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Background: Maintaining a therapeutic level of anticoagulation with unfractionated heparin remains a major challenge for clinicians because of the wide variability of patient responses, which may be explained by variable binding of heparin to plasma proteins. Direct thrombin inhibitors may offer an advantage in more predictable anticoagulation., Methods: Plasma samples from normal volunteers, stable coronary artery disease (CAD) patients, unstable angina patients, and acute myocardial infarction patients were obtained. A fixed concentration of heparin (.13 U/ml) or bivalirudin (1.6 microg/ml) was added to plasma from each of the four study groups and measurement of the APTT was performed. In addition, a pool of plasma from patients with acute MI was diluted in pooled normal plasma, and heparin or bivalirudin was added to the plasma preparation and APTT measurements performed., Results: In heparin-treated plasma samples, mean APTT values were 443 +/- 137% baseline for normal volunteers, 347 +/- 116% for patients with stable CAD, 290 +/- 124% for patients with unstable angina (p < 0.05), and 230 +/- 120% for patients with acute MI (p < 0.05). APTT did not differ across the four groups treated with bivalirudin. There was a much higher degree of variability in APTT values in heparin treated controls (272%-671%, SD approximately 30%) compared to bivalirudin treated controls (284-499%, SD approximately 12%). When the "acute MI pool" was diluted in pooled normal plasma at fixed concentrations of either bivalirudin (1.6 mug/ml) or heparin (0.13 U/ml), there was a sharp decrease in heparin activity from 407% baseline (at 0% acute MI pool) to values as low as 126% baseline (at 100% acute MI pool). A markedly different pattern was seen in the bivalirudin treated samples, where a trend towards decreased APTT values was seen only at the 100% acute MI pool., Conclusion: Both heparin variability and resistance may limit optimal antithrombotic therapy with heparin in patients with ACS and constitutes a potential advantage of direct antithrombin blockade with bivalirudin.

The study evaluated the role of thrombin in activation of vascular smooth muscle cells early after vascular injury. The direct thrombin inhibitor Hirulog (10 mg/kg SQ tid) or vehicle was administered to rabbits over 3 days following balloon injury to the abdominal aorta and the right iliac artery. Hirulog treatment yielded marked systemic anticoagulation as evidenced by an about 3.5-fold prolongation of quantitative thrombin time one hour after an injection, but with a reduction to almost baseline levels at the end of the dosing interval. After 3 days, proliferating cells in the right iliac artery were enumerated. The expression of intercellular adhesion molecule 1, macrophage-colony stimulating factor, tumor necrosis factor alpha, and interleukin-1beta as markers for inflammatory activation of the vessel wall was examined by immunohistochemistry and graded semiquantitatively. Mitotic indices did not differ between control and Hirulog-treated animals. There was also no difference in the expression of markers of inflammatory activation between both groups. In conclusion, thrombin inhibition by Hirulog administration does not reduce acutely (within 3 days) vascular smooth muscle cell proliferation or inflammatory activation after angioplasty. Thrombin inhibitors may therefore limit restenosis in the rabbit by acting later or via other, unknown pathways. The lack of effect of the thrombin inhibitor on the cellular events during the early phase of the response to balloon injury may explain the failure of such strategies to reduce restenosis in recent clinical trials despite effects towards acute thrombotic complications. Together, these results suggest that acute thrombin generation is not a crucial stimulus for early smooth muscle cell proliferation and inflammatory activation after vascular injury.

