Biomedical subjects

Likui Yang

Publications and source records attributed to Likui Yang.

18 recordsLinked to original sources

PubMed2006

Expression, purification and characterization of factor IX derivatives using a novel vector system.

Recent studies have indicated that the loop harboring the S1 specificity site (residues 185-189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na(+)-binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na(+) binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca(2+) with no obvious requirement for Na(+) in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.

Alanine↗

PubMed2006

Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C.

The binding of Ca(2+) to the 70-80 loop of protein C inhibits protein C activation by thrombin in the absence of thrombomodulin (TM), but the metal ion is required for activation in the presence of TM. Structural data suggests that the 70-80 loop is located between two antiparallel beta strands comprised of residues 64-69 and 81-91 on the protease domain of protein C. To test the hypothesis that a salt-bridge/hydrogen bond interaction between Arg-67 of the former strand and Asp-82 of the latter strand modulates the unique Ca(2+)-binding properties of protein C, we engineered a disulfide bond between the two strands by substituting both Arg-67 and Asp-82 with Cys residues. The activation of this mutant was enhanced 40- to 50-fold independent of TM and Ca(2+). Furthermore, the Arg-67 to Ala mutant of protein C was activated in the absence of TM by the Arg-35 to Glu mutant of thrombin with the same efficiency as wild-type protein C by wild-type thrombin-TM complex. These results suggest that TM functions by alleviating the Ca(2+)-dependent inhibitory interactions of Arg-67 of protein C and Arg-35 of thrombin.

Alanine↗

PubMed2005

Mutagenesis studies toward understanding the mechanism of the cofactor function of thrombomodulin.

Thrombomodulin (TM) is as essential cofactor in protein C activation by thrombin. To investigate the cofactor effect of TM on the P3-P3' binding specificity of thrombin, we prepared a Gla-domainless protein C (GDPC) and an antithrombin (AT) mutant in which the P3-P3' residues of both molecules were replaced with the corresponding residues of the factor Xa cleavage site in prethrombin-2. TM is known to interact with GDPC, but not AT in the complex. Thrombin did not react with either mutant in the absence of a cofactor. While the thrombin-TM complex also did not react with the AT mutant, it activated the GDPC mutant with a normal k(cat), but an approximately 4-fold impaired K(m) value. Further studies revealed that the active-site directed inhibitor p-aminobenzamidine acts as a competitive inhibitor of both wild-type and GDPC mutant in reaction with the thrombin-TM complex. These results suggest that the interaction of the P3-P3' residues of GDPC with the active-site pocket of the thrombin-TM complex makes a dominant contribution to the binding specificity of the reaction. Moreover, the observation that the GDPC mutant, but not the AT mutant, functions as an effective substrate for the thrombin-TM complex suggests that GDPC interaction with the thrombin-TM complex may be associated with the alteration of the conformation of the P3-P3' residues of the substrate.

Antithrombins↗

PubMed2005

Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and exosite interactions are required for inhibition.

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.

Alanine↗

PubMed2005

Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin.

Structural data have indicated that the 60-loop of thrombin with 8-9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However,previous deletion of 3-4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired approximately 2-4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved approximately 2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.

Animals↗

PubMed2005

The functional significance of the autolysis loop in protein C and activated protein C.

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

Anticoagulants↗

PubMed2004

The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding.

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.

1-Carboxyglutamic Acid↗

PubMed2004

Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.

Animals↗

PubMed2004

Mutagenesis studies toward understanding the mechanism of differential reactivity of factor Xa with the native and heparin-activated antithrombin.

A unique pentasaccharide fragment of high-affinity heparin activates antithrombin (AT) to enhance its rate of complex formation with factor Xa (FXa) by 200-300-fold. Recent results have indicated that the activation of AT is associated with the exposure of a cryptic exosite on the serpin that is an interactive site for FXa in the complex. Previously, we identified Arg(150) on the autolysis loop of FXa as a candidate residue that may specifically interact with the heparin-activated AT. Three other surface loops on FXa including 39, 60, and the sodium-binding 220 loops have been implicated to be critical for the protease interaction with the activated AT. To determine the extent of the contribution of these loops to the specificity of the FXa interaction with activated AT, several loop mutants of the protease were prepared and their reactivity with AT was studied in both the absence and presence of pentasaccharide. Analysis of the inhibition kinetic data suggests that the residues of both 39 and 60 loop make a minor contribution to the recognition of AT in both the native and activated conformation of the serpin. On the other hand, the reactivity of AT with the sodium loop mutants of FXa in the absence of the cofactor was severely impaired. However, the extent of the rate-accelerating effect of pentasaccharide in the AT inhibition of the mutants was not affected. These results suggest that all three loops play a role in the specificity of the FXa-AT interaction; however, neither loop specifically interacts with the activated conformation of the serpin.

