Session: 301. Vascular Wall Biology, Endothelial Progenitor Cells, and Platelet Adhesion, Activation, and Biochemistry: Poster III
Hematology Disease Topics & Pathways:
sickle cell disease, Diseases, Bleeding and Clotting, Thrombosis, Hemoglobinopathies, Platelet Disorders, Thrombotic Disorders
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 αIIbβ3 (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.
Disclosures: No relevant conflicts of interest to declare.
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