Platelet receptor occupancy with factor IXa promotes factor X activation.

To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the Kd (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 125I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active site-inhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.

To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the K d (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 12'I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active siteinhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.
The interaction between blood platelets and coagulation factors is essential for normal coagulation and hemostasis. Activated platelets promote the catalysis of two sequential reactions in the blood coagulation cascade: the activation of factor X to factor Xa by a complex of factor IXa, factor VIIIa, and calcium ions (1-6), and the conversion of prothrombin to thrombin by a complex of factor Xa, factor Va, and calcium ions (7-12). Platelets possess specific, high affinity, saturable receptors for factor Xa (8,9,12), factor V (Val (8,11,12), factor VI11 (13), factor IX, and factor IXa (14). Although there is ample evidence that factor VI11 and platelets (1-6) or phospholipids (15,16)  the proteolytic activation of factor X by factor IXa, the molecular mechanisms involved in the assembly of the factor X activating enzyme-cofactor complex on the platelet membrane are poorly understood.
Previously we have demonstrated rapid, reversible binding of both factor IX and factor IXa to thrombin-activated, gelfiltered platelets that requires the presence of physiologic concentrations of calcium ions (14). Factor IX was shown to bind to -300 sites/platelet with a Kd -2.5 nM, either in the absence or in the presence of saturating concentrations of thrombin-activated factor VI11 and factor X. In contrast, factor IXa was shown to bind to -550 sites/platelet (including -250 sites/platelet not shared by factor IX) with a Kd -2.5 nM in the absence of factor VIIIa and factor X (or in the presence of either protein alone), whereas in the presence of saturating concentrations of factor VIIIa (0.5-5.0 units/ml) and factor X (0.15-1.5 PM) together, factor IXa was bound to the same number of sites (-550/platelet) but with 5-fold higher affinity (Kd -0.5 nM). These results suggest the hypothesis that the enzymatic species active in factor X activation consists of factor IXa tightly bound (Kd -0.5 nM) to activated platelets in the presence of both factor VIIIa and factor X. To test this hypothesis we have now characterized the bound factor IXa both functionally and structurally.
Proteins-Details of the purification, assay, and characterization of human coagulation proteins, including factor IX, factor IXa, factor VIII, factor X, and a-thrombin were previously published (14). All proteins were >98% pure as judged by polyacrylamide slab gel electrophoresis in NaDodS04. Factor IXa (22.2 p M ) was inactivated by incubation with DEGR-CK (600 p~) for 3 h at 25 "C (dansyl-Glu-Gly-Arg-factor IXa or DEGR-factor IXa) as described by Lollar and Fass (17). For binding studies, 9-labeled factor IXa (2.5 x lo6 cpm/ pg) was prepared as previously described (14). Protein concentrations were determined by the Bio-Rad dye binding assay according to instructions provided by the manufacturer. Polyacrylamide slab gel electrophoresis in NaDodSOl was carried out according to the procedure of Laemmli (18).
Measurements of Rates of Factor Xa Formation-The activation of factor X by platelet-bound factor IXa was determined in the presence of CaC12 and thrombin-activated factor VI11 at concentrations of reactants indicated in the Figure legends. After stopping the reaction by the addition of 6 mM EDTA, the rate of factor X activation was determined by measurement of the amounts of factor Xa at various intervals using the factor Xa-specific chromogenic substrate S2337 as described by vanDieijen et al. (16). Initial rates of factor X activation were linear for at least 20 min, as were rates of p-nitroaniline formation which were read at 405 nm in a VmaX kinetic Microplate Reader" from Molecular Devices.
The results of our kinetic studies were analyzed using two different sets of assumptions. Assuming relatively weak binding (or the situation in which the concentration of binding sites is smaller than the K d ) , the apparent K d can be expressed as: (1) where Eo, total IXa concentration; Co, concentration of total IXa receptors; EC, concentration of IXa-receptor complex. In this case EC is small compared with Co, and EC can be determined as: ( 2) where EC is proportional to initial velocity. However, when binding is tight or Eo is large compared with K d , EC is not insignificant compared with Co and the expression becomes: This can be solved for EC (or velocity) to give: We solved this expression using a nonlinear regression program based on the method of Marquardt (19) and a TRS-80 computer to give a value of K d based on the kinetic data, as previously described (6, 20, 21). Alternatively, the K d was determined from graphical analysis using double-reciprocal plots of rates of factor Xa formation versus added factor IXa or from nonlinear regression analysis (19) of the hyberbola described in Equation 2. Values of K d obtained from graphical analysis and using the two equations were in good agreement (i.e. within 15% of one another). Inhibition of factor X activation by DEGR-factor IXa was analyzed by Dixon plots as previously described by Stern et at. (22).

