Joseph Emmerich

P2Y12 H2 Haplotype Is Associated With Peripheral Arterial Disease A Case-Control Study

Background—We recently described a gain-of-function haplotype, called H2, of the adenosine diphosphate (ADP) receptor P2Y12 gene associated with increased ADP-induced platelet aggregation ex vivo in healthy volunteers. Because platelets play a key role in atherosclerosis and arterial thrombosis, we tested the possible link between the H2 haplotype and the risk of peripheral arterial disease (PAD) in a case-control study. Methods and Results—We studied 184 consecutive male patients under 70 years of age with PAD and 330 age-matched control subjects free of symptomatic PAD and with no cardiovascular history. Mean age was 57.1±7.2 years (cases) and 56.7±7.6 years (control subjects). The H2 haplotype was more frequent in patients with PAD than in control subjects (30% and 21%, respectively; OR, 1.6; CI, 1.1 to 2.5; P =0.02 in univariate analysis). This association with PAD remained significant in multivariate regression analysis (OR, 2.3; CI, 1.4 to 3.9; P =0.002) after adjustment for diabetes, smoking, hypertension, hypercholesterolemia, and other selected platelet receptor gene polymorphisms. Conclusions—These data point to a role of the H2 haplotype in atherosclerosis and raise the possibility of relative thienopyridine resistance in carriers of the P2Y12 H2 haplotype.

Factors V Leiden and II 20210A in patients with symptomatic pulmonary embolism and deep vein thrombosis

PURPOSE Factor V Leiden and factor II 20210A are inherited disorders of the clotting system that occur frequently in patients with deep vein thrombosis. We conducted this study to determine whether these factors are also common in patients with pulmonary embolism. SUBJECTS AND METHODS We determined the prevalence of factor V Leiden and factor II 20210A in 773 consecutive patients with objectively documented symptomatic deep vein thrombosis or symptomatic pulmonary embolism, or with a combination of these disorders. RESULTS Isolated symptomatic deep vein thrombosis occurred in 345 patients; isolated symptomatic pulmonary embolism occurred in 236; and both anomalies occurred in 192. Factor V Leiden was present in 21 (9%) of the patients with isolated symptomatic pulmonary embolism, in 30 (16%) with both manifestations, and in 63 (18%) with isolated symptomatic deep vein thrombosis (P = 0.007). Factor V Leiden was more common among patients with deep vein thrombosis (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.2 to 3.7; P = 0.006) or both pulmonary embolism and deep vein thrombosis (OR = 1.8; 95% CI: 1.0 to 3.3; P = 0.07) than among patients with isolated pulmonary embolism. Factor V Leiden was less common in massive pulmonary embolism (5% [7 of 127]) than in submassive pulmonary embolism (13% [21 of 155], P = 0.03). We found no significant difference in the prevalence of factor II 20210A among the three groups. CONCLUSION Factors V Leiden and II 20210A vary in prevalence among patients with pulmonary embolism and deep vein thrombosis, suggesting that the risk of pulmonary embolization may vary among patients who have different causes of venous thromboses.

HIF‐Prolyl Hydroxylase 2 Inhibition Enhances the Efficiency of Mesenchymal Stem Cell‐Based Therapies for the Treatment of Critical Limb Ischemia

Upregulation of hypoxia‐inducible transcription factor‐1α (HIF‐1α), through prolyl‐hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell‐based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF‐1α were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow‐derived mesenchymal stem cells (hBM‐MSC)‐based therapy. PHD2 silencing enhanced hBM‐MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF‐1α and its target gene, VEGF‐A. In addition, PHD2‐transfected hBM‐MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF‐1α or VEGF‐A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM‐MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD‐2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF‐1α. In conclusion, silencing of PHD2, by the transient upregulation of HIF‐1α and its target gene VEGF‐A, might improve the efficiency of hBM‐MSC‐based therapies. Stem Cells 2014;32:231–243

Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three‐dimensional structure were analysed. The last mutation in exon 4 was associated with a type II ‘reactive site’ deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.

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

Summary. 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 Ala 404 to Thr (both located in the C‐terminal part of the AT III molecule) and also a frameshift in exon IIIa.

A phenylalanine 402 to leucine mutation is responsible for a stable inactive conformation of antithrombin

In a South African family with antithrombin deficiency and unexplained thrombosis, genomic DNA analysis revealed a substitution of Phe 402 by Leu. This mutation involves an amino acid located in the carboxyterminal side of the antithrombin reactive loop and has already been observed in a French family (antithrombin Maisons-Laffitte). In both cases, the expression of the mutation is pleiotropic, i.e. results in a reduction in the circulating concentration of antithrombin and impairs both its anti-thrombin activity and its ability to bind heparin. The effect of a denaturing agent (sodium dodecyl sulfate) on the recognition of the plasma antithrombin by a polyclonal antibody was studied in an immuno-enzymatic assay. The Phe to Leu mutation decreased the sensitivity to denaturation, suggesting that the mutation increases the stability of the protein. Whether this stable conformation is due to a partial insertion of the amino-terminal side of the reactive loop, which would explain how both protease binding and heparin binding are affected, remains to be determined.