Flow cytometric analysis of agonist-induced annexin V, factor Va and factor Xa binding to human platelets

Activated platelets provide a procoagulant surface for the assembly and expression of prothrombinase complex. Expression of activity is associated with the binding of the protease factor Xa (FXa) and the co-factor Va (FVa) to the procoagulant surface. A flow cytometric methodology to measure annexin V-FITC as well as FVa and FXa binding to ionophore A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was calcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to platelets was measured using specific FITC-labelled monoclonal antibodies. The FITC labelled antibodies were displaced by ill-to 20-fold excess of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixation of platelets by formaldehyde caused artificial binding of annexin V, FVa and FXa as well, irrespective of the platelet activation status, Using gel-filtered platelets, the binding of FVa increased with alpha-granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was needed for maximal FVa binding to occur, No binding of FXa from internal platelet stores was observed. Addition of exogenous FVa and FXa resulted in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.