Figure 6.

(a) Platelet activation and platelet–leukocytes interaction (LPI). Diluted blood was incubated with platelet agonist, PAM3CK4 (TLR2 agonist) or lipopolysaccharide (LPS) (TLR4 agonist), then the LPI was measured as the percentage of CD45 cells with aggregated CD41 cells. Thirty MPN patients including 16 ET, eight PV, and six MF (two post-PV-MF, one ET-MF, and four PMF) were compared to seven normal controls. The results showed that unactivated MPN (u-MPN) has more LPI than unactivated controls (u-CTR) with mean values of 13.01 ± 1.12 and 5.01 ± 0.97 in u-MPN and u-CTR, respectively (P  < 0.01). Upon activation by PM3CSK4, patients with MPN also have greater LPI than those by LPS (TLR4 agonist), with mean ± SE of 22.25 ± 0.99 and 10.46 ± 0.44, respectively (P  < 0.0005). (b) TLR Inhibitor in the leukocytes–platelet interactions (LPI). Diluted blood was incubated with platelet agonist, PAM3CK4 (TLR2 agonist) or lipopolysaccharide (LPS) (TLR4 agonist) for 15 min at room temperature in the presence or absence of TLR2 inhibitor C29 or TLR4 inhibitor C34. Fold changes were calculated as LPI after activation by either PM3CK4 (TLR2 agonist) or LPS (TLR4 agonist) or with an added inhibitor of C29 or C34, divided by the LPI before activation. Ten MPN patients were studied, including four ET, two ET-MF, one PV-MF, and two PV patients. The TLR2 inhibitor C29 significantly inhibits the LPI: expressed as (mean ± SE) of fold changes in unactivated MPN (0.38 ± 0.05), activated controls (0.42 ± 0.11), and activated MPN (0.42 ± 0.05). The TLR4 inhibitor C34 had no effects of inhibition: the unactivated MPN value was 0.99 ± 0.02, the activated MPN value was 1.02 ± 0.02, and the value of the activated control was 1.01 ± 0.02. These findings show TLR2 than TLR4 plays a significant role in the interaction of LPI in MPN patients.