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Douglas M Tollefsen - One of the best experts on this subject based on the ideXlab platform.
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proteolytic activation transforms Heparin Cofactor ii into a host defense molecule
Journal of Immunology, 2013Co-Authors: Martina Kalle, Douglas M Tollefsen, Praveen Papareddy, Gopinath Kasetty, Martin Malmsten, Matthias Morgelin, Artur SchmidtchenAbstract:The abundant serine proteinase inhibitor Heparin Cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.
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vascular dermatan sulfate and Heparin Cofactor ii
Progress in Molecular Biology and Translational Science, 2010Co-Authors: Douglas M TollefsenAbstract:Abstract Heparin Cofactor II (HCII) is a plasma protease inhibitor of the serpin family that inactivates thrombin by forming a covalent 1:1 complex. The rate of complex formation increases more than 1000-fold in the presence of dermatan sulfate (DS). Endothelial injury allows circulating HCII to enter the vessel wall, where it binds to DS and presumably becomes activated. Mice that lack HCII develop carotid artery thrombosis more rapidly than wild-type mice after oxidative damage to the endothelium. These mice also have increased arterial neointima formation following mechanical injury and develop more extensive atherosclerotic lesions when made hypercholesterolemic. Similarly, low plasma HCII levels appear to be a risk factor for atherosclerosis and in-stent restenosis in human subjects. These observations suggest that a major function of the HCII–DS system is to regulate the physiologic response to arterial injury.
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vascular dermatan sulfate regulates the antithrombotic activity of Heparin Cofactor ii
Blood, 2008Co-Authors: Li He, Cristina P Vicente, Tusar Giri, Douglas M TollefsenAbstract:Heparin Cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.
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Heparin Cofactor ii modulates the response to vascular injury
Arteriosclerosis Thrombosis and Vascular Biology, 2007Co-Authors: Douglas M TollefsenAbstract:Heparin Cofactor II (HCII) has several biochemical properties that distinguish it from other serpins: (1) it specifically inhibits thrombin; (2) the mechanism of inhibition involves binding of an acidic domain in HCII to thrombin exosite I; and (3) the rate of inhibition increases dramatically in the presence of dermatan sulfate molecules having specific structures. Human studies suggest that high plasma HCII levels are protective against in-stent restenosis and atherosclerosis. Studies with HCII knockout mice directly support the hypothesis that HCII interacts with dermatan sulfate in the arterial wall after endothelial injury and thereby exerts an antithrombotic effect. In addition, HCII deficiency appears to promote neointima formation and atherogenesis in mice. These results suggest that HCII plays a unique and important role in vascular homeostasis.
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selective cleavage and anticoagulant activity of a sulfated fucan stereospecific removal of a 2 sulfate ester from the polysaccharide by mild acid hydrolysis preparation of oligosaccharides and Heparin Cofactor ii dependent anticoagulant activity
Glycobiology, 2005Co-Authors: Vitor H Pomin, Douglas M Tollefsen, Mariana S Pereira, Ana Paula Valente, Mauro S G Pavao, Paulo A S MouraoAbstract:A linear sulfated fucan with a regular repeating sequence of [3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2,4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]][subscript n] is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by Heparin Cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with Heparin Cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by Heparin Cofactor II. Sulfated fucan with [approximately] 45 tetrasaccharide repeating units binds to Heparin Cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with [approximately] 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of Heparin Cofactor II.
Jeffrey I Weitz - One of the best experts on this subject based on the ideXlab platform.
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molecular basis for the susceptibility of fibrin bound thrombin to inactivation by Heparin Cofactor ii in the presence of dermatan sulfate but not Heparin
Journal of Biological Chemistry, 2001Co-Authors: Patricia C Liaw, Debra L Becker, James C Fredenburgh, Alan R Stafford, Jeffrey I WeitzAbstract:Abstract Although fibrin-bound thrombin is resistant to inactivation by Heparin·antithrombin and Heparin·Heparin Cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate·Heparin Cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the Heparin-catalyzed rate of thrombin inhibition by Heparin Cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by Heparin·Heparin Cofactor II reflects Heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary Heparin·thrombin·fibrin complex. This complex, formed as a result of three binary interactions (thrombin·fibrin, thrombin·Heparin, and Heparin·fibrin), limits accessibility of Heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate· thrombin·fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin·fibrin and thrombin· dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by Heparin Cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by Heparin Cofactor II in the presence of dermatan sulfate but not Heparin.
