Xa Binding Research Articles

Assembly of the prothrombinase complex on lipid vesicles depends on the stereochemical configuration of the polar headgroup of phosphatidylserine.

The conversion of prothrombin into thrombin is an imperative step in the sequence of reactions leading to the formation of a hemostatic plug. This reaction is catalyzed by the prothrombinase complex, composed of factors Xa and Va, which is assembled on a phospholipid surface through Ca-mediated interactions with the lipid polar headgroups. In this paper we describe experiments indicative for a major role of the stereochemical configuration of phosphatidylserine in the binding of the prothrombinase complex to a phospholipid surface. Using two stereoisomers of phosphatidylserine, i.e., L-alpha-glycerophosphoryl-L-serine (PLS) and L-alpha-glycerophosphoryl-D-serine (PDS), we demonstrate that membranes containing PLS are appreciably more favorable than membranes containing PDS in promoting assembly of the prothrombinase complex and catalysis of prothrombin conversion. Ellipsometric analysis of the binding of factor Va and factor Xa to a surface composed of phosphatidylcholine and 10 mol % of either PLS or PDS reveals that the apparent Kd for factor Va increases about 25-fold when substituting PDS for PLS. For factor Xa a 5-fold increase in Kd was observed on replacing PDS for PLS. When PLS is replaced by phosphatidyl-beta-lactate (PLac), a phospholipid resembling PS but lacking the amino group, a similar decrease in prothrombinase activity is found as observed with PDS, implicating the importance of both the amino group and the stereoconfiguration of the serine moiety for the assembly of the prothrombinase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

A protease purified from the venom of the elapid snake Naja naja oxiana converts human blood coagulation factor Va into a molecule (factor VaNO) with greatly reduced cofactor activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the venom protease cleaved a small peptide from the heavy chain of factor Va and reduced the apparent M(r) from 105,000 to 101,000. This peptide was isolated by high performance liquid chromatography on a reversed-phase column. Amino acid sequence analysis of the peptide indicated that the venom enzyme cleaved the peptide bond between His682 and Asp683, thus removing 27 amino acids from the carboxyl-terminal part of the heavy chain. The cofactor activities of factors Va and VaNO were compared by measuring their abilities to support factor Xa-catalyzed prothrombin activation in the presence of phospholipids and calcium ions. Both factor Va molecules stimulated the binding of factor Xa to negatively charged phospholipids. However, the amounts of factor Va required for half-maximal incorporation of factor Xa into the membrane-bound factor Xa-Va complex were much lower for native factor Va (0.25 nM) than for factor VaNO (2.01 nM). At saturating concentrations of factor Va or factor VaNO the kcat values for prothrombin activation were 114 s-1 for factor Va and 128 s-1 for factor VaNO. The Km values for prothrombin determined under these conditions were 0.24 and 0.83 microM for prothrombinase complexes with native factor Va and factor VaNO, respectively. Direct binding studies revealed that factors Va and VaNO bind with equal affinity to phospholipids. These data indicate that factor VaNO is impaired in its ability to interact with factor Xa and prothrombin. Together with the structural data this implies that the carboxyl-terminal Asp683-Arg709 domain of the heavy chain is required for optimal interaction of factor Va with factor Xa and prothrombin.

Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa.

