Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500 -600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin-or SFLLRN-activated platelets. Concentrations of these agonists that are halfmaximal or maximal in kinetic studies resulted in only a small subpopulation (4 -20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 ؎ 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (ϳ10%) of PAR 1-stimulated platelets expose ϳ6000 factor IXa binding sites/platelet.

Summary. Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant factors and proteases, is currently unresolved. The interaction between coagulation proteins and platelets is critical to the maintenance of normal hemostasis and is pathogenetically important in human disease.

Annexin V was found to inhibit factor IXa-catalyzed factor X activation on both thrombin-activated human platelets and artificial lipid vesicles containing phosphatidylserine, supporting previous observations of the importance of negatively-charged lipid in potentiating the reaction. Annexin V reduced the Vmax of factor X activation in factor IXa titrations on the platelet surface with an IC50 of 4 nM in the absence of thrombin-activated factor VIII (factor VIIIa), and 4.5 nM in its presence, whereas there was no effect on the EC50,FIXa. This noncompetitive inhibition is consistent with interference of recognition of the factor IXa binding site on the platelet, which was confirmed by equilibrium binding of [125I]-factor IXa to thrombin-activated platelets where, in the absence of factor VIIIa and factor X, annexin V reduced the number of factor IXa binding sites/platelet from 610 to 320, without changing the Kd,app. In the presence of factor VIIIa and factor X, annexin V reduced the number of binding sites, but also raised the Kd,app. Although factor VIIIa improved the affinity of factor IXa for the lipid surface from Kd approximately 60 nM in its absence to Kd 1 nM in its presence, addition of annexin V to factor IXa titrations on lipid vesicles in the presence of factor VIIIa increased the EC50,FIXa with an IC50 of 1.5 nM, without affecting the Vmax. These data provide evidence that factor IXa, although requiring negatively-charged phospholipid for part of its binding site, is accommodated differently on platelets and on artificial vesicles.

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF) FIXa is a serine protease that participates in the intrinsic pathway of blood coagulation. FIXa activates FX as part of the intrinsic FX activating complex. The FX activating complex consists of FIXa, FVIIIa (a nonenzymatic cofactor), and FX (the normal macromolecular substrate) assembled on a procoagulant surface (8, 9). Under physiological conditions, thrombinactivated platelets or endothelial cells can provide this surface (10 -13). Additionally, synthetic phospholipid vesicles containing phosphatidylserine can also support assembly of the FX activating complex and activation of FX (8, 14). Assembly of the surface-bound FX activating complex results in a dramatic increase (ϳ20 million-fold) in the catalytic efficiency (k cat /K m ) of FX activation versus that of FIXa alone in solution (9, 15). The surface localization of FIXa is a requisite step in the catalytic enhancement of FIXa when assembled in the FX activating complex (9, 14).-Recent investigations have focused on the contributions of the EGF-like domains to FIXa biochemistry. Analysis of patient data suggests that residues contained within EGF1 and EGF2 are important for FIX(a) functions. Several point mutations within the EGF-like domains result in dysfunctional FIX(a) activity and a bleeding tendency (3,16). Investigations of the EGF-like domains have found several important functions for these domains. EGF1 is important for interactions with FVIIa/TF (17), FVIIIa (18), and FX (19,20). EGF1 does not appear to be important for binding to surfaces such as phospholipids (21), platelets, (22), or endothelial cells (23). While EGF2 has not been extensively characterized, experiments have indicated its importance for activation of FX. A chimeric FIXa protein in which both EGF1 and EGF2 domains were replaced by those of FX possessed about 4% clotting activity (24). In contrast, a chimeric protein with the FX EGF1 domain but the wild type FIX EGF2 domain was entirely normal (24), suggesting an essential function for EGF2. More recent experiments from our laboratory have indicated that EGF2 is likely involved in mediating surface binding to both platelets and phospholipids (25,26).To further define the contribution of the EGF2 domain to surface binding, we have prepared several chimeric FIXa proteins (see Fig. 1

We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl 3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.An essential event in the hemostatic response to vascular injury is the assembly of the factor X (FX) 1 activating complex on the surface of activated platelets (1,2). The assembly of this important enzymatic complex requires the exposure of coagulation protein binding sites on the surface of platelets activated with thrombin or collagen but not with adenosine diphosphate (3-6). All proteins required for physiologically relevant platelet-supported FX-activation, including the enzyme factor IXa (FIXa), the cofactor factor VIIIa (FVIIIa) and the substrate factor X (FX), must be bound to their respective receptors on activated platelets (7). Thus, the zymogen FIX binds with high affinity (K d ~2.5 nM) to a discreet number of platelet receptors † This study was supported by research grants from the National Institute of Health: HL70683, HL46213 and HL74124. *To whom correspondence should be addressed: Fredda London, Ph.D., Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, Tel: 215-707-4458; Fax: 215-707-3005;...