title: Identification and characterization of variants of tick anticoagulant peptide with increased inhibitory potency toward human factor Xa.
abstract: Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa (fXa), a central enzyme in the blood clotting cascade. As such, TAP is a potential antithrombotic agent. Site-directed mutagenesis studies were undertaken to determine the feasibility of increasing the inhibitory potency of TAP toward fXa. The amino acid substitutions Tyr-1 to Trp (Y1W) and Asp-10 to Arg (D10R) increased inhibitory potency toward human fXa by 2.5- and 4-fold, respectively. The increased inhibitory potency reflected a decrease in the rate constant for dissociation of the final fXa-TAP inhibitory complex. The double mutant, Y1W/D10R, exhibited an inhibition constant of 10 pM, a 37-fold enhancement of inhibitory potency toward human fXa. The improvement in inhibitory potency was less pronounced (12-fold) with dog fXa wherein Kis of 220 and 18 pM were observed for wild-type TAP and the double mutant, respectively. Mutation of Tyr-1 to Glu (Y1E) generated a weaker inhibitor (Ki = 2 nM) that bound human fXa more slowly. However, no change in inhibitory potency toward human fXa was observed when Tyr-1 was replaced by Phe. Taken together, these observations are consistent with the view that a hydrophobic amino acid at the N-terminus of TAP may be a determinant of inhibitory potency. Decreases by 3-4 orders of magnitude in inhibitory potency were noted upon mutation of Asn-2 and Leu-4 of TAP, further implicating the N-terminal domain as an important determinant of inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Mao
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: C
authors.initials: C
authors.lastname: Welebob
authors.forename: M P
authors.initials: MP
authors.lastname: Neeper
authors.forename: V M
authors.initials: VM
authors.lastname: Garsky
authors.forename: J A
authors.initials: JA
authors.lastname: Shafer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D001665:Binding Sites; D065427:Factor Xa Inhibitors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013987:Ticks
chemical_list: D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C063967:tick anticoagulant peptide