The functional properties of a truncated form of endothelial cell protein C receptor generated by alternative splicing.
BACKGROUND
A soluble form of endothelial cell protein C receptor (sEPCR) is generated by shedding of the cellular form. sEPCR binds to protein C and factor VIIa and inhibits both the activation of protein C and the activity of activated protein C and factor VIIa. High sEPCR levels may increase the risk of thrombosis. We wanted to explore the possibility of detecting soluble endothelial cell protein C receptor forms generated by alternative splicing.


METHODS
Reverse transcriptase polymerase chain reaction was used to look for new forms of endothelial cell protein C receptor. A yeast expression system was used to generate sufficient amounts of the distinct sEPCR forms. Surface plasmon resonance experiments, chromogenic assays, clotting assays and immunoassays were subsequently performed to characterize a new form of sEPCR that was found.


RESULTS
We demonstrated, by reverse transcriptase polymerase chain reaction and sequencing, the existence of a new, soluble form of endothelial cell protein C receptor generated by alternative splicing, in which the transmembrane region is replaced by a 56-residue tail (tEPCR). Its cDNA was present in human umbilical vein endothelial cells and in most tissues as well as in lung cancer cells. tEPCR was not located in the membrane of transfected cells. We demonstrated that tEPCR binds to protein C and factor VIIa. tEPCR blocked the generation of activated protein C and inhibited the activity of both activated protein C and factor VIIa. tEPCR was detected, by immunoassays, in the supernatant of lung cancer cells and human umbilical vein endothelial cells.


CONCLUSIONS
A truncated form of alternatively spliced endothelial cell protein C receptor was detected in the endothelium and cancer cells. tEPCR behaves as sEPCR generated by shedding of the cellular endothelial cell protein C receptor.
Molina
Eva
E
Haematology Department and the Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Clínica Universitaria/School of Medicine, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
Hermida
José
J
López-Sagaseta
Jacinto
J
Puy
Cristina
C
Montes
Ramón
R
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000737:chemistry* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D015942:Factor VIIa / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D008969:Molecular Sequence Data; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D001779:Blood Coagulation Factors; D011486:Protein C; D011956:Receptors, Cell Surface; C091963:activated protein C receptor; D015942:Factor VIIa