Hemolysis-derived heme meets protein Z: Insights into a novel heme-binding anticoagulant protein

Introduction Intravascular hemolysis, a consequence of disorders, like sickle cell anemia and paroxysmal nocturnal hemoglobinuria, results in elevated levels of labile heme which surpasses the capability of the natural heme scavenging system, and ultimately leads to proinflammatory and prothrombotic complications. So far, studies have shown that heme can affect the physiological functions and/or stability of coagulation proteins (i.e., factor VIII, fibrinogen) by binding to short, surface-exposed amino acid stretches (heme-binding motifs; HBMs). Up to date, only one endogenous inhibitor of the blood coagulation system, namely activated protein C (APC), has been described to bind heme, which tends to support a prothrombotic state through inhibition of APC by heme. Herein, we describe for the first time direct heme binding to the endogenous anticoagulant cofactor protein Z (PZ) and its functional consequences. Method Heme binding to PZ was analyzed by surface plasmon resonance and UV/Vis spectroscopy. To identify potential heme-binding sites of PZ, the amino acid sequence was screened for potential heme-binding motifs (HBMs) using the webserver HeMoQuest. The predicted HBMs were synthesized as peptide models by solid-phase peptide and their heme-binding capacity was determined using UV/Vis spectroscopy. On protein level, molecular docking and molecular dynamic (MD) simulations were performed to support the experimental findings. To explore the functional consequences of the complex formation, chromogenic and clotting assays were performed. Results SPR spectroscopy showed that specifically one heme molecule binds to PZ. Out of a selection of five predicted potential HBMs, three motifs were demonstrated to bind heme with high affinity. In the course of the MD simulations, the number of binding sites was further specified to one binding site, which correlated well with the UV/Vis spectroscopic heme titration approach. In addition, heme is able to influence the anticoagulant function of PZ, as demonstrated in a selection of biochemical and diagnostic assay systems. Conclusion Taken together, these results indicate that hemolysis-derived heme might have a direct effect on the protein Z pathway through interaction with PZ. Furthermore, this extends the understanding of heme as a regulator within the coagulation cascade, which might support an improved understanding of the development of coagulation abnormalities under hemolytic conditions. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany