Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa.

Extrinsic pathway inhibitor plays a key role in modulating tissue factor-dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 +/- 1.5 nM and 335,000 +/- 84,000 sites/cell when factor Xa was present; a Kd value of 11.9 +/- 3.5 nM and 236,000 +/- 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.5 nM, were found to inhibit factor VIIa-tissue factor-catalyzed release of activation peptide from tritiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI's inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis.     

Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K(i) value 10-100-fold smaller than those of other squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other squash family inhibitors reveal Trp at position 7 (P(2)') and a deletion at position 25 (P(20)') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, we chemically synthesized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P(2)'(Trp) is substituted with Leu, and S-MCTI-II(25N) whose P(20)'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L), and S-MCTI-II(25N) were 1.3x10(-6) M, 2.8x10(-5) M, and 7.3x10(-6) M, respectively. They inhibited factor X activation mediated by factor VIIa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppressed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MCTI-II(25N) did not. Both the Trp at the P(2)' position and deletion at the P(20)' position are thus likely required for the inhibition of factor Xa, trypsin, and factor IXa, while these two positions do not affect factor X activation initiated by the factor VIIa-tissue factor complex.     

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.     

Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation

We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.     

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.     

The use of acetylated factor X to prevent feedback activation of factor VIII during factor X activation: a tool for kinetic studies

The modification of human factor X by 2-sulfo-N-succinimidyl acetate was investigated and shown to produce a factor X species which, when activated, has no activity toward factor VIII. Acylation of factor X (0.9 microM) was carried out in the presence of 1 mM calcium at different reagent concentrations and pH values at 22 degrees C for time courses up to 1 h. Optimal modification was achieved using 0.3 mM reagent at pH 8.0 for 30 min. The modified zymogen, acetylated factor X, is activated at full rates by factor IXa/VIIIa and by the factor X-activating protein of Russell's viper venom. The activated product, acetylated Xa, has an enhanced amidolytic activity (110%) but has almost no detectable clotting activity (0.1%). More importantly, we have shown that acetylated Xa, in contrast to native Xa, does not activate factor VIII. This allows accurate quantitation of factor VIII activation without complications due to positive feedback reactions. We have demonstrated this in an examination of the activation of factor VIII by 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.     

A review of studies of the activation of the blood coagulation mechanism in chimpanzees (Pan troglodytes)

This paper reviews our recent studies of blood coagulation activation in the chimpanzee which were carried out employing sensitive immunoassays that measure activation markers of blood coagulation in plasma. Infused factor VIIa activated both factors IX and X in vivo; this reaction depended on the formation of the factor VIIa-tissue factor (TF) complex. The infusion of endotoxin also led to assembly of the factor VIIa-TF complex, enhancing fibrin formation. This process occurred through the intermediate action of specific cytokines.     

Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.     

Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages

Activation of coagulation factor X via the intrinsic pathway requires the assembly of factors IXa and VIII on lipid membranes. It is known that the platelet expresses membrane sites for assembly of factors IXa/VIII and promotes efficient factor X activation. We now show that human blood monocytes, but not lymphocytes or polymorphonuclear leukocytes, also express appropriate sites for factors IXa/VIII assembly. The maximal rate of factor X activation by factors IXa (0.75 nM) and VIII (1 unit/ml) assembled on monocytes is similar to the maximal rate on platelets. This rate, adjusted per micromole of lipid phosphorus, is 1636 +/- 358 nM factor Xa/min on monocyte, and 1569 +/- 54 nM factor Xa/min on platelets. At physiologic concentrations of factors X and VIII, the activation rate increases with factor IXa concentration asymptotically approaching a maximum. Half-maximal rate is achieved with 1.0 +/- 0.16 nM factor IXa. Monocytes and macrophages, but not platelets, can express membrane tissue factor and thus promote simultaneous assembly of two distinct factor X-activating protease complexes. In these studies, blood monocytes and alveolar macrophages are used as membrane sources in kinetic experiments comparing factor X activation by intrinsic (factor IXa/VIII) versus extrinsic (factor VII/tissue factor) protease complexes. At plasma concentration of factors VIII and VII, apparent Km on the monocyte is 14.6 +/- 1.4 nM for intrinsic and 117.0 +/- 10.1 nM for extrinsic activation. The apparent Km on alveolar macrophages is 12.1 +/- 1.9 and 90.6 +/- 10.2 nM for intrinsic and extrinsic activation, respectively. Maximal rates on monocytes at saturating concentration of factors IXa, VIII, and VII are 48.0 +/- 11.2 nM factor Xa/min, for intrinsic activation, and 16.5 +/- 5.5 nM factor Xa/min, for extrinsic activation. These data show that the monocyte/macrophage is the only blood-derived cell type with membrane sites for both intrinsic and extrinsic pathway assembly. We have exploited this characteristic of the monocyte/macrophage membrane to demonstrate that factor X activation by the intrinsic pathway protease is more efficient than activation via the extrinsic pathway protease complex.     

