Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital model.

BACKGROUND: Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. OBJECTIVES: We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. METHODS: Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe(-/-) mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. RESULTS: Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. CONCLUSIONS: Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo.

Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

BACKGROUND: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). OBJECTIVE: The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. RESULTS: At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). CONCLUSIONS: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.