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 and 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 report, structure based investigations and analysis of the serine proteases profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ (nomenclature of

Schechter and Berger, BBRC, 27:157–162, 1967
) 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 Leu17 (bovine pancreatic trypsin inhibitor/aprotinin numbering) in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with the wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E. Coli, folded and purified to homogeneity. Amino-terminal sequences and mass spectra revealed proper folding of the KD1-WT and KD1-L17R. As compared to KD1-WT, the KD1-L17R neither prolonged the activated partial thromboplastin time of normal plasma nor it inhibited factor XIa, plasma kallikrein or factor VIIa/tissue factor. Further, KD1-L17R inhibited plasmin with ~4-fold increased affinity. In a mouse liver laceration model of bleeding from small vessels, KD1-L17R reduced total blood loss by 84% compared with KD1-WT, which reduced total blood loss by 10%. Moreover, in this bleeding model, KD1-L17R was more effective than aprotinin (70% reduction), which has been used as an antifibrinolytic agent to decrease blood loss during major surgery. In this model, KD1-L17R was also more effective than the lysine analogue tranexamic acid (52% reduction). In additional studies, in a tail transection model of bleeding from a large vessel, KD1-L17R reduced total blood loss by 70% and was more effective than KD1-WT (46% reduction), aprotinin (43% reduction) and tranexamic acid (67% reduction). Notably, as compared to aprotinin, renal toxicity manifesting as multifocal tubular necrosis by histopathology was not observed with KD1-L17R or KD1-WT. In conclusion, KD1-L17R is a specific inhibitor of plasmin without anticoagulant properties and is more effective in reducing blood loss compared with known antifibrinolytic agents in clinical use.

Topics:
antifibrinolytic agents, aprotinin, thromboplastin, engineering, fibrinolysis, hemorrhage, plasmin, anticoagulants, tissue factor pathway inhibitor, factor viia

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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