THE ASSEMBLY OF THE PROTHROMBINASE COMPLEX ON ADHERENT PLATELETS

Prothrombinase complex assembly, in real time, on platelets adherent t o immobilized von Willebrand Factor (vWf) was examined by total intern al reflection fluorescence spectroscopy (TIRFS). Electron microscopy s howed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent plate let membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor fo rm of GPIIb-IIIa detected by antibody binding was seen only after thro mbin stimulation of the adherent platelets. Antibody binding to GPIb w as detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a ch ange in GPIb as a consequence of thrombin stimulation of the platelets . The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studie d individually. Factor Va bound to adherent and thrombin-stimulated ad herent platelets with an estimated K(d) of 58 nmol/L. Minimal factor X a binding was observed on adherent platelets before thrombin stimulati on. Factor Xa binding was, however, readily observed on thrombin-stimu lated adherent platelets. This factor Xa binding was not saturable, an d no K(d) value could be estimated. Direct measurement of prothrombina se complex assembly was demonstrated by using an energy transfer pheno menon between fluorescein-labeled factor Va and rhodamine-labeled fact or Xa. Prothrombinase complex assembly was observed on both adherent a nd thrombin-stimulated adherent platelets. The estimated K(d) for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluore scently labeled factors Va and Xa.