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exosites 1 and 2 are essential for protection of fibrin bound thrombin from Heparin catalyzed inhibition by antithrombin and Heparin Cofactor ii
Journal of Biological Chemistry, 1999Co-Authors: Debra L Becker, James C Fredenburgh, Alan R Stafford, Jeffrey I WeitzAbstract:Abstract Assembly of ternary thrombin-Heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound Heparin binds to exosite 2, produces a 58- and 247-fold reduction in the Heparin-catalyzed rate of thrombin inhibition by antithrombin and Heparin Cofactor II, respectively. The greater reduction for Heparin Cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and Heparin Cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (γ-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 → Ala, Arg97 → Ala, and Arg101 → Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the Heparin-independent inhibitor, α1-antitrypsin Met358 → Arg, is decreased less than 2-fold in the presence of soluble fibrin and Heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-Heparin complex, suggesting that access of Heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by Heparin-catalyzed inhibitors.
Paulo A S Mourao - One of the best experts on this subject based on the ideXlab platform.
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a sulfated fucan from the brown alga laminaria cichorioides has mainly Heparin Cofactor ii dependent anticoagulant activity
Carbohydrate Research, 2007Co-Authors: Seonjoo Yoon, Yuryang Pyun, Jaekwan Hwang, Paulo A S MouraoAbstract:Abstract The major acidic polysaccharide from the brown alga Laminaria cichorioides is a complex and heterogeneous sulfated fucan. Its preponderant structure is a 2,3-disulfated, 4-linked α-fucose unit. The purified polysaccharide has a potent anticoagulant activity, as estimated by APTT assay (∼40 IU/mg), which is mainly mediated by thrombin inhibition by Heparin Cofactor II. It also accelerates thrombin and factor Xa inhibition by antithrombin but at a lower potency. Sulfated fucan from L. cichorioides is a promising anticoagulant polysaccharide and a possible alternative for an antithrombotic compound due to its preferential Heparin Cofactor II-dependent activity.
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selective cleavage and anticoagulant activity of a sulfated fucan stereospecific removal of a 2 sulfate ester from the polysaccharide by mild acid hydrolysis preparation of oligosaccharides and Heparin Cofactor ii dependent anticoagulant activity
Glycobiology, 2005Co-Authors: Vitor H Pomin, Douglas M Tollefsen, Mariana S Pereira, Ana Paula Valente, Mauro S G Pavao, Paulo A S MouraoAbstract:A linear sulfated fucan with a regular repeating sequence of [3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2,4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]][subscript n] is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by Heparin Cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with Heparin Cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by Heparin Cofactor II. Sulfated fucan with [approximately] 45 tetrasaccharide repeating units binds to Heparin Cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with [approximately] 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of Heparin Cofactor II.
Frank C. Church - One of the best experts on this subject based on the ideXlab platform.
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effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by Heparin Cofactor ii and antithrombin
FEBS Letters, 2000Co-Authors: Carrie A Holland, Herbert C Whinna, Alexis T Henry, Frank C. ChurchAbstract:Abstract ‘Thrombin aptamers’ are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin’s anion-binding exosite-I. The effect of aptamer–thrombin interactions during inhibition by the serine protease inhibitor (serpin) Heparin Cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased ∼two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII–Heparin and HCII–dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without Heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII–glycosaminoglycans.
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enhancement of Heparin Cofactor ii anticoagulant activity
Journal of Biological Chemistry, 1999Co-Authors: Susannah J Bauman, Frank C. ChurchAbstract:Abstract Heparin Cofactor II (HCII) is a serpin whose thrombin inhibition activity is accelerated by glycosaminoglycans. We describe the novel properties of a carboxyl-terminal histidine-tagged recombinant HCII (rHCII-CHis6). Thrombin inhibition by rHCII-CHis6 was increased >2-fold at ∼5 μg/ml Heparin compared with wild-type recombinant HCII (wt-rHCII) at 50–100 μg/ml Heparin. Enhanced activity of rHCII-CHis6 was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Δ1–52, Δ1–68, and Δ1–75) in wt-rHCII and rHCII-CHis6. Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis6 deletion mutants were less active compared with full-length rHCII-CHis6. With glycosaminoglycans, Δ1–68 and Δ1–75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to wt-rHCII. Only rHCII-CHis6 had increased activity with Heparin, whereas all three mutants have increased Heparin binding. We generated a carboxyl-terminal histidine-tagged recombinant antithrombin III to study the tag on another serpin. Interestingly, this mutant antithrombin III had reduced Heparin Cofactor activity compared with wild-type protein. In a plasma-based assay, the glycosaminoglycan-dependent inhibition of thrombin by rHCII-CHis6 was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis6, may offer novel reagents for clinical application.
