PubMed Health⌕ Search

Biomedical subjects

W P Sheffield

Publications and source records attributed to W P Sheffield.

At least 19 recordsLinked to original sources
PubMed2001

Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris.

Hirudin is a small, proteinaceous thrombin inhibitor that clears rapidly from the circulation. A hexahistidine-tagged hirudin-rabbit serum albumin (RSA) fusion protein, HLAH6, was characterized following secretion from Pichia pastoris. HLAH6 bound to immobilized nickel, anti-RSA, and anti-hexahistidine antibodies, and contained the expected (ITYTD) N-terminus. Its spectrometric mass was 74,490 (versus the theoretical mass of 74,410 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of 84 kDa). The terminal catabolic half-life in rabbits of HLAH6, recombinant Pichia-derived His-tagged RSA, or plasma-derived RSA did not differ. Injection of 2 mg/kg HLAH6 into rabbits raised the activated partial thromboplastin time (aPTT) above initial values for 4-24 h, while the equimolar dose of unfused hirudin was without significant effect. A higher dose of HLAH6 (3 mg/kg functional HLAH6, equivalent to 37.6 thrombin-inhibitory units/g) raised the aPTT by 2.0- to 2.5-fold; the elevation persisted for > 48 h. Importantly, both HLAH6 and unfused hirudin inhibited clot-bound thrombin. Our results suggest that HLAH6 exhibits not only delayed clearance, but also prolonged biological activity in vivo compared with unfused hirudin.

Amino Acid Sequence↗
PubMed2001

Modification of clearance of therapeutic and potentially therapeutic proteins.

Advances in biochemistry, protein chemistry and molecular biology over the last twenty-five years have spurred the increased use and development of proteins as injectable therapeutic agents. Introduction of proteins into the circulation exposes them to numerous different cells, enzymes and routes of extravasation that contribute to their clearance and their catabolism. Overly rapid clearance, particularly of small proteins, can limit therapeutic efficacy. Many strategies have been devised to retard the clearance of therapeutic or potentially therapeutic proteins, but relatively few proteins with clearance-retarding modifications are in clinical use. Proteins have been chemically modified towards this end by covalent attachment of polyethylene glycol or dextran chains or by protein-protein cross-linking. Genetic modification has also been employed to fuse proteins of interest to long-lived plasma proteins like albumin or immunoglobulins, or portions of these proteins. While all modifications may reduce the biological activity of the protein of interest or elicit antibody formation in recipient animals or patients, there now exists sufficient experience in this area that an optimal clearance-extending strategy can often be designed and successfully executed. With the explosive growth of genomic and proteomic information, an exponentially increasing number of engineered proteins are likely to be developed, with a probable need for clearance-related modification.

Animals↗
PubMed2001

A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro.

Barbourin is a 73 amino acid venom protein that inhibits platelet aggregation. Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahistidine (H6) tags. BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP. BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin alphaIIbbeta3. The terminal catabolic half-life of BLAH6 approached that of RSAH6 [3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 +/- SD)], but was substantially increased relative to that of BARH6 [0.15 +/- 0.03 days (n = 3 +/- SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.

Adenosine Diphosphate↗
PubMed2000

Modulation of clearance of recombinant serum albumin by either glycosylation or truncation.

Albumin is an abundant non-glycosylated plasma protein with a slow clearance profile. It has been employed as a fusion partner in efforts to slow the clearance of small antithrombotic proteins like hirudin. In the present study, the in vivo clearance of recombinant rabbit serum albumin (rRSA), of mutant rRSAs containing consensus sequences for N-linked glycosylation (D494N and V14T variants), and of mutant mini-proteins truncated at albumin domain boundaries (rRSAs 1-185, 1-377, or 378-584) was examined. Mean terminal catabolic half-lives (t(0.5)cat) in rabbits for plasma-derived RSA, rRSA, and the V14T variant did not differ significantly (range 4. 32-4.76 days). In contrast, mean t(0.5)cat was reduced to 2.87 days for the D494N variant and to less than 0.071 days for all mini-proteins. The mini-proteins were found in the urine in tissue distribution experiments, suggesting a renal route of clearance. Our results suggest that all three internally repeated albumin domains are required to maintain the slow in vivo clearance profile of albumin, and that albumin glycosylation can be associated with an acceleration of clearance. This information could be used to design fusion proteins, including those with antithrombotic properties, with predictably altered in vivo half-lives less than that of serum albumin.