Hirulog-1 effectively prevents thrombosis in coronary artery disease and is associated with a low incidence of bleeding complications. Our study characterized the effect of Hirulog-1 on thrombin-induced production of plasminogen activator inhibitor-1 (PAI-1) in cultured baboon aortic smooth-muscle cells (BASMCs). Thrombin increased the steady-state levels of PAI-1 messenger RNA (mRNA) and the release of PAI-1 antigen from BASMCs. Treatments with 10-20 mg/L of Hirulog-1 inhibited >80% of thrombin-induced PAI-1 generation from BASMCs. Hirulog-1 alone did not significantly alter PAI-1 production in the absence of thrombin. Significant reduction of thrombin-induced PAI-1 release was observed in cultures treated with Hirulog-1 for 1 h. The maximal effect of Hirulog-1 on thrombin-induced PAI-1 release was achieved in cultures treated with thrombin plus Hirulog-1 for 3 to 6 h, associated with the normalization of PAI-1 mRNA levels induced by thrombin treatment. Strong inhibition by Hirulog-1 on thrombin-induced PAI-1 release remained in cultures with 8 h of the treatment, but the effect was attenuated 16 h after a single addition of the inhibitor. Our study demonstrates that Hirulog-1 effectively inhibited thrombin-induced PAI-1 production in cultured vascular SMCs at mRNA and protein levels. Vascular SMCs may be exposed to high concentrations of thrombin when endothelium is injured. The information generated from this study suggests that Hirulog-1 potentially prevents intravascular thrombogenesis through inhibiting thrombin-induced PAI-1 production in vascular SMCs, especially when hypercoagulation and endothelial injury occurs.

Background: Thrombin may play an important role in restenosis after balloon angioplasty (BA). Angiographic and pathologic restenosis have been shown to be reduced after BA in an atherosclerotic rabbit model using recombinant desulfatohirudin, a selective and direct thrombin inhibitor. We hypothesized that potent and specific thrombin inhibition with the synthetic peptide hirulog given intravenously at the time of angioplasty would reduce restenosis in rabbits, confirming a specific role of thrombin in restenosis., Methods and Results: Focal femoral atherosclerosis was induced in 27 rabbits by air desiccation endothelial injury followed by a 2% cholesterol diet for 1 month. Rabbits received either heparin (150 units/kg bolus, n = 14) or hirulog (5 mg/kg bolus followed by 5 mg/kg/h for 2 h, n = 13) at the time of BA (2.5-mm balloon with three 60-second, 10-atm inflations 60 s apart). Angiograms performed before and after BA and before sacrifice were analyzed quantitatively. Rabbits were sacrificed 28 days after BA for quantitative histopathologic analysis. Minimum luminal diameter (mm) did not differ between treatment groups before (1.1 +/- 0.2 vs. 1.2 +/- 0.1 mm) or after (1.5 +/- 0.2 vs. 1.6 +/- 0.1) BA in arteries from heparin-versus hirulog-treated rabbits, respectively. At 28 days, however, minimum luminal diameter was significantly less (1.0 +/- 0.4 vs. 1.5 +/- 0.2, p = 0.0001) and percent stenosis was greater (0.46 +/- 0.25 vs. 0.22 +/- 0.08, p = 0.0002) in arteries from heparin- versus hirulog-treated rabbits, respectively. Similarly, quantitative histopathology showed less cross-sectional area narrowing by plaque in the hirulog group (56 +/- 24 vs. 42 +/- 21%, p = 0.04)., Conclusion: A 2-hour infusion of hirulog at the time of angioplasty improved late angiographic luminal dimensions and reduced cross-sectional area narrowing by plaque in rabbits compared with heparin controls. Together with previous studies, this confirms a specific role for thrombin in restenosis after angioplasty.

Hirulog therapy has been studied extensively in numerous settings including prevention of DVT, treatment of unstable angina, treatment of acute myocardial infarction during thrombolysis, and prevention of acute complications of PTCA. Being one of the first direct thrombin inhibitors in clinical development, it has had to 'test the waters', so to speak, of the relationship between pathophysiology and clinical trial design: what are the correct indications, patient entry criteria, endopoints, frequency and duration of dosing, and so on? Our findings validate a role for thrombin in treating arterial thromboembolism and demonstrate clinical activity and tolerability of Hirulog.

A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP-induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells.

