N-glycans and the N terminus of protein C inhibitor affect the cofactor-enhanced rates of thrombin inhibition.
Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.
Sun
Wei
W
Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, Uppsala SE-751 23, Sweden.
Parry
Simon
S
Panico
Maria
M
Morris
Howard R
HR
Kjellberg
Margareta
M
Engström
Ake
A
Dell
Anne
A
Schedin-Weiss
Sophia
S
D050197:Atherosclerosis / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D018046:Protein C Inhibitor / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism; D019851:Thrombophilia / Q000378:metabolism
D011134:Polysaccharides; D018046:Protein C Inhibitor; D018180:Thrombomodulin; D006493:Heparin; D013917:Thrombin