Human Prothrombin Research Articles

Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase.

Binding interactions between the membrane-associated vitamin K-dependent carboxylase and its prothrombin and factor X substrates have been investigated in liver microsomes. Both substrates are firmly attached to microsomal membrane fragments which also harbor the carboxylase. In vitro 14CO2 gamma-carboxylation of these substrates, triggered by reduced vitamin K1H2, resulted in release of 14C-labeled prothrombin precursors from the membrane fragments, but no release of 14C-labeled factor X precursors could be demonstrated, which suggested a difference in early processing of these substrates by the carboxylase. Warfarin treatment of rats resulted in a 3-fold increase in the membrane concentration of factor X antigens and a 20-fold increase in 14C gamma-carboxylation of the membrane pool of factor X carboxylase substrates. There was a dose-response relationship between the amount of drug administered to the rats and 14C labeling of the membrane pool of factor X carboxylase substrates. On the other hand, the membrane concentration of prothrombin antigens did not increase in response to the drug, and 14CO2 gamma-carboxylation of the membrane pool of prothrombin carboxylase substrates was the same in warfarin and saline-treated rats. The results demonstrate significant differences in the interaction between the carboxylase and its prothrombin and factor X substrates. It appears that the different interactions result from binding of the prothrombin and the factor X precursors to separate microsomal membrane proteins that are involved in the gamma-carboxylation reaction. Warfarin appears to induce the factor X precursor-specific but not the prothrombin precursor-specific binding proteins, which suggests a new mechanism for the action of warfarin. These binding proteins may be under different genetic control. Treatment of the prothrombin and the factor X carboxylase substrates with endonuclease H showed that the rat prothrombin and the human factor X carboxylase substrates are high mannose glycoproteins. The human prothrombin and the rat factor X carboxylase substrates did not, on the other hand, change their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after endonuclease H treatment. The data demonstrate differences in the glycoprotein nature of the rat and the human carboxylase substrates.

Metal ion blockage of tritium incorporation into gamma-carboxyglutamic acid of prothrombin. Stoichiometry of gamma-carboxyglutamic acid to Gd3+ for the high affinity sites.

Prothrombin possesses two high affinity and four low affinity gamma-carboxyglutamic acid (Gla)-dependent gadolinium binding sites. Earlier work (Price, P. A., Williamson, M. K., and Epstein, D. J. (1981) J. Biol. Chem. 256, 1172-1176) has shown that tritium can be specifically incorporated at the gamma-carbon of Gla in proteins at pH 5. In the present work we show that inclusion of saturating concentrations of Ca2+ in nondenaturing buffer systems ranging from pH 5.5 to 8.5 prevents the exchange of tritium into all 10 Gla residues of prothrombin. Similarly, saturating concentrations of Gd3+ prevent tritium incorporation into Gla at pH 5.5. Positive cooperativity was observed for the binding of Gd3+ to human prothrombin (at pH 5.5) for the two high affinity sites (Kd congruent to 35 nM). The four low affinity sites bind Gd3+ with a Kd congruent to 5 microM. Incubation of prothrombin ranging in concentrations from 10 to 40 microM with 2 eq of Gd3+ at pH 5.5 prevents 5.7 (average of seven determinations) Gla residues from tritium incorporation. Sedimentation velocity experiments conducted at pH 5.5 indicate that prothrombin in the presence of saturating concentrations of Gd3+ polymerizes, most likely, to a trimer. Further, in the presence of 2 eq of Gd3+, calculated percent weight average concentration of monomer prothrombin is congruent to 100% at 10 microM, approximately equal to 95% at 20 microM, and congruento to 80% at 40 microM protein concentration. Thus, it appears that under conditions in which prothrombin primarily exists as a monomer, occupancy of the initial two metal binding sites by Gd3+ involves six Gla residues.

Inhibition of prothrombin activation by factor X and factor IX Gla-peptides

In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.

Reversibility of prothrombin adsorption on lipid monolayers

Desorption of tritium—labeled human prothrombin adsorbed on lipid monolayers was induced by overall dilution of the aqueous solution by exchange with cold prothrombin and by adding EDTA to remove Ca 2+. The adsorption—desorption isotherms of prothrombin on phosphatidylserine (PS) and on mixed PS—phosphatidylcholine (PC) monolayers at the air/water interface show a very strong hysteresis. Dilution by cold prothrombin decreases the surface radioactivity to less than the dilution factor in the solution. Addition of EDTA displaces prothrombin molecules adsorbed on a pure PS monolayer to the level of adsorption in the absence of Ca 2+; however, the displacement by EDTA from mixed PC—PS monolayers is less effective and the remaining surface concentration is higher than that obtained by adsorption in the absence of Ca 2+. Significant interaction with PC monolayers in the presence of Ca 2+ was observed. Penetration of prothrombin into the monolayer and the involvement of its interaction with the air/monolayer interface in analogy with other interfaces (e.g., mercury/monolayer) are considered. The significance of these results to prothrombin conformation and function on lipid membranes is discussed.

