Page 98 - Haematologica May 2020

M. Chimen et al.
binding are routinely performed ex vivo under the non- physiological condition of stasis in vitro, where the number of platelet-leukocyte aggregates formed is a direct func- tion of the time of incubation.49 Thus, patient blood may have a greater propensity to form aggregates with platelets under static conditions ex vivo, but this probably does not reflect the situation in vivo. Such aggregation may be a surrogate endpoint for the degree of platelet and/or leukocyte activation present in patient blood. In support of this, the patterns of PEV associated with circulating monocytes that we report here are in strong accord with a recent report from Fendl et al. who analysed the effects of pre-analytical blood handling (which included the imposi- tion of shear) on the association of extracellular vesicles with leukocytes.50
Interestingly, upon addition of purified PEV to whole blood, we observed rapid accumulation of GPIbα on monocytes, implying assimilation of PEV was extremely efficient. However, when a platelet activating agonist was added to whole blood the process was continuous and prolonged, leading to an incremental increase in GPIbα expression. The latter profile of accumulation of GPIbα likely reflects the dynamics of PEV formation and release
by platelets in whole blood, implying that the rate-limit- ing step in this thrombo-inflammatory pathway is not PEV-monocyte interaction, but rather the process of PEV release after platelet activation. In addition, accumulation of PEV was more prevalent in monocytes compared to neutrophils and lymphocytes. In a previous study, we observed different patterns of recruitment, migration and reverse migration in vitro between classical and non-classi- cal/intermediate monocytes.43 We characterised a novel process of crosstalk mediated by cytokines between the two subsets that allowed a balanced regulation of endothelial cell activation. Other studies have shown that changes in proportional representation of monocyte sub- sets in the circulation are associated with vascular dis- eases.51,52 However, in this study we observed no preferen- tial binding of PEV between classical and non- classical/intermediate monocytes, which was consistent with similar levels of PSGL1 expression exhibited by all subsets.
CD
E
GPIbα is an adhesion receptor mediating a specialised form of platelet recruitment during haemostasis. Bonds forming between GPIbα and VWF exhibit high on rates, meaning that adhesion can occur between rapidly flowing
A
B
Figure 8. Monocytes accumulate platelet-derived extracellular vesicles (PEV) derived marker CD41 in trauma patients. (A, B) Percentage (A) and median flu- orescent intensity (MFI) (B) of CD41+ monocytes, neutrophils and lymphocytes (PBL) in whole blood in patients at time point (T) <60 minutes (min) after trau- ma, n=31-35. ND: non detectable for lymphocytes. (C, D) Percentage (C) and MFI (D) of CD41+ monocytes in whole blood in patients at T<60 min after trauma plotted against Injury Severity Score (ISS), n=28. (E) Percentage of CD41+ mono- cytes in whole blood in trauma patients at T<60 minutes, 4-12 hours and 48-72 hours, n=33. Data are mean ± standard error of the mean (SEM) (A, B and E). *P≤ 0.05, ***P≤ 0.001 by ANOVA and Bonferroni post-test (A, E), Mann Whitney t-test (B) and linear regression (C, D).
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