Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation

Abstract

Summary. Thrombomodulin (TM) is an endothelial cell glycoprotein that acts as an anticoagulant. Mutation in the TM gene is a potential risk factor for thrombosis. The ®rst TM mutation identi®ed was a heterozygous substitution of T for G at nucleotide position 1456, which predicted Asp468 with Tyr in a Ser/Thr-rich domain. To evaluate the reported TM gene mutation as a possible cause of thrombosis, we transiently tranfected a vector for TM gene carrying the mutation to mammalian COS7 cells.TM antigen levels in lysates of cells transfected with variant TM were comparable to those in preparations of normal TM. The TM cofactor activity for protein C (PC) activation on the variant TM-expressing cells was similar to that of the control. The Michaelis constant K m and V max. of variant TM for PC activation were shown to be similar compared to those of normal TM. The af®nity of each TM for thrombin in PC activation was also similar. We obtained several stable cell lines expressing normal and variant TM. Lysate of the cell lines with normal and variant TM genes had a similar expression level of TM antigen. Pulse-chase analysis showed that normal and variant TM were glycosylated and resistant to endoglycosidase H, indicating that the variant TM was expressed on the cell surface in a mature form.Variant TM protein is apparently expressed on the cell surface with normal cofactor activity for PC activation. It is unlikely that the TM variant directly causes thrombosis by mechanism of reduced expression or impaired cofactor activity for PC activation, which comprises a major anticoagulant activity of TM.

Genetic risk factors of venous thrombosis

“…One reason for this is that simple functional assays are not available to establish that these mutations are detrimental to thrombomodulin gene function. Indeed, in vitro data obtained with the first mutation ever reported in the thrombomodulin gene, Asp468Tyr, did not reveal any abnormality in production levels or functional activity (Nakazawa et al 1999).…”

Non-viral in vivo thrombomodulin gene transfer prevents early loss of thromboresistance of grafted veins

“…A 2.6-kb Xho I-Bal I fragment of the human TM gene containing 5 0 -and 3 0 -untranslated and coding regions was used to construct an expression vector, pPCITMN [10], which was transcribed under the control of the cytomegalovirus enhancer and promoter (Promega, Madison, WI). Synthetic cationic liposomes, (C)-N,N bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide, were purchased from Promega, and human holo-transferrin was from Sigma Chemical Co. (St. Louis, MO).…”

“…After the end was excised, the remainder was closed inside out by continuous 7-0 sutures or clipping (LT100; Ethicon Inc., Cincinnati, OH), so that only endothelium was exposed on the outside. TM cofactor activity in the endothelial cell surface was determined as described previously [10]. Briefly, exogenous protein C (0.16 mM) (Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan) was activated by endothelial cells in the presence of thrombin, and cleavage of substrate S2266 (Chromogenix, Stockholm, Sweden) measured.…”

Potentielle genetische Polymorphismen der venösen Thromboembolie. New Candidate Gene Polymorphisms in Venous Thrombosis

“…Öhlin und Mitarbeiter [40] haben eine Punktmutation in Form einer G zu T-Substitution an der Position 1456 im THBD Gen eines Patienten mit Lungenembolie identifizieren können, die zu einem Asp468Tyr-Austausch führt. Allerdings konnte anhand von Transfektionsuntersuchungen gezeigt werden, daß diese Variante eine vergleichbare Zellexpression und auch Protein C-Kofaktoraktivität mit vergleichbaren Michaelis-Konstanten Kni und Vmax besitzt wie der Wildtyp [41]. In einer italienischen Untersuchung wurde diese Mutation unter Patienten mit venösen Thrombosen nicht gefunden [42].…”