重组水蛭素HV2的稳定性

重组水蛭素ＨＶ２是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质。温度的升高（１００℃水浴）和ｐＨ（１─１３）的改变不影响其活力,在某些变性剂（８ｍｏｌ／Ｌ尿素、１％ＳＤＳ和６ｍｏｌ／Ｌ盐酸胍）存在的条件下也非常稳定,０．１ｍｏｌ／Ｌ的ＤＴＴ在７０℃时使其部分失活,只有ｐＨ和温度同时升高其活力才开始下降,ｐＨ１３、８０℃处理１５ｍｉｎ即完全失活,氨基酸组成和活性分析发现失活样品的Ｃｙｓ和Ｌｙｓ被破坏。重组水蛭素ＨＶ２含有一个结构紧密的Ｎ端核心区和一个无序的Ｃ端尾部。其Ｎ端的３个Ｌｙｓ－Ｘａａ键均不被胰蛋白酶水解;胃蛋白酶及糜蛋白酶消化后,分离所得片段,氨基酸组成分析发现Ｎ端核心区依然保持很高的抗凝血酶活性,继续消化２４ｈ,核心区不被进一步降解。     

Use of fragments of hirudin to investigate thrombin-hirudin interaction

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.     

毕赤酵母发酵生产中的水蛭素降解顺序

水蛭素 (rHV2 Lys4 7)是一个具有 65个氨基酸的抗凝活性肽。在毕赤酵母高密度发酵分泌表达过程中 ,发酵上清中可检出 4个水蛭素活性组份 ,分别为Hir65及其C 末端切除 1～ 3个氨基酸的Hir64、Hir63和Hir62。但目前 4种组份间的衍生关系还不清楚 ,以从发酵上清液中纯化分离所得的 4个组份作为底物 ,加入到菌体裂解液中 ,发现Hir64、Hir63和Hir62组份是由羧肽酶依次降解Hir65肽链C 末端 1个氨基酸后的产物。     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin-specific inhibitor [(1989) J. Biol. Chem. 264, 7141-7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N-terminal core domains (r-Hir1-43 and r-Hir1-52) derived from recombinant hirudins and synthetic C-terminal peptides (Hir40-65 and Hir52-65)--all fragments form complexes with thrombin--we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C-terminal region. Since hirudin C-terminal peptides were shown to bind to a non-catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10-16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.     

Affinity labeling of lysine-149 in the anion-binding exosite of human alpha-thrombin with an N alpha-(dinitrofluorobenzyl)hirudin C-terminal peptide

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.     

Characterization of bothrojaracin interaction with human prothrombin

Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 microM, indicating a approximately 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human prothrombin.     

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.     

Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of alpha-thrombin

The C-terminal peptide of a hirudin acts as an anticoagulant by binding specifically to a noncatalytic (fibrinogen recognition) site of thrombin. This binding has been shown to shield five spatially distant lysines of the thrombin B-chain (Lys21, Lys65, Lys77, Lys106, and Lys107). It was also demonstrated that modification of the sequence of the hirudin C-terminal peptide invariably diminished its anticoagulant activity. The major object of this study is to investigate how the decreased activity of the modified hirudin C-terminal peptide is reflected by the change of its binding properties to these five lysines of thrombin. A synthetic peptide representing the last 12 C-terminal amino acids of hirudin (Hir54-65) was (1) truncated from both its N-terminal and its C-terminal ends, or (2) substituted with Gly along residues 57-62, or (3) chemically modified to add (sulfation at Tyr63) or abolish (Asp and Glu modification with carbodiimide/glycinamide) its negatively charged side chains. The binding characteristics of these peptides to thrombin were investigated by chemical methods, and their corresponding anticoagulant activities were studied. Our results demonstrated the following: (1) the anticoagulant activities of hirudin C-terminal peptides were quantitatively related to their abilities to shield the five identified lysines of thrombin. The most potent peptide was sulfated Hir54-65 (S-Hir54-65) with an average binding affinity to the five lysines of 120 nM. A heptapeptide (Hir54-60) also displayed anticoagulant activity and thrombin binding ability at micromolar concentrations. (2) All active hirudin C-terminal peptides regardless of their sizes and potencies were shown to be capable of shielding the five lysines of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects

The interaction of the C-terminal fragments (residues 52-65 and 55-65) of the thrombin-specific inhibitor hirudin with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution. Thrombin induces specific line broadening of the proton resonances of residues Asp(55) to Gln(65) of the synthetic hirudin fragments H-Asn-Asp-Gly-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH and acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH. This demonstrates that residues 55-65 are the predominant binding site of hirudin fragments with thrombin. Hirudin fragments take on a well-defined structure when bound to thrombin as indicated by several long-range transferred NOEs between the backbone and side-chain protons of the peptides, but they are not structured when free in solution. Particularly, transferred NOEs exist between the alpha CH proton of Glu(61) and the NH proton of Leu(64) [d alpha N(i,i+3)], between the alpha CH proton of Glu(61) and the beta CH2 protons of Leu(64) [d alpha beta(i,i+3)], and between the alpha CH proton of Glu(62) and the gamma CH2 protons of Gln(65) [d alpha gamma(i,i+3)]. These NOEs are characteristic of an alpha-helical structure involving residues Glu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Distance geometry calculations suggest that in the structure of the thrombin-bound hirudin peptides all the charged residues lie on the opposite side of a hydrophobic cluster formed by the nonpolar side chains of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64).     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

