Binding of adenosine diphosphate to intact human platelets

Area of Science:

• Biochemistry
• Hematology
• Platelet Physiology

Background:

• Platelets play a crucial role in hemostasis and thrombosis.
• Adenosine diphosphate (ADP) is a key signaling molecule in platelet activation.
• Understanding ADP uptake mechanisms is vital for developing antiplatelet therapies.

Purpose of the Study:

• To investigate the kinetics and mechanisms of ADP uptake by human platelets.
• To differentiate between initial ADP binding and uptake of its metabolites.
• To characterize the binding sites for ADP on platelets.

Main Methods:

• Incubation of human platelet-rich plasma with radiolabeled ADP ([8-14C]ADP) and albumin.
• Rapid separation of platelets via centrifugation through silicone oil.
• Quantification of radioactivity in plasma and platelet pellets to determine ADP uptake.
• Analysis of ADP metabolites (ATP, ADP, AMP) using radioactivity.
• Inhibition studies with prostaglandin E1, adenosine, ADP, and dipyridamole.
• Scatchard analysis to characterize ADP binding sites.

Main Results:

• Platelets exhibited rapid initial uptake of ADP within 10 seconds, increasing over time.
• Uptake of other nucleotide diphosphates was slower.
• Recovered radioactivity showed 60% as ATP, 30% as ADP, and 10% as AMP.
• Prostaglandin E1 did not affect ADP uptake kinetics or metabolite distribution.
• Heated plasma significantly slowed the rate of ADP uptake.
• Adenosine inhibited later uptake, while ADP inhibited both initial and later uptake.
• Dipyridamole blocked later uptake but not initial uptake.
• [alpha-32P]ADP showed immediate uptake that did not increase further.
• Guinea-pig platelets, lacking adenosine uptake, still showed initial ADP uptake.
• Scatchard analysis revealed approximately 88,000 high-affinity binding sites per platelet (K_A = 5.41 x 10^5 M^-1).

Conclusions:

• The initial rapid uptake of ADP represents direct binding to platelet surface receptors.
• Subsequent uptake is primarily due to the internalization of adenosine, a breakdown product of ADP.
• Platelets possess distinct binding sites for ADP, with a significant number of high-affinity sites identified.