Prothrombin overexpressed in post-natal neurones requires blood factors for activation in the mouse brain

Thrombin is thought to mediate, through protease-activated receptors, both protective as well as cytotoxic effects. As thrombin receptors are expressed in the CNS, an important question arises as to whether the intact nervous system is able to generate thrombin by activation of its precursor prothrombin, derived endogenously or only upon extravasation following brain injury. To address this question, transgenic mice that express C-terminally haemagglutinin tagged human prothrombin in post-mitotic neurones were generated. In situ hybridization and immunohistochemical analysis showed abundant and widespread cerebral expression of the transgene. Amidolytic assays of brain homogenates and hippocampal slice cultures demonstrated that activation of transgenic prothrombin required added factors, such as snake venom or blood components. This strongly suggests that any possible action of thrombin in the adult CNS depends on blood-derived factors that activate prothrombin. Furthermore, the results are consistent with the idea that in the non-pathological situation an as yet unidentified ligand activates thrombin receptors in the nervous system.     

Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.1 M NaCl/0.05 M Tris-HCl buffer (pH 7.4). The affinity of thrombin and prethrombin 2 to fibrinogen-Sepharose depended on ionic strength and reached a maximum at 50 mm concentration. Prethrombin 2 interacts with fibrinogen as well as thrombin; and prothrombin fragment 1.2 is not important in the formation of this complex. Thus, prethrombin 2, which is a precursor of thrombin without measurable enzymatic activity and which lacks the single cleavage at Arg-322-Ile-323 present in thrombin, has the same or very similar structural conformation as thrombin and has the same macromolecular substrate recognition site. These results confirm the earlier results that active center is not necessary in fibrinogen-thrombin interaction.     

The control of prothrombin conversion. Kinetic control by mechanisms inherent in two activation pathways.

It is known that the activation of prothrombin to thrombin can proceed via two pathways: one initiated by the prothrombin-converting complex (factor Xa, factor V, phospholipid, and CA2+ ions) and the other initiated by the product, thrombin. A kinetic study has shown that the pathways do not proceed with equal ease under all conditions. At high levels of the converting complex, both go to completion: some prothrombin is always cleaved by thrombin, but the resulting intermediate is then activated to give quantitative conversion to thrombin. At slower rates of activation, the product-initiated pathway occurs to a relatively greater extent. Moreover, the intermediate then is not cleaved further but accumulates, so that the generation of thrombin is curtailed. The reason the intermediate is productive only at higher levels of activator may be partly that it is a poorer substrate for the converting complex than prothrombin. More importantly, the activity of the complex is also modulated by thrombin, which rapidly destroys the activity of factor V and factor Xa in a feedback reaction. These concerted controls ensure that prothrombin activation damps itself. Thus thrombin production occurs as a burst, the size of which is regulated by the amounts of factor Xa and factor V initially available.     

Isolation and characterization of isoprothrombin in the rat

Isoprothrombin, a protein antigenically related to prothrombin, which accumulates in the hepatic endoplasmic reticulum of vitamin K-deficient or warfarin-treated rats, has been isolated by affinity chromatography employing rat prothrombin antibodies linked to Sepharose. Isoprothrombin has the same molecular weight as prothrombin but a different mobility on disc gel electrophoresis, is not barium adsorbable nor activatable to thrombin by factor X_a. Isoprothrombin is converted to thrombin by $\text{Echis carinatus}$ venom through the same intermediates as prothrombin.     

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.     

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.     

Amide H/2H exchange reveals a mechanism of thrombin activation

Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.     

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.     

Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin

An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.     

STUDIES ON BLOOD COAGULATION : I. THE R?LE OF PROTHROMBIN AND OF PLATELETS IN THE FORMATION OF THROMBIN

1. A method is described for the preparation and titration of prothrombin and thrombin. 2. Confirming the views of Morawitz, Howell (1916–17, 1925), and Bordet, thrombin cannot be regarded as an artificial by-product of coagulation (Wooldridge, Nolf (both quoted from Morawitz)). Calcium, a platelet factor, and a plasma factor (prothrombin) interact to form thrombin, and this then acts upon fibrinogen to form fibrin. The amount and rate of thrombin formation in the first reaction are independent of the presence or absence of fibrinogen. After a variable latent period, thrombin suddenly appears in large quantities, coincident with or immediately preceding the deposition of fibrin if fibrinogen is present. 3. The amount of thrombin formed in a mixture of prothrombin, Ca and platelets is independent of the platelet or Ca concentration, and depends primarily upon the amount of prothrombin used. The platelets (or cephalin) enormously accelerate the transformation of prothrombin to thrombin, and this acceleration seems to be their physiological rôle in the coagulation process. 4. Contrary to previous reports, platelets have not been demonstrated to contain significant quantities of prothrombin. 5. The available data do not allow any definite decision as to whether the platelet factor actually combines with prothrombin to form thrombin, or merely catalyzes the transformation. The very slow formation of thrombin in the complete absence of platelets may be due to dissolved traces of platelet material released during the physical manipulation of the plasma (centrifuging, Berkefeld filtration). 6. There was no evidence for a species-specific activity of platelets in the transformation of prothrombin to thrombin.     

