Characterization of the gamma chain platelet binding site on fibrinogen fragment D.
Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.
Kirschbaum
N E
NE
Sinai Samaritan Medical Center, Milwaukee, WI.
Mosesson
M W
MW
Amrani
D L
DL
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005340:Fibrinogen / Q000302:isolation & purification; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation
D005338:Fibrin Fibrinogen Degradation Products; D010455:Peptides; C015214:fibrinogen D fragment; D000244:Adenosine Diphosphate; D005340:Fibrinogen