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Heparin Cofactor II and thrombin: Heparin-binding proteins linking hemostasis and inflammation
Trends in Cardiovascular Medicine, 1994Co-Authors: Frank C. Church, Maureane HoffmanAbstract:Abstract α-Thrombin is a trypsinlike serine proteinase involved in blood coagulation and wound-healing processes, which interacts with many different macromolecular substances. Heparin Cofactor II is a serpin (serine proteinase inhibitor) superfamily member that specifically inhibits thrombin but no other proteinase in blood coagulation. Both Heparin Cofactor II and thrombin interact with highly negatively charged glycosaminoglycans like Heparin and dermatan sulfate, and they both are leukocyte chemoattractants. The focus of this brief review is structure-function characteristics of Heparin Cofactor II and thrombin and their physiologic participation in uniting hemostatic and inflammatory processes.
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interaction of Heparin Cofactor ii with biglycan and decorin
Journal of Biological Chemistry, 1993Co-Authors: Herbert C Whinna, Haing U Choi, Lawrence Rosenberg, Frank C. ChurchAbstract:Abstract Two small interstitial dermatan sulfate-containing proteoglycans, biglycan and decorin, are present in extracellular matrices of skin, tendon, ligament, and cartilage. We investigated the effects of biglycan and decorin on the inhibition of alpha-thrombin by the serine proteinase inhibitor Heparin Cofactor II. In solution, Heparin Cofactor II inhibition of thrombin is accelerated by intact biglycan or decorin and by the dermatan sulfate-containing glycosaminoglycan (GAG) chains prepared from the proteoglycans, while core protein from cartilage biglycan had no effect. L-Iduronic acid-rich skin decorin and GAG chains had a greater accelerating effect than proteoglycan and GAG chains from cartilage that had lower L-iduronic acid content. Treatment of skin decorin and GAG chains with chondroitinase ABC totally eliminated the ability of these compounds to accelerate thrombin inhibition by Heparin Cofactor II suggesting that dermatan sulfate was responsible for this action. Both biglycan and decorin bound to type V collagen in a saturable and specific manner. Biglycan, decorin, and core protein from biglycan competed for decorin binding to the type V collagen, while only the intact proteoglycans competed for biglycan binding. When bound to type V collagen, both biglycan and decorin accelerated the Heparin Cofactor II/thrombin inhibition reaction as efficiently as the proteoglycans in solution. Our results demonstrate that Heparin Cofactor II in the presence of biglycan or decorin bound to type V collagen provides a "thromboresistant surface," further suggesting a physiological function for these proteins in regulating the extravascular activities of thrombin.
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inhibition of dysthrombins quick i and ii by Heparin Cofactor ii and antithrombin
Journal of Biological Chemistry, 1993Co-Authors: Jeanne E Phillips, Herbert C Whinna, Rebecca A Shirk, Ruth Ann Henriksen, Frank C. ChurchAbstract:Abstract Heparin Cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare Heparin Cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from Heparin-Sepharose suggesting that the putative Heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of Heparin. Also, Quick I was inhibited similarly to alpha-thrombin by Heparin Cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by Heparin Cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by Heparin Cofactor II. We also found that Heparin Cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the Heparin Cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of Heparin Cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.
Debra L Becker - One of the best experts on this subject based on the ideXlab platform.
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molecular basis for the susceptibility of fibrin bound thrombin to inactivation by Heparin Cofactor ii in the presence of dermatan sulfate but not Heparin
Journal of Biological Chemistry, 2001Co-Authors: Patricia C Liaw, Debra L Becker, James C Fredenburgh, Alan R Stafford, Jeffrey I WeitzAbstract:Abstract Although fibrin-bound thrombin is resistant to inactivation by Heparin·antithrombin and Heparin·Heparin Cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate·Heparin Cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the Heparin-catalyzed rate of thrombin inhibition by Heparin Cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by Heparin·Heparin Cofactor II reflects Heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary Heparin·thrombin·fibrin complex. This complex, formed as a result of three binary interactions (thrombin·fibrin, thrombin·Heparin, and Heparin·fibrin), limits accessibility of Heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate· thrombin·fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin·fibrin and thrombin· dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by Heparin Cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by Heparin Cofactor II in the presence of dermatan sulfate but not Heparin.
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exosites 1 and 2 are essential for protection of fibrin bound thrombin from Heparin catalyzed inhibition by antithrombin and Heparin Cofactor ii
Journal of Biological Chemistry, 1999Co-Authors: Debra L Becker, James C Fredenburgh, Alan R Stafford, Jeffrey I WeitzAbstract:Abstract Assembly of ternary thrombin-Heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound Heparin binds to exosite 2, produces a 58- and 247-fold reduction in the Heparin-catalyzed rate of thrombin inhibition by antithrombin and Heparin Cofactor II, respectively. The greater reduction for Heparin Cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and Heparin Cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (γ-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 → Ala, Arg97 → Ala, and Arg101 → Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the Heparin-independent inhibitor, α1-antitrypsin Met358 → Arg, is decreased less than 2-fold in the presence of soluble fibrin and Heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-Heparin complex, suggesting that access of Heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by Heparin-catalyzed inhibitors.