Background-Glycoprotein (GP) IIb/IIIa antagonists inhibit platelet aggregation, an activity attributed to the clinical benefits of these drugs in settings that involve acute coronary thrombosis. However, platelet activation and subsequent aggregation are now known to cause the release of a soluble form of CD40 ligand (sCD40L), a prothrombotic and proinflammatory protein with GP IIb/IIIa binding activity and an established role in atherosclerotic lesion progression. The present study was designed to determine what effect GP IIb/IIIa antagonists have on the release of sCD40L. Methods and Results-Doses of eptifibatide, abciximab, and tirofiban that inhibited platelet aggregation by at least 80% also inhibited sCD40L release in vitro (by 85%, 57%, and 80%, respectively). When platelets were stimulated with a thrombin receptor agonist, inhibition by GP IIb/IIIa antagonists occurred without affecting the release of ␤TG, an ␣-granule protein. Unexpectedly, concentrations of the 3 antagonists that blocked aggregation by only 20% to 50% potentiated the release of sCD40L (by 19% to 26%). Platelets from aspirin-treated individuals were partially protected from sCD40L release, but only when the agonist was collagen, an affect augmented by the addition of GP IIb/IIIa antagonists. Conclusions-These studies suggest that the mechanisms responsible for the clinical benefits of GP IIb/IIIa antagonists (at doses that optimally inhibit aggregation) and of aspirin may not be limited to the inhibition of thrombosis through their blockade of platelet aggregation but may also involve the inhibition of inflammation and thrombosis through their blockade of sCD40L release. These studies also provide a mechanism by which suboptimal doses of GP IIb/IIIa antagonists may be proinflammatory. . Arterial thrombosis in these settings is dependent on platelet aggregation, a process mediated by the binding of soluble fibrinogen to GP IIb/IIIa on the surface of stimulated platelets. GP IIb/IIIa antagonists are effective in these indications because they block the binding of soluble fibrinogen to GP IIb/IIIa and therefore inhibit platelet aggregation. Interestingly, some trials suggest less accrual of events such as myocardial infarction and death in the treatment arm than are observed in the placebo arm during the year after an event. Thus, the clinical benefit extends well past the time of acute administration of GP IIb/IIIa antagonists, which is often less than 20 hours. 2,3 Although it has been considered that thrombosis-mediated ischemia is a culprit for long-term negative consequences, including atherosclerotic lesion progression after acute arterial coronary thrombosis, it is also possible that thrombosis itself could generate such factors, which could be blocked by GP IIb/IIIa antagonists.Platelet stimulation and subsequent aggregation are known to cause the expression or release of several factors that could affect vascular pathology. These include TXA2, a costimulator of platelets that has vasoconstrictive activity; P-selectin, an ␣-granule...

The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide having the tethered ligand sequence (thrombin receptor agonist peptide or TRAP). We conducted a mutational analysis of extracellular residues of the receptor potentially involved in interaction with both the tethered ligand and the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laevis oocytes or inositol phosphate accumulation in COS-7 cells was used to assess receptor activation. We have also examined the binding of a radiolabeled TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses without significantly altering thrombin-induced responses or TRAP binding. Several point mutations and deletion of extracellular domains (DeltaEC3, DeltaNH3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second extracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of thrombin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants with the ability to dissociate receptor-specific binding from functional activity.

Background-Cardiopulmonary bypass (CPB) is known to induce platelet activation, thrombosis, thrombocytopenia, and a systemic inflammatory response. It is known that CD40 ligand (CD40L) exists in platelets, that a soluble form of this protein (sCD40L) is released on platelet activation, that platelets are the primary source of sCD40L in blood, and that sCD40L is involved in thrombosis and inflammation. The present study was designed to determine whether sCD40L is released during CPB. Methods and Results-Blood was obtained from patients undergoing CPB-requiring surgery and analyzed for sCD40L, interleukin-6, and platelet factor 4 and ␤-thromboglobulin (markers of platelet activation). Platelets were also isolated and analyzed for their levels of CD40L. Plasma levels of sCD40L increased Ͼ1.7-fold (from 0.29 to 0.51 ng/mL, Pϭ0.001) within 1 hour on CPB and increased further to 3.7-fold (to 1.08 ng/mL, Pϭ0.03) 2 hours after the procedure. Half of the released sCD40L was cleared in 2 hours, which allowed the sCD40L to return to approximately baseline levels 8 hours after the procedure. The platelet content of CD40L was decreased by 40% (2.675 to 1.64 ng/10 8 platelets, Pϭ0.001) 1 hour after initiation of CPB and was similar to that observed for platelet factor 4 and ␤-thromboglobulin. Interleukin-6, a marker of inflammation, also increased during CPB. Conclusions-The present study demonstrates that CPB causes an increase in the concentration of plasma sCD40L. The corresponding decrease in platelet CD40L suggests that this prothrombotic and proinflammatory protein was derived primarily from platelets and may contribute to the thrombotic and inflammatory complications associated with CPB.

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