Charge reversal at the P3' position in protein C optimally enhances thrombin affinity and activation rate.

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3' Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin-catalyzed activation of D172N, D172G, and D172A was increased 4-9-fold compared to wild-type HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free alpha-thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3' Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3' (D172K), there was a 30-fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5-fold) on the activation rate and affinity for free alpha-thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium-dependent and -independent effects of the acidic P3' residue.     

The activation of bovine protein C by factor Xa

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.     

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.     

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.     

Thrombin Glu-39 restricts the P'3 specificity to nonacidic residues

Residue 39 of serine proteases neighbors positions P'2 to P'4 of the substrate. When Glu-39 of thrombin is replaced with Lys, the resultant enzyme (E39K) retains similar P1, P2, and P3 specificities but has altered P'3 and/or P'4 specificities. These conclusions are based on analysis of both p-nitroanilide and synthetic peptide hydrolysis. The activity of E39K is nearly normal toward 17 p-nitroanilide substrates. In peptide substrates, an acidic residue at either the P3 or P'3 position reduces the rate of cleavage by thrombin. A single substitution of Asp with Gly in either the P3 or P'3 position of a peptide corresponding to the P7-P'5 residues of protein C increases the rate of cleavage by thrombin 2-3-fold. Replacement of both Asp residues with Gly increases the rate of cleavage 30-fold. With E39K, the inhibitory effect of Asp in P3 remains unchanged, but Asp in the P'3 site is no longer inhibitory. Significant differences in the catalytic activity of E39K are also seen with respect to protein C activation. In the absence of thrombomodulin, E39K activates protein C 2.2 times faster than thrombin. In the presence of thrombomodulin, the rate of protein C activation is similar for E39K and thrombin. The second order rate constant of inhibition by antithrombin III, where P'4 is a Glu, is slightly increased (1.4-fold). The clotting activity is reduced 2.4-fold due to a lower rate of fibrinopeptides A and B release where P'3 is Arg. These data show that the P'3 position is a determinant of thrombin specificity and suggest that thrombomodulin may function in part by alleviating the inhibitory effects that may arise from the proximity of the Asp in P'3 of protein C with Glu-39 of thrombin.     

Structure-function studies of the epidermal growth factor domains of human thrombomodulin.

Structure-function relationships in the 6 epidermal growth factor-like domains of human thrombomodulin (TME, residues 227-462) were studied by deletion mutagenesis. Purified and characterised proteins were used for kinetic studies. Deletion of EGF1, EGF2 and residues 310-332 in EGF3 had no effect on thrombin binding (Kd) or on kcat/KM for protein C activation by the thrombin-thrombomodulin complex. Deletion of the rest of EGF3 and the interdomain loop between EGF3 and EGF4 had no effect on Kd but decreased kcat/KM to 10% of TME. Deletion of residues 447-462 of EGF6 had no effect on kcat/KM but increased Kd for thrombin approximately 6-fold. Thus, the region 333-350 in EGF3-4 is critical for protein C activation by the thrombin-thrombomodulin complex and the region 447-462 in EGF6 is critical for thrombin binding.     

Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.     

Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site

Investigations determined the critical amino acids for alpha-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by alpha-thrombin with a Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 microM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km approximately 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.     

The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein

Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.     

The use of sequence-specific antibodies to identify a secondary binding site in thrombin

The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.     

The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C

Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.     

Mutations in autolytic loop-2 and at Asp554 of human prothrombin that enhance protein C activation by meizothrombin

Thrombin acts on many protein substrates during the hemostatic process. Its specificity for these substrates is modulated through interactions at regions remote from the active site of the thrombin molecule, designated exosites. Exosite interactions can be with the substrate, cofactors such as thrombomodulin, or fragments from prothrombin. The relative activity of alpha-thrombin for fibrinogen is 10 times greater than that for protein C. However, the relative activity of meizothrombin for protein C is 14 times greater than that for fibrinogen. Modulation of thrombin specificity is linked to its Na(+)-binding site and residues in autolytic loop-2 that interact with the Na(+)-binding site. Recombinant prothrombins that yield recombinant meizothrombin (rMT) and rMT des-fragment 1 (rMT(desF1)) enable comparisons of the effects of mutations at the Na(+)-binding residue (Asp(554)) and deletion of loop-2 (Glu(466)-Thr(469)) on the relative activity of meizothrombin for several substrates. Hydrolysis of t-butoxycarbonyl-VPR-p-nitroanilide by alpha-thrombin, recombinant alpha-thrombin, or rMT(desF1) was almost identical, but that by rMT was only 40% of that by alpha-thrombin. Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by alpha-thrombin, as already known. Strikingly, however, although meizothrombins modified by substitution of Asp(554) with either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of alpha-thrombin, the activity of these meizothrombins for protein C was increased to >10 times that of alpha-thrombin. It is proposed that interactions within thrombin that involve autolytic loop-2 and the Na(+)-binding site primarily enhance thrombin action on fibrinogen, but impair thrombin action on protein C.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.     

