AN ABNORMAL MRNA SPLICING IS ASSOCIATED WITH THE A3 HAPLOTYPE OF THE EPCR GENE PROCR: A POSSIBLE NEW CONTRIBUTOR TO PLASMA SOLUBLE EPCR LEVELS
Abstract number: O-W-091
Saposnik1 B., Lesteven2 E., Lokajczyk2 A., Emmerich3 J., Esmon4 C.T., Aiach5 M., Gandrille5 S.
11Inserm U765 Université Paris Descartes, and Service d'Hématologie Biologique Hôpital Robert Debré 22Inserm U765 Université Paris Descartes, UFR des Sciences Pharmaceutiques 33Inserm U765 Université Paris Descartes, and Service de Médecine Vasculaire Hôpital Européen Georges Pompidou, Paris, France 44Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, United States 55Inserm U765 Université Paris Descartes, and Service d'Hématologie Biologique A Hôpital Européen Georges Pompidou, Paris, France
How-to-cite Saposnik B, Lesteven E, Lokajczyk A, Emmerich J, Esmon CT, Aiach M, Gandrille S. AN ABNORMAL MRNA SPLICING IS ASSOCIATED WITH THE A3 HAPLOTYPE OF THE EPCR GENE PROCR: A POSSIBLE NEW CONTRIBUTOR TO PLASMA SOLUBLE EPCR LEVELS. J Thromb Haemost 2007; 5 Supplement 2: O-W-091
Introduction: The endothelial cell protein C receptor also exists as a plasma soluble form (sEPCR) resulting from ADAMS17 cleavage. Elevated sEPCR levels are a risk factor for venous thrombosis and are frequently observed in subjects carrying the PROCR A3 haplotype leading to a Ser219Gly substitution in the transmembrane domain, the receptor being more sensitive to cleavage. Since sEPCR production is not completely blocked by metalloprotease inhibition, we looked for another mechanism.
Methods: We genotyped HUVECs from 32 human umbilical cords and compared the EPCR mRNA pattern of A3- and non A3-carrier cells.
Results: Six out of 32 HUVECs were heterozygous for the A3 haplotype and EPCR mRNA analysis revealed the presence of a minor truncated spliced mRNA besides that of a major full-length mRNA. This truncated mRNA reached 3.2% of EPCR mRNA of A3 cells compared to 0.2% only of non A3 cells when evaluated by real time quantitative PCR. This abnormal splicing deleted exon 4 coding part leading to a protein with a unique elongated C-terminus of 50 residues lacking the transmembrane domain, thus assumed to be soluble. The protein corresponding to the truncated mRNA (rsEPCR isoform) and a polypeptide mimicking the plasma sEPCR (rsEPCR) were stably expressed in HEK293 cells. The cDNA corresponding to the truncated mRNA led to the synthesis of a secreted protein of MW 49 kDa (compared to 43 kDa for rsEPCR). Functional studies of purified recombinant proteins revealed that the rsEPCR isoform bound to recombinant protein C (rPC) with affinity similar to that of rsEPCR (appKd 142 and 134 nM, respectively). Using an APTT-based assay, we evidenced that, like rsEPCR, rsEPCR isoform inhibits the anticoagulant activity of aPC.
Conclusions: The truncated form of EPCR mRNA present in high level in A3-carrying cells led to the synthesis of an elongated protein that exhibits functional properties similar to that of sEPCR. This suggests it could act in conjunction with sEPCR in its regulatory effect in plasma.
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Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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