A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

Abstract

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet–vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by ∼89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet–collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

“…Experiments were conducted as described 28 . Platelets were isolated and labeled with 5-carboxyfluorescein diacetate succinimidyl ester as described.…”

“…The right common carotid artery was dissected free and ligated vigorously near the carotid bifurcation for 5 min to induce vascular injury. Adhesion of autogeneous platelets was assessed before and after carotid injury by in vivo video microscopy 28 . Videotaped images were evaluated using a computer-assisted image analysis program 28 (Cap Image 7.4, H. Zeintl).…”

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

“…To directly test the contribution of the collagen GPVI receptor in this thrombus growth phenotype in ceacam1 Ϫ/Ϫ arterioles, we deleted GPVI in vivo using JAQ1 treatment (100 g/mouse) for 5 days before ferric chloride injury and intravital studies. 23 A different cohort of ceacam1 Ϫ/Ϫ mice were similarly treated with control IgG (100 g/mouse). As shown in Figure 6E and F, JAQ1-treated ceacam1 Ϫ/Ϫ mice showed a 3-fold reduction in thrombus volume and 2-fold reduction in stability score compared with control IgG treated and untreated ceacam1 ϩ/ϩ and ceacam1 Ϫ/Ϫ arterioles (n ϭ 10 arterioles from 3 mice/group; P Ͻ .001).…”

“…18 Antirabbit fluorescein isothiocyanate (FITC) was purchased from Dako (Botany, Australia). Anti-mouse GPVI, 19 anti-mouse GPIb␣/IX/V complex, 20 anti-mouse integrin ␣2␤1, 21 anti-mouse CD9, 22 and anti-mouse GPVI (JAQ1) 23 were purchased or obtained from Emfret Analytics (Wü rzburg, Germany). Anti-mouse integrin ␣IIb␤3, 24 anti-mouse CD44 25 and HRP-conjugated anti-phosphotyrosine RC20 antibody were obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ).…”

Platelet Interactions in Thrombosis

“…Congenital deficiency of GPVI in humans is extremely rare, but abnormal response of platelets to collagen in these patients is associated with only mild bleeding tendency. Similarly, depletion of GPVI from mouse platelets has only minimal affect on bleeding, even though platelets are defective in collagen responsiveness (30,32). GPVI-deficient platelets in Figure 1.…”