Animals↗
PubMed2000

Mutation of any site of N-linked glycosylation accelerates the in vivo clearance of recombinant rabbit antithrombin.

Antithrombin (AT) is a plasma protein with four sites of N-linked glycosylation. Asn 135 is incompletely glycosylated, and the resulting 3-glycan AT is cleared more rapidly in vivo than the 4-glycan form. The Asn codons in each of the four sites of glycosylation were altered in turn, to create four mutant rabbit AT cDNAs. Permanently transfected CHO cell lines were generated following transfection of the resulting constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q). Comparison of the five resulting recombinant AT proteins revealed that the major AT species of each variant co-migrated on SDS gels, and migrated more rapidly than the major form of AT-WT. The shift in mobility, from 60 to 57 kDa, was consistent with the loss of one fully sialylated complex N-linked glycan. Neither the amount of AT secreted (range: 1.25 to 4.2 microg/10(6) cells/day) nor the kinetics of secretion differed significantly between cell lines expressing AT-WT or any of the AT variants. All forms of recombinant rabbit AT were capable of forming denaturation-resistant complexes with thrombin. Purification and radioiodination of each of the five recombinant AT proteins permitted pharmacokinetic analysis of their individual clearance in rabbits. While neither the equilibration half-life (t(0.5)alpha) nor the terminal catabolic half-life (t(0. 5)beta) differed significantly between plasma-derived rabbit AT and AT-WT, the t(0.5)beta of all the underglycosylated variants was decreased relative to that of AT-WT (maximum reduction in mean: from 70.1+/-3.2 h to 52.4+/-2.5 h). These results suggest that the overall extent of glycosylation, rather than the location within AT of the glycan chains, is a primary determinant of AT clearance.

Amino Acid Substitution↗
PubMed1997

Cytokeratin 18 is expressed on the hepatocyte plasma membrane surface and interacts with thrombin-antithrombin complexes.

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of 125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996) J. Biol. Chem. 271, 25684-25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101-107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

Animals↗
PubMed1997

Impact of mutations at the P4 and P5 positions on the reaction of antithrombin with thrombin and elastase.

Antithrombin (AT) is a serpin capable of trapping thrombin (IIa) in a stable and covalent complex. Complex formation is prevented by leukocyte elastase (LE) cleavage near the AT reactive centre. We mutated the known LE cleavage sites of AT to explore the possibility of producing an LE-resistant AT molecule. Initially, six rabbit AT variants differing only at residue 390 (P4) were generated in a cell-free system, and gel-based assays were used to assess IIa-mediated complex formation and LE-mediated cleavage of the variants. Substitution of charged residues (Glu or Arg) reduced complex formation by 50-60%, while the Ser variant was incapable of inhibiting thrombin; LE reactivity was less affected. The least (Trp) and most (Ser) affected variants were expressed in COS-1 cells. Again, the Ser variant was incapable of detectably reducing the rate of thrombin-mediated amidolysis while the Trp variant inhibited thrombin at a slightly reduced rate (-28%). LE inactivated the Trp variant and the wild-type AT to a similar extent. Recreation of the Trp mutation in COS-derived human AT showed similar results. Since retention of LE-sensitivity could have arisen due to cleavage at Val389 (P5), we produced and characterized a human AT substitution mutant with Trp at both P4 and P5. This variant showed a slight reduction in thrombin inhibitory activity (-22%), but remained susceptible to LE inactivation. These results suggest either that LE cleaves at secondary sites if its primary cleavage sites are blocked, or that the substrate specificity of LE differs in polypeptides as compared to peptide substrates.

Amino Acid Substitution↗
PubMed1997

Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin.

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.

Amino Acid Sequence↗
PubMed1996

Inhibition of thrombin by hirudin genetically fused to wild-type or mutant antithrombin.