Functional characterization of thrombin Salakta: an abnormal thrombin derived from a human prothrombin variant

The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa- catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl- Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p- nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L- pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.

The genetic relationships between the kringle domains of human plasminogen, prothrombin, tissue plasminogen activator, urokinase, and coagulation factor XII

A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9-23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40-55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle. Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of beta-turns, and very low alpha-helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.

Neutron scattering determination of the binding of prothrombin to lipid vesicles.

Low-angle neutron scattering is used to study the binding of human prothrombin to small single-bilayer vesicles consisting of phosphatidylcholine and phosphatidylserine (1/1 w/w). The radius of gyration of prothrombin indicates that it is an elongated molecule. The vesicles alone were not observed to coalesce, and their molecular weight, outer radius, and average surface area per lipid were respectively (1.6 +/- 0.32) X 10(6), 114 +/- 4 A, and 110 +/- 18 A2. These values were independent of the presence of calcium and were not altered significantly by prothrombin, which binds reversibly to the vesicle outer surface with its long axis projecting approximately radially forming a 90-A thick protein shell. From the titration of the protein-vesicle interaction, the apparent dissociation constant of the binding of prothrombin to these vesicles is estimated to be 0.8 +/- 0.4 microM. At saturation, 57 +/- 7 prothrombin molecules bind, giving 25 +/- 6 lipid residues and an area of 2900 +/- 400 A2 per prothrombin molecule on the vesicle outer surface. This area is about twice that calculated from a prolate ellipsoid model for prothrombin. However, it is close to the maximum cross-sectional area of fragment 1, the lipid binding region of prothrombin, which is coin-shaped in the high-resolution X-ray structure [Park, C.H., & Tulinsky, A. (1986) Biochemistry 25, 3977-3982]. This similarity suggests that prothrombin binding could be sterically limited.

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding.

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.

Nucleotide sequence of the gene for human prothrombin.

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom.

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin.

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.

Expression of completely gamma-carboxylated recombinant human prothrombin.

Human prothrombin cDNA has been expressed in mammalian cells to yield biologically active, fully gamma-carboxylated prothrombin. A 2.0-kilobase cDNA encoding full-length prothrombin was isolated from a human fetal liver library using a cDNA fragment recovered from a lambda gt11 human hepatoma expression library. Prothrombin cDNA was cloned into a mammalian expression vector and transfected into Chinese hamster ovary cells. Selection for expression of dihydrofolate reductase yielded cell lines secreting up to 0.55 microgram/ml of prothrombin. Recombinant prothrombin synthesized in the presence of vitamin K was quantitatively recovered from tissue culture medium by affinity chromatography using conformation-specific antibodies directed against the metal-stabilized, gamma-carboxylated conformer. The purified material migrated as a single band on denaturing polyacrylamide gels with an electrophoretic mobility equivalent to that of plasma-derived human prothrombin. Automated Edman degradation of recombinant prothrombin revealed a single amino-terminal sequence identical to that of plasma-derived prothrombin. Recombinant and plasma-derived prothrombin interacted similarly with antibodies specific for total prothrombin, abnormal des-gamma-carboxyprothrombin, and two metal-stabilized conformers of prothrombin. Recombinant prothrombin exhibited a specific coagulant activity equivalent to that of plasma-derived prothrombin. The gamma-carboxyglutamic acid analysis of recombinant prothrombin demonstrated 9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin. These results represent the first description of the expression of a recombinant vitamin K-dependent protein in which all of the expressed protein is gamma-carboxylated.

Human genes encoding prothrombin and ceruloplasmin map to 11p11-q12 and 3q21-24, respectively.

The gene for human prothrombin, or factor II (F2) has been assigned to 11p11-q12 by the combined use of a panel of somatic cell hybrid DNAs and in situ hybridization, using both cDNA and genomic probes. In addition, the cDNA probe for F2 recognizes a homologous sequence which has been tentatively mapped to the X chromosome. Similar approaches have been used to confirm the assignment of the ceruloplasmin gene, but to regionally localize it more proximally than previously reported (3q21-q24). These results provide further evidence that genes encoding the coagulation factors and related proteins are dispersed throughout the human genome.

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.

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 α-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 α-chymotrypsin. Limited α-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 (Kd=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd=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 (Kd=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, α-chymotrypsin, and elastase.

Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

Determination of magnesium binding to macromolecules

An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity.

Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator.

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.

Prothrombin Tokushima: Characterization of Dysfunctional Thrombin Derived From a Variant of Human Prothrombin

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of "prothrombin" by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of "prothrombin." Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of "thrombin." Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of "thrombin" when Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p'-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as "thrombin" in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than "thrombin" when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as "thrombin" in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.