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Platelet activation with GPIIb/IIIa binding to fibrinogen, aggregation and interaction with leukocytes constitute the principal mediator of thrombosis. Although the clinical benefits of GPIIb/IIIa antagonists have been documented, the relationship between their anti-platelet properties, platelet activation and binding to leukocytes is still debated. We investigated the effects of abciximab, tirofiban, roxifiban, and an anti-P-selectin blocking monoclonal antibody (Mab) on isolated human platelet aggregation using optical aggregometer, and on platelet P-selectin and GPIIb/IIIa expression, and platelet-neutrophil binding using flow cytometry. Thrombin at 0.025 U/ml induced maximal platelet aggregation (76.3 +/- 2.6%), P-selectin expression (88.5 +/- 4%), GPIIb/IIIa activation (PAC-1 binding, 86.2 +/- 8.9%) and platelet-neutrophil binding (58.0 +/- 6.4%). The GPIIb/IIIa antagonists inhibited in a concentration-dependent manner platelet aggregation (IC50 of 100 nM for abciximab and tirofiban and 50 nM for roxifiban) and PAC-1 binding, without any effect on P-selectin. None of these agents affected significantly platelet-neutrophil binding, whereas an anti-P-selectin Mab abolished this binding and amplified the effect of abciximab on platelet aggregation. These results indicate that the effects of these GPIIb/IIIa antagonists on platelet aggregation are not related to inhibition of platelet activation, as P-selectin levels and platelet-neutrophil binding remained unaffected, and highlight the participation of P-selectin with GPIIb/IIIa in platelet aggregation.

The selectin family of cell-adhesion molecules contributes to the interactions of leukocytes and platelets at the site of vascular injury. Such interactions enhance inflammatory reactions and thrombus formation during the arterial response to injury. In this study, we investigated the effects of a selectin inhibitor (Fucoidan) on platelet and neutrophil interactions after arterial injury produced by angioplasty in pigs. [ 51 Cr]-platelet deposition and [ 111 In]-neutrophil adhesion were quantified on intact, mildly, and deeply injured carotid arterial segments, produced by balloon dilation in control (saline, n = 7) and Fucoidan-treated (i.v.; 1 mg/kg, n = 6; 5 mg/kg, n = 5) pigs. In the control group, platelet deposition (×10 6 /cm 2 ) was influenced by the severity of injury and increased significantly (p < 0.05) from 0.06 ± 0.06 on intact endothelium to 3.8 ± 0.6 and 33.6 ± 4.9 on mildly and deeply injured segments, respectively. Fucoidan, 1 mg/kg, had no significant effect, although doses of 5 mg/kg reduced platelet deposition by 73% on deeply injured segments. The level of neutrophil adhesion (×10 3 /cm 2 ) was also influenced by the severity of injury: it increased in the control group from 8.8 ± 2.5 on intact endothelium to 226.6 ± 45.5 and 397.4 ± 61.3 on mildly and deeply injured arterial segments, respectively (p < 0.05). Again, 1 mg/kg Fucoidan had no effect, although doses of 5 mg/kg reduced neutrophil adhesion by 92% and by 84% on mildly and deeply injured segments, respectively. The effects of Fucoidan were associated with a 51% decrease in the vasoconstrictive response at the site of arterial injury. However, Fucoidan had no significant effect on either platelet aggregation or activated clotting time (ACT). In the in vitro perfusion experiments, Fucoidan inhibited both isolated platelet, and neutrophil, adhesion to damaged arterial surfaces. This inhibition was more pronounced in experiments using mixed cell preparations, indicating that Fucoidan interferes with platelet and neutrophil interactions. These results highlight the importance of selectins in the acute physiopathologic reactions related to platelet-neutrophil interactions after arterial injury.