“Thrombin” Receptor-directed Ligand Accounts for Activation by Thrombin of Platelet Phospholipase C and Accumulation of 3-Phosphorylated Phosphoinositides*

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides , i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation.
We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.
* This work was supported by National Institutes of 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 therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8584.
ll To whom correspondence should be addressed. Fax: 802-656-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 are responsible for elevating intracellular [Ca"] and activating PKC, whereas the latter response is associated with mitogenic effects in cells such as fibroblasts (6). The mechanism by which thrombin activates platelets has been a subject of debate (7). Although thrombin must be proteolytically active to be an effective platelet agonist @), a role for saturable binding of thrombin to the platelet, in keeping with occupancy of a thrombin receptor, has been suggested (9). The continuous presence of thrombin has been reported to be necessary for the full activation of PLC, based upon inhibitory studies with hirudin, added at various intervals after thrombin (10, 11). Hirudin, in binding thrombin, renders thrombin unavailable to saturable sites on the platelet and interferes with thrombin's proteolytic function, thereby failing to distinguish between the two potential actions of thrombin. To make such a distinction possible, we have employed in the present studies an inhibitor (DAPA), described by Nesheim et al. (12), which can rapidly inactivate thrombin's proteolytic activity in situ without, we have confirmed, impairing binding (13).
Recently, the cloning of a functional "thrombin" receptor has permitted a major advance in our understanding of how thrombin can activate platelets (14). Thrombin cleaves a seven-transmembrane domain receptor, and the resulting N-terminal tethered ligand at the cell surface is thought to bind to another region of this receptor. A peptide sequence that duplicates a portion of the tethered ligand, SFLL-RNPNDKYEPF, has been found to cause platelet aggregation and secretion (14). The activation of PLC or 3-PPI accumulation in response to this ligand, however, has not been characterized nor has any possible additional role of thrombin in this setting been examined. Such studies are also presented here.

MATERIALS AND METHODS
Radioisotopes and reagents were obtained as described previously (3,4). a-Thrombin (1 unit/ml = 10 nM) and DAPA were generous gifts from Dr. K. G. Mann (University of Vermont, Burlington, VT).
within the Arg-binding pocket (15). Both bind with high affinity to platelets,' comparable with that of 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 incubation with different agonists at varied concentrations for different 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 with 2 p~ U46619 (thromboxane A, mimetic) in the presence or absence of DAPA or hirudin. In experiments with PDBu shown in Fig. 2, aliquots of reaction mixture were removed after 75 s and proteins resolved and quantitated after sodium 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 as described (4).
Resolution and Quantitation of 32P-Labeled Lipids-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 separation of 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 containing PtdOH was extracted with chloroform/methanol/HCl, applied to boric 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 scintillation spectrophotometry. PtdInsP, and PtdInsP,/ATP regions were each scaped from oxalate plates, deacylated, resolved by HPLC, and quantitated as described (4). In some cases, PtdOH was also deacylated and resolved and quantitated after HPLC (3).
Thrombin Platelet Binding Studies-Platelets were isolated by gel filtration using a Sepharose 2B column as published (20, 21).
a-Thrombin was labeled on the day of use with fluorescein by treatment 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-measured fluorescence intensity a t 530 nm as described (20, 21). a-Thrombin was employed at 9 nM and DAPA a t 3 p~. DAPA was either added simultaneously with or 10 s after 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 amino acids (Peninsula Laboratories). The following side chain-protecting groups were used S(benzyl), R(tosyl), D(0-benzyl ester), K(2-chlorobenzyloxycarbonyl), E(O-benzyl ester). Asparagine was coupled as the 1-hydroxybenzotriazole ester, and leucine was double coupled. The peptide was cleaved from the resin and the side 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) acetic acid and by chromatography on a 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 peptide was confirmed by amino acid analysis following acid hydrolysis. Molecular weight was calculated a t 1721.

