Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions, such as migration and 2D and 3D tube formation. Interestingly, we found an increased prevalence of platelets expressing C5aR1 in ischemic tissue in the hindlimb ischemia model compared to control tissue. Furthermore, tissue ischemia also induced complement activation. Growth factor- and hypoxia-driven revascularization was markedly increased in C5ar1^-/- mice, which was reversible upon systemic platelet depletion. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. This secretion occurred in the absence of overall platelet activation and aggregation. Indeed, we could identify a subgroup of alpha-granules responsible for C5a-induced CXCL4 secretion as well as uncover the underlying signaling mechanism. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.

In conclusion, we identified a novel mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4. Furthermore, to our knowledge for the first time, the study exemplifies an agonist-specific secretion mechanism from platelets involving a specific alpha-granule subtype.