Hirulog is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg; serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog. Rat survival to endotoxin was significantly increased in Hirulog pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog at the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog (5-10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a kcat value of 0.01 s-1. The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with Km and kmax values of 0.75 +/- 0.12 microM and 325 +/- 17 s-1. The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 microM in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s-1. A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective thrombin inhibitors are a new class of antithrombotic drugs that, unlike heparin, can effectively inhibit clot-bound thrombin and escape neutralization by activated platelets. Hirulog is a 20 amino acid hirudin-based synthetic peptide that has shown promise in experimental models of thrombosis. Little information is available about the effects of hirulog in patients with coronary artery disease. Forty-five patients undergoing cardiac catheterization, who were taking aspirin, were randomized to receive either (1) hirulog, 0.05 mg/kg intravenous bolus followed by 0.2 mg/kg/hour intravenous infusion until the end of the catheterization; (2) hirulog, 0.15 mg/kg intravenous bolus followed by 0.6 mg/kg/hour intravenous infusion; or (3) heparin; 5,000 U intravenous bolus. Serial activated partial thromboplastin time (APTT), prothrombin time, activated clotting time and fibrinopeptide A were measured. Hirulog produced a dose-dependent prolongation of all coagulation parameters; the 0.6 mg/kg/hour dose prolonged the APTT to 218 +/- 50% of baseline after 2 minutes and 248 +/- 50% of baseline after 15 minutes. The half-life of the effect on APTT was 40 minutes. The hirulog blood level correlated well with the APTT, prothrombin time and activated clotting time (r = 0.77, 0.73, and 0.82 respectively, all p < 0.001). Both doses of hirulog potently suppressed the generation of fibrinopeptide A (p < 0.05). There were no major hemorrhagic, thrombotic or allergic complications in patients treated with hirulog or heparin. Thus, hirulog, a direct thrombin inhibitor, provides a predictable level of anticoagulation and appears to have a potent yet well-tolerated anticoagulant profile in patients with coronary artery disease.

Unabated, the arterial thrombotic process continues to be a major challenge in the management of acute coronary artery disease. Pharmacologic and mechanical revascularization therapies, which have proliferated over the last decade, remain impeded by arterial thrombosis and its clinical sequelae. New antithrombotic drugs aimed at specific points in the arterial thrombotic process offer the potential for substantial improvements in the management of coronary artery disease. The use of these agents as a mainstay of patient care is becoming a reality as controlled clinical studies test their safety and potential benefits.

The X-ray crystallographic structure of the human alpha-thrombin complex with hirulog 3 (a potent, noncleavable hirudin-based peptide of the "hirulog" class containing a beta-homoarginine at the scissile bond), which is isomorphous with that of the hirugen-thrombin crystal structure, was solved at 2.3-A resolution by starting with a model for thrombin derived from the hirugen-thrombin complex and was refined by restrained least squares methods (R = 0.132). Residues of hirulog 3 were well-defined in the electron density, which included most of the pentaglycine linker and the C-terminal helical turn that was disordered in a related structure of thrombin with hirulog 1. The interactions of D-Phe1'-Pro2'-beta-homoArg3' with the active site of thrombin were essentially identical to those of related structures of PPACK- (D-Phe-Pro-Arg chloromethyl ketone) and hirulog 1-thrombin, with the guanidinium function of the arginyl P1 residue forming a hydrogen-bonding ion pair with Asp189 of the S1 site. A noticeable shift in the CA atom of beta-homoArg3' due to the methylene insertion displaces the scissile bond from attack by Ser195, thus imparting proteolytic stability to the beta-homoArg hirulog derivative. Resolution of the pentaglycine spacer, linking N- and C-terminal functional domains into a single oligopeptide bivalent inhibitor, permitted delineation of corresponding S' subsites of thrombin. The position of Gly4' (P1') is stabilized by three hydrogen bonds with His57, Lys60F, and Ser195, while the conformational angles maintained in a strained, nonallowed configuration for non-glycyl amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.

Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095-7101). These peptides, designated 'hirulogs', interact with both thrombin's catalytic center and its anion-binding exosite for fibrinogen recognition. In order to investigate structure-activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic-site binding and anion-binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.

Using the natural protein hirudin as a model, a novel class of synthetic peptide inhibitors were recently designed. These inhibitors, 'hirulogs', retain the carboxy terminal Hir53-64 domain that interacts with the anion binding exosite of thrombin, connected via an oligoglycyl spacer unit to a catalytic site-directed moiety modeled on the sequence [D]Phe-Pro-Arg-X. The scissile Arg-X bond bond of substrate-like inhibitors has been modified to the proteolytic-resistant functions as beta-homo amino acids Arg psi [CH2CONH] X (2) and reduced bond analogues Arg psi [CH2N]X (3). Both classes of compounds demonstrate inhibition of thrombin amidolytic activity, and this active-site inhibition is highly sensitive to the P1' residue X. Thus these hirulog derivatives are resistant to thrombin proteolysis while maintaining substrate-like interactions with the active center. Finally, hirulog derivatives with non-cleavable replacements of the scissile bond are found to be effective anticoagulant agents.

