Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain

Area of Science:

• Biochemistry
• Molecular Biology
• Hemostasis and Thrombosis

Background:

• Tissue factor pathway inhibitor-2 Kunitz domain 1 (KD1) is a protein domain involved in regulating coagulation.
• Plasminogen activation and plasmin activity are critical in fibrinolysis and various pathological processes.
• Modifications to protein domains can alter their inhibitory functions and introduce new biological activities.

Purpose of the Study:

• To investigate the functional consequences of a specific mutation (Leu17Arg) in KD1.
• To determine if the modified KD1 mutant (KD1L17R-KT) exhibits dual inhibitory functions against plasmin and plasminogen activation.
• To evaluate the therapeutic potential of KD1L17R-KT in controlling pathological fibrinolysis.

Main Methods:

• Site-directed mutagenesis of KD1 to create the Leu17Arg mutant.
• Thrombin cleavage to generate smaller molecular variants (KD1L17R-VT and KD1L17R-KT).
• Surface plasmon resonance (SPR) to assess binding kinetics with tissue plasminogen activator (tPA) and Glu-plasminogen.
• In vitro fibrinolysis assays and in vivo mouse liver-laceration model to evaluate anti-hemorrhagic effects.
• Cell-based assays to study inhibition of urokinase-induced plasminogen activation.

Main Results:

• The KD1 Leu17Arg mutant (KD1L17R) showed improved affinity for plasmin active site inhibition.
• Thrombin cleavage yielded KD1L17R-KT, which bound tPA and Glu-plasminogen, unlike KD1L17R-VT.
• KD1L17R-KT demonstrated superior inhibition of tPA-induced fibrinolysis and reduced blood loss in a mouse model compared to KD1L17R-VT and ε-aminocaproic acid.
• KD1L17R-KT, in complex with microplasmin, inhibited urokinase-induced plasminogen activation on U937 cells, whereas KD1L17R-VT did not.

Conclusions:

• KD1L17R-KT possesses a dual mechanism of action: inhibiting plasmin active site and acting as a decoy receptor for plasminogen/plasmin.
• This dual function effectively limits both plasmin generation and activity.
• KD1L17R-KT represents a promising therapeutic agent for controlling pathological plasminogen activation and fibrinolysis.