Mutations in the shutter region of antithrombin result in formation of disulfide‐linked dimers and severe venous thrombosis

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 heparin‐affinity 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.     

Antithrombin Murcia (K241E) causing antithrombin deficiency: a possible role for altered glycosylation

Background

Identification of mutations in the SERPINC1 gene has revealed different mechanisms responsible for antithrombin deficiency. Deletions and nonsense mutations associate with type I deficiency. Certain missense mutations cause type II deficiency by affecting the heparin binding site or the reactive center loop, while others result in type I deficiency by intracellular retention or RNA instability.

Design and Methods

We studied the molecular, biochemical, proteomic and glycomic characterization of a new natural mutant (K241E) that may be classified as pleiotropic.

Results

The mutation caused a significant decrease in the anticoagulant activity mainly due to a reduced heparin affinity and a modification of the electrostatic potential that might explain the impaired ability of the mutant protein to form complexes with the target protease in the absence of heparin. Mass spectrometry and glycomic analyses confirmed an increased molecular weight of 800 Da in the mutant protein possibly due to core-fucosylation, provoking the loss of heparin affinity. Additionally, carriers of this mutation also have a minor mutant isoform that still followed normal glycosylation, retaining similar heparin affinity to wild-type α-antithrombin, and certain anticoagulant activity, which may explain the milder thrombotic risk of patients carrying this mutation. Similar results were observed using recombinant K241E antithrombin molecules.

Conclusions

Our data suggest a new mechanism involved in antithrombin type II deficiency by indirectly affecting the glycosylation of a natural variant. Additional studies are required to confirm this hypothesis.     

Frequency of HLA-A, -B and -DRB1 specificities and haplotypic associations in the population of Castilla y León (northwest-central Spain)

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.     

Causes for cancellation of elective surgical procedures in a Spanish general hospital

Cancellation of scheduled surgery is undesirable for patients and an inefficient use of resources. We prospectively collected data for 52 consecutive months in a public general hospital to estimate the prevalence and causes. The overall cancellation rate was 6.5% (2559 of 39 115 scheduled operations). Cancellation by broad category was for 'medical reasons' in 50%, 'patient-related factors' in 23%, and due to 'administrative/logistic problems' in 25%. The commonest specific causes within these categories were respectively: infections/fever (18%), patient did not attend (20%) and lack of theatre time (23%). This data will help direct resources to target prevention of cancellations as a result of these main problems.     

Pharmacoeconomic considerations in treating iron overload in patients with β-thalassaemia, sickle cell disease and myelodysplastic syndromes in the US: a literature review

Patients with β-thalassaemia, sickle cell disease (SCD) and myelodysplastic syndromes (MDS) require chronic blood transfusions, which can lead to iron overload and substantial morbidity and mortality. To reduce the excess iron and its deleterious effects, available iron chelation therapy (ICT) in the US includes oral deferasirox or infusional deferoxamine (DFO). The aim of this study was to review and synthesize the available pharmacoeconomic evidence on ICT in patients with β-thalassaemia, SCD and MDS in the US. We systematically identified and reviewed pharmacoeconomic studies of ICT in patients with β-thalassaemia, SCD and MDS that either were published in MEDLINE-indexed, English-language journals from 1999 to 2009, or appeared in medical society websites and scientific meeting abstracts. We assessed available cost-of-illness, cost-of-treatment, cost-consequence, cost-effectiveness, utility and patient-satisfaction studies. The majority of the 20 identified studies assessed cost of treatment, mainly focusing on acquisition and administration costs of ICTs. Gaps in the published literature include current data on direct medical costs for patients with MDS, direct medical costs associated with complications of iron overload, direct non-medical costs, indirect costs and patient utilities. Different underlying model assumptions, methodologies and comparators were found in the cost-effectiveness studies, which yielded a broad range of incremental cost-effectiveness ratios for different ICTs. Comprehensive cost-of-illness studies are needed to address data gaps in the published literature regarding the economic burden of iron overload. Comparative-effectiveness studies that evaluate clinical, economic and patient-reported outcomes would help the medical community to better understand the value of different ICTs.