Platelet-Derived Microparticles Promote Clot Stability.

Microparticles (MP) are the plasma membrane fragments. They are formed along with membrane remodelling processes of most stimulated eukaryotic cells or generated upon cellular stimulation including platelets. After having been considered “inert cell debris” previously, recent findings suggested that MP can modulate distinct cellular responses in the related microenvironment. For example, the concentration of circulating platelet-derived MP is increased in acute myocardial infarction and stroke. It is hypothesized that platelet-derived MP promote hemostasis and thrombosis. However, the precise role of MP is still unknown. In this study, we evaluated the influence of platelet-derived MP on clot stability. Anticoagulated blood (3.8% sodium citrate) was obtained from healthy blood donors. Platelet-rich plasma was centrifuged at 1500g (10 min, 22°C). The pelleted platelets were washed three times in PBS (pH 7.4), resuspended in 1 ml of the same buffer and activated with human collagen of type I (10 min, 35°C) at a final concentration of 10 μg/ml. The supernatant (1500g 10 min, 22°C) containing activated platelet MP was centrifuged at 13,000g (30 min, 4°C). The pellet of MP was resuspended in 450 μl of PBS. MP were identified by scanning electron microscopy and flow cytometry following immunolabelling with an anti-GPIbα FITC-monoclonal antibody. The influence of platelet MP onto clot stability, determined as platelet contractile force (PCF) and clot elastic modulus (CEM), was evaluated with a Hemodyne haemostasis analyzer (Hemodyne, Richmond, USA). Mean PCF and CEM in blood of healthy donors (n=7) were 6.5±3 Kdynes and 9.6±6 Kdynes/cm², respectively. Addition of 100 μl of platelet-derived MP increased PCF (forces generated by platelets within a clot) without reaching statistical significance (mean increase of 11% as compared to controls without MP). By contrast, addition of platelet MP significantly enhanced CEM as measure of clot stability from 9.6±6 Kdynes/cm² to 94 ± 62 Kdynes/cm², (p<0.05). In experiments conducted with platelet-rich plasma or platelet-poor plasma instead of anticoagulated whole blood, no influence of added platelet-derived MP on clot stability was observed. In a patient with thrombocytopenia (70,000/μl) supplementation of whole blood with platelet MP increased CEM by 70%. Our ex vivo experiments demonstrate that collagen-induced platelet-derived MP can modulate clot stability. However, this effect is restricted to anticoagulated whole blood and not observed in platelet-rich plasma or plasma alone. Therefore, interaction of platelet-derived MP with other cellular elements than platelets, e.g. monocytes, may be relevant to promote clot stability. In general, microparticles may be a pharmacological target in the management of hemostatic disorders.