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 approximately 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.

Figure 1.

Platelet adhesion and aggregation after vascular injury of the common carotid artery in C57BL/6J mice in vivo. (a) Scanning electron micrographs of carotid arteries before (left) and 2 h after vascular injury (right). Endothelial denudation induces platelet adhesion and aggregation, resulting in the formation of a platelet-rich (lower right) thrombus. (b) Platelet–endothelial cell interactions 5 min after vascular injury were investigated by in vivo fluorescence microscopy of the common carotid artery in situ (solid columns). Animals without vascular injury served as controls (open columns). The left and right panels summarize platelet tethering and firm platelet adhesion, respectively, of eight experiments per group. Platelets were classified according to their interaction with the endothelial cell lining as previously described (refer to Materials and Methods) and are given per mm² of vessel surface. Mean ± SEM. *, significant difference compared with control, P < 0.05. (c) Platelet aggregation after vascular injury was determined by fluorescence microscopy in vivo (solid columns). Animals without vascular injury served as controls (open columns). Mean ± SEM and n = 8 each group. *, significant difference compared with wild-type mice, P < 0.05. The microphotographs (right) show representative in vivo fluorescence microscopy images in control animals (top) or after vascular injury (bottom). White arrows indicate adherent platelets. Bars, 50 μm.

Figure 1.

Platelet adhesion and aggregation after vascular injury of the common carotid artery in C57BL/6J mice in vivo. (a) Scanning electron micrographs of carotid arteries before (left) and 2 h after vascular injury (right). Endothelial denudation induces platelet adhesion and aggregation, resulting in the formation of a platelet-rich (lower right) thrombus. (b) Platelet–endothelial cell interactions 5 min after vascular injury were investigated by in vivo fluorescence microscopy of the common carotid artery in situ (solid columns). Animals without vascular injury served as controls (open columns). The left and right panels summarize platelet tethering and firm platelet adhesion, respectively, of eight experiments per group. Platelets were classified according to their interaction with the endothelial cell lining as previously described (refer to Materials and Methods) and are given per mm² of vessel surface. Mean ± SEM. *, significant difference compared with control, P < 0.05. (c) Platelet aggregation after vascular injury was determined by fluorescence microscopy in vivo (solid columns). Animals without vascular injury served as controls (open columns). Mean ± SEM and n = 8 each group. *, significant difference compared with wild-type mice, P < 0.05. The microphotographs (right) show representative in vivo fluorescence microscopy images in control animals (top) or after vascular injury (bottom). White arrows indicate adherent platelets. Bars, 50 μm.

Figure 1.

Platelet adhesion and aggregation after vascular injury of the common carotid artery in C57BL/6J mice in vivo. (a) Scanning electron micrographs of carotid arteries before (left) and 2 h after vascular injury (right). Endothelial denudation induces platelet adhesion and aggregation, resulting in the formation of a platelet-rich (lower right) thrombus. (b) Platelet–endothelial cell interactions 5 min after vascular injury were investigated by in vivo fluorescence microscopy of the common carotid artery in situ (solid columns). Animals without vascular injury served as controls (open columns). The left and right panels summarize platelet tethering and firm platelet adhesion, respectively, of eight experiments per group. Platelets were classified according to their interaction with the endothelial cell lining as previously described (refer to Materials and Methods) and are given per mm² of vessel surface. Mean ± SEM. *, significant difference compared with control, P < 0.05. (c) Platelet aggregation after vascular injury was determined by fluorescence microscopy in vivo (solid columns). Animals without vascular injury served as controls (open columns). Mean ± SEM and n = 8 each group. *, significant difference compared with wild-type mice, P < 0.05. The microphotographs (right) show representative in vivo fluorescence microscopy images in control animals (top) or after vascular injury (bottom). White arrows indicate adherent platelets. Bars, 50 μm.

Figure 2.