To determine the importance of the thrombin substrate recognition exosite for fibrinogen binding in the formation of both arterial and venous thrombi, we evaluated the antithrombotic effects of the tyrosine-sulfated dodecapeptide from residues 53-64 of hirudin (H peptide) in a nonhuman primate model. This peptide was studied because it inhibits thrombin cleavages of fibrinogen by simple competition without blocking enzyme catalytic-site function. When an exteriorized arteriovenous access shunt model was used in baboons (Papio anubis), thrombus formation was induced by placing a thrombogenic device made of (i) a segment of tubing coated covalently with type I collagen, which generated platelet-rich thrombi under arterial flow conditions, and (ii) two subsequent annular regions of flow expansion that produced fibrin-rich thrombi typically associated with venous valves and veins. Thrombus formation was quantified by measurements of 111In-labeled platelet and 125I-labeled fibrinogen deposition in both arterial-flow and venous-flow portions of the device. Continuous infusion of H peptide (0.5, 15, and 75 mg/kg) proximal to the device for 40 min interrupted, in a dose-response fashion, formation of fibrin-rich thrombus in the regions of disturbed flow and generation of fibrinopeptide A. In contrast, H peptide did not inhibit the capacity of platelets to deposit on the collagen surface (P greater than 0.2 at all doses) or to form hemostatic plugs (as assessed by measurements of bleeding time; P greater than 0.1 at all doses). These findings suggest that, by competitive inhibition of fibrinogen binding to thrombin, fibrin-rich venous-type thrombus formation may be selectively prevented. This strategy may be therapeutically attractive for preserving normal platelet function when conventional anticoagulant therapy is contraindicated.

Thrombin has both beneficial and harmful effects. In order of importance, at very low concentrations, alpha-thrombin firstly amplifies its own generation through the activation of factors V and VIII, which are the primary targets of antithrombotic agents. It secondly functions at the cellular level where, at low concentrations it activates platelets, and at higher concentrations, induces endothelial cell changes (e.g., shape changes, albumin transport release of plasminogen activators and other substances). It thirdly converts fibrinogen into clottable fibrin and becomes actively incorporated into the forming thrombus. In addition, it activates protein C, which in turn degrades factors V and VIII (and/or their activated forms) and causes the shutdown of thrombin generation. When compared to other serine proteinases of the blood coagulation and fibrinolytic systems, alpha-thrombin is unique in that it loses most of its proenzyme activation fragment and has developed multisite short-ranged bridge-binding interactions, which appear to explain thrombin specificity. To understand thrombin is to understand haemostasis.

In addition to its classical active-site regions (catalytic site and adjacent regions), alpha-thrombin has a unique anion-binding exosite, which is functionally independent of the catalytic site and is involved in fibrin(ogen) recognition. This exosite also accounts for adhesion to negatively charged surfaces (e.g., glass), binding to cell surfaces, and interactions with the anionic tail of hirudin. Hirudin (as an apolar, tridisulfide-linked core structure followed by its anionic tail) interacts with alpha-thrombin by apolar (e.g., catalytic-site and adjacent regions of thrombin), as well as by ionic binding (e.g., anion-binding exosite). Circular dichroism measurements reveal a sigmoidal nonadditivity for the hirudin tail fragments, which block fibrinogen-clotting activity without interfering with tripeptide chromogenic substrate activities. Such fragments, however, inhibit factor V activation to much lesser extents than hirudin, where factor V activation is the key step in regulating thrombin generation by hirudin or heparin/antithrombin III. Hirudin-derived antithrombotics may thus have differential modes of action in hemostasis and wound healing processes.

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of alpha-thrombin. These peptides, called "hirulogs", consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 A in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ([D-Phe)-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. In addition, the pentapeptide (D-Phe)-Pro-Arg-Pro-Gly, which comprises the catalytic-site inhibitor moiety of hirulog-1, was determined to have a Ki for thrombin inhibition greater than 2 microM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.

