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Biomedical subjects

R W Carrell

Publications and source records attributed to R W Carrell.

At least 55 records · Page 3Linked to original sources
PubMed1996

Molecular genetics of human antithrombin deficiency.

Human antithrombin is the major plasma inhibitor of thrombin both in the presence and absence of heparin. Its physiological importance is emphasised by the recurrent thromboses that individuals with a deficient or functionally abnormal protein are prone to develop. Such deficiencies are estimated to affect as many as 1:630 of the general population and between 3% and 5% of patients with thrombotic disease. The gene for antithrombin (AT3) has been cloned and shown to map to the long arm of chromosome 1 at 1q23-25. The gene consists of seven exons and six introns and spans 13,477bp of DNA. Advances in molecular genetic techniques have facilitated identification of the underlying DNA mutation(s) in > 80 families with antithrombin deficiency. Such work has proved invaluable in structure-function studies and in helping to provide informed genetic counselling to "at-risk" individuals based upon the natural history of similar variants.

Antithrombins↗
PubMed1996

Inhibitory conformation of the reactive loop of alpha 1-antitrypsin.

The reactive site loop of the serpin family of serine proteinase inhibitors is flexible and can adopt a number of diverse conformations. A 2.9 A resolution structure of alpha 1-antitrypsin-the principal proteinase inhibitor in human plasma-shows the loop in a stable canonical conformation matching that found in all other families of serine proteinase inhibitors. This unexpected finding in the absence of loop insertion into the body of the molecule favours a two-stage mechanism of inhibition and provides a model for the heparin activation of antithrombin. The beta-pleated strand conformation of the loop also accounts for the polymerization of the serpins in disease and for their association with other beta-sheet structures, most notably the beta-amyloid of Alzheimer's disease.

Crystallography, X-Ray↗
PubMed1996

The plasma turnover of transfused antithrombin concentrate in patients with acquired antithrombin deficiency.

Antithrombin concentrate, prepared from human plasma, has been used as replacement therapy in 35 patients with acquired antithrombin deficiency. The inhibitory activity of the concentrate, measured by chromogenic assay, correlates well with the manufacturer's quoted activity. The mean in vivo recovery of the product was 0.0124 iu mL-1 per iu of antithrombin (AT) concentrate administered by kilogram body weight. The recovery was similar in all diagnostic groups studied and did not vary during the course of treatment. Consumption of the antithrombin concentrate was monitored by measuring the production of thrombin-antithrombin complexes and the loss of plasma antithrombin activity. The mean concentration of thrombin-antithrombin complexes was elevated (23 ng mL-1) at the time of admission to the intensive care unit and fell progressively over the next 4 days. The mean time for the decay of half the antithrombin activity was 23 h during the first 24 h of therapy and rose to 42.1 h after day 1. The recovery and half-life measurements are necessary to plan an appropriate dosage regimen for the administration of this antithrombin concentrate in acquired deficiency states.

Antithrombin III↗
PubMed1996

Alpha 1-antitrypsin deficiency. A conformational disease.

The serpin family of protease inhibitors, to which alpha 1-antitrypsin belongs, has the unique feature of a mobile reactive center. Mutations within the critical regions of the molecule that control this mobility can allow premature changes in conformation with consequent abnormalities in folding and accompanying polymer formation. These abnormalities explain the plasma deficiency and liver inclusions associated with the common Z variant, as well as other variants of alpha 1-antitrypsin. The understanding of the molecular mechanisms provides a satisfying explanation for the clinical findings associated with these deficiency variants.

Humans↗
PubMed1996

The biostructural pathology of the serpins: critical function of sheet opening mechanism.