Zymogen factor IX potentiates factor IXa-catalyzed factor X activation

Intrinsic factor X activation is accelerated >10(7)-fold by assembly of the entire complex on the activated platelet surface. We have now observed that increasing the concentration of zymogen factor IX to physiologic levels ( approximately 100 nM) potentiates factor IXa-catalyzed activation of factor X on both activated platelets and on negatively charged phospholipid vesicles. In the presence and absence of factor VIIIa, factor IX (100 nM) lowered the K(d,appFIXa) approximately 4-fold on platelets and 2-10-fold on lipid vesicles. Treatment of two factor IX preparations with active-site inhibitors did not affect these observations. Autoradiographs of PAGE-separated reactions containing either (125)I-labeled factor IX or (125)I-labeled factor X showed that the increased factor X activation was not due to factor Xa-mediated feedback activation of factor IX and that there was increased cleavage of factor X heavy chain in the presence of factor IX in comparison with control reactions but only in the presence of both the enzyme and the surface. Since plasma concentrations of prothrombin, factor VII, protein C, or protein S did not by themselves potentiate factor Xa generation and did not interfere with the potentiation of the reaction of factor IX, the effect is specific for factor IX and is not attributable to the Gla domain of all vitamin K-dependent proteins. These observations indicate that under physiologic conditions, plasma levels of the zymogen factor IX specifically increase the affinity of factor IXa for the intrinsic factor X activation complex.     

A pathway of coagulation on endothelial cells

Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. Factor IX bound to specific endothelial cell sites can be activated by the intrinsic and extrinsic pathways of coagulation. Perturbation of endothelium results in induction of tissue factor which promotes factor VIIa-mediated activation of factors IX and X, thus initiating procoagulant events on the endothelial surface. Cell bound factor IXa, in the presence of factor VIII, promotes activation of factor X. The factor Xa formed can interact with endothelial cell factor V/Va, resulting in prothrombin activation. Thrombin then cleaves fibrinogen and a fibrin clot closely associated with the endothelial cell forms. The perturbed endothelial cell thus provides a focus of localized procoagulant events. This model suggests a simple endothelial-cell-dependent mechanism for initiation of coagulation at the site of an injured or pathological vessel.     

The inhibitor of F VIIa in plasma measured with a sensitive chromogenic substrate assay: comparison with antithrombin, protein C and heparin cofactor II in a clinical material

The plasma inhibitor(s) of factor VIIa-tissue thromboplastin cooperates with factor Xa. This "Extrinsic Pathway Inhibitor" has been quantitated with a sensitive chromogenic substrate assay. Gel filtration of plasma separates 3 EPI peaks. Postoperatively, both EPI and the other coagulation inhibitors decrease. Unlike the other inhibitors, EPI is usually normal in severe liver cirrhosis. In disseminated intravascular coagulation, EPI levels vary considerably.     

