Direct protamine activation of human mast cells is MRGPRX2-dependent and is modulated by heparin

Protamine is a polybasic drug commonly used in cardiac surgery to chemically inhibit, and thereby reverse, the anticoagulant effect of heparin. However, protamine can trigger severe anaphylaxis, with both IgE-dependent and IgE-independent mechanisms implicated. Recent research has linked the receptor MRGPRX2 (Mas-related G protein-coupled receptor X2) to IgE-independent mast cell activation by polybasic drugs, but its direct interaction with protamine has not been studied. This study investigated the role of MRGPRX2 in protamine-induced mast cell activation and explored how heparin influenced MRGPRX2 activation by protamine and other MRGPRX2 agonists. We used an in vitro cultured human mast cell line, Laboratory of Allergic Diseases 2, that constitutively expresses MRGPRX2. Receptor dependence of agonist-mediated signal transduction was demonstrated using a CRISPR MRGPRX2 knock down Laboratory of Allergic Diseases 2 cell line and a MRGPRX2 inverse-agonist. Mast cell activation was quantified using intracellular calcium mobilization, cellular degranulation, and cytokine release. Protamine-induced a concentration-dependent mast cell activation and was characterized as an unbiased partial agonist at MRGPRX2. Protamine-induced responses were significantly reduced by MRGPRX2 knock down and receptor antagonism. Heparin, which is known to complex with protamine, reduced the mast cell activation induced by protamine, compound 48/80, and LL-37, but not by the other MRGPRX2 agonists assessed. Isothermal titration calorimetry demonstrated that this selectivity relates to ligand ability to bind heparin. Recapitulation of clinically used ratios of protamine:heparin revealed that under these conditions, protamine still triggered effective mast cell activation, highlighting the need for further research to better understand the clinical relevance of MRGPRX2 activation by protamine. SIGNIFICANCE STATEMENT: This study identified protamine, a polybasic drug commonly used in cardiac surgery, as a partial agonist at the MRGPRX2 receptor, and that heparin significantly inhibited the receptor activation by protamine and other endogenous MRGPRX2 agonists. However, the commonly used clinical ratio of protamine:heparin still permitted mast cell activation in vitro.