Identification of regions of alpha-thrombin involved in its interaction with hirudin

The contributions of various regions of human alpha-thrombin to the formation of the tight complex with hirudin have been assessed by using derivatives of thrombin. alpha-Thrombin in which the active-site serine was modified with diisopropyl fluorophosphate was able to bind hirudin, but its affinity for hirudin was decreased by 10(3)-fold compared to unmodified alpha-thrombin. Modification of the active-site histidine with D-Phe-Pro-Arg-CH2Cl resulted in a form of thrombin with a 10(6)-fold reduced affinity for hirudin. gamma-Thrombin is produced by proteolytic cleavage of alpha-thrombin in two surface loops corresponding to residues 65-83 and 146-150 in alpha-chymotrypsin [Berliner, L. J. (1984) Mol. Cell. Biochem. 61, 159-172; Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. The gamma-thrombin-hirudin complex had a dissociation constant that was 10(6)-fold higher than that of alpha-thrombin. Treatment of alpha-thrombin with pancreatic elastase resulted in a form of thrombin only cleaved in the loop corresponding to residues 146-150 in alpha-chymotrypsin, and this form of thrombin had only a slightly reduced affinity for hirudin. By using limited proteolysis with trypsin, it was possible to isolate beta-thrombin which contained a single cleavage in the loop corresponding to residues 65-83 in alpha-chymotrypsin. This form of thrombin had a 100-fold decrease in affinity for hirudin. Kinetic analysis of the binding of hirudin to beta-thrombin indicated that the 100-fold decrease in affinity was predominantly due to a decrease in the rate of association of the two molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.     

Difference in enzymatic properties between alpha-thrombin-staphylocoagulase complex and free alpha-thrombin

The steady-state kinetic parameters of human alpha-thrombin and the alpha-thrombin-staphylocoagulase complex as to the chromogenic substrate, H-D-Phe-Pip-Arg-p-nitroanilide (S-2238), were determined. At pH 8.0 and 37 degrees C, the Km values for alpha-thrombin and the complex for S-2238 were 7.9 microM and 7.7 microM, respectively. The kcat of this amidase reaction catalyzed by the complex was 127 s-1, which had apparently decreased from the kcat of 197 s-1 determined for free alpha-thrombin. This difference in the kinetic parameter between alpha-thrombin and the complex was also observed using the fluorogenic substrate, Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. Moreover, the fibrinogen clotting activity of the alpha-thrombin-staphylocoagulase complex was less than half that of alpha-thrombin, suggesting that the alpha-thrombin active site in the complex is different in catalytic ability from that of free alpha-thrombin. Other evidence supporting this view was as follows: The alpha-thrombin-staphylocoagulase complex is insensitive to antithrombin III, the complex shows much weaker binding to hirudin, as compared to free alpha-thrombin, and the amidase pH-profiles of the complex and free alpha-thrombin differ from each other. These results indicate that the microenvironment of the active site of alpha-thrombin is significantly altered by the complex formation with staphylocoagulase.     

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation.

Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.     

Aspartic acid residues 72 and 75 and tyrosine-sulfate 73 of heparin cofactor II promote intramolecular interactions during glycosaminoglycan binding and thrombin inhibition

We used site-directed mutagenesis to investigate the role of Glu(69), Asp(70), Asp(71), Asp(72), Tyr-sulfate(73), and Asp(75) in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin.HCII complex with and without glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 microg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 microg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp(72), Tyr-sulfate(73), and Asp(75) were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either alpha-thrombin/hirugen or gamma(T)-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a "molecular rheostat" to promote thrombin inhibition in the presence of glycosaminoglycans.