The Interaction Between Collagen-Activated Platelets and Erythrocytes Selectively Increases the Release of Subsets of Platelet α-Granules Containing Pro-Angiogenic Substances
Authors: Santos Maria Teresa , Moscardo Antonio , Sanz Miguel A. , Latorre Ana Maria , Dasi Maria Angeles , Valles Juana 
Published: October 2019

Abstract
Activated platelets release a wide range of bioactive molecules including growth factors, cytokines, chemokines, coagulation factors etc., known to influence processes like haemostasis, thrombosis, inflammation, wound healing and angiogenesis. The interaction between activated platelets with intact erythrocytes (RBCs) increases dense (14C-serotonin) and alpha (β-thromboglobulin) platelet granule secretion (1, 2). Platelets have different sets of α-granules which are selectively released depending on the platelet agonist and signaling mechanism (3).
Aims. to study a) if platelet-RBC interactions induce any differential regulation on the secretion of sets of platelet α-granules containing pro- or antiangiogenic compounds and b) the potential influence of aspirin on the release of these substances.
Methods. Washed platelets alone (WP) or WP+RBCs from normal human subjects were incubated (10min, 37ºC) with aspirin (100µM) or solvent. Collagen (or its solvent) were added to samples, mixed (10 sec) and centrifuged (13,000xg, 1min) to obtain a cell-free releasate (1, 2). A semi-quantitative analysis of 43 pro- and anti-angiogenic substances in releasates was performed using antibody arrays (RayBiotech, Inc). In addition, the concentration of VEGF and ENA-78 (pro-angiogenics) and endostatin (anti-angiogenic) were quantified in the cell-free releasates by ELISA (R&D Systems) in 4 donors.
Results. The array data demonstrated a striking selectivity in the effect of RBCs (hematocrit, 40%) for releasing the subset of α-granules containing pro-angiogenic substances. For instances, as compared to WP, RBCs increased the release of CXCL1 (7-fold), angiopoietin (30-fold) and MCP-1 (50-fold), while the release of anti-angiogenic substances such as TIMP-1 or endostatin were not modified by RBCs. Evaluating by ELISA the concentration of two angiogenic stimulators (VEGF and ENA-78) and of one anti-angiogenic compound, endostatin, we observed that RBCs increased the concentration of VEGF and ENA-78 in a hematocrit-dependent manner reaching a 18-fold and 10-fold increase, respectively with 40% hematocrit (p<0.05), while the endostatin release was not increased by RBCs. In samples not stimulated (collagen-solvent) no differences were noted between WP and WP+RBCs. Incubation of samples with aspirin did not modify the release in samples of WP but drastically reduced the collagen-induced VEGF and ENA-78 release in the presence of RBCs (>95% inhibition) while aspirin had a scarce effect on endostatin release.
Conclusions. Cell-cell contact between collagen-activated platelets and RBCs is an important determinant for the specific release of subsets of platelet α-granules containing angiogenesis stimulators, which may play a role in patients with cancer. The aspirin down-regulation of the enhancing effect of RBCs on release of pro-angiogenic substances may be a contributory factor of the beneficial effect of aspirin observed in some patients with cancer.
1.Santos MT et al J Clin Invest 1991;87:571-80; 2. Valles J et al. Blood 1991;78:154-62 ; 3. Italiano JE Blood 2008; 111:1227-33.
Grant support: IIS Carlos III. Fondos FEDER PI13/00016; Red Cardiovascular [RD12/0042/0003]

Disclosures
No relevant conflicts of interest to declare.