Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine.

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.

Platelet function assays.

The roles of platelets in hemostasis and arterial thrombosis involve their adherence to sites of vessel injury or ruptured atherosclerotic plaques, aggregation to form hemostatic plugs or thrombi, and acceleration of the coagulation cascade leading to the formation of thrombin. These roles of platelets are described in this review, hereditary platelet defects and other abnormalities associated with bleeding disorders are listed, and the various aggregating agents are discussed. A number of tests of platelet function are reviewed, including a description of their advantages and disadvantages: bleeding time determination; measurement of platelet aggregation in citrated platelet-rich plasma by recording changes in light transmission; measurement of platelet aggregation in citrated whole blood by impedance aggregometry; measurement of platelet-related hemostasis with the high shear Platelet Function Analyzer (PFA-100) system and the Ultegra Rapid Platelet Function Assay; use of the Cone and Plate(let) Analyzer to measure platelet adherence and aggregation under conditions of high shear; measurement of secretion of granule contents (ATP, 14C-serotonin, platelet factor 4, beta-thromboglobulin) and the formation of thromboxane B(2); and use of flow cytometry to assess the state of platelet activation (including conformational changes in membrane glycoproteins and surface expression of P-selectin and phosphatidylserine) ex vivo and in vitro following addition of agonists, and to measure levels of platelet membrane glycoproteins in the detection of inherited deficiencies.

Calpain inhibition by calpeptin does not prevent APLT activity reduction in PS-exposing platelets, but calpeptin has independent pro-apoptotic effects.

Exposure of procoagulant phosphatidylserine (PS) on the surface of activated platelets is not readily reversible and this may propagate thrombosis. Persistence of PS exposure may be attributed, at least in part, to a continued reduction of the activity of aminophospholipid translocase (APLT), that transports PS from the outer to the inner membrane leaflet. We investigated whether calpain is involved in the inhibition of APLT activity. In flow cytometric investigations, using the inhibitors calpeptin or E64d at a concentration that blocks calpain activation, we found that calpain is not responsible for the reduction in APLT activity that results in persistence of PS exposure. Unexpectedly, we found that the inhibitors had additional effects independent of blocking calpain. Incubation of resting platelets with calpeptin resulted in a subpopulation of platelets with increased intracellular Ca(2+) and persistent PS exposure. The inhibitors also increased the proportion of platelets with persistent PS exposure in suspensions stimulated with thrombin and/or collagen or the Ca(2+)-ionophore A23187 under conditions in which calpain was not activated or in which its activation was completely blocked; P-selectin expression on thrombin and/or collagen-stimulated platelets was inhibited. Furthermore, in stimulated platelets, calpeptin increased the proportion of the PS-exposing platelets expressing a second apoptotic hallmark, collapsed mitochondrial inner membrane potential (DeltaPsi(m)). These additional effects of calpeptin on platelet regulation of intracellular Ca(2+) levels and apoptotic-like events should be taken into account when it is used as an inhibitor of calpain.

Platelet aggregation and adenosine diphosphate/adenosine triphosphate receptors: a historical perspective.

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.

Ethanol enhances the inhibitory effect of an oral GPIIb/IIIa antagonist on human platelet function.

Ethanol is a commonly used substance that can significantly influence platelet responses when combined with therapeutic drugs. In in vitro studies, we combined ethanol with LY309562, a novel 2,6-disubstituted isoquinolone RGD mimic that competes for fibrinogen binding to GPIIb/IIIa. Ethanol inhibits aggregation and secretion, partly by inhibiting thromboxane A(2) formation. We measured aggregation and secretion of dense granule contents by platelets labeled with [(14)C] serotonin in plasma from blood anticoagulated with FPRCH(2)Cl (PPACK). Alone, LY309562 dose-dependently inhibited aggregation induced by 10 micromol/L adenosine diphosphate, 1 microg/mL collagen, 2 micromol/L U46619 (a thromboxane A(2) mimetic), or 15 micromol/L SFLLRN (protease-activated receptor-1-activating peptide); inhibition was complete at 1 micromol/L LY309562 and partial at 0.1 micromol/L (50% inhibitory concentration [IC(50)] 0.19-0.33 micromol/L). Secretion induced by collagen, U46619, and SFLLRN was also inhibited by LY309562 (IC(50) 0.08-0.31 micromol/L). At inhibitory concentrations of LY309562, ethanol (2 or 4 mg/mL) further inhibited responses to collagen, U46619, and SFLLRN (IC(50) for aggregation 0.12-0.16 micromol/L; for secretion 0.04-0.12 micromol/L). Responses of aspirin-treated platelets to U46619 were also inhibited, indicating that ethanol was not acting solely by inhibiting thromboxane A(2) formation. Because it is likely that our results with LY309562 are representative of results with other GPIIb/IIIa antagonists, our in vitro data suggest that the concomitant use of GPIIb/IIIa antagonists and consumption of alcoholic beverages may result in further impairment of platelet participation in hemostasis and thrombosis.