Fatal Dysfunction and Fragmentation of Thrombin-Stimulated Platelets

Introduction: Platelets play a key role in formation of protective hemostatic blood clots and pathological obstructive thrombi. Under (patho)physiological conditions, chemically activated platelets change their morphology, express adhesive molecules, undergo aggregation, secrete procoagulant substances, and induce mechanical contraction (retraction) of the blood clots. Despite the vital importance of these platelet functions, the subsequent fate of activated platelets is largely unknown. We hypothesize that activated platelets undergo late alterations that determine their fate and may have a pathogenic importance in thrombotic and hemostatic disorders.

Methods: We used a combination of confocal microscopy, immunofluorescence, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements to study deferred structural, metabolic, and functional consequences of thrombin-induced activation of viable human platelets, either suspended in platelet-rich plasma or isolated by gel-filtration.

Results: Visualized by confocal microscopy, fluorescently labeled platelets in thrombin-induced plasma clots initially underwent shape changes characteristic of platelet activation, but in about 30 min many platelets and platelet aggregates broke up into organelle-containing vesicular fragments. There were two types of platelet-derived vesicles differing in their size and cellular origin: one smaller type was shedding from the tips of filopodia, while the other type resulted from fragmentation of platelet bodies. Concurrently with the fragmentation, thrombin-activated platelets displayed dramatically altered intracellular distribution of F-actin and septins detected as intense fluorescent clusters with a ~2-fold increase in the intensity of septins 2 and 9 and a ~300-fold increase in the F-actin staining. Synchronously with the structural alterations, thrombin induced a time-dependent reduction of the mitochondrial membrane potential (Δψ_m) in platelets. The overall fluorescence intensity of the Δψ_m-sensitive MitoTracker dye in freshly formed thrombin-initiated plasma clots dropped 2- and 4-fold after 60 min and 90 min, respectively. A drop of Δψ_m inversely correlated with an increase of the fraction of disintegrated platelets (r=-0.93, p<0.01). Flow cytometry showed enhanced phosphatidylserine exposure in thrombin-activated platelets, either with or without mitochondrial depolarization. Thrombin caused a significant 59% decrease of the average ATP content in activated platelets relative to untreated platelets after 60 min of incubation. Remarkably, the initial drop of Δψ_m and ATP content was almost concurred with the termination of contraction of the platelet-rich plasma clot measured as a 90%-decrease of platelet-generated contractile stress. Unexpectedly, no activation of caspase 3/7 was detected in platelets after 90 min of treatment with thrombin. Meanwhile, calpain activity detected in platelets 90 min after thrombin treatment was 6.5-fold higher compared to untreated platelets. Moreover, calpain inhibition caused a ~30-min delay in the commencement of thrombin-induced platelet fragmentation.

Conclusions: Our findings indicate that following thrombin-induced platelet activation, a substantial fraction of platelets undergo time-dependent dysfunction and structural disintegration into subcellular particles. The fragmentation of platelets is accompanied by dramatic rearrangements of platelet cytoskeletal components, including polymerization, clustering, and redistribution of actin and septins. Thrombin-induced platelet fragmentation is concurrent with severe impairment of platelet functionality, including mitochondrial depolarization, ATP depletion, and loss of platelet contractility. The lack of caspase activity and increased calpain activity in energetically exhausted thrombin-treated platelets undergoing fragmentation suggests a calpain-dependent platelet death pathway. These studies indicate that such a form of platelet death may be an underappreciated mechanism for enhanced elimination of platelets from the circulation in (pro)thrombotic conditions or under other conditions once they have performed their functions. Work supported by the Program for Competitive Growth at KFU and AHA grant 17SDG33680177.