Inhibition of GPVI abrogates platelet adhesion and aggregation after vascular injury. (a) Platelet adhesion after vascular injury was determined by intravital video fluorescence microscopy. Fluorescent platelets were preincubated with 50 μg/ml anti-GPVI (JAQ1) Fab fragments or control rat IgG. Platelets without mAb preincubation served as control. The left and right panels summarize transient and firm platelet adhesion, respectively. Mean ± SEM and n = 8 each group. *, significant difference compared with control, P < 0.05. (b) The percentage of platelets establishing irreversible adhesion after initial tethering/slow surface translocation is illustrated. (c) Platelet aggregation after vascular injury in vivo. Aggregation of platelets preincubated with tyrode, irrelevant rat IgG, or anti-GPVI (JAQ1) was assessed by fluorescence microscopy as previously described. Mean ± SEM and n = 8 each group. *, significant difference compared with control, P < 0.05. (d) The photomicrographs show representative in vivo fluorescence microscopy images illustrating platelet adhesion in the absence or presence of anti-GPVI Fab (JAQ1) or control IgG. Bars, 30 μm. (e) Inhibition of GPIbα abrogates platelet recruitment after vascular injury. Platelets were incubated with 50 μg/ml anti-GPIbα Fab fragments (p0p/B) for 10 min. Platelets without mAb preincubation served as control. The left and right panels summarize transient and firm platelet adhesion, respectively. Mean ± SEM and n = 6 each group. *, significant difference compared with control, P < 0.05.

Figure 2.

Inhibition of GPVI abrogates platelet adhesion and aggregation after vascular injury. (a) Platelet adhesion after vascular injury was determined by intravital video fluorescence microscopy. Fluorescent platelets were preincubated with 50 μg/ml anti-GPVI (JAQ1) Fab fragments or control rat IgG. Platelets without mAb preincubation served as control. The left and right panels summarize transient and firm platelet adhesion, respectively. Mean ± SEM and n = 8 each group. *, significant difference compared with control, P < 0.05. (b) The percentage of platelets establishing irreversible adhesion after initial tethering/slow surface translocation is illustrated. (c) Platelet aggregation after vascular injury in vivo. Aggregation of platelets preincubated with tyrode, irrelevant rat IgG, or anti-GPVI (JAQ1) was assessed by fluorescence microscopy as previously described. Mean ± SEM and n = 8 each group. *, significant difference compared with control, P < 0.05. (d) The photomicrographs show representative in vivo fluorescence microscopy images illustrating platelet adhesion in the absence or presence of anti-GPVI Fab (JAQ1) or control IgG. Bars, 30 μm. (e) Inhibition of GPIbα abrogates platelet recruitment after vascular injury. Platelets were incubated with 50 μg/ml anti-GPIbα Fab fragments (p0p/B) for 10 min. Platelets without mAb preincubation served as control. The left and right panels summarize transient and firm platelet adhesion, respectively. Mean ± SEM and n = 6 each group. *, significant difference compared with control, P < 0.05.

Figure 3.

Platelet adhesion after endothelial denudation in GPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. On the top, platelets from mice pretreated with irrelevant control IgG or anti-GPVI (JAQ1) were stained for GPVI and GPIIb/IIIa (top) or GPIa and GPIbα (bottom) and directly analyzed on a FACScalibur™ is shown. Representative dot plots of six mice per group are presented. The expression levels of GPIIb/IIIa, GPIb-V-IX, and GPIa/IIa were not significantly different between the two groups of mice (refer to Table I). On the bottom, whole platelet lysates from three control IgG or JAQ1-treated mice separated by SDS-PAGE under nonreducing conditions and immunoblotted with FITC-labeled JAQ1, followed by incubation with horseradish peroxidase–labeled rabbit anti–FITC antibody is shown. (b) Scanning electron micrographs of carotid arteries 2 h after vascular injury in control animals or GPVI depleted. Endothelial denudation induced platelet adhesion and platelet aggregation in control animals. In contrast, only very few platelets attached along the damaged vessel wall in GPVI-depleted mice. Subendothelial collagen fibers are visible along the denuded area. (c) Platelet tethering and firm platelet adhesion, (d) transition from initial tethering to stable arrest (percentage of tethered platelets), and (e) platelet aggregation after vascular injury of the carotid artery was determined in GPVI-deficient (JAQ1-pretreated mice) or control IgG–pretreated mice (for details refer to Materials and Methods). The panels summarize platelet adhesion (tethering and firm adhesion) and platelet aggregation in eight experiments per group. Mean ± SEM. *, significant difference compared with control IgG, P < 0.05. (f) The photomicrographs show representative in vivo fluorescence microscopy images illustrating platelet adhesion in GPVI-deficient (JAQ1) and control IgG–treated mice. Bars, 30 μm.

Figure 3.

