Enzymatic properties of proteolytic derivatives of human alpha-thrombin

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of hirudin with the dysthrombins Quick I and II

The interaction of hirudin with the dysfunctional enzymes thrombin Quick I and II has been investigated. Natural and recombinant hirudin caused nonlinear competitive inhibition of thrombin Quick I. The results were consistent with thrombin Quick I existing in two forms that have different affinities for hirudin. The affinities of these forms for natural hirudin were respectively 10(4)- and 10(6)-fold lower than that of alpha-thrombin. In contrast, truncated hirudin molecules lacking the C-terminal tail of the molecule caused linear inhibition of thrombin Quick I. These results indicate that different modes of interaction of the two forms of thrombin Quick I with the C-terminal tail of hirudin were the cause of the nonlinear inhibition. Comparison of the dissociation constants of thrombin Quick I with the truncated and full-length forms of hirudin suggested that the interactions that normally occur between the C-terminal tail of hirudin and thrombin were completely disrupted with the low-affinity form of thrombin Quick I. Thrombin Quick II displayed an affinity for natural hirudin that was 10(3)-fold lower than that observed with alpha-thrombin. In contrast, it bound a mutant hirudin with altered N-terminal amino acids only 16-fold less tightly. These results are discussed in terms of structural alterations in the active-site cleft in thrombin Quick II.     

Basis for the reduced affinity of beta T- and gamma T-thrombin for hirudin

Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.     

Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms.

The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.     

Comparison of the catalytic properties of alpha- and beta-forms of thrombin

18-25-fold purified alpha-thrombin, having high esterase activity and coagulating ability of 2500 NIH u per 1 mg of protein, was isolated using chromatography of commercial thrombin through SP-Sephadex C-50. Limited proteolysis of alpha-thrombin on the column with immobilized trypsin resulted in the appearance of beta-thrombin with alpha-thrombin-like esterase activity and tracing coagulating activity (2-5 NIH u per 1 mg of protein). Molecular weight analysis of alpha- and beta-thrombin forms suggests that a peptide (or peptides) with Mr of 1100 is splitted off under proteolysis. Some similarity is revealed in kinetic parameters (Km(app) and kkat) of TAME and BAME hydrolysis by alpha- and beta-thrombin, although Km(app) is somewhat low (approximately 2-fold) for alpha-thrombin. Investigation of TAME hydrolysis kinetics by both thrombin forms at a wide range of substrate concentrations has revealed the effect of substrate activation. Kinetic constants Ks and beta for high substrate concentrations are calculated. It is suggested that the similarity of alpha- and beta-thrombin action on arginine esters and sharp differences in their effect on fibrinogen may be a result of a disturbance of substrate-binding region of beta-thrombin active site.     

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.     

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.     

Enzymic and nonenzymic properties of human beta-thrombin

Autolysis or tryptic hydrolysis converts human alpha-thrombin to its beta-derivative and subsequently to gamma-thrombin. Human beta-thrombin was obtained by tryptic digestion of alpha-thrombin and isolated by BioRex chromatography. The kinetic parameters for human alpha- and beta-thrombins with H-D-phenylalanyl-L-pipecolyl-L-arginine-para-nitroanilide were similar, as well as the rate of inactivation by tosyl-lysine chloromethyl ketone. By contrast, the rate of inactivation by diisopropyl fluorophosphate was reduced by half, and the inhibition constant for benzamidine was increased 2.5-fold. Moreover, the beta cleavages induced a drastic reduction in reactivity toward protein C, affinity for thrombomodulin, and fibrinogen clotting activity. Unlike alpha-thrombin, beta-thrombin was not protected from inhibition by diisopropyl fluorophosphate in the presence of fibrinogen and failed to bind to fibrin-Sepharose. Our results indicate that the beta cleavages induce multiple defects in the functions of human thrombin. Although the three catalytic residues remain in an active configuration, subtle changes are induced in the microenvironment of the active serine. However, the drastic reduction of fibrinogen clotting activity should rather be ascribed to major alterations observed in both the fibrinopeptide groove and the fibrin recognition site. These observations provide further evidence for a double-site mechanism in the interaction of fibrinogen with thrombin.     

Anticoagulant activity of synthetic hirudin peptides

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.     

