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Letters to the Editor
Similar values of kinetic rate constants were determined for hemin binding to human CLEC-2, ka=2.65±0.09×104 mol-1 s-1 and kd=5.06±0.05×10-3 s-1. The interaction of hemin with human and mouse CLEC-2 was further con- firmed by UV-vis absorbance spectroscopy (Figure 3B, D). The differential absorbance spectra profiles revealed sig- nificant shifts of the spectra of hemin towards higher wavelength by human and mouse CLEC-2 demonstrat- ing a direct binding. The differences in the spectral
changes of hemin in the presence of human and mouse CLEC-2 can be explained by distinct residues coordinat- ing heme’s iron. The blue shift in the Soret region observed with mCLEC-2 suggests the involvement of sul- fur containing amino acid (cysteine)11 whereas the red shift observed with human CLEC-2 suggests histidine coordination.
Altogether, these results show that hemin is an endogenous agonist for CLEC-2 leading to platelet activa-
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Figure 1. Hemin mediates human platelet activation through C-type-lectin-like receptor-2. (A, B) Human washed platelets (2x108/mL) were incubated with increasing concentrations of hemin in the presence of Ca2+ (2 mM). Platelet aggregation was assessed for 6 minutes (min) using light transmission aggregom- etry (n=5). (C, D) For flow cytometry analysis, 1x106 platelets were incubated with different concentrations of hemin for 20 min at 37oC. Platelet activation was determined by flow cytometry using (C) anti-P-selectin and (D) GPIIbIIIa PAC-1 antibodies (n=4). Data is shown as fold increase of median of fluorescence (MFI) of treated platelets over control (absence of hemin). (E) Phosphatidylserine exposure was assessed using Annexin V staining. (F) Lactate dehydrogenase (LDH) levels were measured in the supernatant following platelet aggregation (6 min). Platelet lysate was used to detect the total level of LDH in platelets. (G, H) Platelet aggregation by hemin was assessed using Btk inhibitor Ibrutinib (500 nM), Src family kinase inhibitor PP2 (20 mM), Syk inhibitor PRT-060318 (20 mM), TLR4 inhibitor TAK-242 (10 mM), P2Y12 inhibitor Cangrelor (10 mM) or cyclooxygenase inhibitor indomethacin (10 mM). Platelets were pre-incubated with different inhibitors for 5 min at 37 oC prior to the addition of hemin. Dimeric C-type-lectin-like receptor-2 (CLEC-2) (hFc-CLEC-2, 20 mg/mL) or dimeric collagen receptor glycoprotein VI (GPVI) (hFc-GPVI, 20 mg/mL) were pre-incubated with hemin for 20 min at 37 oC prior to addition to platelets (n=5). (H) Histogram data are shown as mean ± standard deviation. (I) Protein levels of the phosphorylation of Syk and PLCγ2, and total Syk and PLCγ2 in platelet lysates were determined by western blotting after 6 min aggregation. Western blots are representative of five independent experiments. The statistical significance was analyzed using a one-way ANOVA with Tukey’s multiple comparisons test using Prism 8 (GraphPad Software Inc, USA). Significance is shown compared to control (absence of hemin), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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