The residues AGDV of recombinant gamma chains of human fibrinogen must be carboxy-terminal to support human platelet aggregation.
The carboxy-terminus of the gamma chain of fibrinogen contains a sequence which is believed to be one of the domains that interacts with glycoprotein (GP) IIb/IIIa to support platelet aggregation. A normal variant of fibrinogen exists in which the four carboxy-terminal amino acids are replaced by 20 amino acids. This variant, known as gamma', has been reported to bind less effectively to platelets. The purpose of the present study was to engineer novel proteins to determine what differences in amino acid sequence between the gamma and gamma' chains influence the interaction of the carboxyterminus with GPIIb/IIIa. In this regard, the gamma chain cDNA in a bacterial plasmid expression vector was modified by oligonucleotide-directed mutagenesis to produce recombinant gamma chains with amino acid changes in the carboxy-terminus which reflect the differences between gamma and gamma'. The recombinant gamma chain with an unmodified carboxy-terminus supported adenosine diphosphate (ADP)-induced platelet aggregation to the same extent as intact fibrinogen. In contrast, the ability of gamma' 427 (the recombinant gamma' variant) and gamma 427 (where the 16 amino acid gamma' extension [412-427] was added to the carboxy-terminus of gamma) to support platelet aggregation was markedly reduced. In addition, the extent of ADP-induced platelet aggregation was decreased in the presence of gamma' 411 (where amino acids 408-411 in gamma were replaced with amino acids 408-411 in gamma'), while gamma 407 (where the four carboxy-terminal amino acids were deleted) was not capable of supporting aggregation. These findings demonstrate that the four residues AGDV are not only required but must be carboxy-terminal to support platelet aggregation.
Hettasch
J M
JM
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Bolyard
M G
MG
Lord
S T
ST
D000595:Amino Acid Sequence; D001483:Base Sequence; D005340:Fibrinogen / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000494:pharmacology*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D005340:Fibrinogen