Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation.
Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (Km = 2.17 +/- 0.13 microM). With EPCR, two classes of sites are apparent (Km = 20 +/- 15 nM and Km = 3.2 +/- 1.7 microM). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM:EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
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D001779:Blood Coagulation Factors; D002460:Cell Line; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism
D001779:Blood Coagulation Factors; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D011486:Protein C; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D018180:Thrombomodulin; C091963:activated protein C receptor