title: 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.
abstract: Essentials Current antifibrinolytics - aminocaproic acid and tranexamic acid-can cause seizures or renal injury. KD1L17R-KT, 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. KD1L17R-KTreduces 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 (KD1L17R-KT) with C-terminal lysine (kringle domain binding) and P2'-residue arginine (improved specificity towards plasmin). KD1L17R-KTstrongly inhibited human plasmin (hPm), with no inhibition of human kallikrein (hKLK) or factor XIa (hXIa). Furthermore, KD1L17R-KTreduced 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 (Ki~ 20 nm) and with measurable affinity for KD1L17R-KT(Ki~ 3 μm); in contrast, KD1WT-VTinhibited 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. KD1L17R-KTand aprotinin were more effective than KD1WT-VTor tranexamic acid in inhibiting tPA-induced mouse plasma clot lysis. Further, KD1L17R-KTwas more effective than KD1WT-VTand 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. KD1L17R-KTis effective in reducing blood loss in a tail-amputation model that mimics extremity injury.
authors: Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.
authors: K
authors: K
authors: Vadivel
authors: Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.
authors: Y
authors: Y
authors: Kumar
authors: Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.
authors: G I
authors: GI
authors: Ogueli
authors: Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.
authors: S M
authors: SM
authors: Ponnuraj
authors: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA.
authors: P
authors: P
authors: Wongkongkathep
authors: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA.|Molecular Biology Institute, University of California, Los Angeles, CA, USA.
authors: J A
authors: JA
authors: Loo
authors: Division of Pulmonology and Critical Care, Department of Medicine, University of California, Los Angeles, CA, USA.
authors: M S
authors: MS
authors: Bajaj
authors: Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.|Molecular Biology Institute, University of California, Los Angeles, CA, USA.
authors: S P
authors: SP
authors: Bajaj
mesh_terms: D000818:Animals; D000933:Antifibrinolytic Agents; D007611:Aprotinin / Q000737:chemistry; D002417:Cattle; D015945:Factor XIa / Q000235:genetics*; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry; D005341:Fibrinolysin / Q000235:genetics*; D005342:Fibrinolysis; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006470:Hemorrhage; D006801:Humans; D007610:Kallikreins / Q000235:genetics*; D007930:Leucine / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D000072417:Protein Domains; D012640:Seizures; D013045:Species Specificity; D014148:Tranexamic Acid / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000933:Antifibrinolytic Agents; D005338:Fibrin Fibrinogen Degradation Products; D006023:Glycoproteins; C036309:fibrin fragment D; C021240:tissue-factor-pathway inhibitor 2; D014148:Tranexamic Acid; D007611:Aprotinin; D010447:Peptide Hydrolases; D007610:Kallikreins; D015945:Factor XIa; D014357:Trypsin; D005341:Fibrinolysin; D007930:Leucine
keywords: TFPI-2; antifibrinolytic agents; aprotinin; fibrinolysis; tranexamic acid