Platelet and leukocyte activation and their interaction : in experimental prothrombotic and inflammatory states

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Sammanfattning: Atherosclerotic disease is associated with inflammation and thrombosis, both of which involve multi-cellular activation and interaction. The present work has investigated the mechanisms of plateletleukocyte cross-talk in vitro, and explored the possibility of multi-cellular activation in vivo in prothrombotic and inflammatory models in healthy volunteers. Whole blood flow cytometric methods for measuring platelet-leukocyte aggregates (PLAs) were developed and evaluated. The methods involved minimal sample manipulation, avoiding centrifugation or red cell lysis, and no or only mild post-incubation fixation, to minimize in vitro artefacts. Antibodies that block ligand-receptor interactions had little effect in unstimulated samples but totally inhibited agonist-induced PLA formation, indicating that the assays reflect circulating PLAs closely. Gating on leukocyte fluorescence improved assay efficiency. Data from these studies show that all leukocyte subtypes can form heterotypic conjugates, and that activation of either platelets or leukocytes can enhance PLA formation. Granulocytes and monocytes predominate among PLAs. Multi-ligand-receptor systems are involved in PLA formation, but P-selectin bridging to PSGL-1/CD15s is the principal bridging mechanism under most conditions. Activation of leukocytes with fMLP evoked platelet activation. The effect did not depend on PLA formation, and was markedly inhibited by PAF antagonism or 5-lipoxygenase inhibition. Collagenactivated platelets evoked leukocyte activation. The effect was not inhibited by a thromboxane A2 receptor antagonist, which abolished collagen-induced platelet P-selectin expression, but was inhibited by blocking PLA formation, indicating that the effect depended on cell-cell contact. Interestingly, GPIIb/IIIa blockade by an nonpeptide inhibitor, SR121566, inhibited platelet-leukocyte cross-talk, and a cocktail containing antibodies to P-selectin, GPIIb/IIIa, and CD 1 1b/CD 18 abolished fMLP-induced leukocyte activation. Thus, platelet-leukocyte cross-talk involved multiple mediators and mechanisms, and adhesion molecules seem to be important in cellular signaling during platelet and leukocyte activation. Strenuous exercise provoked a prothrombotic state, and endotoxin injection induced systemic inflammation in humans. Both interventions resulted in multicellular activation in vivo and enhanced thrombin generation. Platelet activation was shown by increased circulating P-selectin positive platelets and platelet-platelet aggregates, elevated plasma soluble P-selectin levels, and enhanced platelet responsiveness to in vitro stimulation. Leukocyte activation was shown, with increased CD11b expression in circulating leukocytes, elevated plasma elastase levels, as well as enhanced leukocyte reactivity to in vitro stimulation. This resulted in enhanced platelet-leukocyte interaction, as evidenced by increased circulating PLAs and enhanced agonist-induced PLA formation in vitro. Endothelial activation and thrombin generation were shown by markedly increased plasma vWF and F1+2 levels, respectively. Aspirin ingestion at a daily dose of 500 mg reduced platelet P-selectin expression at rest slightly, and inhibited neutrophil responsiveness to fMLP stimulation, but did not counteract the prothrombotic effects of exercise. Adenosine infusion at a dose of 40 [my]g/Kg/min induced leukocytosis in the absence of endotoxemia, probably due to inhibition of leukocyte adhesion and migration on/into the vessel wall. Adenosine infusion attenuated endotoxemia-induced platelet and leukocyte activation moderately, as shown by modest inhibition on platelet reactivity and more marked inhibition of leukocyte adhesion and migration. The latter also resulted in the retention of more activated leukocytes and PLAs in the circulation, and might thus limit leukocyte-mediated tissue damage. Taken together, the present studies suggest that thrombosis and inflammation are closely related pathophysiological processes that involve multicellular activation of platelets, leukocytes, and endothelial cells and enhanced intercellular cross-talk. Aspirin does not counteract prothrombotic effects of exercise. Adenosine is a potentially useful antiinflammatory agent, but the treatment strategy needs to be optimized to achieve clinically relevant effects.

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