Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• The P2T purinergic receptor on human platelets, activated by ADP, is crucial but remains unpurified and uncloned.
• Existing methods for identifying the ADP-receptor (aggregin) lack sensitivity.

Purpose of the Study:

• To develop a novel, highly sensitive immunoaffinity method for identifying and purifying the platelet ADP-receptor (aggregin).
• To explore the potential of this method for purifying other P2-type purinergic receptors.

Main Methods:

• Developed a polyclonal antibody against glutamate dehydrogenase (GDH) modified with 5'-p-fluorosulfonylbenzoyladenosine (FSBA).
• Utilized FSBA and ADP-affinity analogs to label platelet proteins, followed by immunoblotting with the developed antibody.
• Employed immunoaffinity column chromatography for receptor purification.

Main Results:

• The antibody specifically recognized a 100 kDa protein band (aggregin) in FSBA-labeled platelets.
• This recognition was blocked by pre-incubation with ADP, ATP, or ADP-affinity analogs, confirming specificity for the ADP-receptor.
• The immunoaffinity method demonstrated significantly higher sensitivity compared to previous radiochemical techniques.

Conclusions:

• The developed polyclonal antibody effectively identifies FSBA-labeled aggregin, enabling its purification via immunoaffinity chromatography.
• This novel method is a sensitive tool for studying the platelet ADP-receptor and potentially other P2-type purinergic receptors.