From the $Department of Biochemistry, University of. Vermont College of Medicine, Burlington, Vermont 05405-. 0068 and the §Department of Biochemistry, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Communication
Vol. 266,No. 28 Issue of October 5,pp. 18435-18438,1991 0 1991 by The American Society fo; Biochemistry and Molecular Biology, Inc. Printed in U.S. A .
Thrombin is one of the most potent natural agonists known for platelets. It rapidly activates phospholipase C (PLC)’ (1, 2) and, in a manner dependent in part upon the activation of PKC (3), stimulates the accumulation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (4,5),collectively referred to as“3-phosphorylated phosphoinositides” or “3-PPI.’’ The former response generates second messengers that areresponsible for elevating intracellular [Ca”] and activating PKC, whereas the latter response is associated with mitogenic effects in cells such as (Received for publication, June 24, 1991) fibroblasts (6). The mechanism by which thrombin activates platelets has been a subject of debate (7). Although thrombin Ru-song Huang$, Alexander SoriskyS, must be proteolytically active to be an effective platelet agoWilliam R. Church$, Elizabeth R. Simonsg, and Susan E. RittenhouseSll nist @), a role for saturable binding of thrombin to the platelet, in keeping with occupancy of a thrombin receptor, From the $Department of Biochemistry, University of has been suggested (9). The continuous presence of thrombin Vermont College of Medicine, Burlington, Vermont 05405has been reported to be necessary for the full activation of 0068 and the §Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts PLC, based upon inhibitory studies with hirudin, added at various intervals after thrombin (10, 11).Hirudin, in binding thrombin, renders thrombin unavailable to saturable siteson Using three experimental approaches, we have addressed the questions of whether the presence of sat- the platelet and interferes with thrombin’s proteolytic funcurably bound thrombin playsa role in potentiating the tion, thereby failing to distinguish between the two potential activation of plateletphospholipaseC(PLC) and/or actions of thrombin. To make such a distinction possible, we accumulation of the 3-phosphorylated phosphoinosihave employed in the present studies an inhibitor (DAPA), tides (3-PPI), i.e. phosphatidylinositol3,4-bisphosdescribed by Nesheim et al. (12), which can rapidly inactivate phateandphosphatidylinositol3,4,5-trisphosphate, thrombin’s proteolytic activity in situ without, we have conand whether the generation of tethered ligand(Vu, T- firmed, impairing binding (13). K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. Recently, the cloning of a functional “thrombin” receptor (1991) Cell 64, 1057-1068) by thrombin can account has permitted a major advance in our understanding of how fully for thrombin’s proteolytic effects in activating thrombincanactivateplatelets(14).Thrombin cleaves a platelets, as gauged by the above parameters.We have seven-transmembrane domain receptor, andthe resulting 1) measured PLC activation or 3-PPI after we have N-terminaltethered ligand at the cell surface is thought exposed platelets to thrombin for various periodsand to bind to another region of this receptor. A peptide sequence either blocked thrombin’s proteolytic activity without that duplicates aportion of the tethered ligand, SFLLinterrupting its binding or blocked both binding and RNPNDKYEPF, hasbeen found to cause platelet aggregation proteolytic activityof thrombin; 2) attempted to poten- and secretion (14). The activation of PLC or 3-PPI accumutiate 3-PPI accumulation, using combinations of pro- lation in response tothis ligand, however, hasnot been tein kinaseC stimulation, Ca2+ elevation, and saturat- characterized nor has anypossible additional role of thrombin ing but proteolytically inactive thrombins; 3) and com- in this settingbeen examined. Such studies arealso presented pared the activation of platelets by thrombin with here. activation by the “thrombin” receptor-directed peptide,SFLLRNPNDKYEPF(SFLL; a portion of the MATERIALSANDMETHODS tethered ligand created by thrombin’s proteolytic acRadioisotopes and reagentswere obtained as described previously tivity), and examined the effect of thrombin on this (3, 4). a-Thrombin (1 unit/ml = 10 nM) and DAPA were generous latter activation. gifts from Dr. K. G. Mann (University of Vermont, Burlington, VT). We conclude that the initial and sustained effects of Hirudin was purchased from Sigma. The Partisphere SAX HPLC [32P]PtdIns(3,4)P2 and [”PI thrombin in stimulating PLC and the accumulation of column was from Whatman. 3-PPI are completely attributable to thrombin’s pro- PtdIns(3,4,5)P3 were generously contributed by Dr. Peter Downes (University of Dundee, Scotland). PPACK-thrombin and Quick IIteolytic activity. Further, thrombin’s effects in prothrombin were kindly provided by Dr. P. Tracy (University of Vermoting these responses can be accounted for by the mont) and Dr. R. A. Henriksen (East Carolina University School of actions of SFLL peptide,and by implication, formation Medicine, NC), respectively. Both thrombins are proteolytically inof tethered ligand. active, the former because of chemical alteration of the active site
“Thrombin” Receptor-directed Ligand Accountsfor Activation by Thrombin of Platelet Phospholipase C and Accumulation of 3-Phosphorylated Phosphoinositides*
and the latter due to
asingle amino acid substitution at Gly-558
* This work was supported by National Institutesof Health Grants HL-38622 (to S. E. R.), HL-15335 (to E. R. S.), and HL-40467 (to W. R. C.) and a Canadian Medical Research Council award (to A. S.). The blood drawing services of the General Clinical Research Center (GCRC RR109) of the Medical Center Hospital of Vermont are gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll To whom correspondence should be addressed. Fax: 802-6568584.
