Anticoagulation Agents Inhibit Physiological and Pathological Release of Angiogenic Regulatory Proteins From Platelets Usurping the Innate Angiogenic Potential of Platelets.
Authors: Battinelli Elisabeth M. , Markens Beth A. , Kulenthirarajan Rajesh , Italiano Joseph E. 
Published: October 2019

Abstract
Abstract 2174
While the involvement of platelets in hematogenous tumor metastasis has long been recognized, the cause and effect relationship linking the two remains unclear. Platelets are a reservoir for angiogenic proteins that are both sequestered and secreted in a differentially regulated process (Italiano et al., 2009). 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). Due to the propensity for clotting, patients with malignancy are often anti-coagulated with heparin products. While clinical trials suggest that heparin has anti-neoplastic properties that may increase survival, the mechanism by which heparin exerts its anti-metastatic action is unknown. We hypothesized that anti-thrombotic agents, alter the release of angiogenesis regulatory proteins from platelets. Our data reveals that platelets exposed to heparin (UFH) or its derivative low molecular weight heparin (LMWH) release statistically significantly decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells) (Plts +MCF7 85.9 pg/ml, Plts +UFH +MCF-7 18.57 pg/ml; Plts+LMWH+ MCF-7 25.1 pg/ml). 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 (Plts+MCF7 cells 102.6, Plts+UFH+MCF7 5.00, Plts+LMWH +MCF7 13.6) and endothelial cell migration (Plts+MCF7 cells 106, Plts+UFH+MCF7 2.22, Plts+LMWH +MCF7 2.85). 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 an 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. Since, novel anticoagulants including Xa inhibitors are now utilized in the cancer patient population, we also explored their impact on the platelet angiogenic potential. Similar to Heparin, exposure to Fondaparinux led to a significant decrease in platelet releasate VEGF content after exposure to ADP or MCF-7 cells and a decreased angiogenic potential as measured by capillary tube formation and endothelial cell migration. Next we explored the in vivo impact of these medications on the platelet releasate of anticoagulated 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 (no anticoagulation 0.2880 pg VEGF/plt vs LMWH 0.1387 pg VEGF/plt). This effect was not seen with those patients taking Coumadin suggesting that the heparin mechanistically is affecting platelet functional activity. While these data suggest anticoagulation may decrease new blood vessel growth by decreasing pro-angiogenic factors, we also explored their impact on anti-angiogenic factors. Previously, we had shown that activation of platelets with TXA2 produced a releasate that had significantly increased levels of Endostatin and a decreased angiogenic potential. In our experiments, exposure to anticoagulants also produced increased levels of Endostatin. When these experiments were performed with anti-TXA2 antibodies in addition to anticoagulants, the endostatin levels were decreased in the releasate and the angiogenic potential was recovered as demonstrated by increased capillary tube formation. This would suggest that exposure to angiogenic proteins impacts TXA2-related platelet release. 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. Attenuation of platelet angiogenic potential by anti-thrombotic agents may be the mechanism underlying their effects on the improved survival and the decreased metastasis in cancer patients.

Disclosures:
No relevant conflicts of interest to declare.