Thiolester substrates for transamidating enzymes: studies on fibrinoligase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Fibrinoligase (activated Factor XIII) is crucial for fibrin clot stabilization.
• Understanding fibrinoligase's transamidation mechanism is key for hemostasis research.

Purpose of the Study:

• To investigate thiocholine esters as inhibitors of fibrinoligase.
• To explore novel synthetic substrate systems for studying fibrinoligase activity.
• To identify potential esterolytic pathways for fibrinoligase.

Main Methods:

• Synthesis and testing of various thiocholine esters as potential fibrinoligase inhibitors.
• Utilizing a fluorescent amine for detecting enzyme-catalyzed acyl-group transfer.
• Investigating enzyme kinetics and calcium dependency using trans-cinnamoylthiocholine.

Main Results:

• Several thiocholine esters, particularly phenylpropionyl, phenylbutyryl, and trans-cinnamoyl, demonstrated significant inhibition of fibrin crosslinking.
• Successful demonstration of enzyme-catalyzed acyl-group transfers to a fluorescent amine in synthetic systems.
• Discovery of a calcium-dependent esterolytic activity of fibrinoligase using trans-cinnamoylthiocholine.

Conclusions:

• Thiocholine esters are effective inhibitors of fibrinoligase, offering new tools for studying clot stabilization.
• Trans-cinnamoylthiocholine serves as a valuable synthetic substrate for elucidating fibrinoligase mechanisms, including a novel esterolytic pathway.
• These findings advance the understanding of fibrin crosslinking and provide substrates for further enzymatic research.