S2 '-subsite variations between human and mouse enzymes (plasmin, factor XIa, kallikrein) elucidate inhibition differences by tissue factor pathway inhibitor-2 domain1-wild-type, Leu17Arg-mutant and aprotinin

摘要

Background: Using tissue factor pathway inhibitor (TFPI)-2 Kunitz domain1 (KD1), we obtained a bifunctional antifibrinolytic molecule (KD1(L17R)-K-T) with C-terminal lysine (kringle domain binding) and P2'-residue arginine (improved specificity towards plasmin). KD1(L17R)-K-T strongly inhibited human plasmin (hPm), with no inhibition of human kallikrein (hKLK) or factor XIa (hXIa). Furthermore, KD1(L17R)-K-T reduced blood loss comparable to aprotinin in a mouse liver-laceration model of organ hemorrhage. However, effectiveness of these antifibrinolytic agents in a model of hemorrhage mimicking extremity trauma and their inhibition efficiencies for mouse enzymes (mPm, mKLK or mXIa) remain to be determined. Objective: To determine potential differences in inhibition constants of various antifibrinolytic agents against mouse and human enzymes and test their effectiveness in a modified mouse tail-amputation hemorrhage model. Methods/Results: Unexpectedly, mXIa was inhibited with similar to 17-fold increased affinity by aprotinin (K-i similar to 20 nM) and with measurable affinity for KD1(L17R)-K-T (K-i similar to 3 mu M); in contrast, KD1(WT)-V-T inhibited hXIa or mXIa with similar affinity. Compared with hPm, mPm had similar to 3-fold reduced affinity, whereas species specificity for hKLK and mKLK was comparable for each inhibitor. S2'-subsite variations largely accounted for the observed differences. KD1(L17R)-K-T and aprotinin were more effective than KD1WT-VT or tranexamic acid in inhibiting tPA-induced mouse plasma clot lysis. Further, KD1(L17R)-K-T was more effective than KD1(WT)-V-T and was comparable to aprotinin and tranexamic acid in reducing blood loss and D-dimer levels in the mouse tail-amputation model. Conclusions: Inhibitor potencies differ between antifibrinolytic agents against human and mouse enzymes. KD1(L17R)-K-T is effective in reducing blood loss in a tail-amputation model that mimics extremity injury.