Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.     

Ostrich antithrombin III: kinetics and mechanism of inhibition of ostrich thrombin

A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.     

Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine

An hereditary abnormal antithrombin III (ATIII Geneva) with defective heparin cofactor activity was characterized by DNA single strand amplification and subsequent direct sequencing. ATIII Geneva was found to have a G to A transition in Exon IIIa leading to an Arg-129 to Gln mutation. This amino acid is part of the ATIII region comprising residues 114-154, which contains the highest proportion of basic residues (Arg or Lys), and is known from chemical modification studies to be involved in heparin binding. The variant protein did not bind heparin-Sepharose and was isolated from the propositus plasma by immunoaffinity chromatography. High affinity (for ATIII) heparin had only a minimal effect on thrombin and activated factor X inhibition by the purified abnormal ATIII. Taken together, these results demonstrate an important role for Arg-129 in the binding and interaction of ATIII with heparin of high affinity. We propose that a cooperation between Lys-125, Arg-129, Lys-136, and Arg-47 exposed at the surface of the inhibitor allows the binding of the essential pentasaccharide domain of heparin which is specific for the ATIII interaction.     

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.     

Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid sulfopropyl-disk chromatographic purification of bovine and human thrombin

Thrombin, from either a crude commercial preparation (bovine) or a prothrombin activation mixture (human), was purified by sulfopropyl-disk chromatography to homogeneity in a rapid and convenient single-step procedure. The yield of both proteinases was greater than 85%. Purified bovine and human thrombin had sp act of 2500 and 3000 "NIH" units/mg, respectively. Human thrombin was more reactive than bovine thrombin with these active site-directed probes: phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethylketone, and human heparin cofactor II. The sulfopropyl-disk chromatographic method is a useful and rapid technique to prepare milligram quantities of highly purified bovine and human thrombin.     

Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor

Saliva of the cattle tick Boophilus microplus contains two thrombin inhibitors, BmAP and microphilin. This work presents the purification and characterization of microphilin. It was purified from the saliva by gel filtration, ultrafiltration through a 3 kDa cut-off membrane and affinity chromatography in a thrombin-Sepharose column. Analysis by mass spectrometry showed a molecular mass of 1770 Da. Microphilin is the smallest salivary thrombin inhibitor peptide known to date. It inhibits fibrinocoagulation and thrombin-induced platelet aggregation with an IC(50) of 5.5 microM, is temperature resistant and its inhibitory activity was abolished by protease K treatment. Microphilin did not inhibit the amidolytic activity of the enzyme upon a small chromogenic substrate, but inhibited the hydrolysis of a substrate that binds both catalytic site and exosite I. Therefore, we propose that microphilin blocks thrombin at exosite I.     

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the alpha-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified by one-step anion-exchange chromatography in a highly pure form. The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.     

Partial activation of antithrombin without heparin through deletion of a unique sequence on the reactive site loop of the serpin.

Native antithrombin (AT) has an inactive reactive site loop conformation unless it is activated by a unique pentasaccharide fragment of heparin (H(5)). Structural data suggests that this may be due to preinsertion of two N-terminal residues of the reactive site loop of the serpin into the A-beta-sheet of the molecule. Relative to alpha(1)-antitrypsin, the reactive site loop of AT has three additional residues, Arg(399), Val(400), and Thr(401), at the C-terminal P' end of the loop. To determine whether a longer reactive site loop of AT is responsible for loop preinsertion in the native conformation, mutants of the serpin were expressed in which these residues were individually or in combination deleted. Kinetic analysis suggested that deletion of two residues, Val(400) and Thr(401), changed the solution equilibrium of the serpin in favor of the active conformation, thereby enhancing the inhibition of factor Xa by an order of magnitude independent of H(5). Interestingly, the reactivity of this mutant with thrombin was impaired by the same order of magnitude in the absence, but not in the presence of H(5). These results suggest that a longer reactive site loop in AT is responsible for its inactive native conformation toward factor Xa, while at same time AT requires this feature to regulate the activity of thrombin.     

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.     

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.     

Structural and functional studies of differentially O‐glycosylated analogs of a thrombin inhibitory peptide – variegin

Variegin is a 32‐amino acid long thrombin inhibitory peptide isolated from the salivary gland extract of tropical bont tick Amblyomma variegatum. It was identified to be O‐glycosylated on its Thr‐14 side chain, and this glycosylated form was 14‐fold more potent than that of its non‐glycosylated form. However, as the identity of this glycosylation remained elusive, the mechanistic details underlying its functional impact are not yet known. In this report, we synthesized four different O‐glycosylated analogs of variegin bearing physiologically relevant sugars on its Thr‐14. Functional characterization of these analogs by enzyme inhibitory kinetics and surface plasmon resonance methods showed that all the synthesized glycopeptides are strong thrombin inhibitors. Structural studies by macromolecular docking identified that the sugar moiety of these peptides can potentially mediate favorable interactions with amino acids at the base of thrombin's autolysis loop. This report, for the first time, describes the impact of differential glycosylation on the function of a thrombin inhibitory peptide and tries to provide structural insights into the relevance of peptide glycosylation in thrombin inhibition. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibitory plant serpins with a sequence of three glutamine residues in the reactive center

