Aspartic Acid Residues 72 and 75 and Tyrosine-sulfate 73 of Heparin Cofactor II Promote Intramolecular Interactions during Glycosaminoglycan Binding and Thrombin Inhibition

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivationThe blood clotting proteinase, ␣-thrombin (T), 1 possesses two electropositive sites, exosites I and II (1, 2), that play distinct roles in its covalent inactivation by the serine proteinase inhibitors (serpins), antithrombin (AT), and heparin cofactor II (HCII), in the absence and presence of rate-accelerating glycosaminoglycans (GAGs) (3-8 (9,10). Inactivation by AT is accelerated up to 50,000-fold by high molecular weight heparin, which acts as a template, by binding to thrombin exosite II and AT helices A and D (4, 11). Whereas heparin accelerates thrombin inactivation by both AT and HCII, dermatan sulfate (DS), and other polyanions (5, 12-17) selectively enhance HCII inhibitory action. The HCII mechanism was initially thought to utilize GAGs as templates between exosite II and HCII helix D (5, 12, 13), but later hypothesized to involve allosteric, GAG-induced interaction of an acidic N-terminal domain of HCII with exosite I (6, 7, 18 -21). A synthetic HCII-(54 -75) peptide was shown to compete with hirudin-(54 -65) for thrombin binding (18). Residues 52-75 in HCII contain two hirudin-like repeats, the first of which interacts directly with exosite I (21). The crystal structure of the HCII⅐S195A-thrombin Michaelis complex confirmed this exosite I interaction (22). Deletion of the first HCII acidic repeat (6,19), and mutations in exosite I residues Arg 67 and Arg 73 (20) caused large decreases in the rate constants for the heparin-and DS-catalyzed inactivation, further suggesting the importance of the direct HCII-exosite I interaction. Selective thrombin exosite II mutations did not affect the DS-catalyzed inactivation rate significantly (23,24). Blocking exosite II with HD22, a single-stranded DNA aptamer only had a modest effect on this inactivation rate, and DS with a chain length of 12-20 saccharide units was found to be as effective as higher M r DS in catalyzing thrombin inactivation (25). Whereas some studies report DS binding to thrombin (13,23,26), others postulate that DS does not interact with thrombin (27, 28). These findings, and the assumption that DS template action, like heparin, engages exosite II, led to the proposal of a mechanism in which direct interaction of the HCII N-terminal domain with exosite I is predominant, and GAG template action may be less important, or not even obligatory.In the mechanism of high affinity heparin-catalyzed throm-

mentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

The Preferred Pathway of Glycosaminoglycan-accelerated Inactivation of Thrombin by Heparin Cofactor II

Verhamme

Bock

Jackson³

2004

“…A loss of inhibitory activity for uPA and plasmin and an increase in plasmin activity would be predicted to increase promatrix metalloproteinase activation with increased inflammatory cell invasion and aneurysm formation. Certainly, the P1-P1Ј and adjacent RCL amino acid residues have been previously documented to alter activity of other serpins, such as anti-thrombin (1,4,47,48), and additionally other domains of serpins are known to alter serpin-mediated inhibitory actions (1,49,50).…”

Section: Discussionmentioning

confidence: 99%

Identification of Myxomaviral Serpin Reactive Site Loop Sequences That Regulate Innate Immune Responses

Dai

Viswanathan

Sun

et al. 2006

The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1 Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1 Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1 Thr increased thrombosis. Substitution of Serp-1 P2-P7 with Ala 6 allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.

“…The rates were measured under pseudofirst order kinetic reaction conditions as described previously (26). Screening assays were used to compare all the Ala-scanned thrombin mutants for HCII inhibition.…”

Section: Methodsmentioning

confidence: 99%

“…HCII appears to use a novel allosteric process in which glycosaminoglycan binding to the D-helix region allows a more permissive acidic domain to act as a "tethered ligand" for binding to exosite-1 of thrombin (5, 9, 16, 24, 30, 38, 51, 52, 61, 62). There have been two similar mechanisms proposed, with the only difference being the role of glycosaminoglycan acting as a secondary bridge between HCII and thrombin (28,30,38,51,62) versus a purely allosteric model without the need for ternary complex formation between HCII-glycosaminoglycanthrombin (5,6,(23)(24)(25)(26)52).…”

Section: Use Of Ala-scanned Mutants For Thrombin Structure-activitymentioning

confidence: 99%

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

Fortenberry

Whinna²,

Gentry³

et al. 2004

Self Cite

We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln 24 ) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCIIglycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.The blood coagulation cascade is highly regulated with the serine protease thrombin playing an essential role in both procoagulant and anticoagulant pathways. As a procoagulant, thrombin activates platelets, cleaves fibrinogen to fibrin, resulting in the formation of a fibrin clot, activates factors V, VII, and XI via a feedback mechanism, and also activates factor XIII (plasma transglutaminase) (for a review, see Ref. 1 and references cited therein). By contrast, when thrombin binds to the endothelial cell surface receptor thrombomodulin, thrombin activates the anticoagulant zymogen, protein C (for a review, see Ref. 2 and references cited therein). Macromolecular substrate recognition by thrombin is partly mediated by two clusters of positively charged basic amino acids located on opposite sides of the active site, referred to as anionbinding exosite-1 and anion-binding exosite-2 (exosite-1 and exosite-2, respectively) 1 . Exosite-1 binds fibrinogen, fibrin, heparin cofactor II (HCII) (3), and hirudin (4), whereas exosite-2 binds heparin, heparan sulfate, and dermatan sulfate (5-7).Glycosaminoglycan-accelerated inhibition by the serine protease inhibitors (serpins) antithrombin (AT; systematic name SERPINC1) (8), heparin cofactor II (HCII; systematic name SERPIND1) (9), protein C inhibitor (also known as plasminogen activator inhibitor-3; systematic name SERPINA5) (10), and protease nexin-1 (systematic name SERPINE2) (11) is one mechanism for thrombin regulation. Besides thrombin, AT inhibits several proteases in the blood coagulation pathway, including factor IXa, Xa, XIa, XIIa, plasmin, and kallikerin; however, HCII is specific for thrombin in this group of serine proteases. Although both AT and HCII inhibition of thrombin are accelerated by heparin and heparan sulfate, HCII inhibition of thrombin is also accelerated by dermatan sulfate (12, 13). The glycosaminoglycan-binding site for AT and HCII is localized to the D-helix region of these serpins (for a review, see Refs. 8 and 14 -20 and references cited therein). Recent studies show that HCII acts as a thrombin inhibitor in the arterial circulation (21) and that dermatan sulfate proteoglyca...