The abbreviations used are: PLC, phospholipaseC; PKC, protein kinase C; PtdIns, phosphatidylinositol (locants of phosphates indicated in parentheses); 3-PPI, 3-phosphorylated phosphoinositides; PtdOH, phosphatidic acid; FACS, fluorescent-activated cell sorter; DAPA,dansylarginine N-(3-ethyl-1,5-pentanediyl)amide;PPACK, phenylyl-prolyl-arginyl-chloromethylketone; PDBu, P-phorbol dibutyrate; SFLL, SFLLRNPNDKYEPF; P47, platelet protein target of PKC; QII, Quick 11-thrombin; HPLC, high pressure liquid chromatography;FITC, fluorescein isothiocyanate; GTPrS, guanosine 5’-O-(thiotriphosphate).
18435
18436
Phosphoinositide Metabolism and Platelet Thrombin Receptor
within the Arg-binding pocket (15). Both bind with high affinity to platelets,’ comparable with thatof a-thrombin. Preparation and Incubation of Platelets-Human platelets were isolated, labeled with [”PIP,, and washed as described previously (3, 4). ‘”P-Labeled platelets were incubated at 37 “C for 5 min prior to at varied concentrations for differincubation with different agonists ent periods. When DAPA or hirudin was employed, thrombin = 2 units/ml, DAPA = 3 PM, and hirudin = 20 units/ml. In some cases, platelets were activated with2 p~ U46619 (thromboxane A, mimetic) in thepresence or absence of DAPA or hirudin. In experiments with PDBu shown in Fig. 2, aliquots of reaction mixture were removed after 75 s and proteinsresolved and quantitated aftersodium dodecyl sulfate-polyacrylamide gel electrophoresis as described (3). Incubations were terminated with 3.75 volumes of chloroform, methanol, 1 N HCI (1:2.5:0.25, v/v/v) and extracted asdescribed (4). Resolution and Quantitation of 32P-LabeledLipids-PtdOH was used as an index of PLC activation,since we have shown that at least 90% of PtdOH generated in the period of interest arises from the action of diglyceride kinase on diglyceride derived from PLC action on phosphoinositides (17). The initial separationof phosphoinositides and PtdOH was carried out on oxalate-impregnated silica gel TLC plates as described (18). Identification of lipids migrating on TLC was made by comparison with migration of known standards (Sigma, >98% purity), which were located by 1, staining. The area of silica containingPtdOH was extractedwith chloroform/methanol/HCl, applied toboric acid-impregnated LK5 TLC plates (19), and resolved using chloroform/methanol/H20/NHa (120:75:8:4, v/v/v/v). The 32P was quantitated by scintillationspectrophotometry.PtdInsP,and PtdInsP,/ATP regions were each scaped from oxalate plates, deacylated, resolved by HPLC, and quantitated asdescribed (4).In some cases, PtdOH was also deacylated andresolved and quantitated after HPLC (3). Thrombin Platelet BindingStudies-Platelets were isolated by gel afiltration usinga Sepharose 2B column as published(20,21). Thrombin was labeled on the day of use with fluorescein bytreatment with fluorescein isothiocyanate, yielding FITC-thrombin (22, 23). Activity of FITC-thrombin was confirmed by