BHA. These ingredients are heated to 60 C with stirring and water is then added with vigorous stirring to obtain an emulsion, which is adjusted with additional water to 100 ml.

Example 3 Administration Protocol A subject having the disseminated intravascular coagulation condition is treated by administering 20 ml of Ringer's solution containing 100 mg each of PN-I and dextran sulfate two times daily for six days.

Example 4

Effect of pH on PN-I Stability Human fibrόblast protease nexin, purified by heparin sepharose affinity chromatography, was adjusted to pH 3, 4, 5 or 6 by adding 1 M citric acid (adjusted to the correct pH with sodium hydroxide) to a final concentration of 200 mM and to pH 7 or 8 by adding 1 M Tris to a final concentration of 200 mM. The solution was then incubated in the presence of 0.02% sodium azide at 37 C for various times prior to assay. PN was assayed using Tris buffer pH 8.1 + heparin as described herein. Activity was compared to a control sample maintained at pH 6.0 and 4 C. The results are shown in Figure 4. PN-I is rapidly inactivated at pH 3 and 8, and slowly at pH 4 and 7. Maintaining the pH in the range of 5-6 results in a stable composition.

Claims

1. A method of enhancing the ability of PN-I to act as a blood anticoagulant, comprising: combining PN-I with dextran sulfate in blood; and allowing the PN-I and dextran sulfate to interact in the presence of the blood for a time sufficient for the dextran sulfate to enhance the ability ^' of the PN-I to act as an anticoagulant with respect to the blood.

2. The method as claimed in claim 1, wherein 1 to 40 parts by weight of PN-I are combined per 1 to 10 parts by weight of dextran sulfate.

3. The method as claimed in claim 1, wherein the PN-I and dextran sulfate are combined within a period of time of 30 to 40 minutes.

4. The method as claimed in claim 3, wherein the period of time is 1 to 5 minutes.

5. The method as claimed in claim 4, wherein the PN-I and dextran sulfate are combined with each other simultaneously in the blood.

6- The method as claimed in claim 1, wherein the PN-I is added so that it is present in the blood in an amount of 10 to 100 ug of PN-I per ml of blood and further wherein the dextran sulfate is added so that it is present in the blood in an amount of 1 to 10 ug of dextran sulfate per ml of blood.

7. The method as claimed in claim 6, wherein the blood is present in a living subject.

8. The method as claimed in claim 7 , wherein the blood is present in a living human being.

9. A pharmaceutical composition, comprising: PN-I in an amount of about 1 to 40 parts by weight; dextran- sulfate in an amount of about 1 to 10 parts by weight; and a pharmaceutically acceptable excipient material.

10. The pharmaceutical composition as claimed in claim 9, further comprising: a weak acid present in an amount so as to maintain the pH of the composition within the range of 4.5 to 6.5-

11. A treated composition of blood, comprising: PN-I; dextran sulfate; and human blood present in an amount such that the PN-I is present in the blood in an amount of 10 to 100 ug of PN-I per milliliter of blood and the dextran sulfate is present in the blood in an amount of 1 to 10 ug of dextran sulfate per milliliter of blood.

12. The treated composition of blood as claimed in claim 11, wherein the PN-I is present in an amount of about 1 to 40 mg/kg of blood and the dextran sulfate is present in an amount of about 1 to 10 mg/kg of blood.

13. A pharmaceutical composition comprising: protease nexin-I; and a pharmaceutically acceptable buffer carrier wherein the combination of the buffer and protease nexin-I provide the composition with a pH in the range of 4.5 to 6.5.

14. A method of assaying PN-I activity in a biological sample, comprising combining the biological sample with a buffer having a pH of about 7 to 8; adding thrombin and dextran sulfate to the . sample; incubating the sample at a temperature of from about room temperature to about 37 C for 5 to 30 minutes; and assaying for thrombin activity using a calori etric assay and determining PN-I activity based on _. thrombin inhibition.