The binding of factor IXa to cultured bovine aortic endothelial cells. Induction of a specific site in the presence of factors VIII and X

Previous studies have demonstrated a Factor IX and IXa binding site on the endothelial cell surface for which both the zymogen and enzyme compete with equal affinity. In this report, we demonstrate that the affinity of Factor IXa, but not Factor IX, for the cell surface is increased in the presence of both Factors VIII and X. When Factor Xa formation was studied in the presence of saturating concentrations of Factors VIII and X, the half-maximal rate was observed at a Factor IXa concentration of 151 +/- 12 pM. Active site-blocked Factor IXa, 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa, was a more effective inhibitor of Factor X activation (Ki = 124 pM) than was Factor IX (Ki = 3.0 nM). Radioligand binding studies carried out in the presence of Factors VIII and X confirmed the presence of a selective endothelial cell Factor IXa binding site with Kd = 127 +/- 27 pM. In contrast, when Factor IXa binding was studied in the absence of other coagulation factors, or in the presence of Factor VIII (thrombin-activated or unactivated) alone, this new high affinity site was not observed. Competitive binding studies indicated that Factor IXa was 12 times more effective as an inhibitor of Factor IX-endothelial cell binding in the presence of Factors VIII and X. Consistent with the increased affinity of Factor IXa binding in the presence of factors VIII and X, cell-associated Factor IXa coagulant activity decayed 7 times more slowly in the presence of these coagulation factors. These results demonstrate selective Factor IXa-endothelial cell binding in the presence of Factors VIII and X, suggesting this interaction could be a physiologic occurrence.     

Tissue factor-dependent autoactivation of human blood coagulation factor VII.

We recently showed that single-chain zymogen factor VII is converted to two-chain factor VIIa in an autocatalytic manner following complex formation with either cell-surface or solution-phase relipidated tissue factor apoprotein (Nakagaki, T., Foster, D. C., Berkner, K. L., and Kisiel, W. (1991) Biochemistry 30, 10819-10824). We have now performed a detailed kinetic analysis of the autoactivation of human plasma factor VII in the presence of relipidated recombinant tissue factor apoprotein and calcium. Incubation of factor VII with equimolar amounts of relipidated tissue factor apoprotein resulted in the formation of factor VIIa amidolytic activity coincident with the conversion of factor VII to factor VIIa. The time course for the generation of factor VIIa amidolytic activity in this system was sigmoidal, characterized by an initial lag phase followed by a rapid linear phase until activation was complete. The duration of the lag phase was decreased by the addition of exogenous recombinant factor VIIa. Relipidated tissue factor apoprotein was essential for factor VII autoactivation. No factor VII activation was observed following complex formation between factor VII and a recombinant soluble tissue factor apoprotein construct consisting of the aminoterminal extracellular domain in the presence or absence of phospholipids. Kinetic analyses revealed that factor VII activation in the presence of relipidated tissue factor apoprotein can be defined by a second-order reaction mechanism in which factor VII is activated by factor VIIa with an apparent second-order rate constant of 7.2 x 10(3) M-1 S-1. Benzamidine inhibited factor VII autoactivation with an apparent Ki of 1.8 mM, which is identical to the apparent Ki for the inhibition of factor VIIa amidolytic activity by this active site competitive inhibitor. Our data are consistent with a factor VII autoactivation mechanism in which trace amounts of factor VIIa rapidly activate tissue factor-bound factor VII by limited proteolysis.     

In vivo bypass of hemophilia A coagulation defect by factor XIIa implant

Hemophilia A and B coagulation defects, which are caused by deficiencies of Factor VIII and Factor IX, respectively, can be bypassed by administration of recombinant Factor VIIa. However, the short half-life of recombinant Factor VIIa in vivo negates its routine clinical use. We report here an in vivo method for the continuous generation of Factor VIIa. The method depends on the implantation of a porous chamber that contains Factor Xa or XIIa, and continuously generates Factor VIIa bypass activity from the subject's own Factor VII, which enters the chamber by diffusion. Once inside, the Factor VII is cleaved to Factor VIIa by the immobilized Factor Xa or XIIa. The newly created Factor VIIa diffuses out of the chamber and back into the circulation, where it can bypass the deficient Factors VIII or IX, and enable coagulation to occur. In vitro, this method generates sufficient Factor VIIa to substantially correct Factor VIII-deficient plasma when assessed by the classical aPTT coagulation assay. In vivo, a Factor XIIa peritoneal implant generates bypass activity for up to one month when tested in rhesus monkeys. Implantation of such a chamber in a patient with hemophilia A or B could eventually provide a viable alternative to replacement therapies using exogenous coagulation factors.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.