Anticoagulation Inhibits Tumor Cell-Mediated Release Of Platelet Angiogenic Proteins and Disrupts The Platelet Angiogenic Potential

Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. While the involvement of platelets in hematogenous tumor metastasis has long been recognized, the cause and effect relationship linking the two remains unclear. Due to the propensity for clotting, patients with malignancy are often anti-coagulated with heparin products, which paradoxically offer a survival benefit by an unknown mechanism. We hypothesized that anti-thrombotic agents alter the release of angiogenesis regulatory proteins from platelets. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). Our data reveals that platelets exposed to heparin (UFH) or its derivative low molecular weight heparin (LMWH) release statistically significant decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). The angiogenic potential from ADP or tumor cell generated platelet releasate is also significantly decreased as evidenced by dramatically diminished capillary tube branch point formation and endothelial cell migration. To explore the impact of these anticoagulants on the angiogenic protein contents of the releasate, we analyzed the releasate from platelets exposed to LMWH alone or activated with ADP or MCF-7 cells in conjunction with LMWH. Using a angiogenesis protein array to simultaneously sample the angiogenic content of the platelet releasate, we found that exposure to LMWH resulted in a decrease in pro-angiogenic protein content. Fondaparinux (Xa inhibitor) demonstrated similar impact on the platelet angiogenic potential. Since MCF-7 cells are known to secrete thrombin; the main target of anticoagulants, we hypothesized that these drugs disrupt thrombin signaling through the platelet PAR1 receptor. Addition of PAR1 antagonists to platelets decreased VEGF release and angiogenic potential. Exposure to a PAR1 agonist in the presence of anticoagulants rescued the angiogenic potential. Direct evidence of disrupted PAR1 signaling was demonstrated by decreased cleavage (activation) of the PAR1 receptor on anticoagulated platelets in the presence of MCF-7 cells (as measured by SPAN12 using Flow Cytometry). This data establishes a mechanism by which anticoagulants can decrease the angiogenic potential of platelets through impaired PAR1 activation. The impact of anticoagulants was also observed in platelet releasate of patients. We measured the VEGF concentration in the platelet releasate of patients currently medicated with LMWH or Fondaparinux and observed a statistically significant decrease in VEGF release after exposure to MCF-7 cells. Taken together, these data underscore the pivotal role of platelets in regulating tumor angiogenesis and point to a potential source for development of therapeutic intervention targeting the PAR1 receptor.