Platelets Enhance CD4+ Lymphocyte Adhesion to Extracellular Matrix under Flow Conditions: Role of Integrins, CD40 Ligand and P-Selectin Glycoprotein Ligand-1.
Authors: Savion Naphtali , Solpov Alexey , Shenkman Boris , Brill Grigory , Vitkovsky Yuri , Koltakov Alexander , Varon David , Bank Ilan 
Published: October 2019

Abstract
Recent studies established that platelets, beside their haemostatic function play a role in inflammation, immunity and atherosclerosis. In this study, we investigated the role of platelets in CD4+ lymphocyte adhesion to the subendothelial extracellular matrix (ECM) under static and flow conditions. Cultured clone of CD4+ lymphocyte (HVS infected clone) was incubated for 30 min at 37°C under static or flow conditions on tissue culture plates pre-coated with ECM, in the presence or absence of gel-filtered platelets. In separate experiments, the ECM plates were further coated with platelet-poor (PPP) or platelet-rich plasma (PRP) prior to the addition of cells. Flow conditions were applied using the Cone and Plate(let) Analyzer (CPA) device and the results were evaluated by an image analysis system and expressed as the number of adhered T-cells per mm2. Under static conditions, activation of T-cells with phorbol 12-myristate 13-acetate (PMA) led to 2.6-fold increase in cell adhesion. In the presence of platelets the adhesion rate did not change. In contrast, under flow condition (200 s−1), platelets substantially enhanced both resting and PMA-activated T-cells adhesion (12.7- and 18.5-fold, respectively). Moreover, under flow, platelet-T-cell heterotypic clusters appeared on the ECM as revealed by both light and scanning electron microscopy.
When ECM was pre-coated with PPP, adhesion of T-cells was enhanced by 2.3- and 1.8-fold under static and flow conditions, respectively. Pre-coating with PRP did not change T-cells adhesion under static but further enhanced T-cells adhesion under flow (by 80% vs. PPP). In this case platelet-lymphocyte clusters were not observed. Similar pattern of results was seen with natural blood-derived CD4+ lymphocytes cultured for 7 days. In these natural T-cells the adhesion under both static and flow conditions was platelet dependent, however, higher adhesion under flow compared to static conditions (55-fold) due to relatively low T-cells adhesion under static conditions and to the heterotypic cluster formation occurring only under flow conditions was observed. We further investigated the role of different receptors in T-cell-platelet cluster formation on ECM. Blockade of β1-dependent integrins on T-cells was followed by a decrease in adhesion under both static and flow conditions in the presence of platelets (by 37% and 43%, respectively). Combined blockade of CD40 ligand (CD40L) and P-selectin glycoprotein ligand-1 (PSGL-1) receptors on the T-cells decreased their adhesion in the presence of platelets by 72% under high shear (600 s−1) but not under low shear (200 s−1) and under static conditions. Blockage of the platelet integrin αIIbβ3 by tirofiban markedly reduced cluster formation thereby decreasing T-cells adhesion (by 90%) under flow conditions. The results of this study show that platelets support CD4+ lymphocyte adhesion to ECM under flow conditions by formation of heterotypic clusters that are dependent on platelet adhesion and aggregation and mediated by CD40L, PSGL-1 and β1-dependent integrins.