Structure and protein design of a human platelet function inhibitor

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• Salivary apyrase from hematophagous arthropods inhibits platelet activation by degrading ADP.
• ADP is a key mediator of platelet aggregation, released from damaged cells.

Purpose of the Study:

• To determine the X-ray crystal structures of human soluble apyrase.
• To understand the enzyme's substrate binding and active site.
• To engineer a human apyrase with enhanced ADPase activity for antithrombotic applications.

Main Methods:

• X-ray crystallography was used to obtain structures of human apyrase in apo and substrate-bound states.
• Comparative structural biology approaches were employed for enzyme redesign.
• ADPase activity and platelet aggregation inhibition were measured.

Main Results:

• The crystal structures revealed a five-blade beta-propeller nucleotide-binding domain, substrate-binding determinants, and a unique calcium-binding site.
• Engineered human apyrase exhibited over 100-fold increased ADPase activity.
• The enhanced enzyme potently inhibited platelet aggregation.

Conclusions:

• Structural insights into human apyrase facilitate enzyme engineering.
• The engineered apyrase demonstrates significant potential as a novel antithrombotic agent.
• This work may pave the way for new treatments for thrombotic disorders.