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Biomedical subjects

M Aiach

Publications and source records attributed to M Aiach.

At least 145 records · Page 8Linked to original sources
PubMed1992

Molecular abnormalities responsible for thrombosis. Genetic aspects.

The association between congenital deficiencies and recurrent thrombosis strongly suggests that antithrombin III, protein C and protein S play a major role in inhibiting thrombin formation in vivo. Genetic analysis using DNA fragment amplification by polymerase chain reaction and direct gene sequencing has led to the identification of many novel mutations in qualitative and quantitative deficiencies. Elucidation of the molecular basis of these deficiencies is critical to our understanding of natural antithrombotic mechanisms. It not only provides information on the structural features governing protein function, but also permits a better classification, based on genomic abnormalities of hereditary deficiencies responsible for mild to severe phenotypes and may prove of further value to define the most pertinent plasma assays for routine diagnosis.

Antithrombin III Deficiency↗
PubMed1991

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.

Antithrombin III↗
PubMed1991

Molecular basis for antithrombin III type I deficiency: three novel mutations located in exon IV.

Antithrombin III (AT III) type I deficiencies are characterized by a 50% decrease of both immunoreactive and functional protein and carry a high risk of thrombotic complication. We have studied the molecular basis for such deficiencies by asymmetric polymerase chain reaction amplification and direct sequencing of the seven exons and of the intron-exon junction of the AT III gene. Three different mutations were observed in the exon IV: a 4-bp deletion, a 2-bp deletion, and a nucleotide insertion. Each of these mutations results in a frameshift introducing premature stop codons at positions 313, 309, and 232, respectively. These results were confirmed by dot-blot analysis with allele-specific oligonucleotide probes. Furthermore, no mutation was observed in the other six exons. The comparison of the type of mutations observed by our group in six cases of type I deficiencies and in 16 cases of type II heparin binding site variants deficiencies suggests that the former are caused by heterogeneous molecular abnormalities while the latter are caused by recurrent missense mutations.

Adult↗
PubMed1991

Purification of heparin cofactor II from human plasma.

Heparin cofactor II (HCII) is an inhibitor of thrombin in human plasma whose activity is enhanced by heparin and dermatan sulphate. HCII was purified to homogeneity from normal human plasma with an overall yield of 7.5%. After treatment with barium chloride, precipitation with 50% saturated ammonium sulphate and dialysis of the resuspended precipitate against 0.02 M Tris-HCl (pH 7.4), the sample was chromatographed on a heparin-Sepharose CL 6B affinity column, DEAE-Sepharose CL 6B ion-exchange gel and an AcA 34 gel permeation column. For the final steps, a high-performance liquid chromatographic system was used which included ion-exchange chromatography on a Mono-Q column and gel permeation using a Superose column. The purified protein was homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific activity of purified HCII was 12.2 U/mg. The HCII activity was evaluated as antithrombin dermatan sulphate cofactor activity. A specific antiserum against HCII was raised in the rabbit.

Chromatography, Affinity↗
PubMed1991

Heparin cofactor II deficiency in renal allograft recipients: no correlation with the development of thrombosis.

Heparin cofactor II (HC II) is a thrombin inhibitor in human plasma which displays great similarities with antithrombin III (AT III). Hereditary HC II deficiency was recently reported to be associated with thrombophilia. Since thromboembolism constitutes an important post-operative complication after renal transplantation, we measured HC II and AT III in the plasma of 118 healthy renal allograft recipients (RAR) and found stable low HC II and high AT III levels. Administration of azathioprine (AZA), cyclosporine A (CSA) or both as immunosuppressive therapy did not affect HC II levels, but CSA seems to have raised plasma AT III. The proportion of patients with HC II deficiency was significantly higher in RAR than the proportion we previously found (11) in healthy individuals (16.9% vs 1.5%). However, the proportions with low plasma HC II were not different in healthy RAR and in ten RAR with thrombotic events, suggesting that in transplanted patients, HC II deficiency is not in itself a risk factor for the development of thrombosis.

Adolescent↗
PubMed1991

Molecular basis for hereditary antithrombin III quantitative deficiencies: a stop codon in exon IIIa and a frameshift in exon VI.

Antithrombin III (AT III) is an inhibitor of serine protease (serpin) comprising 432 amino acids. Quantitative AT III deficiencies are associated with a high risk of thrombotic disease. Although this risk is smaller in patients with qualitative AT III deficiencies, the molecular defects characterizing the latter have been the subject of many studies. However, in quantitative AT III deficiencies, only three mutations have been described: Pro 407 to Leu and A1a404 to Thr (both located in the C-terminal part of the AT III molecule) and also a frameshift in exon IIIa. Using the asymmetric polymerase chain reaction (PCR) and genomic DNA analysis by direct sequencing, we detected two mutations in three unrelated families: (i) a C----T transition in exon IIIa in two families, leading to the replacement of the codon corresponding to Arg 129 by a stop codon, and (ii) in the third family, insertion of an adenine in the codon corresponding to Phe 408, a highly conserved serpin amino acid. This insertion altered the reading frame and led to the appearance of a premature stop signal. Patients of all three families were heterozygous for their abnormality. These results show that asymmetric PCR and genomic DNA analysis by direct sequencing permit fast identification of the molecular basis of quantitative AT III deficiencies. It is concluded that in many cases the absence of AT III gene product probably results from point mutation, as previously observed for another serpin, alpha-1-antitrypsin.

Adolescent↗
PubMed1991

Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation, transforming the inhibitor into a substrate.

Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.

Amino Acid Sequence↗
PubMed1991

Involvement of heparin cofactor II in chymotrypsin neutralization and in the pancreatic proteinase-antiproteinase interaction during acute pancreatitis in man.

Heparin cofactor II is a proteinase inhibitor which inhibits both chymotrypsin and thrombin, and displays great similarities with antithrombin III, the main inhibitor of thrombin in human plasma. Since acute pancreatitis is known to be associated with modification of the proteinase-antiproteinase equilibrium, we studied heparin cofactor II and antithrombin III as well as other biochemical and haematological parameters in 10 patients experiencing attacks of acute pancreatitis. Heparin cofactor II activity decreased during the first week of illness, while its antigen concentration remained subnormal. This discrepancy between antigen concentration and activity which persisted during the first week of illness was due both to complex formation of heparin cofactor II with its target proteinases and to partial proteolysis of the inhibitor. Heparin cofactor II was shown to form a complex with chymotrypsin in the plasma of such patients. Antithrombin III levels remained unchanged throughout the study, with no discrepancy between its activity and antigen concentration. No modification of haemostasis was shown either, except for a rise in the fibrinogen level during the first days of illness. It is concluded that, unlike antithrombin III, heparin cofactor II is involved in the proteinase-inhibitor equilibrium in patients with acute pancreatitis, and that heparin cofactor II might react as an inhibitor of pancreatic proteinases rather than an inhibitor of thrombin.

Acute Disease↗
PubMed1991

Heparin cofactor II: an acute phase reactant in patients with deep vein thrombosis.

In human plasma, heparin cofactor II (HCII) is a thrombin inhibitor which displays similarities with antithrombin III (ATIII). As previously reported for hereditary ATIII deficiency, cases of recurrent thrombosis were reported in patients with hereditary HCII deficiency. Here, plasma HCII activity was studied in 372 patients with a history of thrombosis, classified according to their anticoagulant therapy. The mean plasma HCII level was significantly higher in patients with acute deep vein thrombosis (DVT) under heparin therapy than in patients with a history of thrombosis, who were studied more than 3 months after the acute event, and were either on, or had been on, oral anticoagulant therapy. HCII and fibrinogen were significantly correlated in all three groups of patients. These results were strengthened by those of a follow-up study in 23 patients with acute DVT. Changes in plasma HCII activity paralleled those of fibrinogen. This suggests that HCII might behave like an acute phase reactant in patients with thrombosis and that the measurement of its plasma level as a risk factor for thrombosis should be performed some time after the acute episode. In conclusion, the prevalence of HCII deficiency in patients with a history of thrombosis might have been underestimated in series which included patients with acute thrombosis.

Acute-Phase Proteins↗
PubMed1990

Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine.

An hereditary abnormal antithrombin III (ATIII Geneva) with defective heparin cofactor activity was characterized by DNA single strand amplification and subsequent direct sequencing. ATIII Geneva was found to have a G to A transition in Exon IIIa leading to an Arg-129 to Gln mutation. This amino acid is part of the ATIII region comprising residues 114-154, which contains the highest proportion of basic residues (Arg or Lys), and is known from chemical modification studies to be involved in heparin binding. The variant protein did not bind heparin-Sepharose and was isolated from the propositus plasma by immunoaffinity chromatography. High affinity (for ATIII) heparin had only a minimal effect on thrombin and activated factor X inhibition by the purified abnormal ATIII. Taken together, these results demonstrate an important role for Arg-129 in the binding and interaction of ATIII with heparin of high affinity. We propose that a cooperation between Lys-125, Arg-129, Lys-136, and Arg-47 exposed at the surface of the inhibitor allows the binding of the essential pentasaccharide domain of heparin which is specific for the ATIII interaction.

Amino Acid Sequence↗
PubMed1990

A plasma clot lysis assay based on the release of fibrin degradation products: application to the diagnosis of hypofibrinolytic states.

Using a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (less than 2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 micrograms of fibrin degradation products per mg of fibrinogen (delta T 200). Two among the 27 patients with effective VO were bad responders with a delta T 200 less than 3 h (whereas all the others had delta T 200 greater than 10 h). These patients had respectively a deficient tPA release (delta tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal delta T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.

Adult↗
PubMed1990

Clinical and biochemical characterization of antithrombin III Franconville, a variant with Pro 41 Leu mutation.

We describe a familial study of AT III, a type III antithrombin III variant which was identified in the propositus by gene analysis as Pro 41 Leu heterozygous mutation. None of the four members of the family who presented with defective heparin cofactor (hep-cofactor) activity, and therefore probably carried the mutation, had experienced deep venous thrombosis. The abnormal AT III was purified from the propositus' plasma, taking advantage of the difference in NaCl concentrations required to elute variant and normal AT III from heparin-Sepharose. The antithrombin and anti-Xa activities of the purified variant AT III were comparable to those observed for normal AT III, but hep-cofactor activity was strikingly reduced. The enhancement by heparin of thrombin and F Xa inhibition by normal and variant AT III was compared in the absence of NaCl and in the presence of normal NaCl concentrations. The difference between the degrees of inhibition by normal and variant AT III was maximal at physiological ionic strength (i.e. at a concentration of 0.15 M). The quantification of heparin AT III interaction with both normal and variant purified proteins in a double reciprocal plot yielded similar dissociation constants but a 9-fold decrease in the maximal pseudo-first order constant. This suggests that Pro 41 is more involved in the molecular changes induced by heparin than in the primary binding of the activator.

Adult↗