) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K D ‫؍‬ 1600 ؎ 300 M, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCIIdependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.The central coagulation proteinase, ␣-thrombin (T), 2 is covalently inactivated by the serpins antithrombin (AT) and heparin cofactor II (HCII), in reactions that are accelerated by sulfated glycosaminoglycans (GAGs) (1-6). Two electropositive sites on thrombin, exosites I and II, are differentially involved in its inactivation by HCII and AT (7,8). High molecular weight GAGs act as templates between thrombin exosite II and the GAG binding sites on AT and HCII (1, 2, 5, 9 -11), and an 18 saccharide unit length is required for template activity (12).The HCII mechanism also utilizes the allosteric interaction of thrombin exosite I with the Glu 53 -Asp 75 acidic sequence in the HCII NH 2 -terminal region that contains two hirudin-(54 -65)-like repeats (3,4,(13)(14)(15)(16)(17). This sequence, not present in AT, becomes available for thrombin interaction upon GAG binding of HCII (3,16). Direct evidence was provided by the crystal structure of the HCII⅐S195A-thrombin Michaelis complex, in which residues 56 -72 of HCII make contact with exosite I (16). Both repeats are required for heparin-and DS-catalyzed thrombin inactivation, as demonstrated by the decreased inhibitory potential of HCII NH 2 -terminal deletion mutants (3,14). Mutation of thrombin exosite I residues Arg 67 and Arg 73 resulted in significantly slower inactivation by native HCII (15). In reactions utilizing template-forming GAGs, both the template and allosteric interactions contribute to the mechanism by binding of GAG to thrombin exosite II and interaction of thrombin-complexed GAG with the heparin binding site in HCII, thereby triggering interaction of the HCII NH 2 -terminal sequence with exosite I. The intermediate T⅐GAG⅐HCII complexes, stabilized by two interactions, are significantly tighter than the T⅐GAG⅐AT complexes (9).Oligosaccharides shorter than 18 saccharide units, such as dermatan sulfate hexasaccharides, and sulfated bis-lactobionic and bis-maltobionic acid amides moderately accelerated inhibition by HCII but not [18][19][20]. These molecules are too small for template action, and it is unknown whether they bind to thrombin. Their mechanism of action is proposed to be solely allosteric, by binding to HCII and triggering interaction of the NH 2 -terminal sequence with exosite I. The sulfated disaccharide, sucrose octasulfate (SOS), a known anti-ulcer drug (21) recently identified as an antitumor...

Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K D ) of 6 ± 1 and 7 ± 1 μM, respectively, ~6-fold tighter than plasma HCII, with K D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K D 4 ± 1 μM, was ~4-fold tighter than for plasma HCII, with K D 15 ± 4 μM. Recombinant HCII, lacking Nglycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn 169 N-glycan with the HCII heparin-binding site. KeywordsRecombinant heparin cofactor II; Serpin; Thrombin; Serine protease inactivation Thrombin, the central proteinase in the coagulation cascade, cleaves soluble fibrinogen to insoluble fibrin which polymerizes to form a blood clot. Thrombin is inactivated by circulating plasma heparin cofactor II (HCII),1 a member of the superfamily of serine * Corresponding author. Fax: +1 615 343 7023. Ingrid.Verhamme@vanderbilt.edu (I.M. Verhamme). 1 Abbreviations used: serpin, serine proteinase inhibitor; HCII, heparin cofactor II; AT, antithrombin; RCL, reactive center loop; SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; T, human α-thrombin; GAG, glycosaminoglycan; IPTG, isopropyl-1-thio-β-D-galactopyranoside; TNS, 2-(p-toluidinyl)naphthalene-6-sulfonic acid; DS, dermatan sulfate; CBS31.39, CH 3 SO 2 -D-LeuGly-Arg-p-nitroanilide; Chromozym TH, Tos-Gly-Pro-Arg-p-nitroanilide; pNA, p-nitroaniline; Hepes, N-(2-hydroxyethyl)-N′-2-piperazine-ethanesulfonic acid; PEG, polyethylene glycol 8000. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript proteinase inhibitors (serpins) [1][2][3]. Serpins possess a reactive center loop (RCL) that is cleaved by their target proteases, with formation of a stabilized, covalent serpin-enzyme complex in which the protease active site is distorted. The thrombin-HCII reaction is accelerated by binding of the sulfated glycosaminoglycans (GAGs), heparin and dermatan sulfate (DS) [4][5][6], and various other polyanions [7][8][9][10] in a ternary complex, and plays an important role in prevention of arterial thrombosis [11]. The thrombin-HCII mechanism utilizes an allosteric interaction of a unique acidic region in the HCII N-terminus with thrombin exosite I [1,...