Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Alpha-thrombin, crucial for blood coagulation, is proteolytically derived from prothrombin.
• The activation of prothrombin involves cleavage at specific sites, exposing the active site and recognition exosites.
• The extent to which proteolytic activation exposes protein recognition exosites on thrombin remains unclear.

Purpose of the Study:

• To investigate the accessibility of the fibrinogen recognition exosite in prothrombin and alpha-thrombin.
• To compare the binding of hirudin peptides to prothrombin and alpha-thrombin using NMR spectroscopy.

Main Methods:

• High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy was employed.
• Interactions between prothrombin, alpha-thrombin, and synthetic hirudin peptides were examined.
• NMR relaxation enhancements, including line broadening and transferred nuclear Overhauser effects, were analyzed.

Main Results:

• Hirudin peptides showed similar NMR relaxation enhancements with both prothrombin and alpha-thrombin.
• These enhancements were specific to the fibrinogen recognition exosite, as demonstrated by control experiments with human serum albumin and gamma-thrombin.
• Binding affinities (Kd) were determined to be approximately 500 microM for prothrombin and < 100 microM for alpha-thrombin.

Conclusions:

• The fibrinogen recognition exosite of thrombin is partially accessible in its proenzyme form, prothrombin.
• The binding mode of hirudin peptides to the fibrinogen recognition exosite is conserved between prothrombin and alpha-thrombin.
• Proteolytic activation of prothrombin may not be entirely required for the initial exposure of certain recognition exosites.