Procoagulant microparticles (MPs) in the circulation are increasingly recognized as playing a role in haemostasis and inflammation and may prove useful biomarkers for clinical studies. Platelets are known to generate MPs in response to stimulation, and platelet-derived MPs (PDMPs) form the majority of the MPs found in the normal circulation and can be elevated in a number of disease states. The current study has focused on the procoagulant activity of platelets and PDMPs following stimulation with the collagen-mimetic peptide CRP-XL. Their activity in accelerating thrombin generation can be accurately measured by the Calibrated Automated Thrombogram (CAT) assay using 1pM TF reagent to initiate the reaction, with the finding that plasma from patients with chronic renal disease has significant thrombin generation due to increased procoagulant activity of MPs. Conversely premature MI patients in a stable condition have thrombin generation comparable to matched healthy control. In all subjects, removal of MPs from plasma by filtration (>0.2 μm) eliminates this procoagulant activity, indicating the importance of MP size in driving the procoagulant response. The procoagulant activity of activated platelets and PDMPs showed a strong correlation with annexin-V binding measured using flow cytometry. Comparison of the regulatory mechanism of the procoagulant activity of platelet and PDMPs upon activation with platelets undergoing apoptosis showed that although both activation and apoptosis resulted in exposure of the procoagulant surface, apoptotic platelets did not release procoagulant MPs or show any markers of activation such as P-selectin expression, fibrinogen binding or aggregation. Reactive oxygen species (ROS) are generated during platelet activation or apoptosis mainly through the NADP(P)H oxidase pathway. The procoagulant activity of platelets and PDMPs was significantly attenuated (as measured by thrombin generation and annexin-V binding) by inhibition of the NAD(P)H oxidase pathway by apocynin, which had similar inhibition on other platelet responses including on 12-HETE generation, TxB2 production and aggregation. However antioxidants only inhibited apoptosis-induced platelet procoagulant activity. These data demonstrate significant involvement of ROS in platelet procoagulant activity induced by both activation and apoptosis and suggest the involvement of lipid peroxidation during apoptosis
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