The Endothelial Protein C Receptor Enhances Hemostasis of FVIIa Administration in Hemophilic Mice in Vivo

Blood. 2014 Aug 14;124(7):1157-65. doi: 10.1182/blood-2014-04-567297. Epub 2014 Jun 23.

Abstract

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Carboxyglutamic Acid / metabolism
  • Amino Acids / genetics
  • Amino Acids / metabolism
  • Animals
  • Binding Sites / genetics
  • Binding, Competitive
  • Blood Coagulation / drug effects
  • Blood Coagulation Factors / genetics
  • Blood Coagulation Factors / metabolism*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Factor VIIa / administration & dosage
  • Factor VIIa / genetics
  • Factor VIIa / pharmacology*
  • Hemophilia A / blood
  • Hemophilia A / drug therapy*
  • Hemostasis / drug effects*
  • Humans
  • Kinetics
  • Male
  • Mice, Inbred C57BL
  • Protein Binding
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology
  • Thrombelastography
  • Thromboplastin / metabolism

Substances

  • Amino Acids
  • Blood Coagulation Factors
  • Receptors, Cell Surface
  • Recombinant Proteins
  • activated protein C receptor
  • 1-Carboxyglutamic Acid
  • Thromboplastin
  • recombinant FVIIa
  • Factor VIIa