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.     

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation

The conversion of prothrombin into thrombin by Factor Xa requires the cleavage of two peptide bonds in prothrombin. Dependent on the order of cleavage, prethrombin 2 or meizothrombin occurs as intermediate. Since prethrombin 2 has as yet been the only observed intermediate, prothrombin activation is generally considered to proceed via prethrombin 2. In this paper we present new methods that allow differentiation between meizothrombin and thrombin formed during the initial phase of prothrombin activation. These methods, which make use of the different reactivities of meizothrombin and thrombin toward fibrinogen and antithrombin III plus heparin, enabled us to show the generation of considerable amounts of meizothrombin during Factor Xa-catalyzed prothrombin activation. Both meizothrombin and thrombin incorporated the active site-directed fluorescent chloromethyl ketone 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-CH2Cl. Gel electrophoretic analysis of chloromethyl ketone-treated aliquots of prothrombin activation mixtures confirmed meizothrombin formation. These observations demonstrate that prothrombin may also be converted into thrombin via meizothrombin.     

Ecarin test in diagnosis of dicoumarol therapy, liver diseases and DIC

The conversion of prothrombin by ecarin is independent of the presence of gamma carboxyglutamic residues on the N terminal of the molecule. Ecarin converts therefore also the acarboxylated precursor-PIVKA II. In a group of 347 patients under dicoumarol therapy of different intensity and duration PIVKA was detected in BaSO4 adsorbed PPP only in samples where prothrombin level (Quick's test) was lower than 50% of normal values. Increase in PIVKA did not correspond to the decrease of prothrombin. In some cases where the treatment was longer than two years no PIVKA was detected even when prothrombin was under 20%. This is explained by synthesis of partially carboxylated prothrombin molecules, which can be adsorbed. In liver diseases the ecarin test and Quick's time in native, untreated PPP showed about identical decrease of prothrombin level. This indicates that no PIVKA is released to plasma. The functional defect of the hepatocyte does not involve gamma carboxylation as long as vitamin K is provided. In patients with DIC ecarin test showed significantly higher level of thrombin activity than those obtained with Quick's test. It is known that during hypercoagulation stage of DIC prethrombin 1 and 2 are formed by the excess of thrombin. These split products of prothrombin are convertible by ecarin to meizothrombin 1 and 2. A positive ecarin test can be also due to acarboxylated precursors which are released during increased proteosynthesis triggered by consumption coagulopathy.     

Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin

An equimolar mixture of phosphatidylserine and (dioleoyl)phosphatidylethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca²⁺ were interacted with prothrombin.In the presence of 25 mM Ca²⁺, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca²⁺ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity.Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca²⁺. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.     

Rat prothrombin: Purification,characterization, and activation

Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor X_a or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor X_a and calcium ion generation of thrombin from rat prothrombin.     

Prothrombin activation by immobilized thrombin]

The ability of thrombin, immobilized on BrCN-activated Sepharose 4B, to split prothrombin, was studied. Immobilized thrombin retained up to 70% of its esterase activity and about 5% of its coagulating activity; it was also found to induce partial proteolysis of prothrombin. Two products of prothrombin degradation isolated, i.e. P1 (m. w. 50.000-52.000) and P2 (m. w. 22.000-24.000), did not show either the thrombin or the prothrombin activities. P1 was converted into thrombin under the action of tripsin or Factor Xa. The rate of conversion was considerably increased after addition of Factor V, thromboplastin and Ca2+ ions. Intravenous administration of P1 to rats resulted in changes in the coagulating system of blood, which may be probably indicative of the stimulation of the anticoagulating system. P2 possessed no thrombogenic activity.     

Thrombin generation and presence of thrombin receptor in ovarian follicles

Prothrombin, once converted to its enzymatically active form (i.e., thrombin), induces a broad spectrum of cellular responses in both vascular and avascular tissues. Bovine ovarian granulosa cells isolated from healthy follicles of various sizes contain both prothrombin mRNA and immunologically reactive prothrombin that appears to be identical to prothrombin in follicular fluid and plasma. When tissue factor, the primary physiological activator of thrombin generation in plasma, is used to initiate thrombin formation, the profile of prothrombin-to-thrombin conversion is similar in follicular fluid and plasma. The conclusion that biologically functional prothrombin is synthesized by granulosa cells is further supported by evidence that mRNA for gamma-glutamyl carboxylase, an enzyme essential for the vitamin K-dependent posttranslational modification of prothrombin, is expressed in granulosa cells in a manner similar to prothrombin mRNA. Thrombin's biological effects are mediated through selective proteolytic cleavage and activation of specific receptors. Bovine granulosa cells possess thrombin receptor (PAR-1) mRNA, and as seen with prothrombin mRNA and gamma-glutamyl carboxylase mRNA, cells isolated from small follicles possess more PAR-1 mRNA than cells from large follicles. Thrombin receptor expression by cells in close proximity to an active thrombin-generating system suggests that these factors may be important mediators of cellular function in the ovarian follicle.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

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.