Isolation and characterization of thrombomodulin from human placenta

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Protein C is a plasma protein crucial for physiological anticoagulation.
• Thrombin activates protein C to its protease form, protein Ca.
• Thrombomodulin acts as a cofactor, accelerating protein C activation by thrombin.

Purpose of the Study:

• To isolate and characterize thrombomodulin from human placenta.
• To determine the role of thrombomodulin in protein C activation.
• To investigate the interaction between thrombin, thrombomodulin, and protein C.

Main Methods:

• Affinity chromatography using thrombin-Sepharose.
• Electrophoresis (SDS-PAGE) for purity and molecular weight determination.
• Western blot analysis.
• Enzyme kinetics to determine kinetic parameters and stoichiometry.

Main Results:

• Thrombomodulin was purified 7,900-fold from human placenta with a 7% yield.
• Purified thrombomodulin demonstrated an 800-fold stimulation of protein C activation by thrombin.
• Thrombin and thrombomodulin form a 1:1 stoichiometric complex.
• The Michaelis constant for protein C activation was determined to be 9.8 µM.
• Calcium ions are required for the reaction, with optimal activity at 1 mM Ca2+.

Conclusions:

• Human placenta is a viable source for thrombomodulin isolation.
• Thrombomodulin is a potent enhancer of thrombin-mediated protein C activation.
• Structural similarities exist between human and rabbit thrombomodulin.
• Thrombomodulin plays a vital role in regulating blood coagulation through the protein C pathway.