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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A.. INC. 900 TIIIRI) ANF:NUE NEW YORK. N. Y. 10022 DONALD H. FORD. PH.D. Assoctwre xsscwecK oixsc-rox May 21, 1987 Memorandum To: Dr. S.C. Sommers and Staff From: D.H. Ford Re: Site visit with Dr. I. Bdrinei`t, University of Pennsylvania, Philadelphia, May 19, 1987 Grant No. 1570A entitled "Characterization of the Platelet Fibrinogen Receptor". Goals: To determine how platelet activation results in the expression of the IIb-IIIa fibrinogen binding sites; to determine why two dissimilar glycoproteins (IIb-IIIa) are both equally deficient in the autosomal recessive disorder, Glanzmann's thrombasthenia; to isolate and characterize the genes containing the message for the synthesis of these two glycoproteins. Results: Since platelets are not suitable for in vitro studies and since the number of megakaryocytes which can be harvested is limited, Dr. Bennett has utilized cells from two human leukemia cell lines, HEL and K562, to study synthesis of GpIIb and GpIIIa. HEL cells contain complexes of Ilb and IIIa, while the K562 cells only express IIIa when stimulated by phorbol ester. RNA from HEL cells directed synthesis in vitro of a 110,000 Mw precursor for GpIIb and a 92,000 Mw precursor for GpIIIa, indicating that the synthesis of each glycoprotein is directed by a separate mRNA. mRNA from the K562 cells directed only the synthesis of a 92,000 Mw protein. These results suggested that GpIIb and GpIIIa are the products of separate genes. (See: "The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa, Silver, etal. Blood 69:1031, 1987). GpIIb and GpI1Ia appear to be members of a family of membrane adhesive proteins which play a role in cell-cell and cell-matrix interaction. GpIIb is the larger of the two units of the platelet receptor and is composed of two disulfide linked subunits. Bennett has prepared cDNA clones for human GpIIb from aAgt 11 expression library

2 prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequenced, revealing a continuous open reading frame encoding both GpIIb subunits. The cDNA encodes 1039 amino acids; 137 constituting the smaller subunit and 871 representing the larger subunit, while 30 represent the NH4+ signal terminal peptide. No homologies were found between the larger and smaller subunits. The smaller subunit contained a 26-residue hydrophobic sequence near its COO- terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids each in the larger unit are homologous to sequences for the calcium binding sites of calmodulin and troponin C. Northern Blot analysis, using HEL cell RNA, indicates that the mature mRNA coding for GpIIb is 4.1 kilobases in size and shows homologies with the sequences for fibronectin and vitronectin receptors. /V//y ~ R R T Slg hA- L pcpTr de <<oo T2ANS Y1EM~f~~/f /~4p -t'Je Fig. 1 Schematic map of the GpIIb precursor: The larger and smaller subunits, are termed a and 8. The dark shaded area represents a possible signal peptide, while the stippled area represents the possible transmembrane region. The 4 cross-hatched bars are regions homologous to the calcium binding sites of calmodulin and troponin C. The region filled with horizontal lines is a likely cytoplasmic region. Possible N-linked glycosylation sites are indicated by solid triangles. Dr. Bennett has also identified a 4 amino acid sequence in the a chain of GpIIb (arginine-glycine-aspartic acid-serine) which blocks fibrinogen binding to its receptor competitively. Ann Butler in Bennetts group has recently isolated a cDNA from the HEL cells expression library for GpIIIa. Two clones have been identified consisting of 3.7 and 3.8 Kilobases. Each contains a subunit of 1.6Kb. She has sequenced the part coding for GpIIIa and found no homologies with the code for the Gpllb glycoprotein. It codes for about 2300 nucleotides of which 1636 are untranslated. There are 762 amino acids in the peptide sequence. The initial sequence of the protein starts with a methionine, which is preceeded by a hydrophobic signal peptide. The transmembrane end is at the COO- terminal. The amino acid chain contains 56 cysteins which are mostly incorporated into 4 cystein-rich repeats, which may represent ligand binding sites homologous to those of the LDL and insulin receptors. `' °< >I < (3 Future nlans: Create mutants of the two glycoproteins expressed in mammalian cells and determine how they influence platelet interactions with fibrinogen. Develop

3 drugs which may interact with GpIIb and GpIIIa. Evaluate the role of the receptor in cancer and examine further how this receptor complex inter-relates with other cell-cell or cell-matrix complexes which contribute to intercellular organization. Comment: Dr. Bennett appears to have made considerable progress in achieving his original goals. It now remains to be seen how he will use this knowledge in his further studies on the role(s) which such adhesive proteins play. The study is still relevant to further understanding of clotting mechanisms and may perhaps better define some role for platelets in metastatic cancer. As such, it would appear to merit continued support by CTR. DHF Publication in Press (See: Structure of the platelet membrane glycoprotein IIb: Homology of the oc subunits of the vitronectin and fibronectin membrane receptors: PONCZ, et al. J. B. C. 262, 1987)