ICAM-1 contributes to the preferential binding of platelets to classical and intermediate monocyte subsets over neutrophils

Abstract

Platelet‐leukocyte interactions have beenlinked to a range of cardiovascular and inflammatory disease states.A variety of mechanisms have been proposed to facilitate platelet–neutrophil and platelet–monocyte interactions, including P‐selectinand ICAM‐1. However, it remains unclear whether platelets preferen-tially bind to particular leukocyte cell types. We therefore set out toaddress this question.Experimental Approach: Blood was collected from healthy volunteersinto sodium citrate (0.32% w/v final). Whole blood (45 μl) was placedinto individual wells of half‐area 96‐well plates containing TRAP‐6amide (0.1–3 μM), U46619 (0.1–3 μM), CRP (0.003–0.1 μg·ml−1)orvehicle (PBS), and mixed (200 rpm for 10 min, 37°C). Following this,samples were immediately fixed and flow cytometric analysis per-formed to determine platelet P‐selectin (CD62P) expression and alsoidentify platelet (CD42b) binding to the following leukocyte subsets:neutrophils (CD66b+), classical monocytes (CD14++CD16−), intermedi-ate monocytes (CD14++CD16+), and non‐classical monocytes(CD14+CD16+). Interactions between platelets and cells were calcu-lated as a percentage (%) of cell subset number and then normalisedfor paired vehicle control. To test for role of ICAM‐1, experimentswere repeated in presence of the inactive competitive ligand fibrino-gen γ‐chain 117‐133 (20 μM). Data presented as mean ± SEM andcompared using t test or one‐way ANOVA with Tukey post hoc test,as appropriate.Key Results: All agonists induced significantly higher platelet P‐selectin expression than vehicle (P < .05, n = 4). TRAP‐6 stimulatedblood exhibited the following order of platelet interactions: intermedi-ate monocytes (55 ± 11%); classical monocytes (46 ± 12%); non‐clas-sical monocytes (12 ± 2%); neutrophils (3 ± 3%). The same order ofbinding was observed when stimulated with CRP (43 ± 9%; 40 ± 9%;11 ± 6%; 0 ± 2%, respectively) or U46619 (27 ± 4%; 20 ± 5%;2 ± 3%; 0 ± 3%, respectively). Competitive antagonism of ICAM‐1 sig-nificantly reduced platelet binding in response to TRAP‐6 to the clas-sical (31 ± 1% vs. 50 ± 3% vehicle, P < .05, n = 3) and intermediate(38 ± 1% vs. 54 ± 3% vehicle, P < .05, n = 3) subsets but not thenon‐classical subset (12 ± 2% vs. 15 ± 3% vehicle, P < .05, n = 3) Our data indicate that following in vitrostimulation with a range of agonists, platelets preferentially bind tointermediate and classical monocytes more than non‐classical mono-cytes or neutrophils. Moreover, as we have previously indicated inmice, ICAM‐1 significantly contributes to mediating these interactions.More work is required to determine the direct downstream influencesthese interactions have on these specific subsets. However, as thesesubsets are commonly characterised as pro‐inflammatory and platelets are known to contain a range of chemokines and cytokines, it may rep-resent a key route for platelets to directly influence the inflammatoryimmune response