Binding of hirudin to meizothrombin

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Prothrombin (coagulation factor II) is the inactive precursor to thrombin (coagulation factor IIa).
• Meizothrombin is an intermediate in thrombin generation, exhibiting limited fibrinogenolytic activity and significant autocatalysis.
• Hirudin is a highly specific inhibitor of thrombin derived from the medicinal leech.

Purpose of the Study:

• To investigate the interaction between meizothrombin and hirudin.
• To characterize the binding of hirudin to meizothrombin and compare it to thrombin-hirudin interactions.
• To elucidate the structural changes induced by hirudin binding to meizothrombin.

Main Methods:

• Production of recombinant D419N-prothrombin and its conversion to D419N-meizothrombin, a catalytically inactive mutant.
• Affinity purification of D419N-meizothrombin using immobilized hirudin-derived peptides.
• Enzyme-linked immunosorbent assay (ELISA) to detect D419N-meizothrombin binding to immobilized hirudin.
• Fluorescence spectroscopy to monitor conformational changes upon hirudin binding.

Main Results:

• D419N-meizothrombin, lacking proteolytic and autocatalytic activity, demonstrated binding to immobilized hirudin.
• Fluorescence titration revealed a significant increase in intrinsic fluorescence upon hirudin binding to D419N-meizothrombin, indicating conformational changes.
• Similar fluorescence enhancement was observed with a double mutant D419N/W468F-thrombin, suggesting hirudin binding induces comparable structural alterations in both meizothrombin and thrombin.
• These structural changes do not involve the flexible gamma-loop.

Conclusions:

• Meizothrombin binds to hirudin, mediated by the anion binding exosite.
• Hirudin binding induces conformational changes in meizothrombin that are similar to those induced in thrombin.
• The flexible gamma-loop is not involved in the structural rearrangements upon hirudin binding to meizothrombin.