Monocytes Down-Regulate Platelet Activation Induced by a Collagen Surface.
Authors: Arnaud Bertrand , Ollivier Veronique , El Benna Jamel , Jandrot-Perrus Martine , Ajzenberg Nadine 
Published: October 2019

Abstract
Damage of blood vessels exposes the subendothelial matrix and results in the adhesion of platelets and monocytes at the site of injury. On eroded atherosclerotic plaques, platelets avidly bind to abundant type I collagen and monocytes are recruited by activated platelets. The purpose of the current investigation was to examine reciprocal interaction between platelets and monocytes upon exposure to a collagen surface. CD14 isolated monocytes and washed platelets were incubated separately or together in a 1/100 ratio in plates coated with type I fibrillar collagen. Platelet activation was assessed by measuring P-selectin expression by flow cytometry and RANTES secretion by ELISA. Platelet adherence and activation on immobilized collagen was analysed by confocal microscopy using FITC-phalloidin. Alternatively, cell-cell contacts were prevented by incubating platelets and monocytes in transwell coculture dishes, both parts of which were coated with collagen. In selected experiments cells were pretreated with the anti-PECAM 1.3 monoclonal antibody or with L-NMMA (NG-methyl-L-arginine, inhibitor of NO synthesis). IL8 was measured as an activation marker of monocytes. In co-incubation studies, collagen-activated platelets triggered IL8 secretion (6-fold increased) by monocytes, in agreement with previous observations indicating that platelets adherent to collagen delivers activating signals to monocytes. We have then focused our attention on the effect of monocytes on platelet activation. Unexpectedly, when monocytes were added five minutes after platelets to the collagen-coated plates, we obtained a decreased platelet expression of P-selectin by 42% (15.2 ± 9.1% positive platelets versus 26.3 ± 11.7% in the absence of monocytes, p = 0.0053, n = 16) and RANTES secretion by 39% (p < 0.0001, n = 6). The inhibitory effect of monocytes on platelet secretion decreased when the time to which they were added to platelets and collagen increased. Platelets incubated with immobilized collagen adhered and formed large aggregates consistent with a strong activation state. When monocytes were added, they established contacts with platelets while the number and size of the aggregates were dramatically decreased and isolated platelets were observed. In experiments performed in transwell coculture dishes, platelet P-selectin expression and RANTES secretion returned to the levels obtained in the absence of monocytes indicating that cell-cell contacts were required to inhibit platelet secretion induced by collagen. Preincubation of monocytes with anti-PECAM 1.3 reduced the inhibition of collagen-induced P-selectin expression and of RANTES secretion by ~ 40 %. Moreover, the inhibitory effect of monocytes on platelet aggregation appeared to be reversed by the anti-PECAM 1.3 antibody with a loss of individual platelets and the presence or large aggregates. In the presence of L-NMMA pre-treated monocytes, RANTES secretion was similar to the value obtained in the absence of monocytes. Together, our data provide evidence that, monocytes limit the initial phase of platelet activation by a collagen surface. The mechanism of this effect is dependent on cell-cell contacts. It is, at least in part, mediated by PECAM-1 with a contribution of NO. The interaction of platelets with monocytes at the surface of a damaged vessel would thus have two different effects:it would limit platelet activation and recruitment andit would increase the contribution of monocytes in inflammatory and procoagulant responses.