title: Inhibition of the amplification reactions of blood coagulation by site-specific inhibitors of alpha-thrombin.
abstract: Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.
authors.affiliation: Canadian Red Cross Society, Blood Transfusion Service, Hamilton, Ont.
authors.forename: F A
authors.initials: FA
authors.lastname: Ofosu
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: J
authors.initials: J
authors.lastname: Maraganore
authors.forename: M A
authors.initials: MA
authors.lastname: Blajchman
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: N
authors.initials: N
authors.lastname: Anvari
authors.forename: M R
authors.initials: MR
authors.lastname: Buchanan
authors.forename: J
authors.initials: J
authors.lastname: Hirsh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects*; D005165:Factor V / Q000037:antagonists & inhibitors; D005170:Factor X / Q000037:antagonists & inhibitors; D005344:Fibrinopeptide A / Q000378:metabolism; D006629:Hirudins / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011516:Prothrombin / Q000037:antagonists & inhibitors; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C062510:hirugen; D005344:Fibrinopeptide A; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D013917:Thrombin; C074619:bivalirudin