membrane potential and cytoplasmic calcium changes induced in FITC-thrombin-exposed platelets. FITC-thrombin binding to human platelets was determined by FACS-measuredfluorescence intensity a t 530 nm asdescribed (20, 21). a-Thrombin was employed at 9 nM and DAPA a t 3 p ~ DAPA . was either added simultaneously with or s10after a-thrombin. Peptide Synthesis-Peptide SFLLRNPNDKYEPF (14)was synthesized as the peptide amide using a Biosearch SAM I1 automatic peptide synthesizer and the solid-phase method of Merrifield (16), with 4-methylbenzylhydrylamine resin (Advanced Chem Tech) and t-butyloxycarbonyl-blocked aminoacids(PeninsulaLaboratories). The following side chain-protecting groups were used S(benzyl), R(tosyl), D(0-benzyl ester),K(2-chlorobenzyloxycarbonyl),E(O-benzyl ester). Asparaginewascoupled as the 1-hydroxybenzotriazole ester, and leucine was double coupled. The peptide was cleaved from the resin and theside chain-protecting groups removed using anhydrous hydrogen fluoride a t 4 “C for 1 h in the presence of 10% (v/v) anisole, 10% (v/v) dimethyl sulfide, 7.5% (v/v) p-cresol, and 2.5% (v/ v) thiocresol. The crude peptide was extracted with 50% acetic acid and lyophilized. Purification of the peptide was by chromatography on a Sephadex G-10 column (100 X 2.5 cm) equilibrated in 10% (v/ v) aceticacid and by chromatography ona preparative Aquapore octyl reverse-phase column (Brownlee) using a 0.05% trifluoroacetic acid/H,O and 0.05% trifluoroacetic acid/acetonitrile gradient elution. T h e composition of the peptidewas confirmed by amino acid analysis following acid hydrolysis. Molecular weight was calculated a t 1721. RESULTSANDDISCUSSION
We have drawn two major conclusions regarding the activation of platelet PLC and 3-PPIaccumulation by thrombin. 1)Stimulation of both eventsis dependent uponthe sustained presence of proteolytically active thrombin, with no detectable role for saturable thrombin binding, and 2) since a peptide portion of the“thrombin” receptor’s tethered ligand can mimic such activationeffects of thrombin completely, without potentiation by thrombin, thrombin’s effects are most probL. Leong, R. A. Henriksen, J. C. Kermode, S. E. Rittenhouse, and P. B. Tracy, manuscript in preparation.
7“
0 10 20 30 40 50 60 70 80 90100
f
f
t
time (s)
FIG. 1. Effects of DAPA or hirudin on accumulations of PtdIns(3,4)P2and PtdOH in human platelets exposed to athrombin. Human platelets (2 x 109/ml),labeled with 3’P as described, were incubated with buffer or 2 units/ml thrombin for up to 90 s a t 37‘C. Enough DAPA (3 p ~ or) hirudin (20 units/ml) to completely inactivate thrombin were added a t times indicated by arrowsaftera-thrombin.PtdIns(3,4)P2andPtdOH were resolved and quantitatedas described under “Materials andMethods.” Values are expressed as the incremental disintegrations/min abovebasal levels and the average & range for a representative of three experiments performed induplicate. A, DAPA; 0,hirudin; W, no inhibitors.