Recombinant fusion proteins consisting of hirudin variant 3 (HV3) fused at its C-terminus to either of two forms of mature rabbit antithrombin (AT) were generated in COS-I cells. HV3 fused to wild-type AT was designated HAT, while a similar chimeric protein in which the P12 residue of AT was mutated from Ala to Thr, was designated HAT(H) for the Hamilton (A382T) mutation. Addition of the HV3 domain resulted in a decreased mobility of both HAT and HAT(H) relative to COS-derived AT (68 kDa versus 60 kDa). Both proteins had a greatly increased ability to inhibit thrombin in amidolytic activity assays, relative to recombinant AT. Addition of heparin to these reactions was without effect. Incubation of conditioned media containing recombinant AT with 125I-labelled thrombin resulted in the formation of SDS-stable AT-IIa complexes; no such complexes were detected in identical reactions containing either HV3-AT fusion protein. The two proteins did not differ significantly in their ability to compete for the binding of 125I-labelled thrombin to immobilized HV1 (CGP 39393). Both proteins were found to bind to heparin-Sepharose, but less tightly than unfused AT. This property was demonstrated by the peak elution of the fusion proteins at 0.65 M NaCl, as compared to that of COS-derived AT at 1.05 M NaCl. We conclude that the fusion proteins inhibit thrombin with similar affinity to unfused hirudin via their hirudin and not their antithrombin domains. The heparin-binding capability of these proteins may indicate the acquisition of vessel wall binding capacity by these novel forms of recombinant hirudin.

Animals↗
PubMed1995

Intracellular events determine the fate of antithrombin Utah.

We sought to determine whether intracellular or extracellular events contribute to the decrease in circulating antithrombin (AT) levels that is seen in subjects with the Utah mutation (Pro 407 to Leu). Site-directed mutagenesis was used to recreate this mutation within a previously characterized rabbit AT cDNA. Cell-free expression of the mutated cDNA yielded an AT protein that failed to react with thrombin. Expression of the rabbit AT-Utah protein in transiently transfected Cos cells resulted in a 10-fold decrease in the amount of AT antigen detected in the conditioned media, as compared with that seen with the wild-type recombinant AT. This effect was not caused by variations in transfection efficiency, because AT levels were normalized to the product of a cotransfected plasmid, chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT mRNA levels were comparable in cells expressing either the rabbit AT-Utah or wild-type recombinant rabbit AT. Immunoblots of conditioned media from the two populations of transfected cells showed that the recombinant AT-Utah protein was intact. The results obtained with Cos cells were reproduced using permanently transfected Chinese hamster ovary (CHO) cells. Pulse-chase experiments with the CHO lines showed that both initial levels of rabbit AT-Utah after the pulse labeling and the rate of subsequent secretion during the chase period were reduced compared with that seen with cells expressing the wild-type AT. The observed reduction in AT secretion was also observed for the AT-Oslo mutation (Ala 404 to Thr) when recreated in the rabbit AT background, and expressed in Cos cells. In these experiments, the media levels of mutant AT were reduced by 50%, compared with wild-type. These results show that intracellular events, as opposed to accelerated clearance or other extracellular causes, contribute to the paucity of AT secretion seen in these strand 1C AT mutants.

Amino Acid Sequence↗
PubMed1995

Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin.

Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation-resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino-terminal regions of the molecular which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: delta 58, delta 81, delta 106, and delta 169, all in an in vitro transcription vector plasmid background. Transcription of the full-length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII-related polypeptides. The delta 106 and delta 169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the delta 58 and delta 82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full-length recombinant HCII; no acceleration of complex formation in the presence of 20 micrograms/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in alpha 1 antitrypsin and other serpins showed that the non-functional delta 106 mutant lacks helix A, while the functional delta 82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino-terminal region of HCII which is not essential for thrombin inhibition.

Animals↗
PubMed1994

Amino acid substitutions of the P2 residue of human antithrombin that either enhance or impair function.

