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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. 900 T2[IRI) AVENUE NEW YORK. N.Y. 10022 Memorandum To: Dr. J. F. Glenn and Staff From: D.H.Ford Re: Site visit with Dr. J.Jesty, Stony Brook, SUNY, Sept. 28, 1988. Grant No. 2255 entitled "Plasma inhibitors of Factor X activation." Goals:To investigate the kinetics of factor X activation in a plasma system, particularly in relation to a tissue factor which he found which inhibits clotting. This goal will be approached in two steps: 1. studies on the control of factor activation in plasma and the isolation of controlling'factors (ie., his tissue inhibitory factors) and 2, the kinetic des- cription of such factors in a pure system. Other studies will relate to possible roles of platelets and endothelial cells in inhibiting activation of factor X. Results: Inasmuch as this program received its initial support from CTR this past July, progress toward the above goals has not proceeded very far. His initial problem has been to obtain enough of factors VIIa and VII and IXa to undertaLce the evaluation of his tissue factor on inhibition of factor R activation. Rather than use plasma, which would require hundreds of liters to obtain enough protein, he is obtaining a factor IX concentrate from a commercial source. He is then able to fractionate this to obtain the necessary factors VII, IX and X.As I understand his progress,he is now ready to undertake the specific studies on factor X activation. The tissue factor which inhibits factor X activation is present in endothelial cells, but also in almost all cell types, includ- the smooth muscle of vessels. He has a fairly pure preparation of the tissue factor, but its nature is still unknown. He is collaborating with other investigators at Stony Brook who are interested in determining the strucure of this factor. So far, he knows that it has a small molecular weight, is possibly a peptide, and not likely to be a lipoprotein. It is a membrane protein, which appears to be on the cytoplasmic side of the membrane unless a cell is damaged. Further, its expression does not require protein synthesis after damage to a cell membrane occurs. (In a sense it sounds similar to the GABA receptor, which exists in two state; one outside the membrane surface and one inside. This receptor can then rotate within the membrane to present either of its two faces, depending on the circumstances). Dr. Jesty has collected 80 plasma samples from entering medical students, which he hopes in the future to evaluate for the possibility of individual variation (Are in the deep freeze).

-2- Note the attached flow sheet diagram for the factors involved in clotting. Dr. Jesty's interest in this area focuses at the site where factor IXa converts to factor X and where the conversion of VIIa to IX or X appears to be inhibited by his tissue factor (TF). A graduate student in the program, Mr. Pierre Neuenschwander is actively involved in culturing human umbilical cord endo- thelial cells for this project, since Dr. Jesty believes that endothelial cells, which possess the tissue factor are important in the hemostatic response. They are currently receiving 4-5 cords/week and have successfully established the endothelial culture.To confirm the quality of the endothelial cells in culture, he is starting to set up a method for the measurement of von Willebrand factor. Comment: Dr. Jesty is a young biochemist whose background is essentially in the field of biochemistry where he appears quite knowledgable in the general field of clotting mechanisms. However, if he is successful in characterizing the tissue factor which inhibits activation of factor X, he will have to establish collaborative connections with clinical hematologists if this factor is to be tested as an anticlotting substance in man. If Dr. Jesty can succeed in characterizing this tissue factor, it should be quite important in the treatment of coronary heart disease. At this time, however, it is too early in the development of this project to judge to what degree he may be successful. DHF

SURFACE PREKALLIKREIN HMW - KININOGEN X1l XIICI ? XI 11.. Xlo Ca Co /OOOTOOF IX~ *w-IXa VIIaE-VII VIo C~ Xa VI co THROMBIN4~~ PROTHROMBIN Xllla~ XIII CROSS-LINKED C0 FIBRIN ~ FIBRIN 4~ FIBRIN~[MONOMER,p~ FIBRINOGEN