Heme-Induced Platelet Mitochondrial Oxidant Production Regulates Platelet Granule Release
Authors: Annarapu Gowtham K , Nolfi-Donegan Deirdre , Reynolds Michael , Shiva Sruti 
Published: November 2021

Background: Patients with Sickle Cell Disease (SCD) are at greater risk for thrombosis and the development of chronic vasculopathy, both of which are major contributors to morbidity and mortality in these patients. While thrombosis and vasculopathy are associated with hemolysis in SCD, the molecular mechanisms by which hemolysis propagates these conditions remains unclear. At the cellular level, we and others have shown that hemolytic components, including hemoglobin and its degradation product, free heme, directly activate platelets. Notably, activated platelets are not only central to thrombosis, but are also implicated in vasculopathy through their degranulation, which results in the release of vasoactive molecules. Though heme and hemoglobin-induced platelet activation has been widely studied, the effect of hemolytic products on platelet degranulation and the identity of the resulting platelet secretome remains less clear. We hypothesize that free heme activates a platelet signaling cascade resulting in platelet degranulation and the release of specific "secretome" molecules.

Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate (n=6). Platelets were treated with heme (2.5µM) in the presence or absence of MitoTEMPO (10 µM) - a mitochondrial oxidant (mtROS) scavenger, or ARQ092 (10 µM), a small molecule that prevents phosphorylation of the serine/threonine kinase Akt at S473. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. Thrombospondin-1 (TSP1), CXCL7, Fibroblast Growth Factor (FGF) basic, TGFβ, IL-1β, PDGF-B, angiostatin, kininogen, CD40L and PAI-1 were quantified in heme treated platelet releasates in the presence and absence of MitoTEMPO by dot blot. The enzymatic activity of mitochondrial electron transport complex V was measured spectrophotometrically by kinetic assay.

Results:

We found that heme stimulated the release of a specific set of molecules from the α-granule of platelets, including TSP1, CXCL7, FGF basic, TGFβ, IL-1β, PDGF-B, angiostatin, and kininogen; but did not stimulate the release of CD40L or PAI-1. Mechanistic studies demonstrate that the release of several of these molecules was dependent on heme-induced activation of platelet Akt which inhibits mitochondrial complex V, resulting in mtROS production. Consistent with this mechanism, the heme-stimulated release of TSP1, CXCL7, FGF basic, IL-1β, PDGF-B, and angiostatin were significantly attenuated by preventing Akt phosphorylation at Ser473 with ARQ092, which also prevented complex V inhibition and mtROS production. Direct scavenging of mtROS with MitoTEMPO also attenuated heme-induced release of these molecules.

Conclusion: These data, for the first time, begin to characterize the platelet secretome released in response to free heme. Further, they demonstrate a novel molecular pathway in which extracellular heme induces the activation of platelet Akt to inhibit mitochondrial complex V, ultimately inducing mtROS. Notably, this study suggests that release of a proportion of the heme-induced secretome is regulated by mtROS production and can be suppressed by mtROS scavengers. Ongoing studies are further characterizing the hemolysis-induced platelet secretome, the downstream effects of secretome products on vascular function, and the extent of regulation of the secretome by mtROS. These studies provide a mechanistic link between hemolysis and platelet degranulation, by which the release of mitogens can lead to the pathogenesis of vascular wall dysfunction. These studies also suggest that platelet mtROS may represent a novel therapeutic target to attenuate vascular dysfunction in hemolytic disorders including SCD.

Note: The finding discussed in the above abstract are available as preprint in bioRxiv; doi: https://doi.org/10.1101/2021.08.02.454816

Disclosures

No relevant conflicts of interest to declare.