Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

Abstract

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560±35 sites for factor IXa with a Kd of 2.75±0.27 mM, compared with 461±60 sites per patient platelet with Kd of 3.2±0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the K. and k,1 were identical for normal and patient platelets in the absence of factor VIlla. In the presence of factor VIIIa, and k,,1, for patient platelets (163 min-') was only 33% of that for normal platelets (491 min-')-This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.

Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

“…The circulating blood cells of patients with Scott syndrome have previously been shown to exhibit a marked defect in their capacity to express procoagulant properties reflecting impaired mobilization of PS to the cell surface through Ca 2 ϩ -accelerated transbilayer movement of plasma membrane PLs (23)(24)(25)(26)(27)(28). Whereas the molecular defect responsible for this disorder remains unknown, the functional defect identified in Scott cells suggests either ( i ) a deficiency or defect in a specific protein that normally confers PL scramblase activity to the plasma membrane or, ( ii ) the expression in defective Scott cells of an inhibitor of the normal PL scramblase pathway.…”

“…Evidence for a platelet membrane protein of similar function has also been reported (22). Scott syndrome is a rare bleeding disorder in which normal cellular PL scramblase activity of blood platelets is defective despite normal secretory and aggregation responses, resulting in markedly reduced mobilization of PS to cell surfaces and reduced expression of membrane catalytic surface for the tenase and prothrombinase enzyme complexes (23)(24)(25)(26)(27)(28). The plasma membranes of these cells contain normal amounts of PS and other PLs, and exhibit normal aminophospholipid translocase activity (24,29).…”

PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

“…*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; e-mail FVIIIa (4,6,11). The physiological relevance of these interactions is emphasized by the fact that occupancy of these binding sites on activated platelets is closely correlated with enhanced rates of FX activation leading to an increase in catalytic efficiency (kcat/K m ) of >2 × 10 7 -fold in the presence of the assembled complex (7), and the fact that severe, spontaneous and post-traumatic bleeding complications occur in patients with deficiencies of FIX (12), FVIII (13), FX (14) and platelet receptors for FVIIIa (15,16).…”

“…*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; e-mail FVIIIa (4,6,11). The physiological relevance of these interactions is emphasized by the fact that occupancy of these binding sites on activated platelets is closely correlated with enhanced rates of FX activation leading to an increase in catalytic efficiency (kcat/K m ) of >2 × 10 7 -fold in the presence of the assembled complex (7), and the fact that severe, spontaneous and post-traumatic bleeding complications occur in patients with deficiencies of FIX (12), FVIII (13), FX (14) and platelet receptors for FVIIIa (15,16).Although platelets respond to many agonists with functional endpoints required for primary hemostasis including adhesion to subendothelial matrix, secretion of granule contents, aggregation and platelet plug formation to stop the flow of blood through breaches of the vasculature (17,18), exposure to strong agonists, such as collagen and thrombin, results as well in membrane surface changes allowing binding and complexation of the coagulation proteins responsible for physiologically relevant intrinsic FXa and thrombin generation (19)(20)(21). Little is known of the platelet signal transduction mechanisms resulting in these membrane surface changes.…”

Production and characterization of transformed B-lymphocytes expressing the membrane defect of Scott syndrome.

“…Scott platelets exhibit diminished capacity to promote plasma clotting due to reduced expression of membrane catalytic activity for the tenase (enzyme complex of coagulation factors VIIIaIXa) and prothrombinase (enzyme complex of coagulation factors VaXa) reactions (3,4). This defect is now known to represent a decrease in the number of newly exposed membrane binding sites for coagulation factors Va and Vllla by the activated platelets, reflecting diminished surface exposure of phosphatidylserine (PS)' and reduced shedding of coagulant plasma membrane vesicles ("microparticles") from the platelet surface (5,6). Although Scott platelets are defective in PS-exposure and microparticle formation in response to either calcium ionophore, complement C5b-9, or agonists such as thrombin and collagen, these platelets exhibit normal agonist-induced secretory and aggregation responses, implying a defect that selectively affects the plasma membrane events that are required for normal expression of platelet coagulant activity (2).…”