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Intravascular hemolysis is a serious complication developed in many severe infections such as malaria, sepsis, typical hemolytic uremic syndrome and congenital diseases such as sickle cell disease. Hemolysis is accompanied by thrombocytopenia, platelet activation, plateletleukocyte aggregates, inflammation, vascular obstruction and organ damage. During hemolysis, red blood cell destruction leads to the release of molecules such as ADP, hemoglobin and heme which exert potent prothrombotic and proinflammatory actions. Under physiological conditions, free heme is scavenged by the plasma protein hemopexin, and is subsequently catabolized by heme oxygenase-1 into carbon monoxide, biliverdin and ferrous iron (Fe). However, acute or/and chronic hemolysis exhausts the scavenging system leading to an increase in free heme in the blood. Upon release, reduced heme is rapidly and spontaneously oxidized in the blood into ferric (Fe) form, hemin, with increased levels observed in hemolytic diseases. It was recently shown that hemin induces human platelet activation and platelet death through ferroptosis, an intracellular iron-mediated cell death. However, the mechanisms and receptors involved in platelet activation by hemin are not known. In this study, we investigated hemin-mediated human and mouse platelet activation in vitro. An increase in labile (i.e., weakly bound and free) heme/hemin is detected in patients with hemolytic diseases with plasma concentrations ranging between 2 mM and 50 mM. Similar concentrations of heme are detected in mice with chronic or acute hemolysis such as sickle cell mice or Escherichia coli-induced hemolysis. In this study, we show that hemin (2-12 mM) induces rapid aggregation of human washed platelets, while aggregation is slower and reduced in magnitude at higher (≥ 25 mM) concentrations (Figure 1A, B). Platelet aggregation was associated with increased P-selectin expression and GPIIbIIIa activation as measured by flow cytometry (Figure 1C, D). High (≥25 μM) but not lower concentrations of hemin significantly increased phosphatidylserine (PS) exposure on platelets as assessed using Annexin V staining (Figure 1E). Moreover, high concentrations of hemin induced toxicity as measured by increased levels of lactate dehydrogenase (LDH) in the supernatant of activated platelets (Figure 1F). Recent data have shown that a high concentration of hemin (25 mM) triggers lipid peroxidation and human platelet death through ferroptosis but not apoptosis or necroptosis. Whether the increase in PS exposure modulates the contribution of platelets to the coagulopathies observed in hemolytic diseases requires further investigation. Hemin is known to bind to Toll-like receptor (TLR) TLR4 on endothelial cells resulting in endothelial cell activation including secretion of Weibel-Palade bodies. However, blocking TLR4 signaling in platelets using TAK-242 did not alter activation by hemin, suggesting a TLR-4-independent pathway of activation (Figure 1H). At low concentrations, human platelet aggregation by hemin was blocked by inhibitors of Syk (PRT-060318), Src family kinases (PP2) and Btk (Ibrutinib), and partially by GPIIbIIIa inhibitor eptifibatide (Integrilin) (Figure 1G, H). These results suggest that hemin induces human platelet activation via an immunoreceptor-tyrosine-based activation-motif (ITAM) receptor-based pathway. Indeed, low concentrations of hemin provoked phosphorylation of Syk and PLCγ2 (Figure 1I). In order to identify the receptor triggering platelet activation by hemin, we used recombinant dimeric forms of C-type-lectin-like receptor-2 (CLEC-2) and collagen receptor glycoprotein VI (GPVI) (hFc-CLEC-2 and hFc-GPVI, respectively) as a blocking strategy. Pre-incubation of hemin with hFcCLEC-2 but not hFc-GPVI abolished platelet aggregation by hemin identifying CLEC-2 as the receptor mediating human platelet activation by hemin (Figure 1G, H). These results also suggest a direct interaction between hemin and CLEC-2. However, while we believe that the inhibition is likely the result of competition between recombinant and platelet CLEC-2, we cannot rule out that recombinant CLEC-2-hemin complex may interfere with CLEC-2 clustering thereby inhibiting platelet activation. Platelet aggregation was not altered by AYP1, 9, an antibody that blocks the interaction between CLEC-2 and podoplanin, providing indirect evidence that hemin binds to a distinct site on CLEC-2 to podoplanin (Figure 1H). Protoporphyrin IX, the precursor of heme, and cobalt hematoporphyrin were shown to bind to CLEC-2 and inhibit podoplanin-CLEC-2 interaction without inducing platelet activation. The type of interactions by which different porphyrins selectively bind to CLEC-2 and induce platelet activation requires further investigation. Platelet aggregation mediated by low concentrations of hemin (6.25 mM) was not altered by cyclooxygenase (COX) (indomethacin) or P2Y12 (cangrelor) inhibitors, showing that, in contrast to podoplanin, secondary mediators are not required for hemin-mediated platelet activation (Figure 1G, H). This may reflect the ability of hemin to induce higher oligomers of CLEC-2 increasing signal transduction. Platelet aggregation does not depend on oxidative stress as hemin-mediated platelet aggregation was not altered by the antioxidant N-acetyl cysteine (NAC) (Figure 1H; Online Supplementary Figure S1). At higher concentrations (>25 mM), platelet aggregation was not inhibited by integrilin or inhibitors of Src, Syk and Btk, suggesting that higher concentrations of hemin induce agglutination (Online Supplementary Figure S2). Increasing the concentrations of inhibitors did not alter platelet agglutination/aggregation (not shown). The distinct mechanisms of platelet activation by low and high concentrations of hemin might be related to formation of hemin aggregates at high concentrations, which might exert different activities as compared to monomeric or dimeric hemin present at low concentrations. Hemin induces mouse platelet aggregation, albeit at a slightly higher concentration (Figure 2A). Similar to human platelets, aggregation by low concentrations of hemin was inhibited by inhibitors of Src, Syk and Btk tyrosine kinases, recombinant mouse Fc-mCLEC-2 and GPIIbIIIa blockade (Figure 2B, C). In contrast, inhibitors of TLR4, COX and P2Y12 had no effect on platelet aggregation by hemin (Figure 2C). Platelet aggregation by hemin was significantly reduced in CLEC-2-deficient platelets confirming a key role for CLEC-2 (Figure 2D, E). However, deletion of CLEC-2 did not inhibit platelet shape change suggesting that one or more other receptors support platelet activation by hemin in mice. The fact that recombinant CLEC-2 inhibits heminmediated platelet aggregation suggests a direct interaction between hemin and CLEC-2. Using surface plasmon resonance-based technique, we demonstrated that hemin binds to both murine and human recombinant CLEC-2 with KD values of ~200 nM in both cases (Figure 3A, C). Hemin binding to mouse CLEC-2 was characterized by kinetic rate association (ka) and dissociation (kd) constants of ka=2.77±0.08×104 mol -1 s and 5.76±0.09×10 s.

Heme induces human and mouse platelet activation through C-type-lectin-like receptor-2