Abstract
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.
Highlights
Platelet Receptor Occupancy with FactorIXa Promotes Factor X Activation*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
To investigate theactivated platelet surface as a locus for factor X activation,the functional consequencesof factor IXa binding to platelets were studied
Functional Characterizationof Bound FactoZr Xa-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 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
Summary
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. 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. We conclude that factor IXa bound tightly to a platelet receptor in thepresence of factor VIIIa is the enzyme active in factorX activation. 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. To test thishypothesis we have characterized the bound factor IXa both functionallyand structurally
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
