MICROVESICLES BIND SOLUBLE FIBRINOGEN, ADHERE TO IMMOBILIZED FIBRINOGEN AND COAGGREGATE WITH PLATELETS

In the present study we have investigated whether platelet derived mic rovesicles can bind soluble fibrinogen, bind to immobilized fibrinogen , and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA well s were used to study binding of microvesicles to immobilized fibrinoge n. Biotinylated microvesicles produced by stimulation with A23187, thr ombin or SFLLRN of platelets which had been surface-labelled with biot in, were used both for the coaggregation experiments and for the bindi ng studies with immobilized fibrinogen. Unlabelled microvesicles and b iotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinyla ted proteins were reacted with avidin or streptavidin which was PE-con jugated, whereas the same substances were conjugated with alkaline pho sphatase for the ELISA studies. The microvesicles formed after stimula tion of platelets by SFLLRN or A23187 clearly bound the soluble, bioti nylated fibrinogen. Moreover, isolated biotinylated microvesicles adde d to washed platelets prior to activation, were associated to the micr oaggregates that formed after stimulation. A significant binding of bi otinylated microvesicles to immobilized fibrinogen could also be detec ted. The binding of microvesicles to soluble and immobilized fibrinoge n and association to platelets was clearly specific and at least partl y dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results i t is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of m icrovesicles in vivo.