Platelets are critical to arterial thrombosis that underlies myocardial infarction and stroke. Activated platelets, regardless of nature of stimulus, initiate energy-intensive processes that sustain thrombus, while adapting to potential adversities of hypoxia and nutrient deprivation within the densely packed thrombus milieu. We report here that stimulated platelets switch their energy metabolism to aerobic glycolysis by modulating enzymes at key checkpoints in glucose metabolism. We found aerobic glycolysis to, in turn, accelerate flux through pentose phosphate pathway and support platelet activation. Hence, reversing metabolic adaptations of platelets could be an effective alternative to conventional anti-platelet approaches that are crippled by remarkable redundancy in platelet agonists and ensuing signaling pathways. Concurring with this proposition, small-molecule modulators of pyruvate dehydrogenase, pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, all of which disfavor aerobic glycolysis and/or pentose phosphate pathway, restrained the agonist-induced platelet responses ex vivo. These drugs, which include the anti-neoplastic candidate - dichloroacetate, and FDA-approved dehydroepiandrosterone, profoundly impaired thrombosis in mice, thereby exhibiting potential as anti-thrombotic agents.

Key Points: Fn-EDA+ promotes arterial thrombosis. Platelet TLR4 mediates the prothrombotic effect of cellular Fn.

Platelets contribute to 95% of circulating amyloid precursor protein in the body and have widely been employed as a “peripheral” model of neurons in Alzheimer's disease. We sought to analyze the effects of amyloid β (Aβ) on platelets and to understand the underlying molecular mechanism. The Aβ active fragment containing amino acid sequence 25–35 (Aβ _25–35 ; 10–20 μM) was found to induce strong aggregation of human platelets, granule release, and integrin activation, similar to that elicited by physiological agonists. Platelets exposed to Aβ _25–35 retracted fibrin clot and displayed augmented adhesion to collagen under arterial shear, reflective of a switch to prothrombotic phenotype. Exposure of platelets to Aβ peptide (20 μM) resulted in a 4.2‐ and 2.3‐fold increase in phosphorylation of myosin light chain (MLC) and MLC phosphatase, respectively, which was reversed by Y27632, an inhibitor of Rho‐associated coiled‐coil protein kinase (ROCK). AP _25–35 ‐induced platelet aggregation and clot retraction were also significantly attenuated by Y27632. Consistent with these findings, Aβ _25–35 elicited a significant rise in the level of RhoA‐GTP in platelets. Platelets pretreated with reverse‐sequenced Aβ fragment (Aβ _35–25 ) and untreated resting platelets served as controls. We conclude that Aβ induces cellular activation through RhoA‐dependent modulation of actomyosin, and hence, RhoA could be a potential therapeutic target in Alzheimer's disease and cerebral amyloid angiopathy.—Sonkar, V. K., Kulkarni, P. P., Dash, D. Amyloid β peptide stimulates platelet activation through RhoA‐dependent modulation of actomyosin organization. FASEB J. 28, 1819–1829 (2014). www.fasebj.org

Limit of platelet life span (8–10 days) is determined by the activity of a putative “internal clock” composed of Bcl-2 family proteins, whereas the role of other molecular players in this process remains obscure. Here, we sought to establish a central role of proteasome in platelet life span regulation. Administration of mice with inhibitors of proteasome peptidase activity induced significant thrombocytopenia. This was associated with enhanced clearance of biotin-labeled platelets from circulation and reduction in average platelet half-life from 66 to 37 h. Cells pretreated in vitro with proteasome inhibitors exhibited augmented annexin V binding and a drop in mitochondrial transmembrane potential indicative of apoptotic cell death and decreased platelet life span. These cells were preferentially phagocytosed by monocyte-derived macrophages, thus linking proteasome activity with platelet survival. The decisive role of proteasome in this process was underscored from enhanced expression of conformationally active Bax in platelets with attenuated proteasome activity, which was consistent with pro-apoptotic phenotype of these cells. The present study establishes a critical role of proteasome in delimiting platelet life span ostensibly through constitutive elimination of the conformationally active Bax. These findings bear potential implications in clinical settings where proteasome peptidase activities are therapeutically targeted.

Thrombospondin 1 (TSP1) has been suggested as a counter receptor to platelet glycoprotein Ibα that supports initial platelet adhesion in absence of von Willebrand factor (VWF). Conversely, several other studies have shown that TSP1 interacts with VWF and may play a mechanistic role in modulating thrombosis. However, the in vivo evidence to support this mechanism remains unclear. Using intravital microscopy, in a 10% FeCl₃-induced thrombosis model, we report similar platelet adhesion in Tsp1^-/-/Vwf^−/− mice compared with littermate Vwf^−/− mice, suggesting that TSP1 does not mediate initial platelet adhesion in the absence of VWF. Tsp1^−/− mice exhibited prolonged occlusion time and a significant decrease in the rate of thrombus growth (P < .05 vs wild-type), but not in the initial platelet adhesion. Complete deficiency of VWF abrogated the rate of thrombus growth in Tsp1^−/− mice; therefore, we generated Tsp1^-/-/Vwf^+/− mice to determine whether TSP1 modulates thrombus growth under conditions of partial VWF deficiency. Tsp1^-/-/Vwf^+/− mice exhibited delayed thrombus growth kinetics and prolonged occlusion time (P < .05 vs Vwf^+/−). Finally, we demonstrate that platelet-derived TSP1 modulates arterial thrombosis in vivo. We conclude that TSP1 released from platelets plays a mechanistic role in modulating thrombosis in the presence of VWF. Key Points Thrombospondin 1 requires the presence of VWF to modulate arterial thrombosis. Platelet thrombospondin 1 contributes to arterial thrombosis.