Recombinant forms of human antithrombin (AT) were expressed in COS-1 cells, and their interaction with human thrombin characterized by comparing the reactivity of two engineered mutant forms of AT with the wild-type recombinant. Both mutant forms contained single amino acid substitutions of Asp (G392D) or Pro (G392P) for the wild-type Gly, at residue 392, termed the P2 position with reference to the adjacent reactive centre bond. All three forms of AT co-migrated on Western blots, with an apparent molecular weight of 58 kD, with endoglycosidase F treatment reducing their mobility to 47 kD. The two mutant forms of AT reacted with thrombin differently from the wild-type molecule, in that the G392D substitution abrogated the thrombin inhibitory capacity of the protein, while the G392P substitution enhanced the reactivity of the recombinant mutant AT with thrombin. Under pseudo-first order conditions, the second order rate constants for the reaction of the recombinant wild-type and G392P mutant AT were determined to be 1.4 x 10(4) L-mol-1 sec-1 and 3.0 x 10(4) L-mol-1 sec-1, respectively, a difference of 210%. In contrast, in the presence of heparin, the reaction rates of the G392P and wild-type AT forms with thrombin, differed by less than 25%. We conclude that the P2 position of AT is an important residue for AT to express its inhibitory activity, alterations to which can either enhance or impair the inhibition of thrombin by AT.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of the P2 residue of human antithrombin.

Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428-1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site-directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild-type recombinant AT, following in vitro transcription and cell-free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild-type in forming complexes with either alpha-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.

Animals↗
PubMed1994

Genetic linkage studies in antithrombin-deficient kindreds using a highly polymorphic trinucleotide short tandem repeat (STR) within the human antithrombin gene.

PCR amplification and analysis of short tandem repeats (STR) have provided a useful tool for genetic linkage studies and for the diagnosis of genetic disorders. We have recently identified a novel trinucleotide STR, (ATT).(TAA), in the fifth intron of the human antithrombin gene (AT3) located on chromosome 1q23. PCR amplification, cloning, and sequence analysis revealed this AT3-STR to be highly polymorphic with repeat units ranging in size from (ATT)5 to (ATT)18. Ten distinct alleles were found in 81 unrelated Caucasian individuals (162 alleles) with an observed heterozygosity of 81%. Genetic linkage studies using the AT3-STR in two previously described antithrombin (AT)-deficient kindreds, AT-Hamilton (Ala 382 Thr) and AT-Amiens (Arg 47 Cys), demonstrate, in a given kindred, that a specific AT3-STR polymorphism is strongly associated with a particular AT mutation. Thus, this highly polymorphic AT3-STR should be very useful in performing linkage studies in AT-deficient kindreds as well as in investigating other chromosome 1-related genetic disorders.

Antithrombins↗
PubMed1994

Defining the heparin-binding domain of antithrombin.

Antithrombin is a serine protease inhibitor that participates in the inactivation and removal from the circulation of thrombin and a variety of other procoagulant serine proteases. Antithrombin is also the major plasma cofactor of heparin which exerts its therapeutic effect primarily through its ability to substantially increase the rate of inactivation by antithrombin of the procoagulant serine proteases. Binding of heparin to antithrombin is thus believed to be a prerequisite for this rate enhancement effect. Heparin binding to antithrombin is mediated by a well-defined unique heparin pentasaccharide sequence. Interaction between this pentasaccharide sequence and antithrombin induces a conformational change in antithrombin, an alteration that appears to be sufficient to explain the enhanced ability of antithrombin to inhibit factor Xa and related serine proteases, but not thrombin. Heparin species with longer polysaccharide chains appear to be required in order to enhance the inhibition of thrombin by antithrombin. This may be because the enhancement of this reaction requires that heparin interacts simultaneously with both the antithrombin and the thrombin molecules. This review describes the interactions between heparin and antithrombin, focusing on the antithrombin residues which are involved in the binding of heparin. The role of the heparin-induced conformational change in enhancing serine protease inhibition by antithrombin is also explored. Then, based on available data, an hypothesis is proposed to explain the mechanisms by which heparin accelerates the rate of inactivation by antithrombin of the various serine proteases.

Animals↗