Platelet adhesion after endothelial denudation in GPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. On the top, platelets from mice pretreated with irrelevant control IgG or anti-GPVI (JAQ1) were stained for GPVI and GPIIb/IIIa (top) or GPIa and GPIbα (bottom) and directly analyzed on a FACScalibur™ is shown. Representative dot plots of six mice per group are presented. The expression levels of GPIIb/IIIa, GPIb-V-IX, and GPIa/IIa were not significantly different between the two groups of mice (refer to Table I). On the bottom, whole platelet lysates from three control IgG or JAQ1-treated mice separated by SDS-PAGE under nonreducing conditions and immunoblotted with FITC-labeled JAQ1, followed by incubation with horseradish peroxidase–labeled rabbit anti–FITC antibody is shown. (b) Scanning electron micrographs of carotid arteries 2 h after vascular injury in control animals or GPVI depleted. Endothelial denudation induced platelet adhesion and platelet aggregation in control animals. In contrast, only very few platelets attached along the damaged vessel wall in GPVI-depleted mice. Subendothelial collagen fibers are visible along the denuded area. (c) Platelet tethering and firm platelet adhesion, (d) transition from initial tethering to stable arrest (percentage of tethered platelets), and (e) platelet aggregation after vascular injury of the carotid artery was determined in GPVI-deficient (JAQ1-pretreated mice) or control IgG–pretreated mice (for details refer to Materials and Methods). The panels summarize platelet adhesion (tethering and firm adhesion) and platelet aggregation in eight experiments per group. Mean ± SEM. *, significant difference compared with control IgG, P < 0.05. (f) The photomicrographs show representative in vivo fluorescence microscopy images illustrating platelet adhesion in GPVI-deficient (JAQ1) and control IgG–treated mice. Bars, 30 μm.

Figure 3.

Platelet adhesion after endothelial denudation in GPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. On the top, platelets from mice pretreated with irrelevant control IgG or anti-GPVI (JAQ1) were stained for GPVI and GPIIb/IIIa (top) or GPIa and GPIbα (bottom) and directly analyzed on a FACScalibur™ is shown. Representative dot plots of six mice per group are presented. The expression levels of GPIIb/IIIa, GPIb-V-IX, and GPIa/IIa were not significantly different between the two groups of mice (refer to Table I). On the bottom, whole platelet lysates from three control IgG or JAQ1-treated mice separated by SDS-PAGE under nonreducing conditions and immunoblotted with FITC-labeled JAQ1, followed by incubation with horseradish peroxidase–labeled rabbit anti–FITC antibody is shown. (b) Scanning electron micrographs of carotid arteries 2 h after vascular injury in control animals or GPVI depleted. Endothelial denudation induced platelet adhesion and platelet aggregation in control animals. In contrast, only very few platelets attached along the damaged vessel wall in GPVI-depleted mice. Subendothelial collagen fibers are visible along the denuded area. (c) Platelet tethering and firm platelet adhesion, (d) transition from initial tethering to stable arrest (percentage of tethered platelets), and (e) platelet aggregation after vascular injury of the carotid artery was determined in GPVI-deficient (JAQ1-pretreated mice) or control IgG–pretreated mice (for details refer to Materials and Methods). The panels summarize platelet adhesion (tethering and firm adhesion) and platelet aggregation in eight experiments per group. Mean ± SEM. *, significant difference compared with control IgG, P < 0.05. (f) The photomicrographs show representative in vivo fluorescence microscopy images illustrating platelet adhesion in GPVI-deficient (JAQ1) and control IgG–treated mice. Bars, 30 μm.

Figure 4.

Role of GPVI in arterial thrombosis after ferric chloride exposure. Vascular injury of the carotid artery was induced by local application of ferric chloride on the carotid artery in GPVI-deficient or control mice. The time to thrombotic occlusion of the carotid artery downstream of the site of injury (n = 10 per group) was assessed in vivo by video fluorescence microscopy. Each symbol represents one experiment.

Figure 5.

Role of GPVI in the regulation of platelet recruitment after wire injury of the carotid artery. Wire-induced endothelial denudation of the carotid artery was induced in GPVI-deficient mice. Untreated animals served as controls. The left shows representative in vivo fluorescence microscopy images illustrating the time course of platelet recruitment to the site of injury in control animals or GPVI-deficient mice (×500). The right summarizes platelet tethering, firm adhesion, and aggregate formation. Mean ± SEM. *, significant difference compared with control, P < 0.05.