The prothrombinase complex converts prothrombin to ␣-thrombin through the intermediate meizothrombin (Mz-IIa). Both ␣-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains ␣-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. ␣-Thrombin can undergo additional proteolysis to ␤-thrombin and ␥-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by ␤-thrombin or ␥-thrombin, and by ␣-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, ␤-thrombin, ␥-thrombin, and ␣-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than ␣-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than ␣-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factorinitiated coagulation and the proteases of the contact system. (Blood. 2011;118(2): 437-445) IntroductionThe trypsin-like protease ␣-thrombin (␣-IIa) is a key contributor to vital host responses to injury, including fibrin formation, 1-3 platelet and endothelial cell activation, 4 and inflammation. 5 During coagulation, prothrombin is converted to ␣-IIa through a series of proteolytic steps catalyzed by factor (F) Xa. 6,7 The process results in formation of a functional active site and expression of 2 anion binding exosites (ABE I and ABE II) that are required for ␣-IIa interactions with many substrates, receptors, and inhibitors (Table 1). [1][2][3]6 In the presence of FVa and phospholipid, FXa initially converts prothrombin to the protease meizothrombin (Mz-IIa; Figure 1A), 7-11 which expresses ABE I. 6 Mz-IIa is rapidly converted to ␣-IIa, which may undergo further proteolysis to form ␤-thrombin (␤-IIa) and ␥-thrombin (␥-IIa) ( Figure 1B), 2 proteases with reduced capacity to catalyze ABE I-dependent reactions. [9][10][11][12] Physiologic roles for ␤-IIa or ␥-IIa have not been established; however, both have been identified in clotting blood. 11 ␣-IIa up-regulates its own generation by activating the cofactors FV and FVIII, [1][2][3]9 and by converting FXI to the protease FXIa. 13,14 FXIa, in turn, sustains ␣-IIa generation by converting FIX to FIXa␤, 15 and possibly by activating FV and FVIII. 16 In the original cascade/ waterfall hypotheses of coagulation, FXI is activated by FXIIa 17,18 ; however, current models deemphasize this reaction based on the observation that FXI deficiency is associated with abnormal bleeding, whereas FXII deficiency is not. 18 FXI activation by ␣-IIa would explain the phenotypic di...

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) 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...

Introduction The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition. Methods MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition. Results and conclusions MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.