Platelet glycoprotein Ibα is an important mediator of ischemic stroke in mice

Stroke is one of the leading causes of death worldwide with limited treatment options [1]. Platelets play a pivotal role in cerebral ischemia/reperfusion injury by adhering to the damaged vessel wall, leading to further platelet recruitment and thrombus formation. The glycoprotein (GP) Ib-IX-V complex is a crucial platelet receptor for initial tethering and adhesion at sites of vascular injury. This abundant complex on the platelet surface (12,500 copies per cell) consists of the leucine-rich repeat glycoproteins GPIbα, GPIbβ, GPIX and GPV in a 2:2:2:1 ratio [2]. The adhesive function of GPIb-IX-V is mainly attributed to the interaction of GPIbα with its major ligand von Willebrand factor (VWF), exposed upon vascular damage. The central role of the GPIbα-VWF interaction in mediating initial platelet adhesion is illustrated by the bleeding disorders Bernard Soulier syndrome [3] and von Willebrand disease [4], caused by deficiency of GPIb-IX-V or VWF respectively. Besides its interaction with VWF, GPIbα can also engage counter-receptors such as αMβ2 (Mac-1) on neutrophils and P-selectin on activated platelets or endothelial cells [2]. Other GPIbα ligands include α-thrombin, clotting factors XI and XII, thrombospondin-I and high molecular weight kininogen [2]. Not surprisingly, the importance of GPIbα far exceeds that of VWF in arterial thrombosis [5]. Thus, by binding a variety of ligands, GPIbα is a central receptor in different vascular processes of thrombosis and inflammation, all of which may contribute to the progression of ischemic stroke. Here, we studied stroke development in transgenic mice expressing GPIbα in which the extracellular domain was replaced by an isolated domain of the α-subunit of the human IL-4 receptor [6]. We found that these mice had better stroke outcome, as evidenced by smaller infarct volumes and better functional scores.

We here report that mice lacking the extracellular part of GPIbα are protected from ischemic stroke as evidenced by 50% smaller infarct volumes and significantly better neurologic outcome when compared to WT mice. These results further establish the notion that GPIbα is critically involved in the pathogenesis of ischemic stroke [12, 13]. Our findings are in agreement with an earlier study using the same mouse tMCAO model in which an anti-GPIbα monoclonal antibody was used to block the GPIbα receptor [14]. Use of this antibody led to a 60% reduction of infarct volumes compared to control mice, which was accompanied by a significant reduction in neurologic deficits [14]. We recently demonstrated that VWF deficient mice were protected from ischemic stroke [8, 15]. Since VWF is the main ligand of GPIbα on platelets, our data support the notion of a pathophysiological role for the GPIbα-VWF interaction in stroke, although we can not rule out that other GPIbα-mediated interactions are also important and abrogated in GPIbα/IL4Rα mice. In full support of this, we showed that VWF-deficient mice that were reconstituted with a VWF mutant defective in binding to GPIbα retained their protection against stroke [9]. Moreover, several conditions, including the GPIbα Kozak polymorphism [16], GPIbα variants with increased binding to VWF [17] and high VWF levels [18, 19], have all been reported to be associated with an increased risk of ischemic stroke in humans.

It has to be noted that GPIbα/IL4Rα mice were reported to have slightly reduced platelet counts [6]. Although not significant, also our measurements indicate a slightly lower platelet count in GPIbα/IL4Rα mice compared to WT animals. Whereas we cannot completely exclude any effect of this small platelet count difference on stroke outcome, we believe that this is unlikely to cause the observed dramatic protection against stroke. Platelet adhesion to ferric chloride-treated mesenteric arterioles was virtually absent in GPIbα/IL4Rα mice, resulting in the complete abrogation of thrombus formation [5]. VWF-deficient mice are still able to form thrombi in this model, although thrombus formation is significantly delayed [20, 21]. Although these studies showed that ligands other than VWF can support GPIbα-dependent platelet adhesion, interfering with the binding of GPIbα to VWF has indeed been proven to have a profound antithrombotic effect [22‐24]. Since an increasing amount of evidence suggests that GPIbα and VWF could also mediate inflammatory processes, further studies are needed to establish whether inhibition of these molecules also reduce inflammation in stroke [25].

The search for safe and efficient drugs for stroke management has been hampered by an unacceptable risk of fatal bleeding as seen e.g. with platelet aggregation inhibitors [26]. In this respect, it is encouraging that we did not observe intracranial hemorrhages after stroke in GPIbα/IL4Rα mice. Also in VWF deficient mice, no increased bleeding was noticed in the tMCAO model [8, 9, 15]. In comparison with inhibition of platelet aggregation, neutralizing the VWF-GPIbα-axis has been shown to have a better benefit-to-risk ratio with respect to bleeding in various models of experimental thrombosis [22‐24]. Interestingly, a recent trial showed that administration of anti-VWF aptamer ARC1779, which blocks the VWF-GPIbα interaction, reduced cerebral emboli signals in patients undergoing carotid endarterectomy [27].

In conclusion, using GPIbα/IL4Rα mice, we demonstrated the crucial role for GPIbα in ischemic stroke. Together with the already existing evidence, our results further warrant larger translational and clinical studies to assess the benefits or drawbacks of interfering with platelet adhesion in stroke.