Related Experiment Videos
Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C
Insights
The heart activates protein C into its anticoagulant form, activated protein C, 20,000 times faster than in solution. This process, crucial for blood clotting regulation, is enhanced by vascular endothelium.
Area of Science:
- Biochemistry
- Cardiovascular Physiology
- Hemostasis
Background:
- Protein C is a vital anticoagulant precursor.
- Thrombin is a key enzyme in the coagulation cascade.
- Understanding protein C activation is critical for thrombosis research.
Purpose of the Study:
- To investigate the myocardium's capacity to activate protein C.
- To identify the mechanism and kinetics of protein C activation in the heart.
- To explore the role of vascular endothelium in protein C activation.
Main Methods:
- Myocardial perfusion studies with protein C and thrombin.
- Assays for activated protein C formation (substrate hydrolysis, anticoagulant activity).
- Experiments using diisopropyl-phospho-thrombin to inhibit activation.
- Studies on cultured human endothelium.
Main Results:
- Myocardium activates protein C to activated protein C at rates significantly exceeding solution-based activation.
- Protein C activation in the heart is thrombin-dependent and maintained for extended periods.
- Vascular endothelium enhances thrombin-catalyzed protein C activation, suggesting a cofactor role.
- Inhibition studies confirm the role of thrombin and identify modulators of activation.
Conclusions:
- The myocardium possesses a highly efficient system for generating activated protein C.
- Vascular endothelium likely provides a surface cofactor that accelerates thrombin-mediated protein C activation.
- These findings highlight a significant physiological mechanism for local anticoagulant control.
Abstract:
Perfusion of the myocardium with protein C in the presence of thrombin (EC 3.4.21.5) elicits a potent anticoagulant activity, which is identified as activated protein C on the basis of synthetic substrate hydrolysis and anticoagulant properties. The rate of activated protein C formation during the transit through the myocardium is at least 20,000 times that of thrombin-catalyzed activation of protein C in the perfusion solution. The capacity of the heart to activate protein C is maintained for at least 1 hr when thrombin is present in the perfusate, but decays (half-life approximately 30 min) once thrombin is omitted. Addition of diisopropyl-phospho-thrombin increases this decay rate more than 10-fold. Coperfusing diisopropylphospho-thrombin with active thrombin lowers the amount of protein C activation in the myocardium. Cultured monolayers of human endothelium enhance the rate of thrombin-catalyzed protein C activation. As with myocardium, the activation rate is inhibited by including diisopropylphospho-thrombin in the medium. It is proposed that the surface of vascular endothelium provides a cofactor that enhances the rate of protein C activation by thrombin.