Essentials Mitochondrial hyperpolarization enhances the conversion of platelets to a procoagulant phenotype. Mitochondrial calcium uniporter (MCU) function is essential in procoagulant platelet formation. Mitochondrial calcium uniporter deletion does not impact other aspects of platelet activation. Ablation of MCU results in the emergence of a permeability transition pore-independent pathway. SUMMARY: Background Procoagulant platelets comprise a phenotypically distinct subpopulation of activated platelets with high-level phosphatidylserine externalization. When initiated by co-stimulation with thrombin and a glycoprotein VI (GPVI) agonist, the transition to the procoagulant phenotype is mediated by extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation. Objectives The intracellular mechanisms coordinating these distinct cytoplasmic and mitochondrial processes remain unclear. The mitochondrial calcium uniporter (MCU) protein is a central component of the transmembrane ion channel that allows the passage of Ca from the cytosol into the mitochondrial matrix. Here we investigate the role of the MCU in the regulation of procoagulant platelet formation. Results Procoagulant platelet formation was directly correlated with pre-stimulatory mitochondrial transmembrane potential, a key determinant of calcium flux from the cytoplasm to the mitochondria. The role of MCU in the regulation of procoagulant platelet formation was investigated using MCU null platelets. Procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation. In contrast, neither granule release nor initial integrin activation was altered in response to stimulation. In the genomic absence of MCU, developmental induction of an alternative intracellular pathway partially rescued procoagulant platelet formation. Conclusion These results identify a key role for the mitochondrial calcium uptake channel in the regulation of mPTP-mediated procoagulant platelet formation and suggest a novel pharmacologic target for procoagulant-platelet-related pathologies.

Our objective was to assess the effect of nitric oxide added to the sweep gas of the oxygenator during cardiopulmonary bypass (CPB) in infants on platelet count, platelet function, clinical outcomes, and safety. A randomized, double‐blinded, placebo‐controlled clinical trial in infants less than a year of age undergoing cardiac surgery requiring CPB was undertaken. Nitric oxide at a dose of 20 ppm was added to the sweep gas in the treatment group. Blood was collected at baseline and prior to separation from CPB to measure platelet count and function as determined by responsiveness to specific agonists. Clinical outcomes were observed through hospital discharge. Methemoglobin levels were measured preoperatively, at the conclusion of CPB, and upon admission to the ICU. Forty patients consented and were randomized in the trial. Eighteen patients were randomized to the treatment group and 22 were included in the placebo group. The groups were similar in terms of age, weight, gender, and surgical complexity. No significant differences were found in measures of platelet count, platelet response to agonist, or clinical outcomes. Patients in the treatment group had higher methemoglobin levels after receiving nitric oxide, but no levels approached toxicity (maximum 2.4%). Nitric oxide added to the sweep gas of the oxygenator during CPB in infants did not have an appreciable effect on the preservation of platelet count, platelet responsiveness to agonist, or clinical outcomes. Methemoglobin levels were increased after receiving nitric oxide but were far below a toxic level of 15%.

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation. To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs. In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibition of SiPP formation in the absence of VWF, in VWF with deletion of the GPIbα-binding A1 domain, and in the presence of antibodies blocking the interaction of GPIb-IX and VWF. Calcium-dependent processes play an essential role in agonist-initiated PSer externalization. However, extracellular Ca2+ is not required for SiPP formation. Inhibition of Ca2+ entry, removal of extracellular Ca2+ and chelation of intracellular Ca2+, all had no effect on SiPP formation. Caspase activation mediates PSer externalization in the apoptotic platelet, but neither pharmacologic inhibition of caspases nor calpain inhibitors prevented SiPP formation. In contrast to the negligible roles of calcium- and caspase mediated events, SiPP formation is abrogated both in the absence of the mPTP potentiator cyclophilin D and in cyclosporine A-treated platelets. Furthermore, SiPP formation was closely correlated with mPTP formation as measured by loss of mitochondrial transmembrane potential. Together, these results identify a new mechanistic pathway of SiPP formation distinct from previously described initiating mechanisms for PSer externalization. Targeting of this distinct pathway may provide a novel pharmacologic strategy for the treatment of high shear hematopathologies, like arterial thrombosis. Disclosures No relevant conflicts of interest to declare.