Identification of Low Molecular Weight GP IIb/IIIa Antagonists That Bind Preferentially to Activated Platelets

  1. Rodney A. Bednar1,
  2. S. Lee Gaul1,
  3. Terence G. Hamill1,
  4. Melissa S. Egbertson3,
  5. Jules A. Shafer2,
  6. George D. Hartman3,
  7. Robert J. Gould1 and
  8. Bohumil Bednar1
  1. 1Department of Pharmacology (R.A.B., S.L.G., T.G.H., R.J.G., B.B.),2Biological Chemistry (J.A.S.) and 3Medicinal Chemistry (M.S.E., G.D.H.), Merck Research Laboratories, West Point, Pennsylvania
     
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    Abstract

    A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [125I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similarKd values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (KdActivated = 5 nM andKdResting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selectiveversus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.

    Platelets play an important role in the pathophysiology of many vascular disorders (reviewed in Coller, 1992; Stein et al., 1989;Harker, 1987). There is considerable biochemical evidence for platelet activation in patients with a variety of vaso-occlusive disorders (Abrams and Shattil, 1991; Rasmanis et al., 1992; Hammet al., 1987; Fitzgerald et al., 1986). The final common step in the formation of platelet thrombi involves the cross-linking of activated platelets by the binding of fibrinogen to the platelet membrane-bound GP IIb/IIIa complex, a member of the integrin superfamily (Plow and Ginsberg, 1989; Phillips et al., 1988; Kieffer and Phillips, 1990; Bennett, 1991). Antagonism of fibrinogen-platelet GP IIb/IIIa interactions is an attractive antithrombotic mechanism for treatment of vaso-occlusive disorders (Coller et al., 1986; reviewed in Coller, 1995, 1997; Gould, 1993). Clinical efficacy of this approach has been validated with the monoclonal antibody abciximab (ReoProTM) (Epic Investigators, 1994; Jordan et al., 1996), which blocks several integrins, and the specific reversible GP IIb/IIIa blocker tirofiban (AGGRASTATTM) (Theroux et al., 1994; Kereiakes et al., 1996). Clinical trials continue with other peptide and nonpeptide antagonists that bind specifically to the GP IIb/IIIa receptors with little or no binding to other integrins (Tcheng, 1996).

    Selective binding of fibrinogen to GP IIb/IIIa on activated platelets and not to resting platelets is critical to fibrinogen’s hemostatic function (Coller, 1992; Phillips et al., 1988). Because inhibition of the binding of fibrinogen to activated receptors is sufficient to prevent thrombosis, effective GP IIb/IIIa antagonists only need to bind to the activated form of GP IIb/IIIa. Selective GP IIb/IIIa antagonists have many theoretical advantages; however, the selectivity of GP IIb/IIIa ligands for binding to resting and activated forms of GP IIb/IIIa has been reported rarely (Kouns et al., 1992; Kunicki et al., 1996). Disintegrins show both selective and nonselective binding (McLane et al., 1994;Hung and Niewiarowski, 1994). We wondered if selectivity was a property exclusive to multivalent protein ligands like fibrinogen and disintegrins or if it was possible to observe selective binding with low molecular weight univalent GP IIb/IIIa antagonists. To identify such compounds, we measured the affinity of antagonists to both the activated and resting forms of purified GP IIb/IIIa. Measurements made directly on resting and activated platelets verified the selectivity that was observed with purified forms of GP IIb/IIIa and demonstrated that it was possible to obtain selective binding with low molecular weight univalent GP IIb/IIIa antagonists. This is the first report of the selectivities of low molecular weight GP IIb/IIIa antagonists, several of which are in clinical development.

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    Methods

    Isolation and purification of GP IIb/IIIa.

    GP IIb/IIIa was purified from outdated human platelets, as shown in fig.1, by a modification of the method ofKouns et al. (1992). Outdated platelet concentrates (Red Cross, Philadelphia) were washed three times in 20 mM Tris-HCl at pH 7.2 containing 150 mM NaCl, and 1 mM EDTA. The platelets, suspended in lysis buffer [1% Triton X-100 (v/v), 20 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 10 μM leupeptin, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 50 μMtransepoxysuccinyl-l-leucylamido-(4-guanidino) butane, pH 7.4], were shaken for 15 hr at 4°C and then centrifuged at 30,000 × g to remove membrane cytoskeletons. The platelet lysate was purified on a Concanavalin A-Sepharose 4B column (Sigma, St. Louis, MO) and the retained proteins eluted in buffer A [0.1% Triton X-100 (v/v), 20 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl, pH 7.4] containing 100 mM methyl-α-d-manopyrannoside (Sigma, St. Louis, MO). Protein eluted from the Concanavalin A column was dialyzed against buffer A and purified further on an RGDS-affinity column. The RGDS-affinity column was prepared by reaction of Sepharose 4B (Sigma, St. Louis, MO) activated with 6-aminohexanoic acid N-hydroxysuccinimide ester with the RGDS peptide. Flowthrough fractions from the RGDS-affinity column were subjected to size-exclusion chromatography on a Sephacryl S-300 (Sigma, St. Louis, MO) column (GP IIb/IIIa fractions did not bind fibrinogen; resting form, form B). Proteins retained on the RGDS-affinity column were eluted with a solution of 3 mM RGDS peptide (Sigma, St. Louis, MO) in buffer A and dialyzed extensively against buffer A. (Pooled fractions bind fibrinogen; activated form, form A.) Purity of the preparations was assessed by SDS-PAGE under nonreduced and reduced conditions (fig.1).

