Thrombin generation in patients with factor XI deficiency and clinical bleeding risk.

SUMMARY: Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty-four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non-bleeders (n = 15). All severe bleeders had a personal history of surgery-related severe bleeding. Thrombin generation (TG) was measured in platelet-rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl(-1), a clinically non-bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl(-1) had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

Fibrinogen angers with a new deletion (gamma GVYYQ 346-350) causes hypofibrinogenemia with hepatic storage.

INTRODUCTION: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing. RESULTS: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra-cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint-like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the gamma chain gene in the 3'end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita's brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in-frame heterozygous 5 amino acid deletion (GVYYQ 346-350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence. CONCLUSION: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.

Molecular basis of severe factor XI deficiency in seven families from the west of France. Seven novel mutations, including an ancient Q88X mutation.

Inherited factor (F)XI deficiency is a rare disorder in the general population, though it is commonly found in individuals of Ashkenazi Jewish ancestry. In particular, two mutations--a stop mutation (type II) and a missense mutation (type III)--which are responsible for FXI deficiency, predominate. The bleeding tendency associated with plasma FXI deficiency in patients is variable, with approximately 50% of patients exhibiting excessive post-traumatic or postsurgical bleeding. In this study, we identified the molecular basis of FXI deficiency in 10 patients belonging to six unrelated families of the Nantes area in France and one family of Lebanese origin. As in Ashkenazi Jewish or in French Basque patients, we have identified a new ancient mutation in exon 4 resulting in Q88X, specific to patients from Nantes, that can result in a severely truncated polypeptide. Homozygous Q88X was found in a severely affected patient with an inhibitor to FXI and in three other unrelated families, either as homozygous, heterozygous or compound heterozygous states. Other identified mutations are two nonsense mutations in the FXI gene, in exon 7 and 15, resulting in R210X and C581X, respectively, which were identified in three families. A novel insertion in exon 3 (nucleotide 137 + G), which causes a stop codon, was characterized. Finally, sequence analysis of all 15 exons of the FXI gene revealed three missense mutations resulting in G336R and G350A (exon 10) and T575M (exon 15). Two mutations (T575M and G350A) with discrepant antigen and functional values are particularly interesting because most of the described mutations are associated with the absence of secreted protein.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways.

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.