Kinetic intermediates in prothrombin activation. Bovine prethrombin 1 conversion to thrombin by factor X

Area of Science:

• Biochemistry
• Enzymology
• Blood Coagulation

Background:

• Alpha-thrombin (alpha-IIa) is proteolytically formed from prothrombin (II) or prethrombin 1 (P1).
• Known activation pathways involve Factor Xa (Xa) alone, with prethrombin 2 (P2F2) intermediates, or Xa with Factor Va and phospholipids, involving meizothrombin (MzIIa(-F1)) intermediates.
• MzIIa(-F1) had not been previously detected during activation by Xa alone.

Purpose of the Study:

• To investigate the activation pathways of prethrombin 1 (P1) by Factor Xa (Xa) alone.
• To quantify the contributions of different pathways to alpha-thrombin (alpha-IIa) formation.
• To identify and characterize intermediates in the activation process.

Main Methods:

• Kinetic analysis of prethrombin 2 (P2F2) and P1 cleavage by Xa alone.
• Determination of rate constants (kc/Km) for intermediate formation and substrate disappearance.
• Active site-affinity labeling of reaction products with a biotinylated inhibitor and Western blotting.

Main Results:

• Prethrombin 1 (P1) activation by Factor Xa (Xa) alone proceeds via two parallel pathways.
• The known pathway via prethrombin 2 (P2F2) and a newly identified pathway involving meizothrombin (MzIIa(-F1)) were kinetically characterized.
• Meizothrombin (MzIIa(-F1)) formation was directly demonstrated as a transient intermediate.

Conclusions:

• Factor Xa (Xa) alone activates prothrombin (P1) through both P2F2 and MzIIa(-F1) pathways.
• Kinetic data and direct detection confirm MzIIa(-F1) as a significant intermediate in Xa-catalyzed P1 activation.
• This study elucidates a more complex mechanism for alpha-thrombin generation than previously understood.