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 10-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.

Platelets deficient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V.

The aggregation of platelets induced by collagens is considered an important step in primary hemostasis. Glycoprotein (GP) IIIb (GPIIIb, GPIV, CD36) has been proposed as a blood platelet receptor for collagen. Platelets from three healthy blood donors were shown to be clearly deficient in GPIIIb. These platelets aggregated normally in response to type I and III collagens. In addition, platelet factor 4, beta-thromboglobulin, and adenosine triphosphate (ATP) secretion in response to type I and III collagens was normal. The findings indicate that GPIIIb is not the major, essential collagen receptor for type I and III collagens. This would explain why all individuals with GPIIIb-deficient platelets examined so far are healthy and, in particular, show no apparent evidence of hemostatic problems. However, in contrast to control platelets, no aggregation and impaired platelet factor 4, beta-thromboglobulin, and ATP secretion was observed in response to type V collagen. Therefore, it is postulated that for type V collagen-induced aggregation both GPIa/IIa and GPIIIb are essential.