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Frank C Church - One of the best experts on this subject based on the ideXlab platform.

  • molecular mapping of the thrombin heparin cofactor ii complex
    Journal of Biological Chemistry, 2004
    Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C Church
    Abstract:

    Abstract 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, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these Exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-heparin when compared with 2.36 × 108 m-1 min-1 with wild-type thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type thrombin) confirmed that the structural integrity of thrombin Exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) 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 HCII-glycosaminoglycan 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.

  • rna aptamer to thrombin binds anion binding Exosite 2 and alters protease inhibition by heparin binding serpins
    FEBS Letters, 2004
    Co-Authors: Martha L Jeter, Herbert C. Whinna, Yolanda M Fortenberry, Bruce A. Sullenger, Rebekah R White, Christopher P Rusconi, Frank C Church
    Abstract:

    We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding Exosite-2 (Exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of Exosite-2. We conclude that a RNA aptamer to thrombin Exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.

  • role of thrombin anion binding Exosite i in the formation of thrombin serpin complexes
    Journal of Biological Chemistry, 1998
    Co-Authors: Timothy Myles, Frank C Church, Herbert C. Whinna, Denis Monard, Stuart R Stone
    Abstract:

    Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding Exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (kon) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77(a)Q, K81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas kon values were decreased 2-3-fold for HCII against the majority of the Exosite-I mutants. The Exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal kon values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the Exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77(a)Q, where kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon values for inhibition of the Exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fold lower than rIIa. Larger decreases in kon values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had kon values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3-7-fold relative to rIIa. The results suggest that ATIII has no major interaction with Exosite-I of thrombin with or without heparin. PN1 bound to heparin uses Exosite-I to some extent, possibly by utilizing the positive electrostatic field of Exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin Exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for Exosite-I, presumably through contact of the "hirudin-like" domain of HCII with Exosite-I of thrombin.

  • inhibition of dysthrombins quick i and ii by heparin cofactor ii and antithrombin
    Journal of Biological Chemistry, 1993
    Co-Authors: Jeanne E Phillips, Ruth Ann Henriksen, Herbert C. Whinna, Rebecca A. Shirk, Frank C Church
    Abstract:

    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.

  • role of thrombin Exosites in inhibition by heparin cofactor ii
    Journal of Biological Chemistry, 1992
    Co-Authors: Susannah J Rogers, Herbert C. Whinna, Charlotte W Pratt, Frank C Church
    Abstract:

    Abstract We determined the role of specific thrombin "Exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding Exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding Exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.

Ingrid M Verhamme - One of the best experts on this subject based on the ideXlab platform.

  • analysis of the factor xi variant arg184gly suggests a structural basis for factor ix binding to factor xia
    Journal of Thrombosis and Haemostasis, 2013
    Co-Authors: Yipeng Geng, Ingrid M Verhamme, Maofu Sun, S P Bajaj, Jonas Emsley, David Gailani
    Abstract:

    Summary Background A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an Exosite required for binding of FIX to activated FXI (FXIa). Objective To test the effects of the Arg184Gly substitution on FIX activation, and to characterize the FIX-binding site on FXIa. Methods Recombinant FXIa and FIX variants were used to identify residues involved in FIX activation by FXIa. Analysis of the FXI structure was used to identify potential FIX-binding sites. Results The Km for FIX activation by FXIa-Gly184 was approximately three-fold higher than for FXIa, suggesting that Arg184 is part of the Exosite. Arg184 and the adjacent residues, Ile183 and Asp185, contribute to charged and hydrophobic areas that are not present in the FXI homolog prekallikrein (PK). Replacing residues 183–185 with alanine abolished Exosite activity, similarly to replacement of the entire A3 domain with the A3 domain from PK (FXIa/PKA3). Reintroducing FXI residues 183–185 into FXIa/PKA3 partially restored the Exosite, and replacing residues 183–185 and 260–264 completely restored Exosite function. FIX in which the Ω-loop (residues 4–11) was replaced with the FVII Ω-loop was activated poorly by FXIa, suggesting that the FIX Ω-loop binds to FXIa. Conclusions The results support a model in which the Ω-loop of FIX binds to an area on FXIa composed of residues from the N-terminus and C-terminus of the A3 domain. These residues are buried in zymogen FXI, and must be exposed upon conversion to FXIa to permit FIX binding.

