Tobacco Documents Online

THE COUNCIL FOR TOBACCO RESr:ARCH-U.S.A., INC. 900 THIRD AVENUE NEW YORK. N.Y. 10022 Memorandum To: Dr. H. McAllister and Staff From: D.H.Ford Re: Site visit with Dr. Katherine Hajjar, Cornell University, August 5, 1993. Visitors: A.Eisenberg, D.Ford and V.Lisanti. Grant No.2169AR2 entitled "Membrane Receptor Function in Cell Surface Fibrinolysis." Aims:To determine the structure and function of the endothelial cell receptor for tissue plasminogen activator (t-PA) and its substrate, plasminogen. Having isolated and clonedthe cDNA.for the receptor, to express it by transfection in a pCMV5 plasmid vector. Finally, to determine what factors might in8.uence binding of t-PA and plasmin- ogen to the receptor. Progress: Dr. Hajjar opened our discusion with an excellent overview of the the role of t-PA and plasminogen in the production of plasmin and subsequent fibrinolysis. Data obtained to date indicate that both t-PA and plasminogen bind to the same receptor, and that plasminogen interacts with C-terminal lysine residues while t-PA interacts with a conformation-dependent epitope sensitive to homocysteine. Details of these results are described in her progress report. The receptor itself appears to be a 40 Kd protein located in the endothelial membrane.which specifically binds t-PA but not urokinase (another enzyme involved in formation of plasmin). Further investigations indicate that this protein is a member of the annexin family of proteins, specifically, annexin II. In another study, Dr. Hajjar transfected the cDNA for annexin II into WEHI cells (mouse B-cells) and observed that these cells now w ould bind.t-PA, which could be blocked when she included an anti-sense nucleotide for the annexin cDNA into the system. Since the presence of annexin II increased the formation of plasmin 13 fold over that which occured with control preparations without this protein, it appears that the receptor (annexin II) participates with t-PA and plasminogen in the formation of plasmin. Thus, while t-PA plus plasminogen will lead to the formation of plasmin, this response is maximized by their association with annexin II. Dr. Hajjar has currently mutated the DNA for annexin in such a way that the lysine binding area for plasmin ogen is converted to an iso- leucine to determine if this interferes with plasmin formation. Dr. Hajjar is also interested in homocysteinuria wherein there is an increase in thromb ormbolic episodes. In a culture system she exposed the endothelial cells to homocysteine and observed a 65% decrease in binding of t-PA associated with a 60% decrease in cell- associated t-PA activity (and thus reduction in plasmin formation). Since most of Dr. Hajjar's studies have utilized endothelial cells from human umbilcal cord veins, we wondered if what she observed also applied to arteries. The only artery she has investigated as yet is the internal mammillary artery where the endothelial cells functioned in a manner compoarable to those from the vein. She is considering determining what occurs with capillaries, but has not yet undertaken this. Intuitively, she feels there may well be differences in capillaries, particularly in the CNS.

2 Comment: This is an exciting program which shows excellent progress. Dr. Hajjar is utilizing molecular biological techniques with great skill to unravel the mechanisms whereby the vessels are protected from thrombotic events. She has recently moved into a larger laboratory area and her lab appears singularly well equipped. Her approach to the project is well thought out, with each phase of the program logically following the previous studies. All-in-all, an excellent, well directed program. DHF