Thrombocytopenia is common in patients with dengue virus (DENV) infections. With a focus on understanding the possible mechanism of thrombocytopenia in DENV infections we described a direct correlation between activation and depletion of platelets in patients. Our data showed a sharp decrease in platelet counts at day 4 of fever in patients. The high DENV genome copies in platelets correlated directly with the elevated platelet activation along with increased binding of complement factor C3 and IgG on their surface at day 4. Recovery in platelet count was observed on day 10 through day 6 and 8 with simultaneous decrease in platelet activation markers. Further, ourin vitrodata supported the above observations describing a concentration-dependent increase in platelet activation by DENV serotype-2. The high copy number of DENV2 genome in the platelet pellet correlated directly with platelet activation, microparticle generation and clot formation. Furthermore the DENV2-activated platelets were phagocytosed in large numbers by the monocytes. The DENV2-mediated lysis and clearance of platelets were abrogated in presence of platelet activation inhibitor, prostacyclin. These observations collectively suggest that platelet activation status is an important determinant of thrombocytopenia in dengue infections. A careful strategy of inactivation of platelets may rescue them from rapid destruction during DENV infections.

Activated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV). Microscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals. Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.

Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37–3 μM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3–6 μM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation.

Intravascular hemolysis increases the risk of thrombosis in hemolytic disorders. Our previous study showed that the binding of adult hemoglobin (HbA) to glycoprotein (GP) 1bα induced the activation of platelets. The elevated plasma Hb or platelet surface bound Hb positively correlated with platelet activation in patients with paroxysmal nocturnal hemoglobinuria (PNH). Furthermore, this study shows that the sickle Hb [HbS, occurs due to single nucleotide polymorphism at A>T of β-globin gene of Hb and causes sickle cell disease (SCD)] also bound to GP1bα and activated platelets in a concentration-dependent manner. The HbS bound to glycocalicin (extramembranous part of GP1bα) with K_D ~ 10.46 ± 3 μM. HbS induced phosphorylation of signaling adapter proteins, such as Lyn, PI3K, Akt and ERK in platelets, and also increased the surface expression of platelet activation markers such as P-selectin (10.7 fold) and PAC1 binding (10.4 fold) in platelet surface in a concentration-dependent manner. HbS also increased the platelet microparticle-generation (4.7 fold) and thrombus-formation (4.3 fold) in a concentration-dependent manner. An elevated level of extracellular Hb in plasma correlated directly with platelet activation markers such as P-selectin (r = 0.7947), PAC1 binding (r = 0.5914) on platelet surface and plasma levels of platelet-derived microparticles (r = 0.7834) in patients with SCD. Our study therefore suggests that the HbS-induced platelet activation may play a crucial role in intravascular clot formation observed in SCD patients characterized by high propensity to vascular occlusion and hypercoagulable states.