title: Stimulation of PAR-1 or PAR-4 promotes similar pattern of VEGF and endostatin release and pro-angiogenic responses mediated by human platelets.
abstract: BACKGROUND
  Platelets mediate angiogenesis through the secretion of several factors, including the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic endostatin. Although previous findings indicated that these molecules are packed into different alpha-granules and selectively released by specific stimulation of protease-activated receptor (PAR)-1 or PAR-4, recent evidences are against this hypothesis.
  
  
  OBJECTIVE
  To elucidate the controversies about the VEGF and endostatin release and the overall angiogenic effect of PARs-stimulated platelets.
  
  
  METHODS
  VEGF and endostatin were quantified by enzyme linked-immunosorbent assay (ELISA). Endothelial proliferation (pNPP assay), wound healing (scratch assay) and tubule formation (matrigel) of human microvascular endothelial cells (HMEC-1) and endothelial progenitor cells (EPC) were determined using supernatants from PAR-1- or PAR-4-stimulated platelets.
  
  
  RESULTS
  Activation of washed platelets (WPs) by PAR-1- or PAR-4-activating peptide (AP) promoted the VEGF and endostatin secretion in a concentration-dependent manner, being PAR-1-AP more potent than PAR-4-AP. The release of both molecules was abrogated by pre-incubation of platelets with PAR antagonists. Activation of platelet-rich plasma (PRP) with either PAR-1-AP or PAR-4-AP induced a significant VEGF secretion. Quantification of platelet-endostatin secretion was not possible in PRP due to the high levels of plasmatic endostatin vs. platelet content. Releasates from PAR-1- or PAR-4-activated WPs promoted similar pattern of angiogenic responses of HMEC-1 or EPC. Moreover, proliferation of HMEC-1 mediated by PAR-stimulated PRP releasates was delayed and significantly lower compared with that induced by PAR-stimulated WPs.
  
  
  CONCLUSIONS
  Our results are in contrast with the previously described differential release of VEGF and endostatin induced by the selective PAR-1 or PAR-4 stimulation, and support the notion that while circulating endostatin accounts for the maintenance of a systemic anti-angiogenic state, locally, the release of platelet alpha-granule content promotes angiogenesis.
authors.affiliation: a Laboratory of Experimental Thrombosis , Institute of Experimental Medicine, CONICET-National Academy of Medicine , Buenos Aires , Argentina.
authors.forename: J
authors.initials: J
authors.lastname: Etulain
authors.affiliation: a Laboratory of Experimental Thrombosis , Institute of Experimental Medicine, CONICET-National Academy of Medicine , Buenos Aires , Argentina.
authors.forename: H A
authors.initials: HA
authors.lastname: Mena
authors.affiliation: a Laboratory of Experimental Thrombosis , Institute of Experimental Medicine, CONICET-National Academy of Medicine , Buenos Aires , Argentina.
authors.forename: S
authors.initials: S
authors.lastname: Negrotto
authors.affiliation: a Laboratory of Experimental Thrombosis , Institute of Experimental Medicine, CONICET-National Academy of Medicine , Buenos Aires , Argentina.
authors.forename: M
authors.initials: M
authors.lastname: Schattner
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D043169:Endostatins / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D018919:Neovascularization, Physiologic; D009842:Oligopeptides / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D044463:Receptor, PAR-1 / Q000819:agonists*; D018179:Receptors, Thrombin / Q000819:agonists*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D043169:Endostatins; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D042461:Vascular Endothelial Growth Factor A; C112908:protease-activated receptor 4
keywords: Angiogenesis; VEGF; endostatin; platelet alpha-granules; protease-activated receptors