Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro-and antiangiogenic proteins are separated in distinct subpopulations of ␣-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct ␣-granules. These observations motivated the hypothesis that distinct populations of ␣-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH 2 ) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH 2 ) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of ␣-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis. IntroductionAngiogenesis, the process of new vessel development, plays an essential role in embryogenesis, but postnatal angiogenesis is limited to sites of abnormal vascular surface. An activated vascular endothelium can be induced by tissue injury or wound healing, by hormonal cycling such as in pregnancy and ovulation, or by tumor-induced vessel growth. In all of these circumstances, platelets act as the initial responder to vascular change and provide a flexible delivery system for angiogenesis-related molecules. [1][2][3][4] The process of postnatal angiogenesis is regulated by a continuous interplay of stimulators and inhibitors of angiogenesis, and their imbalance contributes to numerous inflammatory, malignant, ischemic, and immune disorders. 5 There is a revived interest in the overlap between angiogenesis and platelets 6 because several clinical trials have now shown that anticoagulation can improve cancer survival 7,8 beyond the benefit derived from the treatment of deep vein thrombosis alone.It is known that platelets stimulate endothelial cells in culture and can promote the assembly of capillary-like structures in vitro. 9,10 Platelets may modulate angiogenesis by releasing promoters such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and matrix metalloproteinases (MMPs). 1,6,[11][12][13][14][15][16][17][18] The repertoire o...

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Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocytederived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P ؊ , LAMP-1 ؊ and expressed fulllength filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P ؊ and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet micropar-