Morphological analysis of microparticle generation in heparin-induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) with thrombosis is a serious complication of heparin use. HIT sera can generate platelet-derived microparticles, which are produced in a heparin-dependent manner and are hypothesized to be important initial pathological participants because they promote vascular occlusion. To date, microparticles have been studied using flow cytometric techniques. However, it is uncertain whether the small-sized material seen in flow cytometric studies represents true platelet microparticles shed from activated platelets or whether they are platelets that have contracted after releasing their internal components. This report describes a morphological investigation of platelet-derived microparticles in HIT using, among other techniques, confocal, scanning electron, and transmission electron microscopy. Following incubation with HIT sera, the existence of small membrane-bound vesicles in the milieu of activated platelets was demonstrated. A population of microparticles, expressing platelet-specific glycoproteins, was separated from platelets by centrifugation over a sucrose layer. These microparticles had identical flow cytometric profiles, size heterogeneity, and GPIband GPIIb/IIIa staining intensity as the microparticle population 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 pseudopods on activated platelets. These microparticles appeared to be generated by localized swelling, budding, and release. Collectively, these morphological studies document the existence of true microparticles in platelets activated by HIT sera. The microparticles may play an important role in the pathogenesis of HIT.

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets

“…Microvesicles are shedded by various blood cells in response to activation, maturation, mechanical stress, and other stimuli. Their number is increased under conditions associated with an enhanced thrombotic risk, such as acute coronary disease, heparin-induced thrombocytopenia, and sepsis (17,(24)(25)(26). Our findings suggest that the microvesicle TF contributes to a relevant extent to this prothrombotic state.…”

“…The procoagulant activity was dramatically enhanced, when the microvesicle numbers were increased up to 10-fold, encompassing the range of increases in microvesicles as determined under conditions favoring thrombogenesis (17,20,(24)(25)(26). The enhancement was particularly prominent, when platelets were added to the neutrophil suspensions supplemented with high amounts of microvesicles.…”

Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy

“…According to our images of PMPs and liposomes, ultrastructural deformation caused by immobilization on the mica coverslip reveals that height and XY diameter alone cannot be used to determine the size distributions of PMPs. In contrast, PMPs as imaged by transmission electron microscopy remain suspended in their native state during embedment and processing [32]. To overcome this, we relied upon volumetric analysis to size PMPs, assuming that PMP immobilization will not result in changes to this parameter.…”

Anti‐platelet factor 4/heparin antibodies in patients with impaired graft function after liver transplantation

“…The clinical probability of HIT was determined using the 4Ts pretest clinical scoring system when HIT was clinically suspected [20][21][22]. In the 4Ts system, the risk of HIT is considered to be low (score ≤ 3), intermediate (4)(5)(6), or high (7)(8). In patients with an intermediate to high 4Ts score, anticoagulation with heparin was switched to danaparoid (Orgaran â , Essex Pharma, Munich, Germany).…”