RESULTS AND DISCUSSION
Functional Characterization of Bound Factor ZXa-To characterize bound factor IXa functionally, platelets were incubated with factor IXa (0.18-3.3 nM), thrombin (0.1 unit/ml), and CaClz (5 mM) and after neutralization of thrombin with P P A C K (50 nM), examined for their capacity to support factor X activation in the presence of thrombin-activated factor VI11 as described under "Experimental Procedures." The purpose of the present studies was to determine whether the apparent K d of factor IXa binding from our equilibrium binding experiments corresponds with the concentration of factor IXa required for half-maximal rates of factor X activation in the presence of thrombin-activated platelets. The results (Fig.  l A ) , presented as a double-reciprocal plot of the rate of factor Xa formed uersus the concentration of factor IXa added, indicate that the concentration of factor IXa required for half-maximal rates of factor Xa formation is 0.53 nM. This value is close t o t h e Kd previously reported from equilibrium binding studies of factor IXa in the presence of factor VIIIa the presence of thrombin-activated platelets and factor VIIIa. The rate of activation of human factor X by varying concentrations (0.17 to 3.5 nM) of factor IXa was determined in the presence of 2 to 3 X 108/ml thrombin-stimulated platelets at 37 "C in a reaction volume of 500 pl containing 50 mM Tris, pH 7.9, 175 mM NaC1,5 mM CaC12, 1.5 PM factor X, 2 units/ml of factor VI11 (preincubated for 1 min at a concentration of 250 units/ml with 0.01 unit/ml of human athrombin), and 0.5 mg/ml human serum albumin. Platelets were incubated with 0.1 unit/ml thrombin in the presence of CaClz (5 mM) and factor IXa for 10 min at 37 "C, and PPACK (50 nM) was added immediately prior to addition of factor VIIIa and factor X. For details of the assay, see "Experimental Procedures." The results presented are the means -+ S.E. of three separate experiments, each done in triplicate. B , simultaneous measurements of factor IXa binding to platelets and rates of factor X activation in the presence of factor VIIIa. Gel filtered platelets (2 to 3.5 X 108/ml) were incubated for 10 min at 37 "C with human a-thrombin (0.1 unit/ml), CaCI2 (5 mM), and 1251-factor IX (0.17-3.5 nM). After addition of PPACK (50 nM), factor VIIIa (5 units/ml, preincubated at a concentration of 300 units/ml at 37 "C for 1 min with 0.01 unit/ml of thrombin) and factor X (1.5 mM) were added. Factor IXa binding (closed circles) and factor Xa formation (open circles) were determined as detailed under "Experimental Procedures." Nonspecific factor IXa binding was determined in the presence of excess unlabeled factor IXa (0.44 PM) and was subtracted from total binding to obtain specific binding, which is plotted. The results presented are the means 2 S.E. of three separate experiments, each done in triplicate. The inset (open triangles) was obtained by dividing the rates of factor Xa formation (open circles) by the amounts of factor IXa bound (closed circles). and factor X (14). Analysis of similar experiments carried out at factor X concentrations of 0.15,0.5, and 1.5 pM, and factor VIIIa concentrations of 0.5-5.0 units/ml gave similar results. Thus, the observed rates of factor X activation in this experiment should not reflect a significant contribution from free factor IXa. Consistent with this possibility are the results of a detailed kinetic analysis of the contributions of activated platelets to factor X activation (25). We have shown that the K,,, for factor IXa-catalyzed factor X activation (154 p~, i.e. -1,000-fold higher than the physiological concentration of factor X) is decreased to below the plasma factor X concentration in the presence of thrombin-activated platelets (25).
Since the experiment shown in Fig. L4 was carried out at a factor X concentration (1.5 p~) well below the K,,, in the absence of platelets but well above the K,,, in the presence of platelets (0.1 pM), the observed rates of factor X activation should reflect the platelet-mediated reaction exclusively.
