Platelets possess specific, high-affinity, saturable receptors for factor XI, factor XIa, factor Xa, thrombin, high molecular weight kininogen, and factor Va and promote the proteolytic activation of factor XII, factor XI, factor X, and prothrombin. To investigate further the activated platelet surface as a locus for the molecular interactions of coagulation proteins, we have studied the binding of factor IXa and factor IX to platelets as well as the contribution of platelets to factor-X activation. Factor IX, isolated from human plasma by immunoaffinity purification utilizing a murine monoclonal antibody, was converted to factor IXa by incubation with purified factor XIa. We have demonstrated the specific, high-affinity, reversible binding of both factor IXa and factor IX to thrombin-activated platelets in the presence of calcium ions. Analysis of saturation binding data obtained under equilibrium conditions indicated the presence of 550 plus or minus 70 (mean plus or minus SD) sites per platelet for factor IXa with a dissociation constant of 2.5 plus or minus 0.5 nM, whereas there were 306 plus or minus 57 sites for factor IX with a Kd of 2.68 plus or minus 0.25 nM. Preliminary results suggest that platelet-bound factor IXa promotes half-maximal rates of factor-X activation at a concentration of 0.5 nM. The central hypothesis to be tested is that factor IXa is bound to high- affinity, specific platelet receptors with acceleration of factor-X activation as a functional consequence. Specifically, our objectives are to: 1) elucidate the biochemical basis and physiological significance of factor-IX and factor-IXa binding to platelets including the relationship between factor-IX and factor- IXa receptors; 2) determine the role (if any) of factor VIII and von Willebrand fator in binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 3) determine the state of platelet activation required for binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 4) elucidate the structural characteristics of factor IX and factor IXa required for binding to platelet receptors and for the platelet contribution to factor-X activation; 5) determine the functional consequences of factor- IXa binding to platelets by examining the kinetics of factor-X activation, utilizing chromogenic assays, proteolytic cleavage studies, coagulation assays and the release of an activation peptide from 3H-labeled factor X. These studies will provide essential information about the role of platelets in promoting the assembly of enzyme-cofactor-substrate complexes and the proteolytic activation of coagulation zymogens.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Temple University
Schools of Medicine
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Ho, D H; Baglia, F A; Walsh, P N (2000) Factor XI binding to activated platelets is mediated by residues R(250), K(255), F(260), and Q(263) within the apple 3 domain. Biochemistry 39:316-23
Ahmad, S S; Wong, M Y; Rawala, R et al. (1998) Coagulation factor IX residues G4-Q11 mediate its interaction with a shared factor IX/IXa binding site on activated platelets but not the assembly of the functional factor X activating complex. Biochemistry 37:1671-9
Scandura, J M; Zhang, Y; Van Nostrand, W E et al. (1997) Progress curve analysis of the kinetics with which blood coagulation factor XIa is inhibited by protease nexin-2. Biochemistry 36:412-20
Baglia, F A; Walsh, P N (1996) A binding site for thrombin in the apple 1 domain of factor XI. J Biol Chem 271:3652-8
Scandura, J M; Walsh, P N (1996) Factor X bound to the surface of activated human platelets is preferentially activated by platelet-bound factor IXa. Biochemistry 35:8903-13
London, F; Ahmad, S S; Walsh, P N (1996) Annexin V inhibition of factor IXa-catalyzed factor X activation on human platelets and on negatively-charged phospholipid vesicles. Biochemistry 35:16886-97
Scandura, J M; Ahmad, S S; Walsh, P N (1996) A binding site expressed on the surface of activated human platelets is shared by factor X and prothrombin. Biochemistry 35:8890-902
Baglia, F A; Seaman, F S; Walsh, P N (1995) The Apple 1 and Apple 4 domains of factor XI act synergistically to promote the surface-mediated activation of factor XI by factor XIIa. Blood 85:2078-83
Baglia, F A; Jameson, B A; Walsh, P N (1995) Identification and characterization of a binding site for platelets in the Apple 3 domain of coagulation factor XI. J Biol Chem 270:6734-40
Seaman, F S; Baglia, F A; Gurr, J A et al. (1994) Binding of high-molecular-mass kininogen to the Apple 1 domain of factor XI is mediated in part by Val64 and Ile77. Biochem J 304 ( Pt 3):715-21

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