In order to define structural regions in thrombin that interact with hirudin, the N alpha-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human alpha-thrombin in phosphate-buffered saline at pH 7.4 and 23 degrees C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr approximately 32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr approximately 31,000. Incorporation of the 35S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human alpha-thrombin-DNFB-Hir54-64 complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir54-64 derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 A from the catalytic apparatus.

Thrombin is a serine protease that plays an essential role in blood coagulation and also induces various responses in endothelial cells. The actions of thrombin on the conversion of fibrinogen to fibrin are inhibited by peptides based on the amino acid sequence of hirudin, a natural anticoagulant from leeches. We show in these studies that the peptides Hir45-64 and sulfated Hir53-64 block the effects of thrombin on endothelial cells. These peptides inhibited, in a concentration-dependent manner, the synthesis of prostaglandin I2 and platelet-activating factor, and the acquisition of an adhesive surface for leukocytes that occur in response to thrombin. These actions of the peptides occurred even though the catalytic site of thrombin was not blocked.

Applaggin (Agkistrodon piscivorus piscivorus platelet aggregation inhibitor) is a potent inhibitor of platelet activation. The protein is isolated from the venom of the North American water moccasin snake in three steps, including gel filtration, cation exchange, and reverse-phase HPLC procedures. The purified protein migrates as a 17,700-Da polypeptide by SDS/PAGE under nonreducing conditions and as a 9800-Da peptide in the presence of thiol. The behavior of applaggin on SDS/PAGE would indicate that the protein is a disulfide-linked dimer. Applaggin has been completely sequenced by Edman degradation and consists of 71 amino acids. The sequence is rich in cysteine and contains Arg-Gly-Asp at residues 50-52. Applaggin blocks platelet aggregation induced by ADP, collagen, thrombin, or arachidonic acid with IC50 values ranging from 12 to 128 nM (0.2-2.3 micrograms/ml) depending on the agonist and its concentration. This inhibition is found to correlate with inhibition of thromboxane A2 generation and of dense granule release of serotonin. Inhibition by applaggin of serotonin release induced by ADP, gamma-thrombin, and collagen was monitored in plasma under stirred conditions with [3H]serotonin-loaded platelets, and IC50 values for inhibition are found to range from less than 10 to 145 nM. At saturating concentrations, 125I-labeled applaggin (125I-applaggin) binds to 28,500 sites per unstimulated, washed platelet with a Kd of 1.22 x 10(-7) M. Binding of 125I-applaggin to platelets is inhibited by the synthetic undecapeptide Arg8-Gly-Asp-Val at 200 microM.

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.

We have developed a simple two-step procedure for the separation of monomeric (14,000 mol. wt) and dimeric (28,000 mol. wt) phospholipases A2 from the venoms of Crotalidae family snakes. All venom phospholipases A2 studied thus far exist as monomers under acidic conditions and are chromatographed as such on a column of G-50 Sephadex (superfine) equilibrated in 5% acetic acid. Separation of dimeric phospholipases A2 from any monomeric enzyme(s) in pools of enzyme thus obtained is achieved by chromatography on a second column of G-50 Sephadex (superfine) identical to the first but developed in 1% ammonium bicarbonate. This method has been applied to an investigation of the prevalence of monomeric and/or dimeric enzymes in venoms of the Crotalidae family. The distribution of monomeric and dimeric phospholipases correlates well with phylogeny. In the more primitive Crotalidae genera, such as Trimeresurus and Agkistrodon, monomeric phospholipases A2 are predominant. In the more highly evolved Crotalus genus, only dimeric enzymes are found.

