N-Glycans and the N Terminus of Protein C Inhibitor Affect the Cofactor-enhanced Rates of Thrombin Inhibition

Abstract

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 ⌬6-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 thrombomodulinenhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the ⌬6-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. Protein C inhibitor (PCI)3 is a serine protease inhibitor that belongs to the serpin superfamily of proteins (1, 2). Like other inhibitory serpins, PCI utilizes a unique suicide mechanism for protease inactivation. In this mechanism the inhibitor exposes its reactive site in a loop on the protein surface, called the reactive center loop, RCL. The reactive site is recognized and cleaved by the protease. Instead of being released as with a normal substrate, the protease remains covalently bound to the inhibitor. The N-terminal part of the RCL is subsequently inserted as a sixth strand in ␤-sheet A, while the protease is dragged along the surface to the opposite pole of the serpin. The protease-inhibitor complex is inactive and rapidly cleared from the circulation (3). Protease-serpin specificity is determined by the reactive site and by exosite interactions. PCI is capable of inhibiting diverse proteases, including thrombin, thrombin bound to thrombomodulin, active protein C, factor Xa, factor XIa, plasma and tissue kallikreins, urokinase, and tissue plasminogens (4 -7).The identification of factors that restrict PCI to performing a single task instead of the several tasks it is biochemically capable of carrying out, remain elusive. Some factors have, however, been found...

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