Thrombin-specific inhibition by and slow cleavage of hirulog-1.
Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.
Witting
J I
JI
New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany 12201.
Bourdon
P
P
Brezniak
D V
DV
Maraganore
J M
JM
Fenton
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006629:Hirudins / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C049024:H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide; D014357:Trypsin; D013917:Thrombin; C074619:bivalirudin