    Figure 1
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    Figure 1

    Purification of activated (form A) and resting (form B) forms of GP IIb/IIIa and SDS-PAGE of the purified receptors. The scheme for purification of human GP IIb/IIIa is illustrated. Reduced and nonreduced SDS-PAGE of the purified receptors (2.5 μg/lane) on 8% Tris-glycine gels (Novex) are shown.

    Equilibrium binding of L-736,622 to purified forms of GP IIb/IIIa.

    The fluorescence spectra of L-736,622, GP IIb/IIIa and the increased fluorescence for their complex are shown in figure2A. Purified GP IIb/IIIa in buffer A (final concentration, 140 nM) was transferred to a fluorescence cell and the fluorescence was recorded at 550 nm with excitation at 340 nm. The fluorescent antagonist L-736,622 containing a dansyl moiety (Egbertson et al., 1996) was added stepwise, and 5 min after each addition, the fluorescence of the solution was recorded. Parallel with these measurements, the fluorescence intensity of L-736,622 at the same final concentrations in buffer A was recorded. The fluorescence intensity, representing the fluorescence change upon binding of L-736,622 to the receptor, was calculated by subtracting the fluorescence of L-736,622 alone from the fluorescence measured for the mixtures of GP IIb/IIIa and L-736,622. This fluorescence, representing specific binding of L-736,622 to the receptor, was plotted against the concentration of L-736,622 (fig. 2B). These data were fitted by nonlinear least-squares to the equation F =Fmax (J − (J2 − (4[Ligand][Receptor])0.5)/2[Receptor]), where [Ligand] and [Receptor] are the total concentrations of fluorescent ligand and GP IIb/IIIa receptor, respectively; Jis ([Ligand] + [Receptor] + Kd); andKd is the apparent dissociation constant of the fluorescent ligand (Hulme and Birdsall, 1992). The accuracy of these Kd values depends on the accuracy of the measured GP IIb/IIIa concentrations, because a GP IIb/IIIa concentration in excess of the Kd values was used to get a detectable fluorescent signal for the bound ligand compared with the background fluorescence and light scattering. The concentrations of the receptors were determined by quantitative amino acid analysis.

    Figure 2
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    Figure 2

    (A) Fluorescence spectra of 1 μM solutions of L-736,622, GP IIb/IIIa and their complex in buffer A with excitation at 340 nM. (B) Saturable binding of L-736,622 to purified GP IIb/IIIa. GP IIb/IIIa (0.14 μM form A) was incubated with the indicated concentrations of L-736,622 at 25°C, and the fluorescence signal from the bound complex was determined. A dissociation constant,Kd, of 3.8 ± 1.5 nM, was estimated by a nonlinear least-squares fit of the fluorescence signal as described under “Methods.”

    Stopped-flow kinetic studies of the binding of L-736,622 to purified forms of GP IIb/IIIa.

    The stopped-flow measurements of the binding of L-736,622 (0.6–12.8 μM) to purified GP IIb/IIIa (0.12 μM) in buffer A were carried out under pseudo first-order conditions by use of a DX 17 MW stopped-flow spectrometer with fluorescence detection connected to an Archimedes 420/I computer (Applied Photophysics, Leatherhead, England). The excitation wavelength was 340 nm and emission intensity was recorded with a 530-nm cut-off filter. The changes in the fluorescence intensity upon mixing solutions of GP IIb/IIIa and L-736,622 were recorded repeatedly (n > 10) for each concentration of L-736,622. The average fluorescence data were fitted by nonlinear least-squares to the equation F = a (1 − ekobs t) +b, where t is time in seconds, a is an amplitude, kobs is a first-order rate constant in seconds−1 and b is the intercept with axis y. These values ofkobs were plotted against the concentration of L-736,622 (fig. 3A) and fitted to the equation kobs =kass [L-736,622] +kdiss to determine the values ofkass (association rate constant) andkdiss (dissociation rate constant).

