# Human Protein Z as the Second Known Heme‐Binding Protein from the Endogenous Blood Coagulation Inhibitor System

## TL;DR

This study shows that Protein Z, an anticoagulant, binds heme and changes shape, which could affect blood clotting during hemolysis.

## Contribution

The study identifies a heme-binding site in Protein Z and demonstrates its functional impact on anticoagulation.

## Key findings

- Protein Z binds heme with high affinity and undergoes conformational changes.
- Heme enhances Protein Z's inhibitory effect on thrombin but reduces its anticoagulant function in clotting assays.
- A histidine-based heme-binding motif is identified in Protein Z.

## Abstract

Protein Z (PZ) is a vitamin‐K‐dependent glycoprotein that serves as an anticoagulant cofactor in blood. Although it is homologous to the serine proteases of blood coagulation, PZ has no enzymatic activity. It binds to the PZ‐dependent protease inhibitor and supports inactivation of factor Xa. In addition, an inhibitory effect on thrombin is described. Clinically, changed PZ levels increase the risk for thrombosis. The related activated protein C (APC) is described as the first anticoagulant protein that binds heme under hemolytic conditions leading to its inhibition. However, the network of inhibitors of blood coagulation is still underexplored with respect to their role in heme‐triggered effects and the regulation thereof. PZ seems to be an interesting candidate in this context due to its homology to APC. Using PZ‐derived peptides as models for potential heme‐binding sites in PZ together with in silico studies, one specific heme‐binding site in PZ is identified. Binding studies on protein level demonstrate binding characteristics similar to APC. Finally, the inhibitory effect of PZ toward thrombin is increased in the presence of heme, providing also insights into a potential functional consequence of the PZ–heme interaction. In addition, the anticoagulant function of PZ is dampened in the presence of heme in an aPTT‐based clotting assay, suggesting a tendency toward heme‐induced prothrombotic actions. In future, this will support mapping the diverse effects of heme within blood coagulation.

Protein Z is a vitamin K‐dependent anticoagulant with elusive molecular functions. Herein, it is shown that heme binds to protein Z and induces conformational changes. One histidine‐based heme‐binding motif is identified. High heme‐binding affinity and functional effects are demonstrated on protein level. These findings shed new light on the structural and functional roles of protein Z in hemolysis‐driven coagulation disorders.© 2025 WILEY‐VCH GmbH