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 an d expression of prothrombinase complex. Expression of activity is asso ciated with the binding of the protease factor Xa (FXa) and the co-fac tor Va (FVa) to the procoagulant surface. A flow cytometric methodolog y to measure annexin V-FITC as well as FVa and FXa binding to ionophor e A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was ca lcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to pl atelets was measured using specific FITC-labelled monoclonal antibodie s. The FITC labelled antibodies were displaced by ill-to 20-fold exces s of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixat ion 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 alph a-granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was need ed for maximal FVa binding to occur, No binding of FXa from internal p latelet stores was observed. Addition of exogenous FVa and FXa resulte d in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.