ably accounted for by the generation of tethered ligand. We addressed the hypothesis that, although initiation of one or both aspects of platelet phosphoinositide metabolism by thrombin would be dependent on thrombin’s proteolytic activity, the sustained requirement for thrombin (10) might be dependent upon receptor occupancy, apart from proteolysis. To block thrombin’s proteolytic activity in situ, without impairing thrombin binding, we added DAPA at different intervals after thrombin. To block both thrombin’s binding to and proteolytic interaction with platelets,hirudin was similarly added. DAPA or hirudin, when added mixed with thrombin, completely blocked accumulation of 3-PPI and PtdOH (used as a monitor of PLC activation (17)). Neither DAPA nor hirudin impaired such activationby the thromboxane A2 mimetic, U46619 (not shown), indicating that platelet metabolism, per se, was not inhibited. Analysis by FACS of fluorescently labeled thrombin binding to platelets indicated that maximal binding was achieved within 3 s of mixing of platelets and thrombin and that addition of DAPA to this mixture did not impair the binding of thrombin to platelets. DAPA bound to andthereby inactivatedthrombin maximally in 2-3 s, as did hirudin. This was confirmed by adding thrombin or hirudin + platelets to a mixture of DAPA thrombin uersus adding platelets to DAPA, or hirudin, or buffer, and thrombin in separate droplets. The rate of inactivation by either inhibitor was calculated based upon the initial rate plot of PtdOH formation in response to thrombin, without inhibitors. The amount of PtdOH formed when inhibitors were present, but not premixed, with thrombin corresponded to that observed after 2-3 s exposure to thrombin
+
18437
Phosphoinositide Metabolism and Platelet Thrombin Receptor TABLEI Inhibitory effects of DAPA or hirudin when added a t various intervals post-thrombin Platelets were incubated as in Fig. 1. Inhibition after addition of inhibitor at t , was calculated as 100 - (dpm t~,ll.l+lnh,~,l - dpm t,)/(dpm tyi,,(.,,Inhl~,l - dpm t,)100. In the case of *, total incubation period was 60 s rather than 90 s. Hir, hirudin. Inhibitor at inhibited Event
Exp. 1 PtdOH PtdIns(3,4)P2 Exp. 2* 70 PtdOH 25 PtdIns(3,4)Pz Exp. 3 PtdOH
Inhibitor at
10 s
20 s
DAPA
Hir
DAPA
86 f 7 63 f 5
86 f 5 66 f 5
79 k 5 48 f 7
72 k 4 50 k 3
58 15
62
65 k 1
59 f 10
72 27 76 f 2
16 71 f 1
Hir
Inhibitor a t 30 s
Inhibitor at
60 s
DAPA
Hir
DAPA
Hir
50 f 2
51 + 6
31 k 3
30 f 7
alone. As can be seen in Fig. 1 and Table I, there was no significant difference between the effects of DAPA and hirudin on the inhibition of PtdOH or 3-PPI (as represented by PtdIns(3,4)P2) accumulation. Sustained receptor occupancy (DAPA experiment) thus had no potentiating effect independent of proteolysis. The crucial inhibitoryevent was 3 2 600 clearly blockage of thrombin’s proteolytic function, both inix tially and after lo-, 20-, 30-, or 60-s exposures to thrombin 400 (Table I). Any role for saturable binding of thrombin to the platelet in modulating phosphoinositide metabolism is thus 200 most likely explained in terms of binding to an enzymatic substrate asopposed to a receptor coupled to an intracellular effector. PtdOH accumulation (PLC activation)was consistPPACK-THR THR PDBu+ PDBu+ PDBu+ OR A 2 3A1 28 37 1+A8 27 3+ 1 8 7 ently more impaired by each inhibitor thanwas accumulation TQ II TQ II PPACK-THR of 3-PPI(TableI).Further,addition of excess PPACKFIG. 2. Effects of proteolytically inactive thrombin on 3thrombin, prior to thrombin addition, affected neither PtdOH PPI accumulation. Platelets labeled as in Fig. 1 were incubated for 90 s with buffer, Q11 thrombin (200 nM), PPACK-thrombin (250 nM), nor 3-PPI (not shown). This finding indicates that proteolyteven bind to all saturable sites thrombin (2 units/ml, 20 nM), PDBu (200 nM) + A23187 (1 pM), ically active thrombin need not PDBu + A23187 + QII thrombin, or PDBu + A23187 + PPACK- on the platelet to achieve its agonist effects on phosphoinothrombin ( T H R ) .Incubations were terminatedandphosphoinositides sitide metabolism. and P47 protein resolved and ““Pquantitated as described. Results Since we have demonstrated recently that maximal accuare the average f range of duplicates and are expressed