    Figure 3
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    Figure 3

    Stopped-flow determination of the association and dissociation rate constants for L-736,622 to purified GP IIb/IIIa at 25°C. (A) The dependence of observed association rate constants,kobs, for binding of L-736,622 to purified GP IIb/IIIa (form B) on the concentration of the ligand. Each point represents the average of at least 10 independent measurements. The solid line represents the fit of the data to the equationkobs =kass[L-736,622] +kdiss, where kassand kdiss are the association and dissociation rate constants, respectively. (B) Direct measurement of dissociation rate constants by stopped-flow. An average of ten stopped-flow traces were obtained after mixing the solution of the GP IIb/IIIa (0.64 μM, form B) containing 1 μM L-736,622 with 40 μM antagonist L-739,758 (Kd = 50 pM) to prevent rebinding of the fluorescent ligand. The data were fitted by nonlinear least-squares fit to the equation y =a (1 − ekdisst) + b, where t is time in seconds, a is an amplitude,kdiss is a dissociation rate constant in seconds−1 and b is the intercept with axis y, providingkdiss of 0.0329 s−1(t1/2 = 21 s). A plot of the residuals from the fitted line is shown in the lower part of the panel.

    The dissociation rate constants for L-736,622 from both forms of GP IIb/IIIa were measured directly with a stopped-flow spectrometer. GP IIb/IIIa (0.64 μM) incubated with 1 μM of L-736,622 in buffer A was mixed with 10, 20, 30, 40 and 50 μM potent fibrinogen receptor antagonist L-739,758 (Kd = 50 pM) to prevent rebinding of the fluorescent ligand (fig. 3B). The change in fluorescent intensity as a function of time was fit to the equationF = a (1 −ekdiss t) + b, wheret is time in seconds, a is an amplitude,kdiss is a dissociation rate constant in seconds−1 and b is the intercept with axis y. The calculated dissociation rate constants were independent of the concentration of L-739,758, and equivalent results were obtained with other GP IIb/IIIa antagonists.

    Fluorescence-based assay for the affinity of nonfluorescent antagonists to purified forms of GP IIb/IIIa.

    Displacement of L-736,622 (1000 nM) from the resting and the activated forms of purified GP IIb/IIIa (typically 600 nM) by GP IIb/IIIa antagonists were performed at room temperature. The fluorescence intensity after each of a series of additions of a nonfluorescent fibrinogen receptor antagonist was recorded. The fluorescence measurements were performed either in a fluorimeter or on a fluorescence plate reader. These fluorescence intensities, after correction for the intrinsic fluorescence of GP IIb/IIIa and the fluorescence of free L-736,622 in the buffer (fig. 2A), were plotted as percent of the initial fluorescence (see fig. 4). The IC50 value for the nonfluorescent antagonist was determined by a nonlinear least-squares fit of the fluorescence to the equation, F = (Fmax −Fmin)/(1 + (I/IC50)n) +Fmin, were I is the concentration of the compound tested, n is the Hill slope,Fmax is the maximum binding observed without the test compound and Fmin is the nonspecific binding signal. The Kd for the nonfluorescent compound was calculated from the concentration needed to displace 50% of the fluorescent label (IC50), the Kd value of L-736,622 of 3.7 nM obtained from stopped-flow measurements, the total concentrations of L-736,622 ([L-736,622]TOTAL) and the total receptor concentration (RTOTAL) according to the equation: Kd = (IC50 − RTOTAL/2)/(1 + ([L-736,622]TOTAL −RTOTAL/2)/Kd L-736,622). Kd values into the low nanomolar range can be determined by this method.

    Figure 4
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    Figure 4

    Displacement of dansyl-containing L-736,622 (1000 nM) from purified GP IIb/IIa (600 nM) by antagonists. (A) The displacement of L-736,622 from form A (•) and form B (▪) of purified GP IIb/IIIa by L-734,217 (see fig. 6 for structure) yields dissociation constants of 6 ± 0.5 nM and 620 ± 50 nM, respectively. (B) The displacement of L-736,622 from form A (•) and form B (▪) of purified GP IIb/IIIa by tirofiban (also known as AGGRASTATTM and MK-383) yields dissociation constants of 1.6 nM and 15 nM, respectively.

    In the experiments with human and dog platelets, GFP at a concentration of 2 × 109 cells/ml were used. Because of light scattering from the platelet suspensions, the fluorescence measurements were carried out in a front-face arrangement.

    Scintillation proximity assay for binding of radiolabeled antagonists to purified forms of GP IIb/IIIa.

