Glycosaminoglycans bind factor Xa in a Ca2+-dependent fashion and modulate its catalytic activity.
Recent studies have demonstrated the existence of a Ca(2+)-dependent heparin-binding site on factor Xa. To characterize this heparin-binding site, the extrinsic fluorescence of fluorescein-labeled, active site-blocked factor Xa was monitored as it was titrated with glycosaminoglycans of various sulfate content and chain length. The binding of glycosaminoglycans to factor Xa appears to be charge-dependent because affinity is correlated with degree of glycosaminoglycan sulfation. All glycosaminoglycans bind factor Xa with higher affinity in the presence of Ca(2+) than in its absence. In contrast, when Gla-domainless factor Xa was substituted for factor Xa, glycosaminoglycans bound with similar affinities in the absence and presence of Ca(2+). These results support the hypothesis that the anionic Gla domain impairs glycosaminoglycan binding in the absence of Ca(2+). The changes in fluorescence intensity of factor Xa when titrated with glycosaminoglycans suggest that glycosaminoglycans induce conformational changes in the active site environment of factor Xa. To explore the consequences of these conformational changes, the effect of glycosaminoglycans on the catalytic activity of factor Xa was examined. Glycosaminoglycans influenced the ability of factor Xa to cleave chromogenic substrates and attenuated the capacity of factor Xa to activate factor VII. The potency of glycosaminoglycans in these assays reflected their affinity for factor Xa. These studies suggest that glycosaminoglycan binding perturbs exosites on the surface of factor Xa, potentially modifying interactions with cofactors or substrates.
Henderson Research Centre and McMaster University, Hamilton, Ontario L8V 1C3, Canada.
Lee A
LA
O'Brien
Alan R
AR
Stafford
James C
JC
Fredenburgh
Jeffrey I
JI
Weitz
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D003871:Dermatan Sulfate / Q000737:chemistry; D005167:Factor VII / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D011486:Protein C / Q000737:chemistry; D013379:Substrate Specificity
D000590:Amino Acid Chloromethyl Ketones; D006025:Glycosaminoglycans; D006495:Heparin, Low-Molecular-Weight; D011486:Protein C; D003871:Dermatan Sulfate; D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D006493:Heparin; D015951:Factor Xa; D002118:Calcium