asa percent mulation of 3-PPI is dependent upon PKC activation and of basalvalues (=loo). P47 was includedas a monitor of PKC that PKC activation is necessary but not sufficient for the activation. full 3-PPI response (3), we wondered what the additional requisitefactormightbe,apart from PLC-derived second messengers. We therefore added agents that would increase PKC activity (PDBu), monitored by P47phosphorylation, and elevate cytosolic [Ca”] (A23187) and examined the effect of saturating amountsof two different proteolytically inactive thrombins (PPACK-thrombin and TQII) upon accumulations of 3-PPI. Thiswas compared with the effects of proteolytically active thrombin, which stimulated PKC to an extent similar to that achieved with PDBu. The results summarized inFig. 2 illustrate that the missing factor is clearly not saturably 500 bound thrombin, consistent with the findings in Fig. 1. Nei400 ther form of proteolytically inactive thrombin altered the 3P P I response, even when PLC-dependent second messenger signals were provided independently of PLC activation. Thus, results of both Figs. 1 and 2 point exclusively to the role of proteolysis in implementing thrombin’s effects on both aspects of phosphoinositide metabolism. 600 50 40 Ib 30 20 Finally, we turned to the question of whether thrombin’s TIME (s) essential proteolyticeffects couldbe accountedfor by creation FIG. 3. Effects of a-thrombin, SFLL, or a-thrombin + SFLL of tethered ligand. Fig. 3 shows that, in response to either on accumulations of PtdIns(3,4)P2and PtdOH in human platethrombin ( 2 units/ml) or SFLL (400 P M ) , PtdIns(3,4)PZ inlets with time.Human platelets, as inFig. 1, were incubated with 2 creased in a manner sustainedfor at least 60 s. PtdIns(3,4,5)P, units/ml n-thrombin or 400 p~ SFLL for various times. The data are increased rapidly to a maximum level within 30 s (not shown). representative of two experiments and are expressed as a percent of In contrast, PtdOH accumulatedmore rapidly in response to agonist-free control values (=loo) with error ranges encompassedby leveled off after 20 s, when SFLL symbols. 0, a-thrombin; 0 , SFLL; 0, both; broken line, PtdOH; solid SFLL than to thrombin but line, PtdIns(3,4)P2. was the agonist, whereas PtdOH continued to increase in _1
18438
Phosphoinositide Metabolism and Platelet Thrombin Receptor 900" 700" 500"
SFU(pM)
Thrombin(U/ml)
Effects of a-thrombin or SFLL concentrations on accumulations of PtdIns(3,4)Pzand PtdOH in human platelets. Human platelets, as in Fig. 1, were incubated with indicated concentrations of a-thrombin or SFLL for 60 s. The data are representative of two experiments and are expressed as a percent of agonist-free control values (=loo), with error ranges encompassed by symbols. 0 , PtdIns(3,4)P2;0, PtdOH.
response to thrombin up to(Fig. 3) and beyond (Fig. 1) 60 s. exert its effects on phosphoinositide metabolism. These findThis finding for PtdOH can be explained by the continued ings support the conclusion that the majority of thrombin's generation of tethered ligandby thrombin, eventuallyachiev- effects on phosphoinositide metabolism relate to the generaing a level exceeding that mimicked by SFLL, and by the tion of tethered ligand. greater requirement of PLC uersus 3-PPI activation for agoWe have shown that a tethered peptide analogue activates nist concentration (see below). It is evident (Fig. 4) that the platelet PLC and 3-PPI accumulation in a manner unmodified 3-PPI response is more sensitive to either thrombinor SFLL by proteolytically active thrombin and can achieve maximal than is PLC activation. Concentrationsof agonist producing effects similar to those of thrombin. It is therefore evident a half-maximal 3-PPI responsewere 0.075 units/ml thrombin that future work directed toward an understanding of how and 25 FM SFLL; thoseleading t o half-maximal PtdOHwere thrombin-activated PKC,G protein(s), and other factors regfocus on therecep0.5 units/ml thrombin and 300 PM SFLL. This difference ulate PLC and 3-PPI accumulation should most likely accounts for the lesser sensitivity of 3-PPI accu- tor target (14) of the tethered ligand. Our data would also of DAPA or indicate that it is primarily receptor occupancy by tethered mulation (uersus PLC