Pleiotropic effects of antithrombin strand 1C substitution mutations.

Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to s1C proximity to highly conserved internal beta strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, s1C is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of s1C variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function.

Molecular genetics of antithrombin deficiency.

Antithrombin is the major proteinase inhibitor of thrombin and other blood coagulation proteinases. Antithrombin has two functional domains, a heparin binding site and a reactive centre (that complexes and inactivates the proteinase). Its deficiency results in an increased risk of venous thromboembolism. Appreciable progress has been made in recent years in understanding the structure and function of this protein, the genetic cause of inherited deficiency and its clinical consequence. The structure of antithrombin is now considered in terms of the models derived from X-ray crystallography, which have provided explanations for the function of its heparin interaction site and of its reactive loop. The structural organization of the antithrombin gene has been defined and numerous mutations have been identified that are responsible for antithrombin deficiency: these may reduce the level of the protein (Type I deficiency), alter the function of the protein (Type II deficiency, altering heparin binding or reactive sites), or even have multiple or 'pleiotropic effects' (Type II deficiency, altering both functional domains and the level of protein).

Antithrombin Budapest 3. An antithrombin variant with reduced heparin affinity resulting from the substitution L99F.

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, CTC----TTC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Creation of an additional glycosylation site as a mechanism for type I antithrombin deficiency.

We report the identification of a new mutation resulting in type I antithrombin (AT) deficiency and the mechanism by which the deficiency arose. The single base substitution of G to A at nucleotide 2709 was identified in a proband with a family history of venous thrombosis. The mutation results in a substitution of 82 Ser by Asn, creating a new glycosylation site. Expression studies were then carried out, to confirm Asn-linked glycosylation occurred at this consensus site and that this resulted in the AT deficient phenotype. Cell-free translations using rabbit reticulocyte lysate in the presence of microsomes demonstrated that the 82 Asn variant was post-translationally processed efficiently. The 82 Asn variant protein was of a higher molecular weight than normal AT. consistent with the addition of a fifth glycan chain. Incubation of translation product with endoglycosidase H, confirmed that the higher molecular weight product had resulted from additional carbohydrate. Expression of the 82 Asn variant in COS-7 cells resulted in intracellular accumulation, with a low level of secretion of the protein into culture supernatant, consistent with type I AT deficiency. The addition of an extra carbohydrate side chain to residue 82 of antithrombin may block post-translational folding. trapping the variant intracellulary.

Antithrombin III: a database of mutations.

Elucidation of the molecular defects responsible for antithrombin III deficiency is proceeding rapidly. In order that a record is kept of the new and duplicated mutations that are found, we have compiled a database that we plan to update annually. In this, the first report of the database, we list 6 antithrombin III locus sequence polymorphisms and 94 recorded mutations causing functional deficiency of the protein, 38 of which are novel. As is the case with mutations affecting other protein genes, most mutations of antithrombin III involve a CG to TG or CA change.

Homozygous antithrombin deficiency: report of two new cases (99 Leu to Phe) associated with arterial and venous thrombosis.

Inherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.

The inherited basis of venous thrombosis.

Venous thrombosis represents a manifestation of disordered hemostatic balance. The classical presentation is of pain and swelling of the lower limb, although clinical history and examination are notoriously misleading in reaching a diagnosis. A number of acquired predispositions have been associated with a tendency to thrombosis, such as immobilisation, surgery, malignancy and certain types of oral contraception, but in at least half of the instances no predisposition can be identified. A variety of genetic risk factors have also been identified. Mutations within the genes for antithrombin, protein C and protein S are associated with a venous thromboembolic phenotype. The commonest thrombophilic predisposition however is a variant of coagulation factor V, factor V Leiden, which results from a single amino acid substitution rendering the factor V molecule resistant to activated protein C. Factor V Leiden is present in approximately 5% of individuals of European origin, and is found in up to 40% of those with confirmed venous thrombosis. Increasingly it is recognised that venous thrombosis should be considered a polygenic disorder, with interactions between the various single gene defects which predispose to thrombosis, as well as normal genetic variation between individuals in the levels of both procoagulant and anticoagulant proteins, all determining which individuals will express the phenotype of venous thrombosis.

Blood rheology and myalgic encephalomyelitis: a pilot study.

The blood rheology of EDTA-anticoagulated blood samples from blood donors and subjects considered to have myalgic encephalomyelitis was assessed by multiple shear rate viscometry and by multiple-pressure filterability. Although average viscosities of the two groups were different, the differences did not reach statistical significance. In contrast, the data from multiple-pressure filtration of whole blood showed significant differences between females at the lowest (2.5 cm of water) filtration pressure. It appears that the acute phase of the disorder is associated with changes in blood rheology which could impair microcirculatory blood flow. In contrast, the chronic state does not appear to be associated with rheological abnormalities.

A novel antithrombin gene mutation: slippage and mispairing as a mechanism of genetic disease.

There is a high degree of genetic heterogeneity underlying antithrombin deficiency indicating that a number of genetic mechanisms are responsible for the disorder. We report the identification of a five nucleotide (CAGAA) deletion in exon 2 of the antithrombin gene that results in a shift in the frame of translation of the mRNA and introduces a premature STOP signal in codon 70. The deleted nucleotides represent one repeat of a duplicated sequence within codons 36-39. The deletion may have arisen by slippage and mispairing of the repeated sequences at the replication fork during DNA synthesis.

Red cell and hemorheological changes in multiple sclerosis.

Blood rheology in multiple sclerosis (MS) was investigated in 15 subjects with varying degrees of locomotor difficulties who were members of the local MS Society. Control data were obtained from blood samples from 25 male and 25 female normal blood donors. Whole blood viscosity was measured and blood filterability was assessed. Six MS females provided blood samples for scanning electron microscopy. Erythrocyte membrane fatty acids and phospholipids were assayed. Whole blood viscosity in MS females was higher than controls at 3 of 4 shear rates (p less than 0.001) but in MS males blood viscosity was higher only at shear rate of 1.0 s-1 (p less than 0.05). MS erythrocyte filtration rates were significantly lower than controls (p less than 0.001). Leucocyte counts in MS were greater than controls both in males (p less than 0.01) and females (p less than 0.001). MS erythrocyte morphology was greatly different from controls (p less than 0.0001) and erythrocyte membranes contained less sphingomyelin than controls (p less than 0.01) but more phosphatidylinositol plus phosphatidylserine (p less than 0.02). We conclude that, because our findings indicate an identifiable and potentially correctable abnormality, it is possible to envisage an inhibition of the progressive nature of MS, with the hope of a better prognosis for patients.

Serological and biochemical relationships between the alleged avirulent variant of Corynebacterium kutscheri and streptococci of group N.

Morphological, biochemical and serological observations suggested that no close relationship existed between C. kutscheri and its alleged avirulent variant isolated from mice at the Rockefeller University. However, both capillary and double diffusion precipitin reactions showed the alleged variant to be a streptococcus of group N, indistinguishable from that previously isolated from Rockefeller University mice.

Corynebacterium kutscheri and its alleged avirulent variant in mice.

Corynebacterium kutscheri and its alleged avirulent variant were re-examined in C57Bl/6 and Swiss Lynch mice. It was confirmed that while C57Bl/6 mice were resistant and Swiss Lynch susceptible to C. kutscheri, the alleged atypical variant was avirulent in both mouse strains. However, following immunosuppression of C57Bl/6 mice with hydrocortisone acetate, it was not possible to reactivate latent C. kutscheri or the alleged atypical variant; this was contrary to previous reports. Moreover, sequential hysterectomy derivation over four generations of C57Bl/6 mice did not eliminate their resistance to C. kutscheri compared with conventionally born animals. Vaccination with live attenuated C. kutscheri protected susceptible mice against virulent challenge; vaccination with the alleged atypical variant afforded no such protection. The suggested role of the alleged avirulent variant in resistance to C. kutscheri is challenged and an alternative explanation of such resistance is proposed.

Antithrombin III: summary of first database update.

Antithrombin III is the most important inhibitor of coagulation proteinases such as thrombin and factor Xa. Inherited deficiency of antithrombin III is a well recognised risk factor for the early development of venous thromboembolism. The gene for antithrombin III is located at chromosome 1q 23-25 and its structural organisation has been described. A database of mutations of the antithrombin III gene has been compiled and a recent update lists 184 entries. These entries are listed according to subtype of deficiency and to nucleotide sequence number. There are 68 reports of type I 'classical' and 116 reports of type II 'variant' deficiencies. This summary considers the entries in terms of the number of unique molecular events, the nature of the genetic defects and the role of CpG dinucleotides in deficiency. Sample listings of type I and II deficiency entries are provided.

Respiratory disease in a colony of rats. II. Isolation of Mycoplasma pulmonis from the natural disease, and the experimental disease induced with a cloned culture of this organism.

Mycoplasma pulmonis was isolated from the pneumonic lung of a rat. Two groups of mycoplasma-free rats were inoculated, one with a culture of the M. pulmonis strain which had been cloned four times (group A) and the other with a lung homogenate of the rat from which the strain had been isolated (group B). A third group (C) consisted of uninoculated control animals. Each group was kept in strict isolation and allowed to breed so that the progeny was naturally exposed to any pathogens present in the inoculated animals. After different periods of exposure, rats were autopsied, respiratory tracts and inner ears were cultured for mycoplasmas and bacteria, and sera were tested for complement-fixing antibodies to murine mycoplasmas.In group-A rats, M. pulmonis was consistently isolated from the inner ears or lungs from 50 to 715 days after exposure. Complement-fixing antibody to M. pulmonis was detected 20 days after inoculation, but in the naturally exposed progeny antibody took longer than 50 days to develop. Antibodies to the other known mycoplasmas of murine origin, M. arthritidis and M. neurolyticum, were never found. Purulent otitis interna was consistently found from day 55 onwards, while lung lesions were first observed at 85 days and persisted to 715 days. Pulmonary lesions developed more slowly in inoculated parents than in exposed progeny. Similar results were found in group-B rats, which were examined up to 441 days after inoculation. Uninoculated group-C rats were examined up to 768 days of age, but M. pulmonis was not recovered; of the 54 animals whose serum was tested all were negative to the three species of mycoplasmas, except one which had a titre of 16 with M. pulmonis. Pneumonia, bronchiectasis or lymphoreticular hyperplasia were not seen in any of these control rats. Bacterial respiratory pathogens were not isolated from rats in any of the groups, nor was antibody to Sendai virus detected.The results suggest that M. pulmonis alone can cause pneumonia and bronchiectasis in rats since mechanical carry-over of another pathogen with the initial cloned inoculum is very unlikely and there was no evidence for the participation of any other rat pathogen. The respiratory disease induced by the cloned culture was comparable with that induced by the lung homogenate, and with the well-known syndrome of chronic respiratory disease and bronchiectasis in the rat.