  • fluorescent reporters of thrombin heparin cofactor ii and heparin binding in a ternary complex
    Analytical Biochemistry, 2012
    Co-Authors: Ingrid M Verhamme
    Abstract:

    Abstract Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4′F]FPR-T and [6F]FFR-T, and the Exosite I probe, Hir-(54–65)( SO 3 - ), characterized thrombin Exosite I and II interactions with HCII and heparin in the complex. HCII binding to Exosite I of heparin-bound [4′F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to Exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54–65)( SO 3 - ) binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T·heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin·heparin complex binds HCII and allowing for interaction of thrombin·(heparin)2 complexes with HCII. Hir-(54–65)( SO 3 - ) displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and Exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin·heparin complex is dramatically enhanced compared with heparin binding alone and that Exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.

  • Exosite interactions in factor ix activation by factor xia
    Blood, 2011
    Co-Authors: Yipeng Geng, Ingrid M Verhamme, Maofu Sun, Stephen B Smith, Amanda S Messer, Paul S Bajaj, David Gailani
    Abstract:

    Abstract 2235 Conversion of factor IX (fIX) to the protease factor IXaβ (fIXaβ) is an important reaction during thrombin generation at a site of vascular injury. The physiologic activators of fIX are the proteases factor VIIa and factor XIa (fXIa). The zymogen of fXIa, fXI, is a 160 kDa dimer of two identical subunits linked by a disulfide bond. Each subunit has four apple domains at the N terminus (A1-A4), and a trypsin-like catalytic domain at the C-terminus. Conversion of fXI to fXIa involves cleavage of each subunit at the Arg369-Ile370 bond, generating a heavy chain (the apple domains) and an activated catalytic domain that remains connected to the heavy chain by a disulfide bond. FXIa activates fIX in the presence of calcium ions by sequential cleavage after Arg145 (forming the inactive intermediate fIXα) and then after Arg180 to form fIXaβ. Previously, we showed that an Exosite (a site on fXIa distinct from the active site) on the A3 domain of the fXIa heavy chain is a major determinant of affinity and specificity for fIX activation by fXIa ( J Biol Chem 1999;274:36373 and 2005;280:23523). Evidence has also been presented for a second fIX-binding Exosite on the fXIa catalytic domain. While the catalytic efficiency (kcat/Km) for fIX activation by an isolated fXIa catalytic domain (fXIaCD – no heavy chain) was ∼500 fold lower than activation by fXIa, this was reported to be due to a decrease in kcat, rather than the expected increase in Km that should accompany loss of the A3 Exosite ( Biochemistry 2007;46:9830). To investigate this discrepancy, we used recombinant wild type fXIa (fXIaWT), fXIa missing the Exosite on the A3 domain (fXIa-PKA3) or fXIaCD to activate purified fIX and fIXα. Full progress curves were generated using densitometry of Coomassie Blue stained SDS-polyacryalmide gels imaged at infrared wavelengths. The Km and kcat for cleavage by fXIaWT of fIX after Arg145 (Km 0.09 ± 0.02 μM, k cat = 7.3 ± 0.4 min −1 ) and fIXα after Arg180 (Km 0.12 ± 0.02 μM, k cat = 6.8 ± 0.4 min −1 ) are similar, and agree with published results. FXIa/PKA3 cleaved fIX after Arg145 with a significantly higher Km (>2 μM), consistent with loss of the Exosite, and leading to an ∼100-fold reduction in catalytic efficiency. Because we were not able to reach saturation, it is not clear if the kcat was affected appreciably. Catalytic efficiency for cleavage after Arg180 was ∼3000-fold lower with FXIa-PKA3 than with fXIaWT, but the slow rate of cleavage precluded clearly determining if this was due to an effect on Km or kcat. These results indicate that the A3 Exosite is involved in both cleavages, and loss of the Exosite has a more deleterious effect on the second cleavage after Arg180 that converts fIXα to fIXaβ than the first cleavage after Arg145 that converts fIX to fIXα. This would account for the observation that there is substantial accumulation of fIXα when fIX is activated by FXIa-PKA3, but not by fXIaWT. For fIX cleavage after Arg145 by fXIaCD, Km was again markedly increased (≥ 2 μM) compared to FXIaWT, with a modest (∼3-fold) reduction in kcat resulting in reduced catalytic efficiency that is roughly similar to that for FXIa/PKA3. The catalytic efficiency of cleavage after Arg180 by fXIaCD was ∼4000 fold reduced compared to FXIa-WT. Interestingly, when calcium was removed from the reactions, cleavage of both the Arg145 and Arg180 activation sites by fXIa-WT, but not by fXIa/PKA3 or fXIaCD, were markedly impaired, indicating both cleavages are Ca2+–dependent reactions. Cumulatively, these results indicate that an Exosite on the heavy chain A3 domain is largely responsible for the Ca2+-dependent affinity of fIX and fIXα for fXIa. We used surface plasmon resonance as a complementary approach to look directly at Ca2+-dependent binding of fXIa to fIX. FXIa-WT bound to immobilized fIX with Kd 48nM, in reasonable agreement with results from the kinetic analysis. Isolated fXIa heavy chain (lacking the catalytic domain) bound with similar Kd (53 nM). In contrast, fXIa/PKA3 and fXIaCD bound poorly to fIX (Kd >2 μM). Taken as a whole, the data support the hypothesis that an Exosite on the fXIa A3 domain is largely responsible for affinity and specificity of the fXIa-mediated reactions converting fIX to fIXα, and fIXα to fIXaβ. While the analysis cannot rule out minor contributions of other Exosites to the reactions, they do not support the premise that there is a fIX- or fIXα-binding site on the fXIa catalytic domain that contributes substantially to initial substrate binding. Disclosures: No relevant conflicts of interest to declare.

  • sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor ii
    Journal of Biological Chemistry, 2010
    Co-Authors: Suryakala Sarilla, David Gailani, Sally Y Habib, Dmitri V Kravtsov, Anton Matafonov, Ingrid M Verhamme
    Abstract:

    Abstract Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin Exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH2-terminal segment with Exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (KD) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (kobs). SOS bound HCII with KD 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by Kcomplex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO3−) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO3−) and HCII to Exosite I. Meizothrombin(des-fragment 1), binding SOS with KD = 1600 ± 300 μm, 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 HCII-dependent 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.

  • Exosite mediated substrate recognition of factor ix by factor xia the factor xia heavy chain is required for initial recognition of factor ix
    Journal of Biological Chemistry, 2005
    Co-Authors: Taketoshi Ogawa, Paul E. Bock, Ingrid M Verhamme, Maofu Sun, David Gailani
    Abstract:

    Abstract Studies of the mechanisms of blood coagulation zymogen activation demonstrate that Exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of Exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 ± 2 μm) and aprotinin (Ki 1.13 ± 0.07 μm) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 ± 32 nm) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at Exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 ± 0.52 μm) was non-competitive, whereas inhibition by active site-inhibited factor IXaβ was competitive (Ki 0.33 ± 0.05 μm). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 ± 14 μm) but was not inhibited by factor IX, consistent with loss of factor IX-binding Exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to Exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that Exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.

J E Sadler - One of the best experts on this subject based on the ideXlab platform.

  • heparin cofactor ii is regulated allosterically and not primarily by template effects studies with mutant thrombins and glycosaminoglycans
    Journal of Biological Chemistry, 1994
    Co-Authors: John P. Sheehan, Douglas M Tollefsen, J E Sadler
    Abstract:

