Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels
Authors: Choo HyoJung , Saafir Talib , Wagner Mary , Jobe Shawn 
Published: October 2019

Abstract
Abstract 2024
Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.
It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.
Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.
The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.
Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure.

Disclosures:
No relevant conflicts of interest to declare.