Activation of decarboxyfactor X by a protein from Russell's viper venom. Purification and partial characterization of activated decarboxyfactor X.

1. Incubation of decarboxyfactor X with the factor X-activating enzyme from Russell's Viper venom revealed the generation of amidase activity towards Bz-Ile-Glu-Gly-Arg-pNA, but not of activity in blood coagulation. 2. The rate of activation of both factor X and decarboxyfactor X depends on the ability of the zymogens to bind Ca2+. The relationship between Ca2+ concentration and velocity of the activation reaction is sigmoid in the case of factor X, but hyperbolic with decarboxyfactor X. 3. Activated decarboxyfactor X was purified by powder column electrophoresis. 4. Identical changes of primary structure accompanied the activation of factor X and decarboxyfactor X. Identical molecular weight and common antigenic determinants were found in factor Xa and decarboxyfactor Xa. The amino acid composition was identical except for 12 glutamic acid residues in decarboxyfactor Xa and gamma-carboxyglutamic acid residues in factor Xa. 5. Unlike factor X, activated factor X has a very low electrophoretic mobility in the presence of Ca2+ at pH 8.6. This is probably due to self association of factor Xa under the influence of Ca2+. The electrophoretic mobility of activated decarboxyfactor X is only slightly decreased compared to decarboxyfactor X in the presence of Ca2+.     

Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.     

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.     

The activation of factor X by hepatocyte plasma membranes

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.     

Comparison of coagulation factor Xa and des-(1-44)factor Xa in the assembly of prothrombinase

The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.     

Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.     

Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.     

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.     

Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III

The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Anticoagulant RNA Aptamer That Inhibits Proteinase-Cofactor Interactions within Prothrombinase

The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA_11F7t) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA_11F7t inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA_11F7t to factor Xa with nanomolar affinity in a Ca²⁺-dependent interaction. RNA_11F7t binds equivalently to the zymogen factor X as well as derivatives lacking γ-carboxyglutamic acid residues. We suggest that the ability of RNA_11F7t to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner.     

Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor- dependent coagulation pathway

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low- density, detergent-insoluble microdomains that inefficiently support TF- VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

The tissue factor region that interacts with factor Xa in the activation of factor VII

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.     

Activation of human factor V by factor Xa and thrombin

The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of Mr 220,000 and 105,000. Although thrombin cleaved the Mr 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mr 220,000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the Mr 220,000 peptide. This observation facilitated the study of the kinetics of factor V activation by allowing the activation of factor V to be monitored by the appearance of the Mr 220,000 peptide (factor Xa activation) or the Mr 105,000 peptide (thrombin activation). Factor Xa catalyzed activation of factor V obeyed Michaelis-Menten kinetics and was characterized by a Km of 10.4 nM, a kcat of 2.6 min-1, and a catalytic efficiency (kcat/Km) of 4.14 X 10(6) M-1 s-1. The thrombin-catalyzed activation of factor V was characterized by a Km of 71.7 nM, a kcat of 14.0 min-1, and a catalytic efficiency of 3.26 X 10(6) M-1 s-1. This indicates that factor Xa is as efficient an enzyme toward factor V as thrombin.     

Differentiation of enzyme and substrate binding in the prothrombinase complex

The Ca2+ dependence of factor Xa binding to phospholipid vesicles was measured in the presence and absence of factor Va. The increase in polarization of a fluorescently labeled derivative of factor Xa, [5-(dimethylamino)-1-naphthalenesulfonyl] glutamylglycylarginyl factor Xa (Dns-EGR-Xa), was used as a probe to measure the interaction of factor Xa with phospholipid. The Ca2+ concentration required for half-maximal binding of Dns-EGR-Xa to phospholipid vesicles was 3.5 X 10(-4) M in the presence of factor Va and 9.5 X 10(-4) M in the absence of factor Va. At a Ca2+ concentration of 5 X 10(-4) M, the binding of Dns-EGR-Xa to phospholipid-bound factor Va was near maximal, whereas there was no detectable interaction of Dns-EGR-Xa with phospholipid alone at this Ca2+ concentration as detected by fluorescence polarization. These results were qualitatively confirmed by high-performance liquid chromatography. The rate of hydrolysis of the factor Xa synthetic substrate, benzoylisoleucylglutamylglycylarginine p-nitroanilide, by factor Xa in the presence of factor Va and phospholipid decreased in a Ca2+-dependent manner. These data were analyzed as fraction of factor Xa bound to the phospholipid. A Ca2+ concentration of 2.7 X 10(-4) M resulted in half-maximal binding by this technique. The relationship observed between rates of prothrombin activation and Ca2+ concentration could be predicted quantitatively from calculations of local enzyme and substrate concentrations.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Interaction of prothrombin with factor Va-phospholipid complexes

The effects of factor Va and the phospholipid-binding fragment of factor Va [factor Va light chain (LC), Mr 80000] on the binding of prothrombin, factor X, and factor Xa to phospholipid vesicles are reported. Equilibrium binding experiments were performed that utilized large-volume vesicles, which can be removed from the bulk solution by centrifugation. Factor Va decreased the dissociation constant of the prothrombin-phospholipid complex 50-fold, from 2.0 X 10(-7) M to 4.0 X 10(-9) M. For the factor X-phospholipid complex the decrease was 60-fold (1.8 X 10(-7) M to 3.0 X 10(-9) M) and for factor Xa, 160-fold (1.6 X 10(-7) M to 1.0 X 10(-9) M). The ratios of moles of protein bound to moles of total added factor Va at saturation of phospholipid-bound factor Va indicate an 1:1 stoichiometric complex of either factor Xa, factor X, or prothrombin and phospholipid-bound factor Va. In the presence of factor Va LC, the dissociation constants of factor Xa- and prothrombin-phospholipid complexes were increased, while the maximal protein-binding capacities of the vesicles were not affected by factor Va LC. The data suggest a competitive interaction between factor Xa and factor Va LC binding as well as between prothrombin and factor Va LC binding at the phospholipid surface. From this, it is concluded that the phospholipid-binding fragment of factor Va alone does not serve as the binding site for interactions of factor Xa and prothrombin with factor Va.     

Interaction of factor Xa with heparin does not contribute to the inhibition of factor Xa by antithrombin III-heparin

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.     

The acceleration of the inhibition of platelet prothrombinase complex by heparin.

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 X 10(7) M-1 X min-1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 X 10(8) M-1 X min-1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 X 10(7), 1.4 X 10(7) and 9.8 X 10(6) M-1 X min-1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.     

Inhibition of prothrombinase complex by plasma proteinase inhibitors

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.