    Abstract Besides its critical role in hemostasis, the serine protease thrombin also participates in wound healing, inflammation, and atherosclerosis. Thrombin is inhibited by the serpins antithrombin and heparin cofactor II (HCiI) in reactions that are accelerated markedly by specific glycosaminoglycans. Following vascular injury, thrombin must be inhibited at both intravascular and extravascular sites that impose different constraints on the recognition of thrombin by these inhibitors. The present study examines the role of anion-binding Exosite II of thrombin in the interaction with glycosaminoglycans and HCII. Acceleration of thrombin inhibition by serpins in the presence of glycosaminoglycans is proposed to occur by a template mechanism, in which inhibitor and protease bind simultaneously to the same glycosaminoglycan chain, facilitating their interaction. According to the template model, disruption of protease binding to glycosaminoglycan should significantly reduce acceleration of the inhibition. Specific mutations in Exosite II (R89E, R245E, K248E, and K252E) disrupted thrombin binding to both dermatan sulfate and heparin, indicating that both glycosaminoglycans bind to a common site in Exosite II. The same mutations markedly decreased the rate constant for thrombin inhibition by antithrombin-heparin (up to 100-fold) but had little effect on the rate constant for thrombin inhibition by HCII-heparin (7-fold maximal reduction) and no effect on the rate constant for thrombin inhibition by HCII-dermatan sulfate. These results are incompatible with a template model for thrombin inhibition by HCII and dermatan sulfate. In the presence of glycosaminoglycan, HCII and antithrombin interact with opposing thrombin Exosites and use distinct mechanisms of glycosaminoglycan catalysis. Antithrombin employs a template mechanism that requires heparin to interact with thrombin Exosite II, whereas HCII employs an allosteric mechanism that requires thrombin Exosite I but is largely independent of Exosite II. These findings have potential implications for glycosaminoglycan therapy and for the respective physiologic roles of HCII and antithrombin.

  • heparin cofactor ii is regulated allosterically and not primarily by template effects studies with mutant thrombins and glycosaminoglycans
    Journal of Biological Chemistry, 1994
    Co-Authors: John P. Sheehan, Douglas M Tollefsen, J E Sadler
    Abstract:

    Abstract Besides its critical role in hemostasis, the serine protease thrombin also participates in wound healing, inflammation, and atherosclerosis. Thrombin is inhibited by the serpins antithrombin and heparin cofactor II (HCiI) in reactions that are accelerated markedly by specific glycosaminoglycans. Following vascular injury, thrombin must be inhibited at both intravascular and extravascular sites that impose different constraints on the recognition of thrombin by these inhibitors. The present study examines the role of anion-binding Exosite II of thrombin in the interaction with glycosaminoglycans and HCII. Acceleration of thrombin inhibition by serpins in the presence of glycosaminoglycans is proposed to occur by a template mechanism, in which inhibitor and protease bind simultaneously to the same glycosaminoglycan chain, facilitating their interaction. According to the template model, disruption of protease binding to glycosaminoglycan should significantly reduce acceleration of the inhibition. Specific mutations in Exosite II (R89E, R245E, K248E, and K252E) disrupted thrombin binding to both dermatan sulfate and heparin, indicating that both glycosaminoglycans bind to a common site in Exosite II. The same mutations markedly decreased the rate constant for thrombin inhibition by antithrombin-heparin (up to 100-fold) but had little effect on the rate constant for thrombin inhibition by HCII-heparin (7-fold maximal reduction) and no effect on the rate constant for thrombin inhibition by HCII-dermatan sulfate. These results are incompatible with a template model for thrombin inhibition by HCII and dermatan sulfate. In the presence of glycosaminoglycan, HCII and antithrombin interact with opposing thrombin Exosites and use distinct mechanisms of glycosaminoglycan catalysis. Antithrombin employs a template mechanism that requires heparin to interact with thrombin Exosite II, whereas HCII employs an allosteric mechanism that requires thrombin Exosite I but is largely independent of Exosite II. These findings have potential implications for glycosaminoglycan therapy and for the respective physiologic roles of HCII and antithrombin.

  • activation induced exposure of the thrombin anion binding Exosite interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin Exosite specific antibody
    Journal of Biological Chemistry, 1994
    Co-Authors: Virginie Picard, Martine Aiach, J E Sadler
    Abstract:

    Abstract The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding Exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an Exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding Exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the Exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the Exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin Exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.

