Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

The effect of bovine thrombomodulin on the specificity of bovine thrombin

Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.     

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Staphylococcal Superantigen-like Protein 10 (SSL10) Inhibits Blood Coagulation by Binding to Prothrombin and Factor Xa via Their γ-Carboxyglutamic Acid (Gla) Domain

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10⁻⁷ m in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca²⁺ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Decarboxylation of bovine prothrombin fragment 1 and prothrombin

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.     

Role of the N-terminal epidermal growth factor-like domain of factor X/Xa

The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.     

Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase

Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680-709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Delta680-709)) and a mutant molecule with the (695)DYDY (698) --> AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va (Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Delta680-709) displayed an approximately 39% increase in k cat, while prothrombinase assembled with factor Va(4A) exhibited an approximately 20% increase in k cat for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.     

Metal and phospholipid binding properties of partially carboxylated human prothrombin variants

To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.     

Multiple active forms of thrombin. IV. Relative activities of meizothrombins

The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes. These results suggest meizothrombin generation is targeted toward anticoagulant function such as protein C activation, whereas alpha-thrombin generation is targeted toward procoagulant functions, such as fibrinogen clotting, factor V activation, and platelet aggregation.     

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.     

Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.     

Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S

Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and localized the thrombin cleavage sites. Protein S contains 11 gamma-carboxyglutamic acid residues in the amino-terminal region (residues 1-36). This part of protein S is highly homologous to the corresponding parts in the other vitamin K-dependent clotting factors, whereas the region between residues 45 and 75 is not at all homologous to the other clotting factors. Thrombin cleaves two peptide bonds in this part of protein S, first at arginine 70 and then at arginine 52. The peptide containing residues 53-70 is released from protein S after thrombin cleavage. The amino-terminal fragment, residues 1-52, is linked to the large carboxyl-terminal fragment by a disulfide bond, which involves cysteine 47. After residue 78, protein S is again homologous to factors IX and X and to proteins C and Z, but not to prothrombin. Position 95 is occupied by a beta-hydroxyaspartic acid residue.     

Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.     

Acetaldehyde-ethanol exchange in vivo during ethanol oxidation.

Bovine thrombin was immobilized on sepharose 4B through an oxidized sialic acid group on its B chain. The immobilized thrombin was reduced with β-mercaptoethanol in 8 m urea under conditions that were shown to be sufficient to reduce the disulfide bond connecting the A and B chains. The immobilized B chain that remained after the A chain was washed away was allowed to refold, and the disulfide bonds were reoxidized with a mixture of oxidized and reduced glutathione under anaerobic conditions. The refolded immobilized B chain exhibited 15–25% of the tosyl-l-arginine methyl ester esterase activity of the immobilized thrombin and a significant amount of fibrinogen clotting activity. The immobilized B chain behaved qualitatively like immobilized thrombin towards two oligopeptide fibrinogen-like substrates and showed no activity towards lysine or arginine peptide bonds in a fragment of ribonuclease.Since the recovered activity was greater than that computed for a random refolding of seven -SH groups to form three SS bonds, it is concluded that the B chain retains a sufficient number of interacting groups to refold correctly, and it is suggested that prothrombin might fold in localized domains with only weak interactions between domains. The behavior of the B chain towards the substrates tested suggests that the A chain does not play a significant role in determining the catalytic specificity of thrombin or in distinguishing its specificity from that of trypsin.     

Characterization of the catalytic defect in the dysthrombin, Thrombin Quick

The dysthrombin, Thrombin Quick, is chromatographically separable into two components designated Thrombin Quick I and Thrombin Quick II. Thrombin Quick II lacks observable catalytic activity toward thrombin substrates. The steady-state kinetics of hydrolysis of benzoylarginine ethyl ester and Tos-Gly-Pro-Arg-p-nitroanilide by Thrombin Quick I are equivalent to those of thrombin. These results, in addition to binding studies with the active site titrant N2-(5-dimethylaminonaphthalene-1-sulfonyl)arginine N-(3-ethyl-1,5-pentanediyl)amide, indicate that binding interactions at the catalytic site of Thrombin Quick I are unaltered. Thrombin Quick I is inhibited by anti-thrombin III at the same rate as thrombin. Steady-state kinetic parameters for the release of fibrinopeptide A indicate defects in both kcat and Km for Thrombin Quick I with kcat/Km equal to 0.012 of the value for thrombin, corresponding to the relative fibrinogen clotting activity of 0.013. The results are interpreted as indicating a defect in Thrombin Quick I at a binding site, external to the catalytic site, which is essential for determining specificity toward fibrinogen. The defect in kcat may result secondarily from small perturbations in the steric relationship of the catalytic triad residues. The rate of hydrolysis by Thrombin Quick I of the protein substrates bovine prothrombin and bovine protein C (in the absence of cofactors) is about one-third of that observed for thrombin, indicating that hydrolysis of these substrates by thrombin involves different specificity determinants than does the hydrolysis of fibrinogen.     

Prothrombin--substrate for blood plasma kallikrein and factor XIIa

A study was made of the interaction between prothrombin and enzymes: blood plasma kallikrein and factors alpha-XIIa and beta-XIIa immobilized on enzacryl-AH. Kallikrein-induced prothrombin proteolysis was accompanied by a decrease in prothrombin activity, appearance of BAME-esterase and poor clotting activity. As a result of fractionation of products on the column with DEAE-Sephadex A-50, some fractions that have thrombin amidase activity (splitting of the substrate S-2238) and high antithrombin activity were obtained. Antithrombin activity manifested in the inhibition of fibrinmonomer aggregation during fibrin formation. During incubation with prothrombin, factors alpha-XIIa and beta-XIIa also stimulated the appearance of BAME-esterase activity. None of the immobilized enzymes activated factor X.     

Cleavage of prothrombin bound in immune complexes results in high thrombin enzymatic activity.

Thrombin plays a pivotal role in blood clotting as well as in the regulation of vascular remodeling and oxidative stress. Recent evidence suggests that auto-antibodies directed against prothrombin, may play an important role in the pathogenesis of atherosclerosis. It is however not clear, if prothrombin bound in an immune complex retains its clotting and regulatory properties or acts solely by increasing vascular inflammation. In order to answer this question, we used a newly developed stain for the detection of thrombin activity of such complexes. Plasma and serum samples were subjected to rocket immunoelectrophoresis in an anti-prothrombin antiserum containing agarose gel. Gel plates, covered with a nitrocellulose membrane were soaked with chromogenic thrombin substrate. The product of thrombin activity was diazotized to red azo dye bound to nitrocellulose. Activity stain revealed barely discernible rockets in plasma, but heavily stained ones in serum. Pre-incubation with trypsin enhanced activity of immunoprecipitates deriving from plasma, but not from serum. Densitometric analysis showed, that the trypsin-enhanced activity in plasma derived immune complexes was twice as high as in serum derived immunoprecipitates. Thrombin active centre is not blocked by anti-prothrombin antiserum allowing to retain thrombin activity. Moreover, prothrombin in immunoprecipitate is readily cleaved by proteolytic enzymes. This cleavage could potentially be enhanced by antibody binding, although these results need to be confirmed using different antibodies.