Summary. Background: Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. Objectives: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. Patients and methods: We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparinaffinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. Results: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. Conclusions: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.
Summary. It is now apparent that the inactivated latent and cleaved conformers of antithrombin (AT) are of pathological significance. Using a single-run electrophoretic technique that allows the quantitative assessment of these conformers in 2 lL plasma, we show that near 3% of the total AT in the circulations of normal individuals is in latent conformation. Only trace amounts of cleaved AT were observed. The slow decline in AT activity on incubation of plasma at 37°C was shown to be almost wholly due to a transition of native AT to its inactive latent form. Also initial studies in the rabbit indicate that the latent form, like the cleaved, has an identical circulatory half-life to that of native AT. We deduce that the steady concentration of latent AT in the circulation is due to the transition of some 10 12 molecules of AT per second balanced by an equivalent clearance of the latent form. Examples of clinical applications of the new technique include its use as a comprehensive single-step screen for genetic variants associated with AT deficiency, and notably the potential it provides to monitor the changes responsible for the loss of AT in the shock syndromes.
Cambridge II (A384S) is a highly prevalent antithrombin variant in the British population (1.14 per 1000) and predisposes carriers to a mild but significant increased risk of thrombosis. To determine if the association of Cambridge II with thrombophilia is due to a perturbation of the antithrombin inhibitory mechanism, we expressed and characterized the variant. Antithrombin Cambridge II was found to be normal in its affinity for heparin, its ability to form sodium dodecyl sulfate-stable complexes with factor Xa and thrombin, and its uncatalyzed stoichiometries and rates of inhibition. However, in the presence of full-length heparin there was a 3-and 7-fold increase in stoichiometry of inhibition of factor Xa and thrombin. The stoichiometries were not affected by pen
Many disorders, including Alzheimer’s, the prion encephalopathies and other neurodegenerative diseases, result from aberrant protein aggregation. Surprisingly, cellular toxicity is often due not to the highly-ordered aggregates but to the oligomers that precede their formation. Using serpins as a paradigm, we show how the active and infective interface of oligomers is inherently toxic and can promiscuously bind to unrelated peptides, including neurotransmitters. Extension of the oligomer and its eventual sequestration as amyloid can thus be seen as a protective response to block the toxic interface. We illustrate how the preferential self-association that gives this protection has been selectively favoured.
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