In ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization and assessed its role if diseases featuring tissue ischemia in patients. 

We assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in coronary artery disease and found a correlation between specific but not all markers of platelet activation and C5aR1 expression on platelets. Furthermore, we have assessed the role of C5aR1 in peripheral artery disease (PAD) patients. Interestingly, we have recruited a cohort of PAD patients with proven high-grade arterial stenoses of the leg or groin arteries but not ischemic walking pain. In the LUERPAD-Immuno-study conducted at the University Heart Centre Luebeck, we have matched these patients with respect to gender and age with patients with PAD and typical symptoms. We characterized both groups clinically in-depth and found them to be very similar except for a large difference in pain-free walking distance. We hypothesized that lack of symptoms might be due to a better capacity for collateral artery growth in asymptomatic PAD patients. Strikingly, the anti-angiogenic agent CXCL4 in plasma of these patients correlated inversely with pain-free walking distances in this cohort. The same could be found for C5aR1 expression on platelets and platelet C5aR1 and CXCL4 in plasma also correlated with each other. Thus, the C5aR1-CXCL4 axis seems to play a role in adaption to diseases featuring tissue ischemia also in cardiovascular patients. Furthermore, we employ genetic mouse models to substantiate this. Indeed, the presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model, a mouse model mimicking PAD. Ischemia-driven angiogenesis was significantly improved in C5aR1-/- mice, but not in C5-/- mice suggesting a specific role of C5aR1. Experiments using supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. Using C5aR1-/-CXCL4-/- mice, we observed no additional effect in the revascularization response, underscoring a strong dependence of CXCL4 secretion on the C5a-C5aR1-axis.

We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by release of antiangiogenic CXCL4. Importantly, we can present evidence for the significance of this mechanism in cardiovascular patients suffering from diseases featuring tissue ischemia.