We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo. Platelets are normally regarded as effectors of haemostasis, but proteomic analyses suggest they may play a wider role in wound healing, tumour growth, inflammation and regeneration. Our data show a differential role for platelets in mediating two distinct mechanisms of in vivo angiogenesis, namely capillary sprouting and longitudinal splitting. A failure to rescue platelet depletion by VEGF overexpression suggests the mechanism is more complex than a-granule secretion alone. It is difficult to directly assess platelet granule release in vivo, and monocyte-derived VEGF may play a role in some forms of angiogenesis, but these data are consistent with VEGF levels per se not being responsible for angiogenesis ablation following platelet depletion. Maintenance of angiogenesis following loss of GPVI expression, although partial, indicates collagen-induced platelet activation is not the primary stimulus, while BrdU pulse-labelling suggests platelets do not affect endothelial cell proliferation. Sprouting angiogenesis induced by muscle overload causing elevated mechanical deformation of vessels and a resulting reduction in capillary FSS, was LEE011 abolished by platelet depletion. In contrast, splitting angiogenesis is induced by elevated capillary FSS, and was unaffected by platelet depletion. Together, these data may suggest a shear-related response to platelet mediation of physiological angiogenesis. It is well known that higher FSS levels induce upregulation of vasoprotective genes in EC. Indeed, while we observed significantly decreased levels of PGF1a with capillary sprouting there was no alteration in levels with longitudinal splitting. An alternative hypothesis is a difference in signalling between the angiogenic forms. For example, sprouting is dependent on matrix metalloprotease activity while both forms require VEGF. VEGF is found in both platelets and granulocytes, particularly neutrophils. However, capillary sprouting was normal following granulocyte/monocyte depletion, demonstrating that despite the initial fall in immune cell numbers during platelet clearance after anti-GPIba administration, it was depletion of platelets rather than immune cells that mediated angiogenesis. Inhibition of capillary sprouting following treatment with a dual regimen of clopidogrel/ASA confirmed the platelet depletion result. ASA was identified as the active agent at both high and low dose regimens. The data are consistent with overload-induced angiogenesis acting through COX signalling, and implicate COX inhibition of TXA2 or PGI2 synthesis as the anti-angiogenic target. Although ASA is not platelet specific, treatment may have a greater effect on platelets than EC depending on dose treatment regimen, since anucleate platelets are unable to initiate COX gene transcription. In addition, EC are primarily responsible for production of PGI2, and platelets TXA2.