Inhibition of ADP-induced platelet responses by covalent modification of aggregin, a putative ADP receptor, by 8-(4-bromo-2,3-dioxobutylthio)ADP.
ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.
Puri
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RN
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Kumar
A
A
Chen
H
H
Colman
R F
RF
Colman
R W
RW
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D000345:Affinity Labels / Q000494:pharmacology; D001345:Autoradiography; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010974:Platelet Aggregation / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D013873:Thionucleotides / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology
D000345:Affinity Labels; D007476:Ionophores; D010761:Phosphorus Radioisotopes; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C055422:aggregin; C058329:8-((4-bromo-2,3-dioxobutyryl)thio)adenosine 5'diphosphate; D000001:Calcimycin; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D013917:Thrombin