The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin. Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex. Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold. The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation. To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli. Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined. Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan. Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52. However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII. Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII. The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin. Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans. These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.     

Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys47), was found to be a competitive inhibitor of human alpha-thrombin with respect to peptidyl p-nitroanilide substrates. These results contrast with those of Degryse and coworkers that suggest that recombinant hirudin variant-2(Lys47) inhibited thrombin by a noncompetitive mechanism [Degryse et al. (1989) Protein Engng, 2, 459-465]. gamma-Thrombin, which can arise from alpha-thrombin by autolysis, was shown to have an affinity for recombinant hirudin variant-2(Lys47) that was four orders of magnitude lower than that of alpha-thrombin. It was demonstrated that the apparent noncompetitive mechanism observed previously was probably caused by a contamination of the thrombin preparation by gamma-thrombin. Comparison of the inhibition of alpha-thrombin by recombinant hirudins variant-2(Lys47) and variant-1, which differ from one another in eight out of 65 amino acids, indicated that the two variants have essentially the same kinetic parameters.     

The structures and proteolytic specificities of autolysed human thrombin.

Three Arg/Lys-Xaa bonds in the B-chain of human alpha-thrombin were found to be the major autolytic sites. Under the conditions of 1 mg of alpha-thrombin/ml in 50 mM-ammonium bicarbonate solution at 25 degrees C, the 50% cleavage times of Lys-Gly (residues 154-155), Arg-Tyr (residues 70-71) and Arg-Asn (residues 73-74) were 32 h, 72 h and 96 h respectively. Fragments generated from these three major autolytic sites were purified and analysed. In addition, minor and random autolytic cleavages occurred simultaneously that eventually led to the complete breakdown of the enzyme. These results reveal several novel aspects about the process of autolysis and the structure of autolysed human thrombin. It identifies a major autolytic site at Arg-Tyr (residues 70-71) that has not been previously reported. It demonstrates that beta-thrombin is not an obligatory intermediate during the process of conversion of alpha-thrombin into gamma-thrombin. There exists a new form of autolysed thrombin, designated as beta'-thrombin (with cleavage at Lys-Gly only), which also serves as the intermediate in the conversion of alpha-thrombin into gamma-thrombin. It shows that autolysis of human alpha-thrombin does not proceed in an absolutely clear-cut manner. Numerous minor cleavages, which amount to approx. 20% of the three major autolytic sites, occur simultaneously. It is the first time that several autolytic sites of human alpha-thrombin have been quantitatively analysed, and that it has been shown that formation of beta-, beta'- and gamma-thrombins can be quantitatively followed by the h.p.l.c. method. Furthermore, the data demonstrate that alpha-thrombin and the autolysed thrombin (mixture of beta-, beta'- and gamma-thrombins) have comparable proteolytic activity and specificity towards various sizes of non-fibrinogen polypeptide substrates with relative molecular masses ranging from 3000 to 25,000.     

Platelet responses to compound interactions with thrombin.

Catalytic and noncatalytic interactions of thrombin with platelets are investigated with use of thrombin variants with altered specificities and with ligands of thrombin receptors on platelets. Both alpha-thrombin and weakly coagulant meizothrombin-des-fragment-1 (mu-thrombin) hydrolyze proteolytically activated receptor 1 for thrombin (rPAR1(T), recombinant) with catalytic efficiencies of >10(7) M(-)(1) s(-)(1), whereas rPAR1(T) is not a substrate for weakly coagulant beta-thrombin. In contrast, both mu-thrombin and beta-thrombin are weak agonists of platelet dense body (ATP) secretion. Antibodies that block rPAR1(T) cleavage strongly inhibit the secretory reaction to alpha- and mu-thrombins but not to beta-thrombin or to thrombin receptor activating peptide (TRAP). However, catalytically inactive FPR-thrombin, which binds glycoprotein Ib but does not inhibit rPAR1(T) cleavage, inhibits responses to TRAP as well as those to alpha- and mu-thrombins, which indicates that binding of the inactive enzyme to platelets influences the function of PAR1(T). An antibody that inhibits binding of thrombin to platelet glycoprotein Ib inhibits secretory responses to thrombin but not to TRAP, so occupancy of glycoprotein Ib per se accounts for only part of the attenuation. All three thrombins stimulate a rise in cytosolic Ca(II), and the dose response to beta-thrombin is congruent with that for ATP secretion. However, the response of cytosolic Ca(II) is 10-100 times more sensitive to mu-thrombin and alpha-thrombin than ATP secretion is, and is inhibited by neither anti-PAR1(T) Ig nor FPR-thrombin. Thus, alpha-thrombin appears to have an activity not shared by either mu- or beta-thrombins. This activity is owed to more than coupling of independent signals from cleavage of two proteolytically activated receptors, as there is no synergism when mu-thrombin and beta-thrombin costimulate secretion. It is concluded either that alpha-thrombin has a third interaction site on platelets with which neither mu-thrombin nor beta-thrombin interacts or that dual receptors are coordinately cleaved. In either case, the strong secretory response to thrombin appears to be moderated, independently of cytosolic Ca(II), by occupancy of a noncatalytic interaction site such as glycoprotein Ib.     