A gene encoding a 113-amino acid, NH2-terminal fragment of CD4, rsT4.113, was constructed and expressed in Escherichia coli under the control of the tryptophan operon promoter. Following induction, rsT4.113 is produced at 5-10% of total E. coli protein, and it is found in inclusion bodies. The protein is purified in two steps under denaturing and reducing conditions. Solubilized rsT4.113 is first purified on a column of Q-Sepharose to remove low molecular weight contaminants and then purified to greater than 95% homogeneity by gel filtration. Renaturation of rsT4.113 is achieved at approximately 20% yield by dilution and dialysis. High performance liquid chromatography analysis of renatured rsT4.113 reveals a less than 15% contaminant of reduced protein. Purified and renatured rsT4.113 contains epitopes for both OKT4a and Leu3a, anti-CD4 monoclonal antibodies which block CD4-gp 120 association, but lacks measurable affinity toward a nonblocking anti-CD4 monoclonal antibody, OKT4. By comparison to a longer form (375 amino acids) of recombinant soluble T4 produced in mammalian cells that contains the entire extracellular domain, rsT4.113 has a comparable affinity for binding to OKT4a and Leu3a in a radioimmunoassay. Analysis of antiviral activity of rsT4.113 demonstrates that the E. coli-derived protein inhibits human immunodeficiency virus-induced syncytium formation with an IC50 of 5-10 micrograms/ml. These data demonstrate that the human immunodeficiency virus-binding domain of CD4 is localized within the NH2-terminal 113 amino acids of CD4 and is contained within a structure homologous to the kappa variable-like domain of immunoglobulins.

The aspartyl residue at position 49 in phospholipases A2 (PLA) has been viewed as a component of the catalytic apparatus because of its involvement in binding the essential cofactor, calcium. We recently discovered a new class of PLA's in which, among other changes in highly invariant residues, Asp-49 is replaced by a lysine (Maraganore et al. (1984) J. Biol. Chem. 259, 13839). These Lys-49 PLA's are also calcium-dependent, but, in contrast to the Asp-49 enzymes, they bind phospholipid strongly in the absence of calcium. Lys-49 PLA's are, therefore, ideal for studying structural and mechanistic aspects of these enzymes. Attempts to modify Lys-49 with the amino group-specific reagent, trinitrobenzenesulfonic acid (TNBS) led to the inactivation of the PLA, but reaction occurred not as expected at position 49, but at Lys-53. These findings lead us to propose a model, applicable to PLA's in general, in which cationic side chains at position 53 in these enzymes participate in phospholipid binding on the path to formation of the catalytic complex. This model serves to explain a number of unresolved observations in the current literature relating to enzyme-substrate interactions in the PLA's.

The basic Lys-49 phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus venom is homologous to the basic Asp-49 PLA2 from the same venom as well as other snake venom PLA2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent Vmax of the Lys-49 PLA2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA2, compared to the Asp-49 PLA2 had less than 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA2. Evaluation of the present results, along with earlier findings using Asp-49 PLA2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: The A. p. piscivorus Asp-49 PLA2 enzyme resembles the Asp-49 enzymes from N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. In contrast to some previous suggestions, basic PLA2 enzymes are not necessarily more toxic than neutral or acidic enzymes. Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA2 enzymes exert effects independent of phospholipid hydrolysis.

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis.

Lipoprotein lipase from bovine milk reacted stoichiometrically with diisopropylphosphorofluoridate (DFP), an inactivator of serine esterases, resulting in the loss of enzymatic activity against triacylglycerols. The reaction obeyed first-order kinetics with a rate constant of 0.69 h-1. In order to isolate the peptide containing the diisopropylphosphoryl moiety (DIP), partially purified lipoprotein lipase was covalently labeled with [3H]DFP, and the labeled protein was reduced, carboxymethylated, and further purified to about 90% homogeneity. Cyanogen bromide cleavage followed by gel filtration yielded a radioactive peptide of 6-8 kDa. This peptide was succinylated and then digested with Staphylococcus aureus V8 proteinase. From this digest, a peptide containing 0.95 mol of [3H] DIP/mol of peptide was isolated by gel-permeation chromatography followed by reverse-phase high performance liquid chromatography. Automated Edman degradation provided the following sequence: Ala-Ile-Gly-Ile-His-Trp-Gly-Gly- (DIP)Ser-Pro-Asn-Gln-Lys-Asn-Gly-Ala-Val-Phe-Ile-Asn-(Ser, Leu)-Glu. Analysis of the sequence for secondary structure suggests that the reactive serine of lipoprotein lipase is in a beta-turn, a structure similar to those of the active sites of most other serine proteinases. Lipoprotein lipase appears to share this secondary structure with other serine hydrolases despite significant differences in the primary structure of this domain.

Three monomeric phospholipases A2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air-water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.