Heme Induced Platelet Mitochondrial Oxidant Production Regulates Thrombospondin-1 Release from Platelets

Background: Sickle Cell Disease (SCD) patients develop chronic vasculopathy leading to conditions such as pulmonary hypertension (PH), a major cause of morbidity and mortality. Vasculopathy is tightly associated with hemolysis in SCD. We and others have shown that in SCD, hemolysis activates platelets, and activated platelets are implicated in vasculopathy through their release of vasoactive molecules, such as thrombosponding-1 (TSP1), from storage granules. In SCD, free heme is accumulated in the circulation as a consequence of oxidative degradation of hemoglobin secondary to intravascular hemolysis. Under homeostatic conditions, albumin, haptoglobin, hemopexin, and high- and low-density lipoproteins clear plasma heme from the circulation. However excessive hemolysis can overwhelm these clearance mechanisms, resulting in elevated levels of free heme, which can lead to platelet activation, aggregation and thrombus formation. The role of activated platelets in these thrombotic events is well studied, but the mechanisms by which heme induces platelet activation and platelet-dependent signaling to the vessel wall remain unclear. We previously showed that hemolysis induces platelet mitochondrial reactive oxygen species (mtROS) production, but the mechanism by which heme induces mtROS production and downstream consequences are less clear. We hypothesize that heme induces platelet mtROS through Toll-like Receptor 4 (TLR4) signaling, which regulates platelet activation and granule secretion.

Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate anticoagulant (n=6). Platelets were treated with heme (2.5µM) in presence or absence of TLR4 neutralizing antibody, IRAK1/4 inhibitor, TBK1/IKKε inhibitor, MitoTEMPO or DS16570511 (Mitochondrial calcium uniporter inhibitor). Platelet activation was measured using flow cytometry by staining for CD62P (P-selectin) on the platelet surface and the active confirmation of GPIIb/IIIa. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. The granule secretion function of platelets was measured by assessing TSP-1 and Platelet Factor 4 (PF4) levels in platelet releasates by ELISA.

Results: We found that heme activates platelets and induces mtROS production, which can be mitigated by TLR4 neutralizing antibody, a molecule that makes platelet surface TLR4 unavailable to heme. In heme-treated platelets, pharmacological inhibition of the interleukin-1 receptor kinase-4 (IRAK4), a key kinase recruited by the TLR4 adaptor protein MyD88, had no significant effect on platelet activation. In contrast, inhibition of tank binding kinase-1 (TBK1), a TLR4 effector molecule independent of MyD88 signaling, significantly attenuated heme-mediated platelet activation and mtROS production. Scavenging mtROS with MitoTEMPO did not alter platelet activation, instead it decreased the TSP1 release from platelets, indicating heme induced platelet mtROS tightly regulates the granule secretion function of platelets. In addition, blocking mitochondrial calcium uptake by Mitochondrial calcium uniporter (MCU) inhibitor significantly decreased mtROS production and TSP-1 release from heme treated platelets.

Conclusion: Our data demonstrate that heme activates human platelets and induces mtROS production via a TLR4 -MyD88-independent signaling pathway. In addition, heme also induces mitochondrial calcium uptake that enhances mtROS production in platelets. Heme induced platelet mtROS tightly regulates platelet granule secretion function. Ongoing studies are further elucidating this pathway and determining the role of mtROS in heme-dependent platelet function. These studies will provide a mechanistic link between hemolysis and thrombosis, as well as platelet-vascular wall signaling. These studies suggest that platelet mtROS may potentially be a novel therapeutic target to prevent thrombotic events and vascular dysfunction in hemolytic disorders including SCD.