Morphological analysis of microparticle generation in heparin-induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) with thrombosis is a serious complic ation of heparin use. HIT sera can generate platelet-derived microparticles , which are produced in a heparin-dependent manner end are hypothesized to be important Initial pathological participants because they promote vascula r occlusion. To date, microparticles have been studied using flow cytometri c techniques. However, it is uncertain whether the small-sized material see n in flow cytometric studies represents true platelet microparticles shed f rom activated platelets or whether they are platelets that have contracted after releasing their internal components. This report describes a morpholo gical investigation of platelet-derived microparticles in HIT using, among other techniques, confocal, scanning electron, and transmission electron mi croscopy. Following incubation with HIT sera, the existence of small membra ne-bound vesicles in the milieu of activated platelets was demonstrated. A population of microparticles, expressing platelet specific glycoproteins, w as separated from platelets by centrifugation over a sucrose layer. These m icroparticles had identical flow cytometric profiles, size heterogeneity, e nd GPlb(alpha) and GPIIb/IIIa staining intensity as the microparticle popul ation in unfractionated samples. When microparticles were generated in situ and fixed onto grids, they were demonstrated to be distinct membrane-bound vesicles that originated near the platelet body and terminal ends of pseud opods on activated platelets. These microparticles appeared to be generated by localized swelling, budding, and release. Collectively, these morpholog ical studies document the existence of true microparticles in platelets act ivated by HIT sera. The microparticles may play an important role in the pa thogenesis of HIT. (Blood. 2000;96:188-194) (C) 2000 by The American Societ y of Hematology.