Role of Erythrocytes in Signal Transduction Mechanisms of Recruiting Platelets: Effects on Platelet Tyrosine Phosphorylation and Cytoskeletal Reorganization.
Authors: Valles Juana , Santos Maria T. , Moscardo Antonio , Dasi Maria A. , Aznar Justo 
Published: October 2019

Abstract
Platelet activation induces the release of granular components and metabolic products. This platelet releasate is a complex physiological agonist that upon interaction with other platelets induces platelet recruitment and thrombus growth. The agonistic potency of the platelet releasate is increased when platelets are stimulated in the presence of intact erythrocytes (RBCs) (Santos et al J. Clin Invest1991; 87: 571; Valles et al Blood1991;78:154). The result is enhanced recruitment, α IIbβ3 receptor activation and P-selectin exposure in recruiting platelets (Valles et al Blood2002;99:3978). Receptor-mediated platelet activation initiates mechanisms of signal transduction that culminate in platelet functional responses. The biochemical mechanisms regulating the effects of cell-releasates in promoting platelet recruitment have not yet been elucidated. The aims of this study are: a) to characterize the effects of releasates from collagen-stimulated platelets on protein tyrosine phosphorylation (PTP), cytoskeletal reorganization and translocation to cytoskeleton of proteins in platelets being tested for recruitment; and b) to study the effects of platelet-erythrocyte interactions in these processes. Washed platelets (WP) or WP+RBCs (Htc. 40%) were stimulated with fibrillar collagen (1μg/mL) and rapidly centrifuged to obtain a cell-free collagen-free releasate within 1 min. An aliquot of this releasate was used as platelet agonist and the proaggregatory response (recruitment) was monitored by optical aggregometry as in the references above. For kinetic studies the reaction was halted at 15–180 sec., and PTP, cytoskeletal reorganization and cytoskeletal-associated tyrosine phosphorylated substrates were evaluated (Santos et al Circulation2000; 102:1924–1930). Our results demonstrate for the first time that the platelet releasate induces PTP in the platelets tested for recruitment. This effect is remarkably amplified with releasates from platelet-erythrocyte mixtures especially at earlier time points. Of interest is the strong FAK phosphorylation induced by this releasate, a kinase thought to play a role in αIIbβ3 clustering and focal adhesion formation. The greater potency of the platelet-erythrocyte releasate was also evident in cytoskeletal reorganization (actin, ABP, talin). The platelet-erythrocyte releasate induced more PTP and cytoskeletal reorganization than 20 m M ADP. Cytoskeletal reorganization and translocation of tyrosine phosphorylated substrates to the cytoskeleton induced by the platelet-erythrocyte releasate were quicker than those induced by 1 U/ml thrombin and were of comparable final intensity. Interestingly, erythrocytes markedly enhanced translocation of FAK kinase and αIIbβ3 to the platelet cytoskeleton. The data reveal new biochemical mechanisms regulating platelet recruitment and demonstrate the remarkable role of erythrocytes in the signal transduction mechanisms of platelets during the recruitment process, which is a limiting step in mural thrombus formation. (Grant FIS 03/0270).