HMGB1-Mediated Platelet Activation Is Independent of Platelet Mitochondrial Reactive Oxygen Species Generation

Background: Sickle cell disease (SCD) is a hemolytic disorder that exhibits pathologic platelet activation. Notably, hemolysis is tightly associated with platelet activation and thrombotic complications of SCD such as stroke, leg ulceration, and pulmonary hypertension. To this end, we and others have shown that free hemoglobin (Hb) released into the plasma via hemolysis directly activates healthy platelets ex vivo in a concentration-dependent manner. Treatment with Hb stimulates the production of mitochondrial reactive oxygen species (mtROS) within the platelet, resulting in thrombotic activation. Accordingly, scavenging mtROS prevents Hb-induced platelet activation. While it is established that hemolysis mediates platelet activation, the factors that modulate this response remain unknown. High mobility group box 1 (HMGB1) is an inflammatory mediator, which signals through toll-like receptor-4 (TLR4), to instigate thrombosis. Notably, we previously showed that HMGB1 is elevated in the plasma of SCD patients and augments platelet activation on its own. Importantly, complementary interactions between Hb and other pro-thrombotic plasma components such as HMGB1 have not been investigated. Preliminary data show that HMGB1, like Hb, stimulates platelet mtROS generation. Thus, we hypothesized that elevated levels of HMGB1 and Hb synergize in SCD to stimulate platelet mtROS production and downstream platelet activation.

Methods: Whole blood samples were collected from healthy human control subjects or SCD subjects in steady state (n=5 per group). Plasma HMGB1 concentrations were quantified by ELISA. Platelets were isolated and treated with reagents including HMGB1, ferric Hb, or the mtROS scavenger MitoTEMPO. Platelet activation was measured by flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and two markers of platelet activation: PAC1 to detect the activation-dependent conformational change in integrin α_IIbβ₃ (GP IIb-IIIa), and APC to detect exposure of surface CD62P (P-selectin). Platelet mtROS were estimated using MitoSOX Red and fluorescence spectroscopy. Data was analyzed using FlowJo software and nonparametric statistical tests.

Results: The baseline level of platelet activation in SCD was 7-fold higher than in controls (P=0.009). However, there was no difference in platelet activation levels between isolated SCD and control platelets in response to ex vivo treatment with agonists ADP (5 uM), collagen (50 ug/ml), or thrombin (0.1 U/ml) (P=0.38), suggesting that SCD platelets have the same agonist-induced response as control platelets when they are removed from their native plasma. We found that the concentration of HMGB1 in SCD plasma is 1.5 fold higher than controls (P=0.04), and that plasma HMGB1 concentrations positively correlate with increased platelet activation (P=0.04). Co-incubation of isolated healthy control platelets with HMGB1 (10 ug/ml) and major hemolytic byproduct Hb (10 uM, 30 uM, 50 uM) enhanced platelet activation at all doses of Hb by 3-fold compared to that of Hb alone. Mechanistically, platelets exposed to Hb alone generated mtROS which are necessary for Hb-induced platelet activation. We found that HMGB1 similarly induced platelet mtROS generation, but scavenging with MitoTEMPO did not prevent HMGB1-mediated platelet activation. However the addition of MitoTEMPO to platelets co-treated with HMGB1 + Hb attenuated platelet activation 2-fold, indicating that scavenging mtROS neutralizes the enhanced effect of Hb + HMGB1.

Conclusions: We show that HMGB1 synergizes with Hb to enhance platelet activation in SCD, but unlike Hb, HMGB1-dependent platelet activation is independent of mtROS generation. Accordingly, we observed that MitoTEMPO partially attenuates the enhanced activation from co-stimulation by HMGB1 + Hb. These data characterize the synergy between HMGB1 and Hb for the first time and demonstrate differential platelet signaling between the two agonists.