Thrombin clotting activity measurement and standardization with lyophilized plasma

Area of Science:

• Biochemistry
• Hematology
• Enzymology

Background:

• Thrombin plays a crucial role in hemostasis by converting fibrinogen to fibrin.
• Accurate measurement of thrombin activity is essential for clinical diagnostics and research.
• Standardization of clotting assays requires reliable and stable reagents.

Purpose of the Study:

• To characterize the kinetics of thrombin-induced fibrin clot formation.
• To evaluate lyophilized human plasma as a source of clottable fibrinogen for clotting assays.
• To establish a method for standardizing thrombin activity using plasma.

Main Methods:

• Measurement of thrombin-induced clotting times across a range of fibrinogen concentrations.
• Application of enzyme kinetic principles to define clotting parameters (Km(clot), kclot).
• Correlation of clotting times with alpha-thrombin concentrations for standardization.

Main Results:

• Thrombin-induced clotting times showed an inverse relationship with fibrinogen concentrations (98-2900 nmol/L).
• Kinetic parameters (Km(clot) and kclot) were determined, indicating lyophilized plasma is a suitable source of fibrinogen.
• Lyophilized plasma demonstrated stability in clottability over 15 months.
• Clotting times correlated strongly with reciprocal alpha-thrombin concentrations, enabling standardization.

Conclusions:

• Lyophilized human plasma is a stable and effective source for fibrinogen in thrombin clotting assays.
• The study provides a kinetic framework and standardization method for thrombin activity measurement.
• Autoproteolytic forms of thrombin did not interfere with alpha-thrombin clotting activity.