Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca2+-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.

Objective-In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and the various platelet receptors for thrombin. Methods and Results-We demonstrate that procoagulant activity is related to a sustained increased [Ca 2ϩ ] i , which in turn depends on extracellular Ca 2ϩ influx. Increased PtdSer exposure coincides with increased [Ca 2ϩ ] i and was observed in a subpopulation (Ϸ14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIb␣ made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca 2ϩ ] i and procoagulant activity induced by collagen plus thrombin. Key Words: thrombin Ⅲ collagen Ⅲ platelets Ⅲ procoagulant activity Ⅲ protease-activated receptors P latelet membranes have an asymmetrical distribution of phospholipids over the two membrane leaflets with aminophospholipids almost exclusively present in the cytoplasmic leaflet. This asymmetrical distribution can be dissipated by the action of a phospholipid scramblase, resulting in surface exposure of phosphatidylserine (PtdSer). Presence of PtdSer in the exoleaflet of the platelet plasma membrane is of physiological importance because it enhances thrombin formation, which is essential to the formation of a stable hemostatic plug or which contributes to the formation of stable thrombi that may occlude blood vessels. 1 For years it has been shown that a combination of collagen and thrombin is a far more potent agonist in generating procoagulant platelet surfaces than each of the individual agonists alone. 2 The mechanism behind this synergistic action of thrombin and collagen, however, remains to be elucidated. A key element in the process is the level of the platelet cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ). 3 It has been suggested that the synergistic effect of thrombin and collagen in exposing PtdSer is based on the ability of this combination of agonists to induce a high calcium influx that persists over a sufficiently long period to stimulate scramblase that transfers PtdSer to the outer-leaflet and to inhibit aminophospholipid translocase to prevent PtdSer from being pumped back to the inner leaflet. 4,5 We hypothesize that thrombin and collagen must act together through distinct platelet receptors and signaling pathways that reinforce their individual capacities in elevating the platelet intracellular calcium level. Conclusions-TheGlycoprotein (GP)VI is the maj...

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