The Effects of Antiplatelet Agents on Endocytosis
Authors: Roweth Harvey G. , Malloy Michael , Forward Jodi A. , Ceglowski Julia , Flaumenhaft Robert C. , Italiano Joseph E. , Battinelli Elisabeth 
Published: November 2019

Almost every platelet-derived protein originates from either megakaryocytes (MKs), or the endocytosis of factors within blood circulation. These endocytosed factors can be locally released upon platelet activation to regulate hemostasis, or to promote the growth and neovascularization of solid tumors. Although platelet endocytosis has long been recognized, our mechanistic understanding remains largely confined to receptor-mediated endocytosis of fibrinogen via integrin αIIbβ3. Whether antiplatelet therapy and/or different disease states influence platelet endocytosis remain understudied areas of investigation. In this project, we examined how various antiplatelet agents affect the endocytosis of vascular endothelial growth factor (VEGF) and endostatin, which are broad pro-angiogenic and anti-angiogenic regulators, respectively.
For our initial experiments, human platelets in platelet-rich plasma were exposed to either recombinant VEGF or endostatin for 1 hour, then washed to remove residual/unbound protein. Platelets were then lysed and changes in VEGF or endostatin concentration determined by ELISA. Using this approach, we demonstrated that platelets endocytose VEGF and endostatin in a concentration-dependent manner. We next established that following platelet activation, lysate concentrations of endocytosed VEGF and endostatin decreased and supernatant concentrations increased. This finding implies that endocytosed VEGF and endostatin are packaged into platelet alpha granules and are released following their activation, which we aim to confirm by measuring co-localization with endosomal and alpha granule markers. We have also developed two assays to track endocytosis into platelets, one where proteins are conjugated to pH-sensitive dyes that increase their fluorescence upon endosomal compartmentalization, and another utilizing fluorophore-targeting antibodies to quench eternal fluorescence of labeled factors.
We next assessed if antiplatelet agents modulated endocytosis into platelets. We found that pre-treating platelets with aspirin, vorapaxar or ticagrelor resulted in dose-dependent inhibition of VEGF and fibrinogen but not endostatin endocytosis. This finding suggests that antiplatelet agents selectively inhibit the uptake of pro-angiogenic regulators, a statement we aim to support by measuring the endocytosis of additional angiogenic proteins. Our observations also affirm previous studies, claiming that fibrinogen and VEGF are differentially packaged into a subset of alpha granules distinct from endostatin. Experiments are ongoing to measure platelet levels of VEGF and endostatin in human subjects before and after aspirin intervention, to assess if our findings hold physiological relevance.
The fact that several antiplatelet agents impaired VEGF endocytosis suggests a common inhibitory mechanism linked to suppressing basal levels of platelet activation. We tested this hypothesis by either simulating platelets with low-dose adenosine diphosphate or by chelating intracellular calcium to prevent basal activation. Neither of these processes influenced VEGF or endostatin endocytosis, suggesting that the process is not associated with basal platelet activation. We are currently investigating if antiplatelet agents alter the activity of small GTPases Arf6 and Dynamin-2, which have been shown to regulate receptor-mediated endocytosis of fibrinogen by platelets. Future experiments will aim to elucidate the mechanism by which antiplatelet agents inhibit protein uptake.
It has previously been shown that platelets endocytose tumor-derived factors and RNA that may promote disease progression or act as biomarkers for liquid biopsies. We are currently studying if the daily administration of aspirin prevents the endocytosis of tumor-derived factors in a mouse model of tumor generation.
In summary, our data show platelets endocytose and release key angiogenic regulators, a process which can be blocked through pre-treatment with various antiplatelet agents. Given the important role of platelets to tumor neovascularization, preventing the endocytosis of pro-angiogenic mediators could represent a novel mechanism that contributes to the inhibitory effects of antiplatelet agents on tumor growth and metastasis.

Disclosures
Flaumenhaft: Relay Therapeutics: Consultancy; PlateletDiagnostics: Consultancy, Other: Founder. Italiano:Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.