The serpins illustrate the way in which the study of a protein family as a whole can clarify the functions of its individual members. Although the individual serpins have become remarkably diversified by evolution they all share a common structural pathology. We have previously shown how plotting of the dysfunctional natural mutations of the serpins on a template structure defines the domains controlling the mobility of the reactive centre loop of the molecule. Here we compare these natural mutations with reciprocal mutations in recombinants that restore the inhibitory stability of a labile member of the family, plasminogen activator inhibitor-1 (PAI-1). The combined results emphasise the critical part played by residues involved in the sliding movement that opens the A-sheet to allow reactive loop insertion. It is concluded that changes in these residues provide the prime explanation for the ready conversion of PAI-1 to the inactive latent state. The consistency of the overall results gives confidence in predicting the likely consequences of mutations in individual serpins. In particular the two common polymorphic mutations present in human angiotensinogen are likely to affect molecular stability and hence may be contributory factors to the observed association with vascular disease.

Animals↗
PubMed1996

Type I antithrombin deficiency: five novel mutations associated with thrombosis.

The genetic basis of Type I antithrombin deficiency has been investigated in six unrelated kindred with positive histories of thrombosis using a PCR amplification/direct sequencing approach. Four frameshift mutations, all introducing premature translation termination codons were identified. Thus, deletions, of a C at nucleotide position 2599 or 2600, a G at position 2601-2602 and a CT dinucleotide at position 7428-7429 were detected in three kindred and confirmed by restriction enzyme analysis. The identical insertion, of a T at nucleotide 2770, was observed in two apparently unrelated families. This finding may have been due to a founder effect since antithrombin gene polymorphism analysis showed all affected individuals to share a common haplotype. An in frame deletion of 6 bp at nucleotide position 2690-2696 causing the removal of codons 76 and 77 encoding Ile 76 and Phe 77 was also detected indicating that these amino acids are essential for stability of the mature antithrombin.

Adult↗
PubMed1995

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

Amino Acid Chloromethyl Ketones↗
PubMed1995

alpha 1-Antitrypsin Mmalton (Phe52-deleted) forms loop-sheet polymers in vivo. Evidence for the C sheet mechanism of polymerization.

The Z (Glu342-->Lys) and Siiyama (Ser53-->Phe) deficiency variants of alpha 1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a beta-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A beta-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation. Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.

Amino Acid Sequence↗
PubMed1995

Development of a novel recombinant serpin with potential antithrombotic properties.

Recombinant alpha 1-antitrypsin with a P1 arginine residue (Arg-alpha 1-antitrypsin) is a rapid inhibitor of both thrombin and activated protein C (APC). A series of mutants were made in an attempt to increase the specificity of this serpin for thrombin over APC. Initially, P2 and P'1 residues of Arg-alpha 1-antitrypsin were replaced in single and double mutations by the corresponding residues in antithrombin and C1 inhibitor which are very poor inhibitors of APC. No improvement in selectivity was achieved by these mutations. In fact, all P2/P'1 substitutions led to a decrease in selectivity for thrombin over APC. For example, replacement of the P2 proline of Arg-alpha 1-antitrypsin by glycine decreased the association rate constant (kass) with thrombin by 37-fold while the kass value with APC was reduced by only 16-fold. Cooperative effects were observed with the double P2 and P'1 substitutions; the mutational effects were not additive. The decrease in the kass for thrombin caused by the mutation of the P2 proline to alanine or glycine was 3-fold greater when threonine was present in the P'1 position instead of the normal serine. In contrast to the disappointing results with the P2/P'1 mutations, replacement of the P7 to P'3 residues of alpha 1-antitrypsin by those of antithrombin led to a dramatic increase in selectivity. Although this substitution only affected the kass value with thrombin by 10-fold, a 12,500-fold decrease in this value with APC was observed. Substitution of proline for the P2 glycine of this chimeric serpin increased the kass values with thrombin and APC by 7- and 90-fold, respectively. The effect of the P2 substitution was again found to depend on the sequence surrounding the residue; the change in the kass for APC caused by the P2 Pro-->Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of the kass values of the chimeric serpin with a P2 proline in light of the likely rates of inhibition of thrombin and APC during antithrombotic therapy with heparin suggested that this serpin may have kinetic parameters suitable for an antithrombotic agent.

