Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.
Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.
Liaw
P C
PC
Department of Medicine, McMaster University and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, L8V 1C3 Canada.
Austin
R C
RC
Fredenburgh
J C
JC
Stafford
A R
AR
Weitz
J I
JI
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D003871:Dermatan Sulfate / Q000494:pharmacology*; D014644:Genetic Variation; D006025:Glycosaminoglycans / Q000494:pharmacology; D015844:Heparin Cofactor II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D003871:Dermatan Sulfate; D006025:Glycosaminoglycans; D015844:Heparin Cofactor II; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D013917:Thrombin