The role of Glu(192) in the allosteric control of the S(2)' and S(3)' subsites of thrombin

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir(52-65)) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S(2)' and S(3)' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P(2)' and P(3)'. Regardless of the amino acids, Hir(52-65) decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu(192) participates in this modulation. We have examined the role of Glu(192) by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P(2)' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the "least favorable" P(2)' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P(3)' residues and its strong aversion for negatively charged P(3)' residues. Furthermore, both Hir(52-65) and heparin increased the k(cat)/K(m) value of substrate hydrolysis. We conclude that Glu(192) is critical for the P(2)' and P(3)' specificities of thrombin and for the allostery mediated through exosite 1.     

Characterization of proexosite I on prothrombin

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed.     

The functional domain of hirudin, a thrombin-specific inhibitor

Hirudin is a thrombin-specific inhibitor of Mr 8000 (65 amino acid residues). Native hirudin contains 3 disulfide linkages within the first 39 amino-terminal residues, and a highly acidic C-terminal segment which is freely accessible to enzyme digestion by both endo- and exo-peptidases. Removal of the acidic C-terminal amino acids of native hirudin by both chemical and enzymatic methods resulted in a concomitant loss of hirudin inhibition activity. It is concluded that this acidic C-terminal segment of hirudin is essential for hirudin-thrombin interaction. The implication of the hirudin-thrombin interaction for the enzymatic specificity of thrombin is also discussed.     

Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin

Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.     

The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity.

A thrombin receptor has recently been cloned and the sequence deduced. The sequence reveals a thrombin cleavage site that accounts for receptor activation. The receptor also has an acidic region with some similarities to the carboxyl-terminal region of the leech thrombin inhibitor, hirudin. Synthetic peptides corresponding to the receptor cleavage site (residues 38-45), the hirudin-like domain (residues 52-69), and the covalently associated domains (residues 38-64) were evaluated for their ability to bind to thrombin. Peptides 38-45 and 38-64 were competitive inhibitors of thrombin's chromogenic substrate activity (Ki = 0.96 mM and 0.6 microM, respectively. Residues 52-69 altered the chromogenic substrate specificity, resulting in accelerated cleavage of some substrates and inhibited cleavage of others. The same peptide binds to thrombin and alters the fluorescence emission intensity of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-thrombin in which the dansyl is attached directly to the active site serine (Kd = 32 +/- 7 microM). Residues 52-69 displace the carboxyl-terminal peptide of hirudin, indicating that they share a common binding site in the anion exosite of thrombin. These data suggest that the thrombin receptor has high affinity for thrombin due to the presence of the hirudin-like domain and that this domain alters the specificity of thrombin. This change in specificity may account for the ability of the receptor to serve as an excellent thrombin substrate despite the presence of an Asp residue in the P3 site, which is normally inhibitory to thrombin activity.     

Mechanism of the inhibition of alpha-thrombin by hirudin-derived fragments hirudin(1-47) and hirudin(45-65)

The kinetic mechanism of the inhibition of alpha-thrombin by hirudin was analyzed using the hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Previously, these fragments have been shown to interact with alpha-thrombin at distinct sites inhibiting thrombin-mediated clot formation. Binding to the active site the N-terminal fragment hirudin(1-47) competitively inhibits hydrolysis of the substrates Tos-Gly-Pro-Arg-NH-Mec (Tos, tosyl; NH-Mec, 4-methylcoumaryl-7-amide) and fibrinogen with Ki values of 420 +/- 18 nM and 460 +/- 25 nM, respectively. Interacting with the anion-binding site of alpha-thrombin the C-terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 +/- 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide-NH-Mec substrate. According to the Botts-Morales scheme for enzyme inhibition, the parameters Ki = 710 +/- 38 nM, K'i = 348 +/- 22 nM, as well as alpha = beta = 0.49 of thrombin inhibition by the C-terminal fragment hirudin(45-65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.     

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.     

Antithrombin activity of the hirudin N-terminal core domain residues 1–43

Hirudin N-terminal core domain residues 1–43 (r-Hir^1–43) were prepared from limited proteolysis of recombinant hirudin by V8 Staphylococcal protease followed by purification with reversed-phase HPLC. r-Hir^1-43 lacks the putative reactive site of hirudin (Lys⁴⁷), but binds to thrombin (with K_i of 300 nM) and blocks the catalytic activity of the protease. The structural element which accounts for the thrombin inhibitory activity of r-Hir^1–43 is analyzed in this report.     

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.