Purpose: To characterise morphological and phenotypical differences between small-size platelet MPs (sPMP) released from resting platelets, thrombin activation and platelet disruption (termed as platelet debris [PD]) using our novel technological approach. Besides, we aimed to assess their prothrombotic properties by evaluating the ability to induce thrombus formation, platelet and monocyte activation ex vivo.
Methods: sPMP produced in resting conditions and thrombin stimulation were analysed by high-resolution flow cytometry and transmission electron microscopy. Platelet debris (PD) were produced by platelet disruption. Plasmatic counts of sPMP were analysed in coronary artery disease (CAD) patients (n=40) and healthy controls (HC) (n=40). The effect of sPMP in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry.
Results: New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Higher sAMP counts were released under thrombin stimulation compared to resting conditions (p=0.002). CD42b+, CD61+ and CD63+ counts were not altered after thrombin stimulation. Platelets and sPMP after thrombin stimulation did express CD62P (p=0.03). PD and residual platelets after disruption and resting did not express CD62P. Patients with CAD had 2-fold higher sAMP counts compared to sex- and age-matched HC (p=0.003). Addition of sPMP in these pathophysiological doses resulted in platelet degranulation and P-selectin redistribution to the membrane (p=0.019) in a dose and time-dependent manner. Addition of sPMP had no effect in PAC-1 binding. Blood clotting time (CT) decreased after addition of sPMP (p=0.005), but was not affected by PD. Blocking CD62P+ sPMP before blood incubation markedly reverted the effect on thrombus kinetics (p=0.035). In addition, exposure to sPMP stimulated monocytes (different monocyte subsets) expression of intercellular adhesion molecule-1 (all p<0.03) and decreased monocyte interleukin-6 receptor density (all p<0.01). No effect of sPMP was observed on monocyte CXCR4 and CD163 expression.
Conclusions: There are significant phenotypic differences between sPMP depending on the stimulus which should be taken into account for future sPMP quantification. The findings of this study demonstrate that, far from platelet debris, sPMP have complex implications as a direct source of downstream platelet, monocyte activation and thrombogenesis, in part through P-selectin expression.