RESULTS AND DISCUSSION
We have drawn two major conclusions regarding the activation of platelet PLC and 3-PPI accumulation by thrombin. 1) Stimulation of both events is dependent upon the 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 activation effects of thrombin completely, without potentiation by thrombin, thrombin's effects are most prob-L. Leong, R. A. Henriksen, J. C. Kermode, S. E. Rittenhouse, and P. B. Tracy, manuscript in preparation. 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 activation by 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 and thereby inactivated thrombin maximally in 2-3 s, as did hirudin. This was confirmed by adding platelets to a mixture of DAPA + thrombin or hirudin + 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  Fig. 1. Inhibition after addition of inhibitor at t , was calculated as 100 -(dpm s. Hir, hirudin. t~,ll.l+lnh,~,ldpm t,)/(dpm tyi,,(.,,Inhl~,ldpm t,)100. In the case of *, total incubation period was 60 s rather than 90  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 inhibitory event was clearly blockage of thrombin's proteolytic function, both initially and after lo-, 20-, 30-, or 60-s exposures to thrombin (Table I). Any role for saturable binding of thrombin to the platelet in modulating phosphoinositide metabolism is thus most likely explained in terms of binding to an enzymatic substrate as opposed to a receptor coupled to an intracellular effector. PtdOH accumulation (PLC activation) was consistently more impaired by each inhibitor than was accumulation of 3-PPI (Table I). Further, addition of excess PPACKthrombin, prior to thrombin addition, affected neither PtdOH nor 3-PPI (not shown). This finding indicates that proteolytically active thrombin need not even bind to all saturable sites on the platelet to achieve its agonist effects on phosphoinositide metabolism.
Since we have demonstrated recently that maximal accumulation of 3-PPI is dependent upon PKC activation and that PKC activation is necessary but not sufficient for the full 3-PPI response (3), we wondered what the additional requisite factor might be, apart from PLC-derived second messengers. We therefore added agents that would increase PKC activity (PDBu), monitored by P47 phosphorylation, and elevate cytosolic [Ca"] (A23187) and examined the effect of saturating amounts of two different proteolytically inactive thrombins (PPACK-thrombin and TQII) upon accumulations of 3-PPI. This was compared with the effects of proteolytically active thrombin, which stimulated PKC to an extent similar to that achieved with PDBu. The results summarized in Fig.  2 illustrate that the missing factor is clearly not saturably bound thrombin, consistent with the findings in Fig. 1. Neither form of proteolytically inactive thrombin altered the 3-PPI 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.
Finally, we turned to the question of whether thrombin's essential proteolytic effects could be accounted for by creation of tethered ligand. Fig. 3 shows that, in response to either thrombin ( 2 units/ml) or SFLL (400 PM), PtdIns(3,4)PZ increased in a manner sustained for at least 60 s. PtdIns (3,4,5)P, increased rapidly to a maximum level within 30 s (not shown). In contrast, PtdOH accumulated more rapidly in response to SFLL than to thrombin but leveled off after 20 s, when SFLL was the agonist, whereas PtdOH continued to increase in response to thrombin up to (Fig. 3) and beyond (Fig. 1) 60 s. This finding for PtdOH can be explained by the continued generation of tethered ligand by thrombin, eventually achieving a level exceeding that mimicked by SFLL, and by the greater requirement of PLC uersus 3-PPI activation for agonist concentration (see below). It is evident (Fig. 4) that the 3-PPI response is more sensitive to either thrombin or SFLL than is PLC activation. Concentrations of agonist producing a half-maximal 3-PPI response were 0.075 units/ml thrombin and 25 FM SFLL; those leading to half-maximal PtdOH were 0.5 units/ml thrombin and 300 PM SFLL. This difference most likely accounts for the lesser sensitivity of 3-PPI accumulation (uersus PLC activation) to the addition of DAPA or hirudin post-thrombin (Table I). Significantly, when SFLL and thrombin were added simultaneously to platelets, stimulation of 3-PPI was not additive but was only marginally greater than when either was added alone (Fig. 3). Maximum platelet enzymatic capacity had not been reached, however, since we have shown that the potent G protein-directed agonist, GTPyS, can achieve a stimulation that greatly exceeds that for thrombin (3,4). Thus, binding of thrombin or additional proteolytic targets for thrombin apart from tethered ligand generation contributed nothing more in promoting PLC activation and 3-PPI accumulation.
Making a comparison between thrombin/tethered ligand and SFLL in terms of stoichiometry is complicated by three factors. 1) Tethered ligand, as a function of thrombin's sustained proteolytic activity on platelets, is apparently generated over the time course (at least 60 s) of platelet incubations with thrombin, whereas SFLL is present maximally at the outset; 2) SFLL may not be the optimal size or in optimal conformation for binding to the platelet receptor; and 3) tethered ligand is generated near its receptor, and therefore its efficiency of binding or binding rate is most likely much greater than that of SFLL. Indeed, were all of 400 FM SFLL to bind to 2 X lo9 platelets/ml, there would be about 12 x lo7 binding sites for this peptide on the platelet. This number is probably 3-4 orders of magnitude too high for a functional receptor. Thus, in the absence of an assay to quantitate the amount of tethered ligand generated over the course of incubation with thrombin and measurement of specific binding of SFLL, stoichiometries cannot be compared. However, lack of potentiation of SFLL's effects by thrombin would indicate that thrombin is most likely not acting either proteolytically at additional sites distant from the cleavage site which generates the tethered peptide or as a direct ligand in order to exert its effects on phosphoinositide metabolism. These findings support the conclusion that the majority of thrombin's effects on phosphoinositide metabolism relate to the generation of tethered ligand.
We have shown that a tethered peptide analogue activates platelet PLC and 3-PPI accumulation in a manner unmodified by proteolytically active thrombin and can achieve maximal effects similar to those of thrombin. It is therefore evident that future work directed toward an understanding of how thrombin-activated PKC, G protein(s), and other factors regulate PLC and 3-PPI accumulation should focus on the receptor target (14) of the tethered ligand. Our data would also indicate that it is primarily receptor occupancy by tethered ligand that, in addition to activating PLC/PKC, plays a direct role in promoting 3-PPI accumulation.