INTACT PLATELET MEMBRANES, NOT PLATELET-RELEASED MICROVESICLES, SUPPORT THE PROCOAGULANT ACTIVITY OF ADHERENT PLATELETS

The possibility that platelets release microvesicles on adherence to e ither von Willebrand factor (vwf) or collagen was examined by flow cyt ometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the tota l platelet mass was released as microvesicles after thrombin stimulati on of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as m icrovesicles subsequent to platelet stimulation with the nonphysiologi cal agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteol ysis of platelet cytoskeletal proteins was not responsible for microve sicle shedding under the conditions studied. Examination of the vWf-ad herent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle bind ing to the adherent platelets was observed, indicating that the majori ty of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to su pport procoagulant activity was measured with a prothrombinase activit y assay and was compared with the activity supported by the adherent p latelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for uns timulated platelets and platelets stimulated with physiological agonis ts. Furthermore, the residual activity found in the buffer fraction co ntaining detached platelets and any released microvesicles could be at tributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf- or collagen -adherent platelets did not promote increased procoagulant activity re lative to the unstimulated adherent platelets, even though microvesicl e release was detected as a result of agonist addition. Neither full p latelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.