Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1.

Kanagasabai Vadivel,S Paul Bajaj,William H Velander,Mark A Arbing,Wallace G Buchholz,Anne K Zaiss,Ayman E A Ismail,Yogesh Kumar,Frank M Fabian

doi:10.3390/jcm9113684

Abstract

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE…IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.

Highlights

In severe trauma and during major surgical procedures, such as cardiac surgery, the fibrinolytic system is hyperactivated, resulting in severe hemorrhaging [1,2,3]
The currently approved therapeutic agents, tranexamic acid (TXA) and ε-aminocaproic acid (EACA), are lysine analogues, which avert the binding of plasminogen and tissue plasminogen activator to the fibrin clot [13,14]
An improved antifibrinolytic agent is needed that is devoid of adverse effects of aprotinin and lysine analogs

Summary

Introduction

In severe trauma and during major surgical procedures, such as cardiac surgery, the fibrinolytic system is hyperactivated, resulting in severe hemorrhaging [1,2,3]. Uncontrolled bleeding is the leading cause of preventable death in trauma and often leads to the need for extensive blood transfusions during surgeries [4,5]. Aprotinin (bovine pancreatic trypsin inhibitor, BPTI), a potent inhibitor of the plasmin active site, had been the leading antifibrinolytic agent to reduce blood loss during cardiac surgery and extremity trauma [8]. Aprotinin is of bovine origin, and its anaphylactic potential is a major concern [11]. EACA and TXA are not as effective as aprotinin in reducing blood loss [15] Like aprotinin, they cause kidney failure [16], and recent evidence indicates that TXA, and to a lesser extent EACA, are associated with a significant incidence of seizures [16,17]. An improved antifibrinolytic agent is needed that is devoid of adverse effects of aprotinin and lysine analogs

Methods

Results

Conclusion