  • localization of the single stranded dna binding site in the thrombin anion binding Exosite
    Journal of Biological Chemistry, 1992
    Co-Authors: M Tsiang, J E Sadler
    Abstract:

    Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding Exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the Exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding Exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the Exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.

Sriram Krishnaswamy - One of the best experts on this subject based on the ideXlab platform.

  • active site independent recognition of substrates and product by bovine prothrombinase a fluorescence resonance energy transfer study
    Journal of Biological Chemistry, 2004
    Co-Authors: Danilo S Boskovic, Thomas Troxler, Sriram Krishnaswamy
    Abstract:

    Abstract The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (Exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green488 at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the Exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of Exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.

  • nematode anticoagulant protein c2 reveals a site on factor xa that is important for macromolecular substrate binding to human prothrombinase
    Journal of Biological Chemistry, 2002
    Co-Authors: Sai K Buddai, Larisa Toulokhonova, Peter W. Bergum, George P. Vlasuk, Sriram Krishnaswamy
    Abstract:

    Abstract The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K d ∼1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (Exosites) on the enzyme complex. A weaker interaction (K d = 260–500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential Exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with Exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.

  • the contribution of factor xa to Exosite dependent substrate recognition by prothrombinase
    Journal of Biological Chemistry, 2002
    Co-Authors: Matthias Wilkens, Sriram Krishnaswamy
    Abstract:

    Abstract Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (Exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (αBFX-2b) to investigate the contributions of surfaces on the proteinase to Exosite-mediated protein substrate recognition by prothrombinase. αBFX-2b bound reversibly to a fluorescent derivative of factor Xa (K d = 17.1 ± 5.6 nm) but had no effect on active site function of factor Xa or factor Xa saturably assembled into prothrombinase. In contrast, αBFX-2b was a slow, tight binding inhibitor of the cleavage of either prethrombin 2 or meizothrombin des-fragment 1 by prothrombinase (K = 0.55 ± 0.05 nm). Thus, αBFX-2b binding to factor Xa within prothrombinase selectively leads to the inhibition of protein substrate cleavage without interfering with active site function. Inhibition kinetics could adequately be accounted for by a kinetic model in which prethrombin 2 and αBFX-2b bind in a mutually exclusive way to prothrombinase. These are properties expected of an Exosite-directed inhibitor. The site(s) on factor Xa responsible for antibody binding were evaluated by identification of immunoreactive fragments following chemical digestion of human and bovine Xa and were further confirmed with a series of recombinantly expressed fragments. These approaches suggest that residues 82–91 and 102–116 in the proteinase domain contribute to αBFX-2b binding. The data establish this antibody as a prototypic Exosite-directed inhibitor of prothrombinase and suggest that the occlusion of a surface on factor Xa, spatially removed from the active site, is sufficient to block Exosite-dependent recognition of the protein substrate by prothrombinase.

  • Exosite interactions determine the affinity of factor x for the extrinsic xase complex
    Journal of Biological Chemistry, 2000
    Co-Authors: Robert J Baugh, Craig D Dickinson, Wolfram Ruf, Sriram Krishnaswamy
    Abstract:

    The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (Exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with Exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at Exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.

Mark Banaszak M Holl - One of the best experts on this subject based on the ideXlab platform.

  • substrate triggered Exosite binding synergistic dendrimer folic acid action for achieving specific tight binding to folate binding protein
    Biomacromolecules, 2016
    Co-Authors: Junjie Chen, Mallory A Van Dongen, Rachel L Merzel, Casey A Dougherty, Bradford G Orr, Ananda Kumar Kanduluru, Philip S Low, Neil E G Marsh, Mark Banaszak M Holl
    Abstract:

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered Exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein Exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP Exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  • Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein
    2016
    Co-Authors: Junjie Chen, Rachel L Merzel, Casey A Dougherty, Bradford G Orr, Ananda Kumar Kanduluru, Philip S Low, Neil E G Marsh, Mallory A. Van Dongen, Mark Banaszak M Holl
    Abstract:

    Polymer–ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered Exosite binding of a generation five poly­(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein Exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP Exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly­(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching