Biomedical subjects

R W Carrell

Publications and source records attributed to R W Carrell.

At least 19 recordsLinked to original sources

PubMed1998

Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation.

The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main beta-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6 degreesC) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0 degreesC), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband's plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.

Amino Acid Substitution↗

PubMed1998

Angiotensinogen cleavage by renin: importance of a structurally constrained N-terminus.

Angiotensinogen, a plasma serpin, functions as a donor of the decapeptide angiotensin I, which is cleaved from the N-terminus by renin. To assess the contribution of the serpin framework to peptide cleavage we produced a chimaeric molecule of alpha1-antitrypsin carrying the angiotensinogen N-terminus and determined the kinetic parameters for angiotensin I release. The Km for plasma angiotensinogen was 18-fold lower than for the chimaeric protein while the catalytic efficiency was four-fold higher. We also show that Cys-18 participates in a disulphide bond and propose that constraints on the N-terminus profoundly affect the interaction with renin.

Amino Acid Sequence↗

PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗

PubMed1998

Conformational changes and disease--serpins, prions and Alzheimer's.

Some of the most perplexing disorders in medicine are each now known to arise from the conformational instability of an underlying protein. The consequence is a continuum of pathologies with typically a change in fold leading to ordered aggregation and tissue deposition. The serpins provide a structural prototype for these pathologies and give a perspective on the assessment of current proposals as to the conformational basis of both Alzheimer's disease and the transmissible prion encephalopathies.

Alzheimer Disease↗

PubMed1998

Antithrombin cambridge II (Ala384Ser): clinical, functional and haplotype analysis of 18 families.

Thirty-one individuals from 18 unrelated families with antithrombin deficiency have been identified as having a single point mutation within codon 384 (13268 GCA-->TCA) resulting in an alanine to serine substitution. Six families (11 individuals) were identified by the screening of individuals with thromboembolic disease or with a family history of thromboembolic disease, whilst the remaining 12 families (20 individuals) were identified by screening of asymptomatic blood donors. Four individuals had a history of venous thrombotic disease, a further 2 gave a history of superficial thrombophlebitis but the remaining 25 individuals were asymptomatic. Affected individuals demonstrated normal immunological levels of antithrombin but a decrease in anti-IIa activity in the presence of heparin. Haplotype analysis was used to examine the possibility of a founder effect to explain the high frequency of this non-CpG mutation. 29/31 individuals showed a single common "core" haplotype, the only variation existing in the number of copies of an (ATT)n repeat polymorphism--13, 14, 15 or 17. The results suggest that at most there are four independent origins for this mutation.

Alanine↗

PubMed1998

Recombinant antitrypsin Pittsburgh undergoes proteolytic cleavage during E. coli sepsis and fails to prevent the associated coagulopathy in a primate model.

During severe sepsis there is dramatic activation of both contact proteases and the coagulation pathway. These processes contribute to the development of shock and disseminated intravascular coagulation (DIC) respectively. The Pittsburgh mutant of antitrypsin (358Met-Arg) is a novel protease inhibitor with activity against both thrombin and the contact proteases and should therefore prove beneficial as a therapeutic agent in the management of septic shock. This hypothesis was supported by an earlier study in a pig model where recombinant antitrypsin Pittsburgh (rAT Pittsburgh) at a concentration of 1 microM alleviated some of the features of shock, but did not improve survival. In order to reduce the lethal effects of E. coli sepsis we postulated that a higher concentration of antitrypsin Pittsburgh would be necessary. To test this hypothesis we used rAT Pittsburgh in a primate model. This was chosen in preference to another species as E. coli sepsis in the primate has been well characterised and closely resembles the changes seen in man. Surprisingly this treatment did not alleviate the features of shock and unexpectedly appeared to exacerbate the associated coagulopathy. We propose two possible mechanisms for this unforeseen outcome. The first results from the broad spectrum of activity of antitrypsin Pittsburgh. As well as inhibiting thrombin and the contact proteases, the Pittsburgh mutant also inhibits activated protein C. Inhibition of the protein C system is known to exacerbate septic shock. Secondly, a significant quantity of inactive antitrypsin Pittsburgh, cleaved at the reactive centre, was detected in the plasma of the treated animals. Proteolytically altered serpins, including antitrypsin. have been shown to enhance the inflammatory process. Therefore the accumulation of cleaved rAT Pittsburgh might be expected to exacerbate septic shock.

Animals↗

PubMed1997

The anticoagulant activation of antithrombin by heparin.

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-A structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin-protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.

Anticoagulants↗

PubMed1997

Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.

The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.

