Translocation of spin-labeled phospholipids through plasma membrane during thrombin- and ionophore A23187-induced platelet activation

Abstract

After incorporation of spin-labeled phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine analogues in the outer leaflet of the plasma membrane in resting platelets, more than 90% amino-head analogues accumulated within 30 min in the inner leaflet by aminophospholipid translocase activity, while choline analogues mostly remained on the outer leaflet. Platelets were then activated by thrombin or Ca2+ ionophore A23187. No outward movement of internally located spin-labeled aminophospholipids was observed during thrombin-induced activation, whereas the influx of externally located probes increased slightly. During A23187-mediated activation, similar slightly increased influx was observed, while 40-50% of the initially internally located aminophospholipids could then be extracted from the outer leaflet. This sudden exposure on the outer face was dependent on an increase in intracellular Ca2+ and achieved in less than 2 min at 37 degrees C. Inhibition of translocase activity by N-ethylmaleimide did not induce any aminophospholipid outflux. When probes were incorporated on the outer face of the plasma membrane in resting platelets, they were still fully accessible from the extracellular medium after A23187-induced activation. Moreover, they were distributed between the vesicles and remnant platelets in proportion to the external membrane phospholipidic content in each structure. This suggested that no scrambling of plasma membrane leaflets occurred during the vesicle blebbing. Moreover, the spin-labeled aminophospholipids exposure rate and amplitude were unchanged when vesicle formation was inhibited by the calpain inhibitor calpeptin. These results indicate that loss of asymmetry thus inducing generation of a catalytic surface is not the consequence of vesicle formation. Conversely, we propose that vesicle shedding is an effect of PL transverse redistribution and calpain-mediated proteolysis during activation.

Loss of phospholipid asymmetry in human platelet plasma membrane after 1–12 days of storage

“…The rapid outflux of Pam(dox)GroPSer observed in < 9-day-old platelets after A23 187 stimulation was previously described in fresh platelets for Pam(dox)GroPSer and a spin-labeled analogue of phosphatidylethanolamine, l-palmitoyl- [4] with the same velocity and amount of PLs re-exposed on the outer leaflet. Furthermore, the lack of redistribution of Pam(dox)GroPSer after stimulation of > 9-day-old platelets was not only due to loss of the initial asymmetric distribution of the probe between the two leaflets.…”

“…Indeed, we showed that 50% of the initial inside probes were always re-exposed on the outer leaflet of fresh platelets after A231 87 activation regardless of the initial distribution of spin-labeled PL [e.g. Pam(dox)GroPEtn] [4].…”

Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury

“…Of the best characterized calpain isoforms in neurons, calpain I (-calpain) and calpain II (m-calpain) require the presence of micromolar and millimolar Ca 2ϩ ions, respectively, for their activation and subsequent translocation to their target substrates (Suzuki et al, 1992;Basse et al, 1993). To date, many substrates have been identified for calpain, and some are participants in cell death pathways (Gao and Dou, 2000;M.…”

Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

“…This asymmetric distribution of PL is maintained by an aminophospholipid translocase, an Mg 2 ϩ -dependent ATPase that transports PS and PE, but not PC, from outer to inner plasma membrane leaflet (2)(3)(4)(5). Whereas the rate of spontaneous movement of PL between membrane leaflets is normally quite slow, a substantial increase in intracellular Ca 2 ϩ resulting either from agonistinduced activation, or secondary to apoptosis or immune injury, initiates rapid transbilayer migration of all plasma membrane PL, collapsing the normal PL asymmetry (1,(6)(7)(8)(9). The resulting exposure of PS and PE at the surface of activated or injured blood cells or endothelium serves to promote blood coagulation by providing binding sites for coagulation enzyme complexes including factor VIIIaIXa (tenase) and factor VaXa (prothrombinase) (1,10,11).…”

“…Elevated cytosolic Ca 2 ϩ has been shown to initiate rapid bidirectional transbilayer redistribution of all plasma membrane PL (6-8, 14, 15), although evidence for concomitant selective and vectorial egress of PS and PE to the cell surface has also been reported (9,16). Proposed mechanisms for this effect of intracellular Ca 2 ϩ on the movement of PL between plasma membrane leaflets include ( i ) Ca 2 ϩ -induced vesiculation of the plasma membrane (11,17); ( ii ) calpain-mediated proteolysis of the submembrane cytoskeleton, liberating PL headgroups from interaction with endofacial proteins (18); ( iii ) loss of polyamine-membrane associations that might serve to stabilize PL asymmetry (15,19); ( iv ) destabilization of the PL bilayer by the complex of Ca 2 ϩ and the polyanionic PL, phosphatidylinositol 4,5-bisphosphate (19,20); ( v ) action of a Ca 2 ϩ -dependent membrane protein with PL scramblase activity.…”