Hirudin: amino-terminal residues play a major role in the interaction with thrombin

Amino acid substitutions within the amino-terminal 5 residues of the thrombin-specific inhibitor hirudin dramatically alter its ability to inhibit the thrombin-catalyzed hydrolysis of both a chromogenic substrate and fibrinogen. Replacing the highly conserved Tyr-3 residue with Trp or Phe increases hirudin's affinity for thrombin 3-6-fold (decreases the inhibition constant, Ki) whereas Thr results in a 450-fold increase in Ki. A more extensive modification involving deletion of the amino-terminal Val, and Tyr-3----Val, Thr-4----Gln, and Asp-5----Ile replacement, results in a large reduction in thrombin inhibitory activity corresponding to greater than a 10(7)-fold increase in Ki and a 10(3)-fold increase in IC50, using D-Phe-L-pipecolyl-Arg-p-nitroanilide (S-2238) and fibrinogen, respectively, as substrates. Kinetic analysis of these mutant proteins and synthetic peptide fragments and available structural information on thrombin and hirudin derived from protein crystallography and two-dimensional NMR studies indicate that the amino-terminal region of hirudin binds at the apolar binding/active site region of thrombin, with Tyr-3 occupying the S3 specificity site. The large effect of these modifications on hirudin activity suggests that alteration of the amino-terminal segment can destabilize the interaction of other regions of hirudin with thrombin.     

Influence of chain shortening on the inhibitor properties of hirudin and eglin c

To find out minimal sizes of the proteinase inhibitor proteins hirudin and eglin necessary for their biological activity the inhibitors were incubated with exopeptidases. From the incubation mixtures shortened derivatives were isolated and characterized. Eglin c can be N-terminally shortened by up to 6 amino-acid residues without any loss of affinity towards chymotrypsin. The complex of thrombin with hirudin lacking 3 C-terminal amino-acid residues showed a 15-20-fold increased Ki value as found previously for desulfato-hirudin and desulfato-hirudin shortened by 2 amino-acid residues. Obviously, the C-terminal part of the hirudin molecule has a positive influence on its affinity to thrombin.     

Bifunctional thrombin inhibitors based on the sequence of hirudin45-65

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other.     

Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor

Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.     

The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.     

Activation of factor XIII by beta-thrombin]

It was found that similar to alpha-thrombin, beta-thrombin (possessing a high esterase and only a trace coagulating activities) converts plasmic transglutaminase (factor XIII) into its active form, thus promoting stabilization of fibrin. Activation of pure and plasmic preparations of factor XIII after incubation with beta-thrombin was observed in vitro. alpha-Thrombin at concentration corresponding to the trace coagulating activity of beta-thrombin had no activating effects. An intravenous injection of beta-thrombin to animals with aminazine-inhibited anticoagulating system reflectory arc resulted in an increase of factor XIII activity in the same way as was observed in vitro. On the other hand, an intravenous injection of beta-thrombin to intact animals did not increase factor XIII activity, which may be accounted for by a decrease in the level of factor XIII due to activation of the anticoagulating system.     

Refined structure of the hirudin-thrombin complex

The structure of a recombinant hirudin (variant 2, Lys47) human alpha-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ile1' to Tyr3' penetrates to the catalytic triad. The alpha-amino group of Ile1' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ile1' and Tyr3' occupy the apolar site, Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ile1' to Tyr3' form a parallel beta-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3(10) helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and D-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode.