Purification of factor XIa inhibitor from human platelets

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Activated factor XI (FXIa) plays a role in coagulation.
• Platelets are implicated in hemostasis and thrombosis.
• Amyloid beta-protein precursor (APP) has been suggested to have other functions beyond amyloidogenesis.

Purpose of the Study:

• To identify and characterize an inhibitor of FXIa present in human platelets.
• To determine the biochemical properties and inhibitory activity of the purified FXIa inhibitor.
• To investigate the relationship between the FXIa inhibitor and amyloid beta-protein precursor (APP).

Main Methods:

• Purification of FXIa inhibitor (XIaI) from activated human platelet supernatant using a six-step column chromatography.
• Assessment of XIaI amidolytic activity inhibition toward FXIa.
• Determination of the inhibitor's molecular weight and amino-terminal sequence.
• Enzyme kinetic analysis (Ki value) and effect of ZnCl2 addition.

Main Results:

• Approximately 90 micrograms of XIaI were purified from 200 units of platelet suspension.
• The purified XIaI exhibited potent inhibitory activity against FXIa, with a Ki value of 526 +/- 120 pM.
• The molecular weight of XIaI was determined to be 94,000.
• The amino-terminal sequence of XIaI matched a specific region of APP751 and protease nexin-2.
• Inhibition was enhanced by the addition of ZnCl2.

Conclusions:

• Amyloid beta-protein precursor (APP) functions as a significant inhibitor of activated factor XI (FXIa) in human platelets.
• The purified platelet FXIa inhibitor (XIaI) is identical to APP, specifically protease nexin-2.
• This discovery highlights a novel role for APP in regulating coagulation pathways via platelet FXIa inhibition.