Mechanism of tissue factor activation on cells

Area of Science:

• Biochemistry
• Cell Biology
• Hematology

Background:

• Tissue Factor (TF) initiates the extrinsic pathway of blood coagulation.
• TF procoagulant activity (PCA) is latent on intact cells.
• TF PCA can be rapidly expressed upon cellular stimulation.

Purpose of the Study:

• To investigate the mechanism of TF PCA activation.
• To determine the role of calcium and calmodulin in TF activation.
• To elucidate the structural changes in TF associated with PCA.

Main Methods:

• Cellular stimulation with ionomycin (a Ca2+ ionophore).
• Inhibition studies using calmodulin inhibitors.
• Protein crosslinking with 3-3'-dithiobis(sulphosuccinimidylpropionate).
• Analysis of TF-FVIIa complex binding with pseudosubstrate inhibitors.

Main Results:

• Ionomycin treatment increased TF PCA over 100-fold within seconds.
• Calmodulin inhibitors blocked the ionomycin-induced TF PCA.
• TF quaternary structure changed from dimeric to monomeric upon ionomycin treatment.
• Pseudosubstrate inhibitors selectively bound TF-FVIIa on ionophore-treated cells.

Conclusions:

• Ca2+ influx triggers TF PCA activation via a calmodulin-dependent pathway.
• TF PCA activation involves a conformational change in TF, exposing substrate-binding sites.
• This process converts cryptic TF dimers to active TF monomers, initiating coagulation.