To study further the relationship between factor IXa binding and factor X activation, we carried out experiments in which both factor IXa binding and factor X activation were examined in the same incubation mixture, consisting of thrombin-activated platelets, '251-labeled factor IXa, thrombin-activated factor VIII, factor X, PPACK (added to neutralize thrombin after platelet activation and before addition of factor VIIIa), and CaCI2. The results (Fig. 1B) demonstrate a close correspondence between the amount of fractor IXa bound and the rates of factor Xa formation, both showing a hyperbolic relationship to the concentration of factor IXa added. By dividing rates of factor Xa formation by the amount of factor IXa bound at each concentration of added factor IXa, we could calculate turnover numbers (in moles of factor Xa formed per min/mol of factor IXa bound), which proved to be independent of enzyme concentration (Fig. 1B, inset)  ; i

OEGR-FlXo (nM)
FIG . 3. A, competition by unlabeled factor IXa and DEGR-factor IXa for 'Wabeled factor IXa binding sites on thrombin-activated platelets in the absence and presence of factor VIIIa and factor X. Gel filtered platelets (3.8 X 10n/ml) were incubated for 20 min at 37 "C with human a-thrombin (0.1 unit/ml), CaCI2 (5 mM), ' ' ' Ilabeled factor IXa (3.3 nM), various concentrations of unlabeled factor IXa or DEGR-factor IXa, and PPACK (50 nM) in the presence or absence of thrombin-activated factor VI11 (2 units/ml) and factor X (0.15 PM), as described in the legend to Fig. 1. Binding was determined as described under "Experimental Procedures." Maximal binding (100%) was determined by subtracting the nonspecific binding, i.e. the binding determined in the presence of excess unlabeled factor IXa or DEGR-factor IXa (0.4 pM) from total binding. The results shown represent residual factor IXa binding in the presence of unlabeled factor IXa in the absence (open circles) or presence (closed circles) of factor VIIIa and factor X and in the presence of unlabeled DEGR-factor IXa in the absence (open triangles) and presence (closed triangles) of factor VIIIa and factor X. The results presented are the mean 2 S.E. of three separate experiments, each done in triplicate. B, inhibition of platelet-bound factor IXa (FIXa)-factor VIIIa-mediated factor X activation by DEGR-factor IXa. The rate of factor Xa formation was determined in the presence of various concentrations (0.5-50 nM) of DEGR-factor IXa in the presence of 5 X lO'/ml of thrombin-activated (0.1 unit/ml), gel filtered platelets at 37 "C in a reaction volume of 100 pl containing 50 mM Tris, pH 7.9, 175 mM NaCI, 5 mM Ca&, 1.5 pM factor x , 5 units/ml of factor VI11 (activated with thrombin as described in the legend to Fig. 11, and 0.5 mg/ml human serum albumin. Platelets were stimulated with 0.1 unit/ml thrombin in the presence of CaCI2 (5 mM). Factor IXa, 0.5 nM (open circles), 1.5 nM (closed circles), and DEGR-factor IXa (0.5-50 nM) were preincubated with platelets for 10 min at 37 "C. Excess thrombin was neutralized with 50 nM PPACK prior to addition of factor VIIIa and performance of the assay.
close correlation between factor IXa binding and factor X activation reported herein suggests that the specific factor IXa binding site induced on the surface of thrombin-activated platelets in the presence of factor VIIIa and factor X is functionally active in factor X activation.
Structural Characterization of Bound Factor IXa-To char-Factor X Activation by Platelet-bound Factor I X a 20015   FIG. 4 . Hypothesis depicting platelet receptor-mediated factor X activation by factor IXa. The symbols RI and R2 refer to platelet membrane binding sites for factor IXa and factor VIII, respectively. Each reversible equilibrium is designated by arrows with dissociation constants ( K d ) or Michaelis constants (K,) indicated. Roman numerals refer to coagulation proteins. Unidirectional arrows are enzymatic reactions characterized by catalytic constants (kcat). Details are discussed in the text.

min"
+ acterize the bound factor IXa structurally, platelets were incubated with thrombin, CaC12, factor VIIIa, and factor X for 10 min at 37 "C and centrifuged through 20% sucrose to separate bound from free ligand. Platelet pellets were solubilized in NaDodSOl and were analyzed by polyacrylamide gel electrophoresis and autoradiography. More than 95% of lZ5I was solubilized and applied to the gel. The bound radioligand migrated as an M, 44,000 protein on nonreduced gels (Fig. 2, lane 3 ) and was indistinguishable from free factor IXa (Fig.  2, lane 2 ) . After reduction, the bound factor IXa migrated as two polypeptides of M, 27,000 and 17,000 (Fig. 2, lane 4 ) . lZ5I-Labeled factor IX, which was a single band at M, 57,000 is shown for comparison (Fig. 2, lane 1 ). When factor X and factor VIIIa were excluded from the incubation mixture, identical results were obtained. This experiment provides no evidence for the formation of high molecular weight covalent complexes or for proteolytic degradation of factor IXa by platelets, and confirms that the bound radioactivity consists entirely of factor IXa and not a radiolabeled contaminant. The fact that the bound factor IXa is structurally intact and indistinguishable from free factor IXa provides strong evidence that the functional factor X activating complex consists of factor IXa bound to platelets in the presence of factor VIIIa.
