Physiological and Pathological Agonists Induce Differential Release of Angiogenesis Regulatory Proteins From Platelet Alpha Granules Influencing the Angiogenic Response

In addition to their primary roles in hemostasis and thrombosis, platelets participate in other physiological and pathological processes, including inflammation, wound healing, and tumor metastasis. Although platelets are presumed to contribute to angiogenesis by providing numerous pro-and anti-angiogenic factors, the cellular and molecular basis by which platelets regulate angiogenesis is poorly understood. Previously, we have shown that platelets differentially package specific angiogenesis regulatory proteins into distinct populations of alpha-granules. Using selective agonists for the PAR1μ and PAR4 receptor, we demonstrated that these distinct subpopulations of alpha-granules are susceptible to differential release upon platelet activation (Italiano et al. Blood 2008). To investigate whether other platelet receptors facilitate differential release of alpha-granules, we treated human platelets with physiological agonists and assayed for differential alpha-granule release. Previously we have shown that activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF (an angiogenesis stimulator) in vitro, but not endostatin (an angiogenesis inhibitor). We now have evidence that the agonist Thromboxane A₂ (TXA₂) acts in an opposite manner; leading to increased endostatin release, but not VEGF release. To more broadly establish the angiogenic potential of the platelet releasate stimulated by exposure to different platelet agonists, including ADP and TXA₂, we used a number of well-established angiogenic assays including endothelial cell migration, capillary tube formation, and also an angiogenic protein array panel. All platelet releasates generated by activating platelets with ADP led to increased angiogenic potential with statistically significant increased cell migration, capillary tube formation, and an overall pro-angiogenic protein content of the releasate. Conversely, treatment with TXA₂ led to decreased endothelial cell migration and inhibited the formation of capillary tube structures by human umbilical vein endothelial cells. Although it is established that tumor cells can interact with platelets, the mechanisms involved are not known. This prompted us to investigate the hypothesis that cancer cells may also have the capacity to induce differential alpha-granule release during the pathological angiogenesis associated with tumor growth. To investigate whether tumor cells facilitate differential release of pro-angiogenic alpha-granules, we treated human platelets with different tumor cell lines and assayed for differential alpha-granule release. Exposure of platelets to the breast cancer cell line, MCF-7 cells stimulated significant release of VEGF. The angiogenic potential of this releasate was increased as measured using the endothelial cell migration assay, capillary tube formation assay, and protein arrays. This study provides mechanistic evidence to support an interplay between tumor cells and platelet agonists to orchestrate differential release of angiogenic factors from platelets, and illustrates a key role for platelets in tumor angiogenesis through the delivery of specific angiogenic factors.

No relevant conflicts of interest to declare.