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TiAt: Cur.vcii. Fot:71'oBacco Rr•.Sr•.Axcil-U.S.A.. Iwc. NETMORANIDLIN! Tn: W. U. Gardner and Staff FRCM: D. H. Ford and D. Stone HKI1920005 March 26, 1980 SOB.7FX,T: Grant 1261. JaMes C. lee, Ph.D., St. Louis University, St. Louis, Nb. "Effects of Ligands on Platelet Mic'rotu~te Assenbly." SITE VISIT on March 20, 1980. The first studies perfozmed by Dr. $ee in this progran have utilized neurotubulin because it is readily available in pure form and the best characterized. He has used~it mainly to establish his technical procedures, widch has taken about the first six months of the grant period. Since antibod.ies to neuro and platelet tubulin cross-react well, he feels that the early data abtained -witta neurotubulisl will prolasbly relate well with the data he will be eollecting soon fran human platelet tubulin, even though the two proteins are not ecmpletely identical. Brain tubulin is very similar to platelet tubulin: antibodies cross'-react and they exhibit the same stoidrnietiy to ligands, i.e., nicotine, vinbiastine, and colchicine. He expeats qualitative similarity but anticipates quantitative differences. He is now in the process of preparing tubulin fran haoan platelets obtained frun the Red Cross, which can provide data a's to age, sex and snrsxing history. He will also attenpt to determine to what degree alcohol, coffee, tea, etc., are used. This material will be used to test the hypothesis that platelet aggregation is related irrevocably to integrity of the microtubule bundles, i.e., microtubu).es must disassetnble for aggregatiari to occur. Believes nicotine (N) Turnover of Microtubule Tubulin hicotr.,L Ac4i oh 0- s Tublin Nicotine ma;r effect either/both assembly or disassembly, but not the preformed microtubule, ie. effects either end but probahly mostly assembly. In contrast colchicine complexes with tubulin and inhibits assembly.

timulates microtubule diasse,nbly leading to platelet aggregation. Nicotine prevents ,olymerization of tubulin into microtubules (see page 1). Microtubule assembly protein MAP) believed to protect against disaggregation caused by nicotine as shown in ?reliminary work (see progress report), while cAMP should stabilize the microtubule bundles. N f{®1920d06 Nicotine has been shown to cause disaggregation of micrmtubules. This is apparently caused by a binding of 4 molecules of N to tubulin before it beoanes incorporated at the end of a microtiabulp bundle. (Such bundles are always in a dynami.c equilibrium, in a sense fonning at one end and disaggregating at the other end.) As the N-labeled tubulin incorporates into the tubule, two Ns are given off and there occurs a confoimatiarnal change in the newly incorporated tubulin at the end of the microtubule unit which then prevents aoc;uisition of additional tubulin molecules, thus breaki.ng the secuence of tubulin formation. Since depolymerization continues at the other end, the tubule is eventually all disaggregated. Lee's evidence is that the primazy effect of N is at the polymerization end of the tubule, blocking developxnent of the tubule. (He utilizes turbidity measuresnents to determine the equilibrium constant of tubulin growing into the forniation of mtcrotuba].es. 7he reauested (;tiilford spectxgphotometer is essential for this measurement. He has an old Beclanan, which is over-used that is available, but not cc.nparable to the G:ilford for speed and accuracy. Does N alter microtubule function before the tubules have totally disaggrbgated? Doesn't know yet, but plans to check, but will do this with the platelet tubulin rather than neurotubulin. Will be interested in detennining if N alters ability of platelets to foan psexiopodia prior to aggregation, which occurs when the microtubules disaggregate. This aspect of the prorject will_ require the Aggregation meter specifically and cannot be done without it. Will cAtP counteract the N effect and favor tubulin polymerization? This is shown in Table II of the pro3ress report which indicates that increasing c1M ooncentrat.:.cn favors microtubule asserhbly, thus inhibiting platelet aggregation, and inhibiting the N effect of facilitating microtubule disassenbly. tM and dW did very little in this systen. Figure 5 illustrates that cRNP is firmly bound to tubulin since tubulin fluorescence decreases as more cAPIP is bound. Bignami has reported in Thrombosis and Hemostasis (37:423, 1977) that platelets aggregate faster in young than in older snokers and that younger smokers are mdre affected by N; therefore, with platelet tubulin Lee plans to campare effects of N on tubulin and citicxotubules in smokers and nonstakers in relation to age, sex and snoking history. Lee believes one of the regulating proteins involved in microtutz_ile asse.nbly (MAP) may be missing or in low amounts in younger individuals. Therefore, he will do an in vitro assay (see Figure 2 of progress report) to determine to what degree MAP inh bits the N interference in microtubul.e assembly and will also determine levels of NIl4P present in relation to age. Since writing the progress report he has been investigating the effect of N on the number of sH groups exposed in tubulin, using BlIn-ans reagent (5,5'-dithio-2-nitro benzoic acS.d). This reacts tdth sulfydril groups to give a color at 412nn, thus measuring the number of such groups available. As the Figure below shows, N increases the number of such groups exposed:

