Rapid Procoagulant Phosphatidylserine Exposure Relies on High Cytosolic Calcium Rather Than on Mitochondrial Depolarization

Abstract

Objective-Relationships between intracellular Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) and apoptotic events, such as mitochondrial depolarization (⌬⌿m loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. Methods and Results-Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), andJurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. ⌬⌿m loss occurred when platelets were stimulated in Ca 2ϩ medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca 2ϩ ] cyt increase, and ⌬⌿m loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca 2ϩ release from the endoplasmic reticulum and, consequently, external Ca 2ϩ influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not ⌬⌿m loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. Conclusions-Whether ⌬⌿m loss occurs or not, PS exposure is triggered by a high [Ca 2ϩ ] cyt increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca 2ϩ ] cyt increase, independently of its effect on mitochondrial permeability transition pore. Key Words: platelets Ⅲ Jurkat Ⅲ apoptosis Ⅲ procoagulant phosphatidylserine Ⅲ thrombin plus convulxin P hospholipid scrambling, leading to phosphatidylserine (PS) exposure to the external leaflet of cell membranes, characterizes blood platelet procoagulant activation. The essential role of phospholipid reorganization on the surface of stimulated blood cells is illustrated by the existence of Scott syndrome, a rare bleeding disorder caused by defective membrane scrambling in platelets and hematopoietic cells such as lymphocytes (reviewed in 1 ).PS exposure also characterizes apoptosis and occurs during platelet storage with other typical morphological and biochemical features of apoptosis (reviewed in 2 ), including loss of the mitochondrial transmembrane potential (⌬⌿m). [3][4][5] ⌬⌿m loss is caused by opening of a channel, the mitochondrial permeability transition pore (mPTP), and requires an increase in mitochondrial Ca 2ϩ . 6 The mPTP is composed of several proteins, including the adenine nucleotide translocator (ANT), the voltage-dependent anion channel, and cyclophilin D (CypD). 7 mPTP opening is blocked by cyclosporin A (CsA), which binds to CypD and dissociates it from the translocator. 8 In platelets, the physiological procoagulant activity is triggered by dual agonist stimulation with thrombin (Thr) plus convulxin (Cvx), the latter stimulating GPVI, a platelet collagen receptor. 9 This leads a subpopulation of platelets, named coated-platelets, to express PS and high levels of several procoagulant proteins on their surface. 10 Studies have shown that both ⌬⌿m loss and ...

Cellular Mechanisms Underlying the Formation of Circulating Microparticles

“…108 The results demonstrated that-regardless of the loss of membrane mitochondrial permeability-PS exposure is triggered by a marked increase in cytosolic calcium ions concentrations. Challenging previous observations, these findings clearly indicate that PS exposure can occur even without loss of membrane mitochondrial permeability.…”

“…99 Accordingly, PS exposure is triggered by a marked increase in cytosolic Ca 2ϩ concentrations in platelets regardless of a loss of membrane mitochondrial permeability. 108 In any case, cyclosporin A holds promise for the pharmacological modulation of MP shedding in the clinical setting of kidney transplantation because treatment with cyclosporin A results in an earlier reduction of endothelial-derived MPs levels than in patients treated with tacrolimus/mycophenolate. 110 The above evidence indicates that different molecular pathways (caspase-dependent mechanisms, increased mitochondria permeability, sustained increase in intracellular calcium ions) can mediate membrane lipid scrambling and ultimately lead to PS exposure.…”

Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

“…Effective inducers of the latter response are the combination of the glycoprotein VI agonist convulxin (Cvx) with thrombin (Thr), or otherwise Ca 2ϩ ionophores such as A23187 and ionomycin (16,17). Common to these agonists is that they induce a high and sustained rise in cytosolic Ca 2ϩ , which is considered to trigger the Ca 2ϩ -operated membrane protein, TMEM16F, which regulates the scrambling of phospholipids and exposure of PS (18,19).…”

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ib - 14-3-3 ] association

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