Progressive development of a thrombin inhibitor binding site.

Area of Science:

• Biochemistry
• Protein Chemistry
• Enzymology

Background:

• Thrombin is a key enzyme in blood coagulation.
• Thrombin is synthesized as an inactive precursor, prothrombin.
• Understanding the activation process of prothrombin is crucial for hemostasis research.

Purpose of the Study:

• To investigate the interaction of thrombin precursors with a fluorescent inhibitor, DAPA.
• To determine which cleavage events in prothrombin lead to inhibitor binding.
• To characterize the binding of DAPA to prethrombin 2 and thrombin.

Main Methods:

• Utilized dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) as a fluorescent, reversible thrombin inhibitor.
• Studied binding interactions using fluorescence spectroscopy (emission intensity, lifetime, polarization).
• Determined dissociation constants (Kd) and stoichiometry of DAPA binding.

Main Results:

• Prothrombin and prethrombin 1 did not bind DAPA, indicating intact cleavage sites prevent interaction.
• Prethrombin 2 and Meizo thrombin, with single cleavage events, bound DAPA.
• DAPA binding to prethrombin 2 and thrombin caused distinct changes in fluorescence properties, with thrombin binding ~30 times more tightly.

Conclusions:

• Cleavage at Arg274-Thr275 is implicated in the progressive formation of the thrombin active site.
• DAPA binding to prethrombin 2 demonstrates interaction with an inactive zymogen.
• Further cleavage at Arg323-Ile324 is necessary for full enzymatic activity, but initial cleavage facilitates inhibitor binding.