title: Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu271-Ala277 of domain 3 in the heavy chain of high molecular weight kininogen.
abstract: The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM. A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292. Peptides Phe279-Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity. A heptapeptide Leu271-Ala277 inhibited thrombin-induced aggregation of platelets with an IC50 of 65 microM. The effect is specific for the activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.
authors.affiliation: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Kunapuli
authors.forename: H N
authors.initials: HN
authors.lastname: Bradford
authors.forename: B A
authors.initials: BA
authors.lastname: Jameson
authors.forename: R A
authors.initials: RA
authors.lastname: DeLa Cadena
authors.forename: L
authors.initials: L
authors.lastname: Rick
authors.forename: R P
authors.initials: RP
authors.lastname: Wassell
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D003094:Collagen / Q000494:pharmacology; D005091:Exons; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007704:Kininogens / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
chemical_list: D007704:Kininogens; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D003094:Collagen; D013917:Thrombin