Amino Acid Substitution↗

PubMed2004

Zymogenic and enzymatic properties of the 70-80 loop mutants of factor X/Xa.

The Ca(2+) binding 70-80 loop of factor X (fX) contains one basic (Arg(71)) and three acidic (Glu(74), Glu(76), and Glu(77)) residues whose contributions to the zymogenic and enzymatic properties of the protein have not been evaluated. We prepared four Ala substitution mutants of fX (R71A, E74A, E76A, and E77A) and characterized their activation kinetics by the factor VIIa and factor IXa in both the absence and presence of cofactors. Factor VIIa exhibited normal activity toward E74A and E76A and less than a twofold impaired activity toward R71A and E77A in both the absence and presence of tissue factor. Similarly, factor IXa in the absence of factor VIIIa exhibited normal activity toward both E74A and E76A; however, its activity toward R71A and E77A was impaired approximately two- to threefold. In the presence of factor VIIIa, factor IX activated all mutants with approximately two- to fivefold impaired catalytic efficiency. In contrast to changes in their zymogenic properties, all mutant enzymes exhibited normal affinities for factor Va, and catalyzed the conversion of prothrombin to thrombin with normal catalytic efficiencies. However, further studies revealed that the affinity of mutant enzymes for interaction with metal ions Na(+) and Ca(2+) was impaired. These results suggest that although charged residues of the 70-80 loop play an insignificant role in fX recognition by the factor VIIa-tissue factor complex, they are critical for the substrate recognition by factor IXa in the intrinsic Xase complex. The results further suggest that mutant residues do not play a specific role in the catalytic function of fXa in the prothrombinase complex.

Amino Acid Substitution↗

PubMed2003

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin.

Exosite 1 of thrombin consists of a cluster of basic residues (Arg-35, Lys-36, Arg-67, Lys-70, Arg-73, Arg-75, and Arg-77 in chymotrypsinogen numbering) that play key roles in the function of thrombin. Structural data suggest that the side chain of Arg-35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin (TM). To study the role of these residues in TM-mediated protein C (PC) activation by thrombin, a charge reversal mutagenesis approach was used to replace these residues with a Glu in separate constructs. The catalytic properties of the mutants toward PC were analyzed in both the absence and presence of TM and Ca2+. It was discovered that, with the exception of the Arg-67 and Lys-70 mutants, all other mutants activated PC with similar maximum rate constants in the presence of a saturating concentration of TM and Ca2+, although their affinity for interaction with TM was markedly impaired. The catalytic properties of the Arg-35 mutant were changed so that PC activation by the mutant no longer required Ca2+ in the presence of TM, but, instead, it was accelerated by EDTA. Moreover, the activity of this mutant toward PC was improved approximately 25-fold independent of TM. These results suggest that Arg-35 is responsible for the Ca2+ dependence of PC activation by the thrombin-TM complex and that a function for TM in the activation complex is the allosteric alleviation of the inhibitory interaction of Arg-35 with the substrate.

Allosteric Regulation↗

PubMed2003

Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin.