Protein S is a vitamin K-dependent non-enzymatic coagulation factor involved in the regulation of activated protein C (APC). In this paper we report an APC-independent anticoagulant function of protein S. We observed an inhibition of prothrombinase activity on endothelial cells and platelets which was half-maximal at physiological concentrations of free protein S in plasma. On endothelial cells, thrombin-cleaved protein S (PSt) as well as protein S in complex with C4b-binding protein (C4BP) inhibited prothrombinase activity to the same extent as protein S did. In solid-phase binding assays, direct binding of protein S and PSt to factor V and factor Va were observed. Protein S-C4BP complex did not bind to factor V or factor Va, implicating that the factor V(a) binding site on protein S is lost by the interaction with C4BP. A direct inhibition of factor Xa activity by protein S was also observed. Incubation of factor Xa with protein S revealed a noncompetitive inhibition of factor Xa by protein S with a Ki of (4.9 +/- 0.8) x 10(-7) M. Both protein S and protein S-C4BP complex were able to inhibit factor Xa to the same extent, whereas PSt had lost its inhibitory activity. This suggests that the conformational change induced by cleavage of the amino-terminal thrombin-sensitive loop results in a loss of a factor Xa binding site on protein S. The inhibitory effect of protein S involves interactions with both factor Va and factor Xa. The interaction of protein S with factor Xa is influenced by cleavage of the thrombin-sensitive loop, whereas C4BP blocks the interaction of protein S with factor Va. Inhibition of the prothrombinase complex by either forms of protein S might be an important mechanism in regulating thrombin generation in blood coagulation. The importance of the APC-independent anticoagulant action of protein S was emphasized by experiments in which the addition of protein S to normal plasma induced a prolongation of clotting time in a dilute activated partial thromboplastin time (dAPTT) assay. Furthermore, inhibition of endogenous protein S by the addition of monoclonal antibodies against protein S to normal plasma, induced a shortening of the clotting time in a dAPTT assay.

Transport Rate Limited Catalysis on Macroscopic Surfaces: The Activation of Factor X in a Continuous Flow Enzyme Reactor

Blood coagulation is initiated on cells which present a macroscopic surface to the flowing blood stream. We have used a continuous flow enzyme reactor to model this system and to investigate the effects of shear rate and mass transport on the activation of factor X by the complex of the transmembrane protein, tissue factor, and the serine protease, factor VIIa. This initial step of blood coagulation was found to be half-maximal at very low enzyme densities (0.03-0.06%) on the wall of the capillaries. In agreement with hydrodynamic theory, the apparent Km in the flow reactor was correlated with the cube root of the wall shear rate. These data indicate that at high tissue factor densities (> 0.6%) the activation of 150 nM factor X is controlled by the flux of X toward the surface, which is controlled by wall shear rate and substrate concentration. The appearance of the product, Xa, in the effluent was delayed to 8-12 min, which was caused by high-affinity binding of Xa to the phospholipid. This delay was considerably shortened by embedding tissue factor into PC or by coating the PS/PC surface with the phospholipid binding protein, annexin V. At low tissue factor densities, annexin V inhibited X activation by 45%, while no inhibition was observed at high densities. We demonstrate that when the reaction is limited by substrate flux, addition of further enzyme does not increase reaction rates. This contrasts with classical three-dimensional catalysis in which the initial velocity is ordinarily linear with the enzyme concentration.

Molecular cloning of effector cell protease receptor-1, a novel cell surface receptor for the protease factor Xa.

Cellular receptors for blood proteases regulate chemotaxis, extracellular proteolysis, and growth behavior of normal and malignant cells. Binding of the coagulation protease factor Xa to leukocytes is contributed by a recently identified molecule, denominated Effector cell Protease Receptor-1 (EPR-1). Monoclonal antibodies were generated against EPR-1+ MOLT13 lymphocytes and selected for reactivity with lymphocyte surface proteins by flow cytometry and with affinity-purified EPR-1 in Western blots. Antibody-based functional cloning of the cDNA for EPR-1 reveals the sequence of a novel molecule encoded by a prominent 1.9-kilobase mRNA. The cDNA predicts a glycoprotein of 337 amino acids, characterized by a unique cysteine-rich extracellular module, a single membrane-spanning domain, and a serine-rich (26%) cytoplasmic tail featuring at least 15 potential phosphorylation sites. Genetically engineered EPR-1 transfectants were recognized by monoclonal antibodies to EPR-1 and bound 125I-factor Xa in a specific and saturable manner (Kd approximately 10-15 nM). In the absence of factor V/Va, EPR-1 transfectants promoted prothrombin activation in a factor Xa concentration-dependent reaction, inhibited by a monoclonal antibody to EPR-1. These findings define EPR-1 as a novel cell surface receptor for factor Xa potentially implicated in protease-dependent cellular effector functions.

Binding of factor Xa to fibrinogen, fibrin and fibrin degradation products (FDP): its comparison with thrombin binding

Binding of factor Xa to fibrinogen, fibrin and fibrin degradation products (FDP): its comparison with thrombin binding

Expression and Assembly of Procoagulant Complexes by Human Pleural Mesothelial Cells

Many pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor VIIa to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

The assembly of the prothrombinase complex on adherent platelets.

