Attenuation of fibrinolysis through heme binding to the serine protease plasmin.

Hemolytic disorders are characterized by increased concentrations of circulating labile heme, a redox-active metalloporphyrin increasingly recognized as a modulator of protein function. Here, we identify the fibrinolytic serine protease plasmin as a previously unrecognized high-affinity heme-binding protein and delineate the molecular basis and functional consequences of this interaction. Surface plasmon resonance and UV/visible spectroscopy revealed nanomolar binding affinity and an overstoichiometric heme-binding capacity of plasmin, respectively. Systematic mapping with plasmin-derived peptides uncovered six discrete heme-binding motifs, which were further evaluated by molecular docking and molecular dynamics simulations, suggesting stable coordination at three surface-exposed sites proximal to functional regions. Importantly, heme binding resulted in a pronounced, concentration-dependent inhibition of plasmin-mediated fibrinolysis at heme levels consistent with pathological hemolysis. Collectively, these findings characterize plasmin as a transiently heme-binding protease and provide a molecular framework linking excess labile heme to impaired fibrinolysis and the prothrombotic phenotype associated with hemolytic disorders.