THE DIFFERENCE BETWEEN PLATELET AND PLASMA FXIII USED TO STUDY THE MECHANISM OF PLATELET MICROVESICLE FORMATION

The formation of microvesicles from platelets was induced either by ac tivation of the complement system by a monoclonal antibody to CD9, or by incubation of platelets with the calcium ionophore A23187. A filter technique to isolate the microvesicles without plasma contamination i s described. The microvesicles contained FXIIIa2 from the platelet cyt oplasm which shows that these particles contain significant amounts of intracellular material. This was shown by the use of crossed immunoel ectrophoresis with rabbit antibodies to total human platelet proteins in the second dimension get and polyclonal antibodies against the a- a nd b-subunit of FXIII in the intermediate gel. The FXIIIa2 in the micr ovesicle was found to be functional as an enzyme. To prove this, it wa s shown that FXIII in its immunoprecipitate arc could catalyze the inc orporation of monodansylcadaverine into casein as identified by fluore scence of this arc in ultraviolet light. The observation that the plas ma form of FXIII (FXIIIa2b2) was absent from the microvesicles collect ed by the filtration technique, whereas it was present in platelet fra gments obtained by mechanical disruption by ultrasonication, indicates that the activation-dependent microvesicles are formed by a true budd ing process with the inclusion of intracellular, but not extracellular material.