Blood Platelets Organize Pro- and Anti-Angiogenic Factors into Separate, Distinct Alpha Granules: Implications for the Regulation of Angiogenesis.

Abstract

In addition to contributing to hemostasis, platelets are increasingly being viewed as major contributors to wound healing and tumor growth. This can be attributed to the many angiogenesis regulatory proteins selectively taken up and stored within the alpha granules of platelets and deposited in the local environment of a tumor or wound upon platelet aggregation and adhesion. Platelets contain both pro and anti-angiogenesis regulators, and the mechanisms by which they modulate angiogenesis are unclear. To address this question, we examined the localization of both pro-and anti-angiogenic factors in platelets and megakaryocytes using immunostaining and confocal microscopy. Double immunofluorescence microscopy revealed that VEGF (an angiogenesis stimulator) and endostatin (an inhibitor), are localized in separate and distinct alpha granules. Immunofluorescence for thrombospondin 1, basic FGF, platelet factor 4, and placental growth factor subsequently confirmed the separate and distinct alpha granules. These observations, in combination with a recent report that proteinase-activated receptors (PARs) selectively modulate the release of angiogenesis regulators from platelets (Ma et al., PNAS 102:216), motivated the hypothesis that these distinct populations of alpha granules could undergo selective release as well. To investigate whether PARs facilitate differential release of alpha granules, we treated human platelets with PAR agonists or antagonists and assayed for differential alpha granule release. The selective PAR1 agonist stimulated the release of endostatin-containing alpha granules and suppressed the release of VEGF-containing alpha granules. Furthermore, confocal microscopy studies revealed that in the presence of the PAR1 antagonist (SCH79797), thrombin stimulated the release of endostatin-containing alpha granules, but not VEGF-containing granules. These observations suggest that platelets may be organizing angiogenesis regulatory proteins into pharmacologically and morphologically distinct populations of alpha granules, which are then susceptible to differential regulation upon platelet activation.