Differential Localization and Conditioned Release of Pro- and Anti-Angiogenic Cytokines from Resting and Stimulated Platelets.

Mechanisms regulating a selective release of pro- or anti-angiogenic cytokines from platelets have not been yet established. In this work, we studied the potential regulative role of shear and collagen in the release of VEGF and endostatin from human platelets. We present data suggesting that subjection of platelets to adhesive proteins under shear, results in selective secretion of VEGF but not endostatin. Furthermore, we demonstrate compartmentalized distribution of these cytokines inside platelets. Platelets subjected to flow (shear rate of 200–2000 s⁻¹) for 4 min on surface-immobilized collagen, selectively released VEGF (from undetectable level to more than 80 pg/ml), whereas the endostatin level remained unchanged (about 65 ng/ml). The release of VEGF was independent of the presence of RBCs or plasma as was demonstrated in experiments with WBC-depleted whole blood, PRP, or washed platelets. A similar yet moderate effect was observed on fibrinogen-coated surface, whereas application of shear force only or incubation of platelets on immobilized collagen or fibrinogen under static conditions did not induce the cytokine release. Soluble collagen promoted release of VEGF that could be inhibited by aspirin in a dose-dependent fashion ranging below effective anti-aggregation doses, which is in accordance with the observed anti-angiogenic effect of NSAIDs. VEGF release was Ca²⁺-dependent and could be prevented by EDTA or PGE1. In contrast to its inhibitory effect on VEGF release, EDTA markedly increased secretion of endostatin suggesting that calcium depletion confers an anti-angiogenic profile to platelets. The difference in the release regulation of these two cytokines led us to examine their localization in platelet granules. Confocal microscopy evaluation of permeabilized fluorescently double-stained (for VEGF and endostatin) platelets showed a distinct staining of each cytokine with almost no co-localization. For better resolution we prepared sections of resting gluteraldehyde-fixed platelets for electron microscopy. Immunogold labeling of the two cytokines revealed localization of VEGF in α granules and of endostatin in sub granular structures. This differential localization of VEGF and Endostatin is consistent with thier conditioned and constitutive release patterns respectively. In conclusion, we propose a novel phenomenon of selective cytokine release from cytokine-dedicated platelet granules according to environmental signals, which further elucidates the control over the pro and anti angiogenic roles of platelets.