activation) to the addition ligand that, in addition to activating PLC/PKC, plays adirect hirudin post-thrombin (Table I). Significantly, when SFLL and thrombin were added simultaneously to platelets, stimu- role in promoting 3-PPI accumulation. lation of 3-PPI was not additive but wasonlymarginally Acknowledgments-We wish to thankLaurie A. Ouellette for techgreater thanwhen either was added alone (Fig. 3). Maximum nical assistance with peptide synthesisand purification, Theresa platelet enzymatic capacity had not been reached, however, Davies for assistance with fluorescently labeled thrombin and FACS since we haveshown thatthepotent G protein-directed analysis, S h a m Coughlin for providing an initial sample of SFLL agonist, GTPyS, can achieve a stimulation that greatly expeptide, and Lisa McNaney for assistance with the manuscript. ceeds that for thrombin (3, 4). Thus, binding of thrombin or REFERENCES additional proteolytic targets for thrombin apart from teth1. Rittenhouse-Simmons, S. (1979) J. Clin. Znuest. 63, 580-587 ered ligand generation contributed nothing more in promoting 2. Tarver, A. P., King, W. G., and Rittenhouse, S. E. (1987) J. Biol. Chem. 262,17268-17271 PLC activation and 3-PPI accumulation. 3. King, W. G., Kucera, G. L., Sorisky, A,, Zhang, J., and Rittenhouse, S. E. Making a comparison between thrombin/tethered ligand (1991) Biochem. J., 278,475-480 53454. Kucera, G. L., and Rlttenhouse, S. E. (1990) J. Biol. Chem. 265.,~ and SFLL in termsof stoichiometry is complicated by three 5348 factors. 1) Tethered ligand, as a function of thrombin's sus5. Nolan, R. D., and Lapetina, E. G. (1990) J. Biol. Chem. 265,2441-2445 6. Williams, L. T. (1989) Science 243, 1564-1570 tained proteolytic activity on platelets, is apparently gener7. Berndt, M. C., and Phillips, D. R. (1981) Platelets Biol. Pathol. 2,43-75 ated over the timecourse (at least 60 s) of platelet incubations 8. Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974) J. Biol. Chem. 249,2646-2651 with thrombin, whereas SFLL is present maximally at the 9. Harmon, J. T., and Jamieson, G. A. (1986) J. Biol. Chem. 261, 15928outset; 2) SFLL may not be the optimal size or in optimal 15933 IO. Huang, E. M., and Detwiler, T. C. (1987) Eiochem. J. 242,11-18, conformation for bindingtotheplateletreceptor;and3) 11. Tam, S. W., Fenton, J. W., 11, and Detwller, T. C. (1979) J. B~ol.Chem. 254,8723-8725 tethered ligand is generated near its receptor, and therefore M. E., Prendergast, F. G., and Mann, K. G. (1979) Biochemistry its efficiency of binding or binding rate is most likely much 12. Nesheim, 18,996-1003 greater than thatof SFLL. Indeed, were all of 400 FM SFLL 13. Knupp, C. L. (1988) Thromb. Res. 49,23-36 T-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell t o bind to 2 X lo9platelets/ml, there would be about 12 x lo7 14. Vu,64, 1057-1068 Henriksen R. A., and Mann, K. G. (1989) Biochemistry 28, 2078-2082 15. binding sites for this peptide on the platelet. This number is 16. Merrifield,'R. B. (1963) J. Am. Chem. Soc. 85,2149-2154 probably 3-4 orders of magnitude too high for a functional 17. Huang, R.-S., Kucera, G. L., and Rittenhouse, S. E. (1991) J. Biol. Chem. 266,1652-1655 receptor. Thus, in the absence of an assay to quantitate the 18. Vadnal, R. E., and Parthasarathy, R. (1989) Biochem. Biophys. Res. Comamount of tethered ligand generated over the course of incumun. 163,995-1001 19. Rittenhouse, S. E..(1983) Proc. Nptl. Acad. Sci. U. S. A . 80. 5417-5420 bation with thrombin and measurement of specific binding of 20. 265. Davies. T. A,. Wed. G. J.. and Slmons E. R. (1990) J. Bzol. Chem.. _", SFLL, stoichiometries cannot becompared. However, lack of 11522-11526 T. A., Drotts, D. L., Weil, G. J., and Simons E. R. (1989) J. Biol. potentiation of SFLL's effects by thrombin would indicate 21. Davies, Chem. 264,19600-19606 that thrombin is most likely not acting either proteolytically 22. Lundblad, R. L., Uhteg, L. C., Vogel, C. N., Kingdon, H. S., and Mann, K. G: (1975) Biochem. Biophys. Res. Commun. 66,482-489 at additional sites distant from the cleavage site which gen23. Davles, T. A,, Drotts, D. L., Weil, G. J., and Simons,E. R. (1988) Cytometry erates the tethered peptide or as a direct ligand in order to 9, 138-142
