Figure 3.

Homophilic Interaction Between Transmembrane-JAM-A and sJAM-A Exerts the Prothrombotic Effect

(Ai) Immunofluorescence confocal microscopic images showing phalloidin (green) stained platelets adhered to collagen and immobilized recombinant truncated sJAM-AD1 and full-length sJAM-AD1D2. Bar = 2 μm. (Aii) Fluorescence intensity from platelets adhered to collagen (100 μg/mL), sJAM-AD1 (10 μg/mL), and sJAM-AD1D2 (30 μg/mL), in presence of anti-JAM-A-antibody (10 μg/mL) or IgG control (10 μg/mL) (n = 7). (Bi) Immunofluorescence confocal microscopic images showing enhanced expression of JAM-A (red) on inflamed endothelial cells. Bar = 5 μm. (Bii) sJAM-A (∗∗∗P < 0.001) substantiated platelet adhesion on JAM-A–enriched endothelial surface (n = 5) counteracted by anti-JAM-A-antibody (αJAM-A) (10 μg/mL) (###P < 0.001). (C) Synergistic effect of sJAM-AD1D2 (30 μg/mL) and sJAM-AD1 (10 μg/mL) on TRAP induced α_IIbβ₃-integrin activation (PAC-1) and platelet degranulation (CD62P) ±anti-JAM-A-antibody (αJAM-A) (10 μg/mL) or IgG control (10 μg/mL). ∗P < 0.05; ∗∗P < 0.01. (D) Synergistic effect of sJAM-AD1D2 (30 μg/mL) and sJAM-AD1 (10 μg/mL) on TRAP-induced platelet aggregation is counteracted by anti-JAM-A-antibody (αJAM-A) (10 μg/mL). ##P < 0.01; ∗∗∗P < 0.001. (C and D) Data represent mean ± SEM from n = 5 experiments with technical replicates. (E) Flow cytometric histogram overlay and bar diagram (n = 4 with technical replicates) showing the synergistic effect of sJAM-AD1D2 (30 μg/mL) on TRAP-induced Annexin-V binding ±anti-JAM-A-antibody (αJAM-A) (10 μg/mL). ∗∗∗P < 0.001; #P < 0.05. Abbreviations as in Figure 1.