Abstract
Asymmetry of phospholipids across the plasma membrane bilayer is a feature of all eukaryotic cells. When platelets are stimulated with certain agonsists, phospholipids are randomized by the action of a Ca2+-dependent scramblase enzyme, resulting in exposure of the anionic aminophospholipid phosphatidylserine (PS) on the outer leaflet that provides a procoagulant surface, catalyzing thrombin formation. We have previously demonstrated that the procoagulant surface of activated platelets persists in vitro for at least 4 hrs (Blood100:63b, 2002). Such persistence may propagate thrombosis in vivo when activated procoagulant platelets re-enter the circulation after fibrinolysis. There is currently little information concerning the mechanisms by which the procoagulant surface persists on activated platelets. In this in vitro study, the Ca2+-chelator BAPTA (0.1 μmol/109 platelets) was used to investigate the role of intracellular Ca2+ (Ca2+i) in procoagulant surface expression and persistence; PS expression was determined flow cytometrically by the binding of annexin A5-FITC. Unexpectedly, chelation of Ca2+i resulted in a 2–2.5x-fold increase in PS expression on the surface of platelets 5 min after activation with thrombin or thrombin+collagen (T+C), and this persisted for up to 4 hrs (last time point tested). Since PS expression is a hallmark of apoptosis in nucleated cells, we also examined another platelet apoptosis marker, the collapse of the mitochondrial inner membrane potential (ΔΨm), by flow cytometry using the potential-sensitive dye TMRM; PS expression was measured concurrently. This allowed us to distinguish between activated platelets expressing PS with an intact ΔΨm and apoptotic platelets expressing PS with a dissipated ΔΨm. 70–85% of the thrombin- or T+C-activated platelets expressing PS had an intact ΔΨm, which persisted for up to 4 hrs after activation. Thus, PS expression can occur independently of ΔΨm loss. However, chelation of Ca2+i with BAPTA resulted in 60–70% of the thrombin- or T+C-activated platelets persistently expressing PS to also have a collapsed ΔΨm, indicating that apoptotic pathways similar to those found in nucleated cells may modulate PS expression in platelets and may depend on Ca2+i concentrations. Caspases and calpain are centrally involved in apoptotic signaling and execution in nucleated cells. Caspases-9 and -3 have been identified in human platelets and may be responsible for downstream activation of calpain. We examined the effects of Ca2+i chelation in thrombin- and T+C- activated platelets on the activation of procaspases and calpain by Western blotting. In keeping with our observations of increased PS expression with concurrent ΔΨm loss in activated platelets with Ca2+i chelation, we observed cleavage of both procaspase-9, procaspase-3 and calpain, which did not occur in activated platelets without Ca2+i chelation. Taken together, our results indicate that Ca2+i levels in activated platelets may serve as a decisional checkpoint for the apoptotic pathway in human platelets, where procaspase-9 and procaspase-3 along with downstream calpain may function in a Ca2+-sensitive manner to protect platelets against PS exposure and ΔΨm collapse.
The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation
