Background: Rhabdomyolysis can lead to severe acute kidney injury (AKI); however, there is no effective treatment at present. According to a recent study, heme (hemin) released from damaged muscles activates platelets, and the activated platelets induce macrophage extracellular traps (METs), resulting in exacerbation of AKI. We hypothesized that C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) on platelets are involved in hemin-induced platelet activation, and we have reported in the last ISTH that both CLEC-2 and GPVI contributes to hemin-induced platelet aggregation in mice.

Aims: The first aim is to reveal the detailed mechanism of hemin-induced platelet activation in human platelets. The second is to investigate the role of CLEC-2 and GPVI in rhabdomyolysis-induced AKI by using a mouse model.

Methods: We performed human platelet aggregation assay and western blotting to identify the signaling cascade activated by hemin stimulation. We next evaluated the direct binding between hemin and recombinant CLEC-2 or GPVI by surface plasmon resonance. Using a mouse model of rhabdomyolysis, we biochemically and histologically compared the renal function of CLEC-2-depleted FcRγ-deficient (equivalent to CLEC-2/GPVI double knockout and referred to as DKO) mice with that of wild type (WT) mice.

Results: Hemin induced human platelet aggregation in a dose-dependent manner with increasing phosphorylation of SYK and PLCγ2, which was blocked by SFK inhibitor. In addition, surface plasmon resonance analysis revealed that hemin directly binds to both CLEC-2 and GPVI (Figure 1). Lower serum creatinine level and less injured renal tubules (Figure 2) and METs-like structures were observed in DKO mice compared with WT mice.

Conclusions: Hemin is a novel ligand of both CLEC-2 and GPVI and activates platelets through the SFK-SYK-PLCγ2 pathway. Less prominent renal dysfunction in DKO mice suggested that CLEC-2 and/or GPVI may be new therapeutic targets for rhabdomyolysis-induced AKI patients.

[Figure 1 Direct binding of hemin to immobilized human CLEC-2 (left) and human GPVI (right)]

[Figure 2 Renal tubular injury evaluated by PAS staining]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/heme-activates-platelets-and-exacerbates-rhabdomyolysis-induced-acute-kidney-injury-via-clec-2-and-gpvi-fcr%ce%b3/