Serpins appear to be ubiquitous in eukaryotes, except fungi, and are also present in some bacteria, archaea and viruses. Inhibitory serpins with a glutamine as the reactive-center P1 residue have been identified exclusively in a few plant species. Unique serpins with a reactive center sequence of three Gln residues at P3-P1 or P2-P1' were isolated from barley and wheat grain, respectively. Barley BSZ3 was an irreversible inhibitor of chymotrypsin, with a second-order association rate constant for complex formation k(a)' of the order of 10(4) M(-1) s(-1); however, only a minor fraction of the serpin molecules reacted with chymotrypsin, with the majority insensitive to cleavage in the reactive center loop. Wheat WSZ3 was cleaved specifically at P8 Thr and was not an inhibitor of chymotrypsin. These reactive-center loops may have evolved conformations that are optimal as inhibitory baits for proeinases that specifically degrade storage prolamins containing Gln-rich repetitive sequences, most likely for digestive proteinases of insect pests or fungal pathogens that infect cereals. An assembled full-length amino acid sequence of a serpin expressed in cotton boll fiber (GaZ1) included conserved regions essential for serpin-proteinase interaction, suggesting inhibitory capacity at a putative reactive center P2-P2' with a sequence of four Gln residues.     

Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.     

Elimination of P1 arginine 393 interaction with underlying glutamic acid 255 partially activates antithrombin III for thrombin inhibition but not factor Xa inhibition

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu(255) on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg(393)-Glu(255) hydrogen bond and ionic interactions were eliminated by converting Glu(255) to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu(255)-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all.     

Probing reactive center loop insertion in serpins: a simple method for ovalbumin

Insertion of the reactive center loop in beta-sheet A in serpins has been typically inferred from the increased stability of the cleaved form to thermal- and urea-induced denaturation. We describe a convenient and rapid fluorescence-based method that differentiates the loop-inserted form from the loop-exposed form in ovalbumin, a prototypic noninhibitory serpin. Recombinant wild-type and R345A ovalbumins in the intact form bind ANS with equilibrium dissociation constants of 116 and 125 microM and a maximal fluorescence increase of 200 and 264%, respectively, in pH 6.8 buffer. Cleavage of the two proteins with porcine pancreatic elastase results in a 1.6- and 2.6-fold increase in the ANS-binding affinity. While cleavage of the reactive center loop in rR345A ovalbumin results in a approximately 200% increase in the ANS fluorescence, the rWT protein exhibits a approximately 50% decrease. Similar experiments with alpha(1)-proteinase inhibitor and antithrombin, two inhibitory serpins that exhibit reactive center loop insertion, show a decrease in ANS fluorescence on cleavage with porcine pancreatic elastase and thrombin, respectively. Denaturation studies in guanidinium hydrochloride indicate that the reactive center loop is inserted in the main body of the serpin in the cleaved form of rR345A mutant, while it is exposed in the cleaved form of rWT ovalbumin. These results demonstrate that ANS fluorescence change is an indicator of the loop-inserted or loop-exposed form in these recombinant ovalbumins, and thus could be advantageously used for probing reactive center loop insertion in ovalbumins. The major increase in fluorescence for the rR345A mutant on cleavage primarily arises from a change in ANS binding rather than from the generation of an additional ANS-binding site.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Effects of different heparins on the enhancement of the thrombin-antithrombin reaction

Kinetic analyses were made of the thrombin/antithrombin III (ATIII) reaction in the presence of catalytic amounts of three kinds of mammalian HA-heparin (bovine, pig and whale), which have the same affinities for ATIII. In the absence of NaCl, the first-order rate constant was higher with whale HA-heparin than with the other two HA-heparins. However, the strength of the interactions of thrombin with the HA-heparins was in the order, bovine greater than pig greater than whale. Thus, the slow reactions in the case of bovine and pig HA-heparins were probably due to preferential binding of the two HA-heparins to thrombin rather than to ATIII, thus causing the HA-heparins to be inhibitory for the reaction by reducing the turnover rates of the polysaccharides as catalysts. In the presence of 0.15M NaCl, the first-order rate constants were slightly higher in the case of pig HA-heparin than of the other two HA-heparins. Since the strength of the interactions of these HA-heparins with thrombin was in the order, pig greater than or equal to bovine greater than whale, the faster reactions were probably due to higher associations of the enzyme with the essential HA-heparin-ATIII complex.