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

Essentials Current antifibrinolytics - aminocaproic acid and tranexamic acid-can cause seizures or renal injury. KD1_L17R -K_T , aprotinin and tranexamic acid were tested in a modified mouse tail-amputation model. S2'-subsite variations between human and mouse factor XIa result in vastly different inhibition profiles. KD1_L17R -K_T reduces blood loss and D-dimer levels in mouse with unobserved seizures or renal injury.

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 ~ 17-fold increased affinity by aprotinin (K_i ~ 20 nm) and with measurable affinity for KD1_L17R -K_T (K_i ~ 3 μm); in contrast, KD1_WT -V_T inhibited hXIa or mXIa with similar affinity. Compared with hPm, mPm had ~ 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 KD1_WT -V_T 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.