Amino Acid Sequence↗
PubMed1995

Mutations which impede loop/sheet polymerization enhance the secretion of human alpha 1-antitrypsin deficiency variants.

alpha 1-Antitrypsin plasma deficiency variants which form hepatic inclusion bodies within the endoplasmic pathway include the common Z variant (Glu342-->Lys) and the rarer alpha 1-antitrypsin Siiyama (Ser53-->Phe). It has been proposed that retention of both abnormal proteins is accompanied by a common mechanism of loop-sheet polymerization with the insertion of the reactive center loop of one molecule into a beta-pleated sheet of another. We have compared the biosynthesis, glycosylation, and secretion of normal, Z and Siiyama variants of alpha 1-antitrypsin using Xenopus oocytes. Siiyama and Z alpha 1-antitrypsin both duplicated the secretory defect seen in hepatocytes that results in decreased plasma alpha 1-antitrypsin levels. Digestion with endoglycosidase H localized both variants to a pre-Golgi compartment. The mutation Phe51-->Leu abolished completely the intracellular blockage of Siiyama alpha 1-antitrypsin and reduced significantly the retention of Z alpha 1-antitrypsin. The secretory properties of M and Z alpha 1-antitrypsin variants containing amino acid substitutions designed to decrease loop mobility and sheet insertion were investigated. A reduction in intracellular levels of Z alpha 1-antitrypsin was achieved with the replacement of P11/12 alanines by valines. Thus a decrease in Z and Siiyama alpha 1-antitrypsin retention was observed with mutations which either closed the A sheet or decreased loop mobility at the loop hinge region.

Animals↗
PubMed1995

Preparation and characterization of latent alpha 1-antitrypsin.

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride.

Amino Acid Sequence↗
PubMed1995

Two antithrombin mutations in a compound heterozygote: Met20Thr and Tyr166Cys.

The molecular basis for a family with Type I antithrombin deficiency has been established. Amplification and sequencing of the antithrombin gene identified two mutations: Met20Thr (2523T-->C) within exon 2 and Tyr166Cys (5493A-->G) within exon 3a. Further analysis indicated that the propositus was a compound heterozygote but in addition provided evidence for phase disruption during the amplification and/or cloning procedure. The Met20Thr mutation appears to be a neutral mutation with no functional consequences. In contrast, the Tyr166Cys mutation is associated with a Type I phenotype.

Antithrombin III↗
PubMed1995

What do dysfunctional serpins tell us about molecular mobility and disease?

Proteinase inhibitors of the serpin family have a unique ability to regulate their activity by changing the conformation of their reactive-centre loop. Although this may explain their evolutionary success, the dependence of function on structural mobility makes the serpins vulnerable to the effects of mutations. Here, we describe how studies of dysfunctional variants, together with crystal structures of serpins in different forms, provide insights into the molecular functions and remarkable folding properties of this family. In particular, comparisons of variants affecting different serpins allow us to define the domains which control this folding and show how spontaneous but inappropriate changes in conformation cause diverse diseases.

Angioedema↗
PubMed1995

Two novel antithrombin variants, Asn187Asp and Asn187Lys, indicate a functional role for asparagine 187.

Three unrelated families have been identified with mutations involving asparagine 187. Two of these families are asymptomatic and were identified during the screening of random blood donors, whilst the third has a history of recurrent thromboembolic disease. In two families the mutation (6460 AAC-->GAC) results in an asparagine to aspartate substitution and is associated with normal immunological levels of antithrombin but a reduction in functional activity. In the third family the mutation (6462 AAC-->AAA) results in an asparagine to lysine substitution at residue 187 and is associated with a parallel reduction in both immunological and functional antithrombin levels. Asparagine 187 is located in the middle of the F helix of antithrombin and forms the major link between the F helix and strand 3 of the A sheet. The F helix is seen to overlie the A sheet of the molecule and moves with strands 2 and 3 of this sheet as they open to allow entry of the reactive site loop to form strand 4. Substitutions of asparagine 187 are, therefore, likely to disrupt this sliding movement leading to a loss of inhibitory activity.

Adult↗
PubMed1994

The effect of the Z mutation on the ability of alpha 1-antitrypsin to prevent neutrophil mediated tissue damage.

Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.

Connective Tissue↗