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant K d = 140 ± 27 nM (mean ± S.E.) and maximal surface concentration Γ max = (8.9 ± 0.8) · 10 −3 mole of protein per mole of lipid, were obtained. For factor Xa, these values were K d = 49.6 ± 6.3 nM and Γ max = (23.0 ± 1.4) · 10 −3 mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti- factor V IgG and to a large extent (approximately 70%) was calcium- independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti- factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti- TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.

Tissue Factor Pathway Inhibitor and Protease Nexin-1 Are Major Factor Xa Binding Proteins on the HepG2 Cell Surface

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti-factor V IgG and to a large extent (approximately 70%) was calcium-independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2-bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L-arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti-tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti-factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti-TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.

Factor Xa interacts with two sites on monocytes with different functional activities

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa- catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

Factor Xa Interacts With Two Sites on Monocytes With Different Functional Activities

AbstractStudies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IXα through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (α-HFV1). At the monocyte surface, the factor Va/Xa-catalyzed activation of prothrombin was maximally inhibited with 0.25 µmol/L α-HFV1, whereas 1.0 µmol/L α-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did hot cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

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.

The effect of leukocyte elastase on tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa-TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.

The effect of leukocyte elastase on tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa- dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa- TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.

Platelet factor Xa receptor

The assembly and function of the prothrombinase complex on the bovine and human platelet membrane is mediated through binding interactions in which factor Va bound to the platelet surface forms at least part of the "receptor" for factor Xa in a 1:1 stoichiometric complex. A model depicting these binding interactions is shown in Fig. 12. Data from our laboratory indicate that the prothrombinase catalyst assembles in an analogous manner on the surface of monocytes, lymphocytes, neutrophils, and well-defined phospholipid vesicles employed in model systems. The 74,000-Da subunit of factor Va, component E, which mediates the binding of factor Va to either bovine platelets, human monocytes, or phospholipid vesicles, is shown binding to the cell membrane through its putative "receptor." The 94,000-Da subunit of factor Va, component D, is associated with the membrane surface through its metal ion-dependent interaction with component E. Factor Va forms at least part of the receptor that mediates the binding of factor Xa to an appropriate membrane surface, because component E has been shown to contribute significantly to the interaction of factor Xa with either the platelet, monocyte, or vesicle membrane surface. Our data do not preclude the possibility that component D contributes to the binding of factor Xa and the function of the prothrombinase complex. Component D appears to be important for several reasons. Cleavage of component D by activated protein C results in the complete loss of factor Va cofactor activity. An interaction between factor Xa and component D is implied from the observation that factor Xa protects factor Va from activated protein C inactivation. Furthermore, the binding of factor Xa to platelet-bound factor Va results in the time-dependent cleavage of components D and D'. Because component D is not required absolutely for prothrombinase complex assembly, we would speculate that it may be important in mediating prothrombin binding (depicted as a three-domain molecule) and increasing the catalytic efficiency of the enzymatic complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane-mediated assembly of the prothrombinase complex.

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.

The association of coagulation factor Xa and factor Va

The binding of factor Xa to factor Va in the presence of Ca2+ ions and phospholipid is fundamental for the activation of prothrombin to thrombin. Nevertheless, the biochemistry of the intrinsic association between factors Xa and Va is poorly understood. In the present study we have measured the formation of the protein-protein complex in the absence of phospholipid by using analytical ultracentrifugation. Factor Xa or factor Va were respectively modified with a chromophore-peptidyl-chloromethyl ketone or a thiol-specific chromophore, which permitted selective evaluation of the sedimentation of either component by virtue of its unique absorbance properties. Regardless of which protein was labeled, a factor Xa-Va complex (s20,w = 9.8) was formed. The interaction is specific and reversible. In 2 mM Ca2+ and at 20 degrees C, the dissociation constant for the binding of factor Xa to factor Va is 0.8 microM with a 1:1 stoichiometry. The association has multiphasic Ca2+ dependence. At concentrations of Ca2+ below 1 mM or above 2 mM, a weaker protein-protein equilibrium is maintained.

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets.

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.