Competition Studies with Factor I X a and DEGR-Factor IXa-We carried out competition studies with unlabeled factor IXa and DEGR-factor IXa by incubating thrombin-stimulated platelets in the presence of CaClz for 20 min at 37 "C with 1251-labeled factor IXa and various concentrations of unlabeled proteins in the presence or absence of factor X and thrombin-activated factor VIII. When residual binding of Iz5Ilabeled factor IXa was determined (Fig. 3A), it was apparent that excess factor IXa and DEGR-factor IXa prevented >95% of 1251-labeled factor IXa binding. The presence of factor VIIIa and factor X significantly decreased the concentration of unlabeled factor IXa required to displace bound lZ5I-labeled factor IXa from thrombin-activated platelets. Addition of excess unlabeled DEGR-factor IXa in the presence of factor VIIIa and factor X showed displacement curves identical to those observed with excess unlabeled factor IXa, suggesting that active site-blocked factor IXa competes equally well with unlabeled factor IXa and that the active site is not required for binding of factor IXa to thrombin-stimulated platelets. From the results presented in Fig. 3A, it is estimated that the concentration of factor IXa and DEGR-factor IXa required for half-maximal inhibition of factor IXa binding in the absence of factor VIIIa and factor X were 2.5 and 2.6 nM, respectively, and the concentration of factor IXa and DEGR-factor IXa required for a similar effect in the presence of factor VIIIa and factor X were 0.5 and 0.6 nM, respectively.

Furthermore, we have determined the inhibition constant (Ki)
for both factor IXa and DEGR-factor IXa both in the presence and absence of factor VIIIa and factor X using a computer fit displacement curve as described by Rodbard (23) and modified to calculate the K, using the formula of Cheng and Prusoff (24). These values indicate that the Ki for factor IXa and the Kt for DEGR-factor IXa in the absence of factor VIIIa and factor X were 2.2 and 2.3 nM, respectively, and the K, for factor IXa and for DEGR-factor IXa in the presence of factor VIIIa and factor X were 0.38 and 0.43 nM, respectively.
Finally, we examined the effect of DEGR-factor IXa on the activation of factor X by factor IXa in the presence of thrombin-activated platelets and factor VIIIa (Fig. 3B). The resulting Dixon plot suggests that DEGR-factor IXa is a competitive inhibitor of factor IXa with a K, of 0.4 nM. Thus, both in competition binding studies (Fig. 3A) and in factor X activation (Fig. 3B), DEGR-factor IXa behaved as a competitive inhibitor of factor IXa binding. These results also indicate that factor IXa binding to platelets is essential for and is closely correlated with factor X activation. Equally important is the conclusion that the conversion of factor X to factor Xa is not required for the high affinity binding of factor IXa to its platelet receptor.
The studies reported herein and previously by our laboratory (1, 2, 14, 25) and by other investigators (3-6, 13) give rise to the hypothesis depicted schematically in Fig. 4. This presents a two-receptor model in which factor IXa and factor VI11 can bind independently to two separate and distinct sites on the surface of activated platelets. As shown by Nesheim et al. (13), factor VI11 can bind saturably, reversibly, and specifically to -450 sites/platelet with a Kd of -3.0 nM. The effects of factor IXa and factor X on factor VI11 binding have not been reported, nor have studies of factor VIIIa binding been published. The requirement of factors VIIIa and X that we have reported (14) for the induction of high affinity binding sites for factor IXa on platelets is similar to the requirement for factors VI11 and X reported by Stern et al. (22) for the induction of high affinity factor IXa binding sites on endothelial cells. Direct comparative experiments have not yet been reported to examine the relationship between factor VI11 (or VIIIa) receptor occupancy and rates of factor X activation. However, we have shown that, whereas by itself, factor VIIIa has no effect on the kinetics of factor X activation by factor IXa, the addition of saturating concentrations of factor VIIIa (13,14) to incubation mixtures containing thrombin-activated platelets decreases the K , of factor IXa-catalyzed factor X activation, while increasing the kcat -2000-fold (25). Our present experiments (Fig. 1B) suggest that the true kcat ( i e . the maximal rate of factor Xa formed in moles/min/mol of factor IXa bound or -2400 min") may be considerably higher than that previously estimated (i.e. -500 moles of factor Xa formed per min/mol of total factor IXa added; Ref. 25). Although further details of this hypothesis (Fig. 4) remain to be worked out, we believe its central features are supported by our present and previous studies (1, 2, 14, 25) and those reported by others (3-6, 13), i.e. that factor X activation by factor IXa is a platelet-receptor-mediated process tightly coupled to receptor occupancy by factor IXa and factor VIIIa.