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The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.

Mutation of 79 highly exposed amino acids that comprise approximately 62% of the solvent accessible surface of thrombin identified residues that modulate the inhibition of thrombin by antithrombin III, the principal physiological inhibitor of thrombin. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence and absence of heparin (W50A, E229A, and R233A) also decreased hydrolysis of a small tripeptidyl substrate. These residues were clustered around the active site cleft of thrombin and were predicted to interact directly with the "substrate loop" of antithrombin III. Despite the large size of antithrombin III (58 kDa), no residues outside of the active cleft were identified that interact directly with antithrombin III. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence but not in the absence of heparin (R89A/R93A/E94A, R98A, R245A, K248A, K252A/D255A/Q256A) in general did not also affect hydrolysis of the tripeptidyl substrate. These residues were clustered among a patch of basic residues on a surface of thrombin perpendicular to the face containing the active site cleft and were predicted to interact directly with heparin. Three mutations (E25A, R178A/R180A/D183A, and E202A) caused a slight enhancement of inhibition by antithrombin III.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.

Vitamin K-dependent carboxylation activity measured with pentapeptide substrate (Phe-Leu-Glu-Glu-Leu) gradually decreases upon in vivo injection of vitamin K to vitamin K-deficient rats. A decrease in pentapeptide carboxylation can also be observed by the in vitro addition of antibodies against prothrombin and other vitamin K-dependent proteins to the soluble system derived from vitamin K-deficient rat liver microsomes. In both cases, adding back in vitro partially decarboxylated vitamin K-dependent proteins or purified hepatic prothrombin precursor restores the level of pentapeptide carboxylation. After warfarin treatment, a 3-fold increase in carboxylation results, which can be abolished by giving cycloheximide along with the warfarin. However, the resulting decreased activity is restored by the in vitro addition of partially decarboxylated vitamin K-dependent proteins. These data are consistent with the hypothesis that (after warfarin treatment) increased peptide carboxylation is primarily due to activation of the system by precursor proteins, rather than synthesis of an increased amount of enzyme.

Vitamin K is required in an enzymatic reaction which carboxylates glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid residues. The partial requirements of this microsomal, vitamin K-dependent carboxylase system have been determined. A requirement of the system for cytosolic factors appears to be due primarily to the presence of reduced pyridine nucleotides or a reduced pyridine nucleotide-generating system in the cytosol. The hydroquinone of vitamin K has been demonstrated to be the enzymatically active form of the vitamin. When vitamin K1 hydroquinone is added to the carboxylase system, no NAD(P)H is needed for maximum activity. The carboxylase activity is half-maximally stimulated by 0.25 mug of vitamin K1/ml in the presence of cytosolic components but requires at least 10 times as much vitamin when microsomes are incubated in a cytosol-free buffer. Menadione is inactive as a vitamin source in this system, and the carboxylase activity is inhibited by the 2-chloro analog of vitamin K1 and by Warfarin. The ATP analog, AMP-P(NH)P, inhibited the carboxylase activity, but a dependence on exogenous ATP or an ATP-generating system could not be demonstrated. Carboxylase activity was found to be dependent on an O2-containing gas phase, and upon the HCO3- concentration.

Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.