Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.

Ex vivo inhibition of platelet aggregation in patients with severe limb arteriopathy treated with BAY U3405, a specific TX A2 receptor antagonist.

A double-blind, placebo-controlled randomized study with BAY U3405, a specific thromboxane A2 (TX A2) receptor blocker, was performed in patients suffering from severe stade II limb arteriopathy. BAY U3405 or placebo was administered in 16 patients at 20 mg four times a day (from day 1 to day 3). Hemostatic studies were done before therapy, and on day 2 and day 3 under therapy. On day 3, BAY U3405 was shown to induce a highly statistically significant decrease of the velocity and the intensity of the aggregations mediated by arachidonic acid (56 +/- 37% for the velocity, 58 +/- 26% for the intensity) or by U46619 endoperoxide analogue (36 +/- 35% for the velocity, 37 +/- 27% for the intensity). Similar results were already observed on day 2. By contrast, such a decrease was not noticed with ADP mediated platelet aggregation. Furthermore, plasma levels of betathromboglobulin and platelet factor 4 remained unchanged. Peripheral hemodynamic parameters were also studied. The peripheral blood flow was measured using a Doppler ultrasound; the pain free walking distance and the total walking ability distance were determined under standardized conditions on a treadmill. These last two parameters show a trend to improvement which nevertheless was not statistically significant. All together these results encourage further in vivo studies using BAY U3405 or related compounds on a long-term administration.

Familial variant of antithrombin III (AT III Bligny, 47Arg to His) associated with protein C deficiency.

The association of a variant of antithrombin III (AT III Bligny) and protein C deficiency is described in a 36-year-old patient having suffered from severe thrombotic episodes. His mother has protein C deficiency and showed a single episode of thrombophlebitis following surgery. His father, sister and daughter have the variant AT III and are asymptomatic. The abnormal AT III was characterized in plasma by the discrepancy between a normal progressive activity and a reduced heparin cofactor activity. This variant AT III was purified, separated from the normal protein by heparin-Sepharose chromatography and was eluted with increased NaCl concentrations. At pH 7.4, the variant AT III eluted at lower (0.3 to 0.5 M) NaCl concentrations than normal (1 to 1.5 M) AT III, thus demonstrating a decreased affinity for heparin. At pH 6.0, however, the abnormal molecule bound more avidly to heparin-Sepharose and was eluted like normal AT III at pH 7.4. Similarly, the heparin enhancement of intrinsic fluorescence of the variant AT III, markedly reduced at pH 7.4, was normalized at pH 6.0. The abnormal AT III showed a normal antiprotease activity, a normal molecular weight by SDS-PAGE, but displayed only a partial immunological identity with the normal protein. Analysis of amplified genomic DNA from this patient by dot-blot demonstrates a heterozygous substitution of arginine by histidine at position 47.

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.

[Homocysteine, 5,10-methylenetetrahydrofolate reductase and deep venous thrombosis. Survey of 120 patients in internal medicine]. / Homocystéine, 5,10-méthylènetétrahydrofolate réductase et thrombose veineuse profonde. Enquête auprès de 120 patients en médecine interne.

BACKGROUND: In the last few years, the association of deep vein thrombosis with frequent biological risk factors and genetic polymorphisms has significantly modified the field of venous thrombosis. In this study, we measured plasma homocysteine levels and tested the C677T methylenetetrahydrofolate reductase (MTHFR) mutation. PATIENTS AND METHODS: Plasma homocysteine levels and test for C677T MTHFR mutation were performed in 120 consecutive patients with objectively diagnosed deep vein thrombosis, and in 120 controls. RESULTS: We found a strong association between hyperhomocysteinemia and thrombosis (odd ratio: 2.43 IC 95% [1.27-4.7]). Conversely the C677T MTHFR gene polymorphism is only associated with hyperhomocysteinemia but not associated with thrombosis. CONCLUSION: This is a preliminary study to the ongoing international multicentric study of SNFMI (Société nationale française de m0+edecine interne) concerning hyperhomocysteinemia and venous thrombosis.

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.

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.