Genetic and antibody‐induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl3‐induced thrombosis

At sites of vascular injury, platelet adhesion, activation and aggregation occurs on exposed subendothelial collagens and contributes to hemostasis but also to occlusion of diseased vessels leading to a myocardial infarction or ischemic stroke. The activating platelet collagen receptor, glycoprotein (GP) VI [1], is a platelet-specific transmembrane type I receptor that non-covalently associates with the Fc receptor (FcR) c-chain which contains an immunoreceptor tyrosine-based activation motif (ITAM) [2]. Upon ligand-induced GPVI clustering, the ITAM becomes tyrosine phosphorylated and initiates a series of phosphorylation events finally resulting in cellular activation [3]. GPVI has been proposed as an attractive antithrombotic target, particularly because anti-GPVI antibodies have been shown to induce irreversible downregulation of the receptor through ectodomain shedding in circulating platelets in mice, resulting in long-term antithrombotic protection but overall normal tail bleeding times [4–7]. Different mouse models of GPVI-deficiency (FcR c-chain-deficient which also lack GPVI [8], GPVI-immunodepleted or Gp6 mice) have been used to determine the functional relevance of the receptor in thrombus formation in different experimental thrombosis models (laser-, mechanically and chemically induced injuries). AlthoughGPVI is generally regarded as a critical regulator of pathological thrombus formation [9], partially contradictory results have been reported on the relative importance of GPVI in some of these models. For example, GPVI was found to play no [10,11] or only a minor [12,13] role in models of laser-induced vascular injury where thrombus formation is suggested to be largely dependent on thrombin generation, although different results were reported by others [14]. Ferric(III)chloride (FeCl3)-induced vascular injury is a widely used model in thrombosis research because it allows variable levels of injury in different vascular beds and can be monitored by microscopic visualization or blood flow measurements. Previous studies yielded evidence for a role of GPVI in FeCl3-induced thrombus formation in mice [10,15,16], but this was questioned in a very recent study assessing the mechanisms underlying thrombus formation in that model in detail [17]. The authors demonstrated that in their model, FeCl3 diffuses through all vessel wall layers and severely injures the endothelium without damaging the internal elastic lamina (IEL), and only exposes the basement membrane to the vessel lumen. They further report that in their hands GPVI-immunodepleted mice are not protected from vessel occlusion after FeCl3-induced injury of mesenteric arterioles or carotid arteries by measuring thrombus area and time to occlusion. Based on these data, the authors state that FeCl3-induced vascular injury may not be an appropriate model to study the role of subendothelial adhesive proteins and their platelet receptors in thrombosis [17]. This conclusion, if correct, may have major implications for the interpretation of a large number of previous results obtained with this model and also significantly influence its acceptance as a valid experimental system to assess the mechanisms of arterial thrombosis. Therefore, here we assessed the role of GPVI-mediated platelet activation/ adhesion in FeCl3-induced thrombus formation systematically. To do so, we subjected control, Gp6 (details of the knockout generation will be published elsewhere) and GPVIimmunodepleted (100 lg JAQ1 i.p., analysis on day 6 post injection) mice to a model of FeCl3-injury of mesenteric arterioles and carotid arteries, and monitored thrombus formation by intravital fluorescence microscopy or blood flow measurements, respectively. In addition, thrombus formation in these mice was tested in a model of mechanical injury of the abdominal aorta. A drop of 20% FeCl3 was topically applied on small mesenteric arterioles of 4to 5–week-old mice and thrombus formation of fluorescently labeled platelets was monitored for 40 min or until complete occlusion of the vessel had occurred. Kinetics of platelet adhesion (data not shown) and the beginning of first thrombus formation were similar between control, Gp6 and GPVI-immunodepleted mice (Fig. 1A, Correspondence: Bernhard Nieswandt, University Hospital Wurzburg and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Josef-Schneider-Str. 2, 97080 Wurzburg, Germany. Tel. : +49 931 31 80405; fax: +49 931 201 61652. E-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de