Amino Acid Sequence↗

PubMed1997

Heparin-dependent modification of the reactive center arginine of antithrombin and consequent increase in heparin binding affinity.

Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-beta-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P1 Arg of alpha-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P1 Arg becomes accessible. Furthermore, the deimination of P1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P1 His variant, due to a lowered dissociation rate constant for the antithrombin-pentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P1 Arg.

Antithrombin III↗

PubMed1997

Conformational disease.

Several diverse disorders, including the prevalent dementias and encephalopathies, are now believed to arise from the same general disease mechanism. In each, there is abnormal unfolding and then aggregation of an underlying protein. The gradual accumulation of these aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the clinical disease. The understanding of these processes at the molecular level is opening prospects of more rational approaches to investigation and therapy.

Amyloid↗

PubMed1997

The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site.

The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 A, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-beta-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.

Antithrombin III↗

PubMed1997

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Amino Acid Sequence↗

PubMed1997

Five antithrombin variants, four associated with thrombosis.

We have identified five mutations in antithrombin by direct sequencing of exons amplified using polymerase chain reaction. Four of these mutations are associated with thrombosis, three cause type I antithrombin deficiency and one has features of a type II deficiency. The fifth variant appears to have no functional consequences. The type I mutations are in exon 2, exon 3b and exon 4. The first of these is a nonsense mutation causing substitution of a Tyr-->stop at position -16 within the secretion signal sequence. The second is a missense mutation resulting in the substitution Cys-->Ser at position 247. This disrupts the disulphide bond with Cys 430 leaving a free cysteine residue and the C-terminus unconstrained. The third type I mutation is an in-frame deletion resulting in the loss of Ile 186. This is a highly conserved residue in the serpin superfamily and will predictably result in the disruption of the F-helix. The fourth mutation, in exon 3a, results in the substitution of Ser 162 by Asn. This residue is sited in the E-helix and the replacement of the buried side chain of serine by the larger asparagine side chain will predictably cause structural perturbation. The last example, Val 415-->Asp, was an incidental finding as a follow up investigation of a nephrotic patient. Although one other member of the family also had the mutation there was no linked history of thrombotic disease.

Adult↗

PubMed1997

Mechanisms of antithrombin polymerisation and heparin activation probed by the insertion of synthetic reactive loop peptides.

Incubation of antithrombin with a series of synthetic reactive loop peptides showed that 6-mer and 7-mer peptides, P14-P9 and P14-P8 of antithrombin respectively, induced loop-sheet polymerisation and binary complex formation. These peptides are likely to anneal to the upper part of the dominant A-sheet, favouring sheet opening and allowing insertion of a second reactive loop in the lower part of the A-sheet to form polymers. The insertion of longer peptides filled the A-sheet beyond the P7 position and prevented polymerisation. Heparinised antithrombin was more resistant to polymerisation and peptide insertion, indicating that heparin induces a conformational change that closes the A-sheet and expels the reactive loop.

Antithrombin III↗

PubMed1997

Commercial plasma alpha1-antitrypsin (Prolastin) contains a conformationally inactive, latent component.

Fractionated plasma alpha1-antitrypsin is widely-used as replacement therapy in patients with Z alpha1-antitrypsin deficiency-related emphysema. We have recently shown that purified antitrypsin may be induced to adopt an inactive latent conformation by heating at high temperatures in stabilizing concentrations of sodium citrate. Such a conformation was predicted to be present in commercial preparations of antitrypsin, as these require heating under similar conditions for viral inactivation. Native antitrypsin was purified from plasma, and commercial antitrypsin (Prolastin) was obtained from Bayer Corporation. Western blot analysis of transverse urea gradient (TUG) gels showed that commercial antitrypsin migrated as two bands: one with an unfolding profile of native antitrypsin and the second with a profile of latent antitrypsin. A latent fraction, comprising approximately 8% of the total antitrypsin, was separated from the native antitrypsin in Prolastin by anion exchange chromatography. The specific activity of this latent form against bovine alpha-chymotrypsin increased from 1 to 2% to 50% over 3 h after refolding from 6 M guanidine hydrochloride. These data show that commercial antitrypsin contains a latent component. The significance of this conformation in vivo is unknown, although Prolastin has shown few adverse side-effects in prolonged clinical usage.

Animals↗

PubMed1996

Probing serpin reactive-loop conformations by proteolytic cleavage.

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10-P9, P8-P7 and P7-P6 of anti-trypsin was distorted by binary complex formation. The P1'-P2' bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4-P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1-P1' bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15-P10) of either protein, whether native or as binary complexes. these results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A beta-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.

Amino Acid Sequence↗

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↗