3 HKI192aoo7 s C 4+dwst T&ere4'~, w,err k+r~owf IV -SH.fre..pt a"& N vXyesnwve I i I ne (i-cc) Similar studies done to measure the effect of N on exposure of tryptophane molecules . due to perturbation in neighboring parts of the protein molecule at 317m showed that N had no effect. Note: All of the studies performed thus far have beenk done with very high levels of N. He will use doses ocaparable to those encountered in smoking with the human tubulin studies. He anticipates human p].atelet tubulin will be much more responsive than neurotubulin. Preliminary work has established his procedures. Techniques used to purify bovine tubulin will be used for obtaining hunan platelet tubulin, as follaas: (1) Differential centrifagation -10, platelet rich fraction. (2) Hotngenize fraction in Viritis. (3) Centrifuge and remove supernatant containing tpbulin. (4) Heat at 370 C to form microtubules. (5) Centrifuge to form pellet of microtubules. (6) Resuspend pellet and chill to 0° C to disaggregate tubules. (7) Centrifuge and reheat supernatant at 370 C to reform microtubules. (8) Spin and resuspend pellet and chill to disaggregate again. Repeat as often as necessar_v• to obtain cxaplete purification of tubulin. With the pure tubulin he will be in a position to study the effects of age and sex in relation to ssroking history on microtubule formation. Platelet aggregation would presumably be studied in platelets before ramving tubulin and the responses observed related to how nicotine, cANP and MAP affect the parifie3 tubulin later. once nicotine work is canpletefl, Lee will still have the study with cotinine and aspirin to undertake. We do not see this study being oampleted in less than three years. Budget. The aggregation meter is essential for ccmplieting the platelet aggregation study. The new Guilford spectrometer is also essential, although he has an eld BecJanan available. These two instruments are not carpar,able, though they do the same job. The Guilford is much faster and more accurate. Half of the Guilford will be paid for by another grant. Deborah Fields is a graduate student who is only doing part-time lab work this year (fu11-time next year). 17r.•hee indicated that he has no other source of funds for her and the stipend which she receives includes $2,000 which goes forr tuition and taxes. Lee indicated that this arran9enPnt was standard at St. Louis U. However, $6,000 does sesr like a very hanwisam graduate student stipend, but cutting it by $2,000 to make it more oampzrable to sc2xools elsewhere would not appreciably reduce the cost of the grant. over . . . .

HK®1920008 Cament. We would continue support at amuzt requested. The two iten.s of e.; ,;pmmt are essential for the investigatioh to be perfonned. Denying them would probably resuit in enasculation of the project. Lee appears to be a bright young man who has approached his problem intelligently ard who should pravide sa~i~e interesting data on nicotine effects on platelet function by the time he is through with the study. D. H. Ford D. Stone /ek