Engineering kunitz domain 1 (KD1) of human tissue factor pathway inhibitor-2 to selectively inhibit fibrinolysis: properties of KD1-L17R variant.
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; 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 study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' 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 Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.
Department of Medicine, Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.
Madhu S
MS
Bajaj
Godwin I
GI
Ogueli
Yogesh
Y
Kumar
Kanagasabai
K
Vadivel
Gregory
G
Lawson
Sreejesh
S
Shanker
Amy E
AE
Schmidt
S Paul
SP
Bajaj
D019943:Amino Acid Substitution; D000818:Animals; D000933:Antifibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology; D007611:Aprotinin / Q000737:chemistry / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000737:chemistry; D004195:Disease Models, Animal; D004926:Escherichia coli; D005342:Fibrinolysis / Q000187:drug effects*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006470:Hemorrhage / Q000188:drug therapy*; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014148:Tranexamic Acid / Q000737:chemistry / Q000494:pharmacology
D000933:Antifibrinolytic Agents; D001779:Blood Coagulation Factors; D006023:Glycoproteins; D011994:Recombinant Proteins; C021240:tissue-factor-pathway inhibitor 2; D014148:Tranexamic Acid; D007611:Aprotinin