Thrombomodulin (TM), or its epidermal growth factor-like domains 456 (TM456), enhances the catalytic efficiency of thrombin toward both protein C and protein C inhibitor (PCI) by 2-3 orders of magnitude. Structural and mutagenesis data have indicated that the interaction of basic residues of the heparin-binding exosite of protein C with the acidic residues of TM4 is partially responsible for the efficient activation of the substrate by the thrombin-TM456 complex. Similar to protein C, PCI has a basic exosite (H-helix) that constitutes the heparin-binding site of the serpin. To determine whether TM accelerates the reactivity of thrombin with PCI by providing a binding site for the H-helix of the serpin, an antithrombin (AT) mutant was constructed in which the H-helix of the serpin was replaced with the same region of PCI (AT-PCIH-helix). Unlike PCI, the H-helix of AT is negatively charged. It was discovered that TM456 slightly (<2-fold) impaired the reactivity of AT with thrombin; however, it enhanced the reactivity of AT-PCIH-helix with the protease by an order of magnitude. Further studies revealed that the substitution of Arg35 of thrombin with an Ala also resulted in an order of magnitude enhancement in reactivity of the protease with both PCI and AT-PCIH-helix independent of TM. We conclude that TM enhances the reactivity of PCI with thrombin by providing both a binding site for the serpin and a conformational modulation of the extended binding pocket of thrombin.

Allosteric Regulation↗

PubMed2003

Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex.

Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54-65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a KD of 0.5-0.7 microm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2-30 microm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex.

Binding Sites↗

PubMed2003

Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa.

The autolysis loop (residues 143-154 in chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation proteases. This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the protease specificity of factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant activated factor X normally both in the absence and presence of factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with antithrombin (AT) in both the absence and presence of the cofactor, heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the autolysis loop are recognition sites for FX independent of factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the serpin is in the heparin-activated conformation.

Arginine↗

PubMed2003

The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C.

Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.

Amino Acid Sequence↗

PubMed2002

Localization of the heparin binding exosite of factor IXa.

Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.

Amino Acid Sequence↗

PubMed2002

Role of basic residues of the autolysis loop in the catalytic function of factor Xa.

The autolysis loop of factor Xa (fXa) has four basic residues (Arg(143), Lys(147), Arg(150), and Arg(154)) whose contribution to protease specificity of fXa has not been examined. Here, we substituted these basic residues individually with Ala in the fX cDNA and expressed them in mammalian cells using a novel expression/purification vector system. Following purification to homogeneity and activation by the factor X activator from Russell viper venom, the mutants were characterized with respect to their ability to assemble into the prothrombinase complex to activate prothrombin and interact with target plasma fXa inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin. We show that all mutants interacted with factor Va with normal affinities and exhibited wild-type-like prothrombinase activities toward prothrombin. Lys(147) and Arg(154) mutants were inhibited by TFPI approximately 2-fold slower than wild type; however, both Arg(143) and Arg(150) mutants were inhibited normally by the inhibitor. The reactivities of Arg(143) and Lys(147) mutants were improved approximately 2-fold with antithrombin in the absence but not in the presence of heparin cofactors. On the other hand, the pentasaccharide-catalyzed reactivity of antithrombin with the Arg(150) mutant was impaired by an order of magnitude. These results suggest that Arg(150) of the autolysis loop may specifically interact with the activated conformation of antithrombin.

Antithrombins↗

PubMed2002

Contribution of basic residues of the 70-80-loop to heparin binding and anticoagulant function of activated protein C.

The role of basic residues of the 70-80-loop, Arg(74), Arg(75), and Lys(78) (chymotrypsin numbering) in the catalytic function of activated protein C (APC) was investigated by expressing mutants of protein C in which these residues were replaced with Ala in three separate constructs. Following purification to homogeneity and activation by thrombin, the catalytic properties of the mutants were characterized with respect to their ability to cleave the chromogenic substrate Spectrozyme PCa, react with protein C inhibitor (PCI), and inactivate factor Va. Relative to wild-type APC, the mutants cleaved Spectrozyme PCa with identical or improved catalytic efficiencies. Similarly, PCI inhibited mutants with identical or improved second-order rate constants (k(2)) in the absence of heparin. However, the heparin-catalyzed inhibition of mutants by PCI was impaired approximately 10-fold. Analysis of k(2) values by a ternary complex model revealed that the affinities of mutants for heparin were impaired to a similar extent. Moreover, analysis of the NaCl gradient elution profiles of APC derivatives from Heparin-Sepharose supported this conclusion. An oligosaccharide containing 14 residues efficiently catalyzed the PCI inhibition of APC by a template mechanism. Further studies revealed that the ability of Arg(74) and Arg(75) mutants to inactivate factor Va was markedly impaired. We conclude that basic residues of the 70-80-loop are critical for the catalytic function of APC.

Amino Acid Substitution↗