Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets

Abstract

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca 2+ levels ([Ca 2+] i), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca 2+ two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca 2+ vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca 2+] i and binding of annexin V. However, a small fraction of the adherent platelets (∼1%) responded much faster. Ionomycin-enhanced influx of Ca 2+ in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0±0.7 μm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca 2+] i and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca 2+-induced generation of procoagulant sites and Ca 2+-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.