Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa.
We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a Kd in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP- and thrombin-stimulated gel filtered platelets caused a 2-3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.
Ramsamooj
P
P
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103.
Doellgast
G J
GJ
Hantgan
R R
RR
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000918:Antibody Specificity / Q000276:immunology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects / Q000276:immunology; D010980:Platelet Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013917:Thrombin / Q000494:pharmacology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin