Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis: PROPERTIES OF KD1-L17R VARIANT

• Published in: The Journal of biological chemistry 2011-02, Vol.286 (6), p.4329-4340
• Main Authors: Bajaj, Madhu S, Ogueli, Godwin I, Kumar, Yogesh, Vadivel, Kanagasabai, Lawson, Gregory, Shanker, Sreejesh, Schmidt, Amy E, Bajaj, S. Paul
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Antifibrinolytic Agents - chemistry; Antifibrinolytic Agents - pharmacology; Aprotinin - chemistry; Aprotinin - pharmacology; Blood Coagulation Factors - chemistry; Disease Models, Animal; Escherichia coli; Fibrinolysis - drug effects; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - pharmacology; Hemorrhage - drug therapy; Humans; Mice; Mutation, Missense; Protein Folding; Protein Structure and Folding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Structure-Activity Relationship; Tranexamic Acid - chemistry; Tranexamic Acid - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.191163

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2' residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ~6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ~8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.