    One bottle of SPA fluomicrospheres (500 mg, Amersham RPN 143, Type 1 yttrium silicate) was suspended for 5 min in 50 ml of HN Buffer (20 mM HEPES, 150 mM NaCl, pH 7.5) containing 1% Triton X-100 in a 50-ml centrifuge tube. After centrifugation, Triton X-100 was removed from the fluomicrospheres by four washes with HN buffer. After the last wash, the fluomicrospheres were suspended in 12.5 ml of HN buffer containing 5 mM CaCl2. Either form A or form B of purified GP IIb/IIIa (∼1 mg/ml in buffer A containing 0.1% Triton X-100) was added dropwise in 25-μl aliquots every 5 min to the stirred suspension of fluomicrospheres. After a total of 0.2 mg of GP IIb/IIIa was added, the suspension was allowed to incubate an additional hour. The fluomicrospheres were then pelleted, washed with HN buffer and resuspended in 12.5 ml of HN before aliquoting and storage at −70°C.

    Binding of a radioligand (e.g., [125I]L-692,884) to the GP IIb/IIIa coated on SPA fluomicrospheres was measured conveniently without the need for separation of bound and free ligand. The decay of a bound radioligand produces excitation of the yttrium silicate solid scintillant in the fluomicrosphere and the concomitant production of photons that were detected with a Top Count scintillation counter (fig.5A). A nonspecific binding signal was measured by adding either a large excess of nonradiolabeled GP IIb/IIIa antagonist or by adding EDTA to prevent specific binding of the radioligand. The apparent nonspecific binding is directly proportional to the concentration of fluomicrospheres (independent of added GP IIb/IIIa) and to the concentration of [125I]L-692,884 (data not shown) and likely is caused by a nonproximity effect resulting from excitation of the yttrium-coated beads by solution-phase Iodine-125 ligand, which decays within 35 μm of the bead (Bosworth and Towers, 1989; Cook et al., 1992). Use of a smaller amount of SPA fluomicrospheres loaded with a greater density of GP IIb/IIIa could maximize the specific binding signal relative to the nonspecific binding signal. The efficiency of loading GP IIb/IIIa onto SPA fluomicrospheres is enhanced by use of more concentrated slurries of SPA fluomicrospheres in the loading process because this leads to more efficient trapping of the precipitated receptor on the glass surface of the beads. Presumably the hydrophobic transmembrane regions of GP IIb/IIIa bind to the glass surface of the bead (Carrell et al., 1985).

    Figure 5
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    Figure 5

    Binding of [125I]L-692,884 to purified GP IIb/IIIa coated onto SPA fluomicrospheres and displacement by selected ligands. (A) GP IIb/IIIa coated onto fluomicrospheres (0.2 nM form B) was incubated with a range of concentrations of [125I]L-692,884 in a Packard Optiplate in the presence and absence of 10 mM EDTA. The bound counts were determined in a Packard Topcount microplate scintillation counter and corrected for the nonspecific binding signal as described under “Methods.” The increase in specifically bound counts as a function of added [125I]L-692,884 yields a Kd of 0.6 nM. (B) The displacement of [125I]L-692,884 (0.2 nM) from GP IIb/IIIa-coated fluomicrospheres (0.2 nM receptor) by a series of ligands was used to determine their binding affinities for an activated form of purified GP IIb/IIIa. The EC50 values and hill slopes (in parentheses) obtained from these data are: 2.8 nM (n = 0.7) for L-734,217, 0.12 nM (n = 1.4) for L-738,167, 0.85 nM (n = 0.9) for L-692,884 and 8.7 nM (n = 1.8) for fibrinogen.

    The amount of GP IIb/IIIa which is associated productively with the SPA fluomicrospheres was determined by titration with the very high affinity GP IIb/IIIa antagonist [3H]L-738,167 (Kd ∼ 0.1 nM). A range of concentrations of [3H]L-738,167 (12, 10, 8, 6, 4, 2, 1, 0.5, 0.24, 0.12, 0.063, 0 nM) in HN buffer containing 10 mM CaCl2 were incubated with GP IIb/IIIa-coated SPA fluomicrospheres (10 mg fluomicrospheres/ml) in 96-well Packard Optiplates (Packard no. 6005190) in both the presence and absence of 15 mM EDTA (final volume, 200 μl). Plates were heat-sealed and shaken overnight before counting in a Packard Topcount microplate scintillation counter. Nonspecific binding was determined from the incubations in the presence of EDTA. A plot of specifically bound counts against the concentration of added [3H]L-738,167 shows a sharp break at the concentration of radiolabeled compound equal to the concentration of productively bound receptor (data not shown), and thus provides a measure of the concentration of active receptors.

    The radiochemical purity of [125I]L-692,884 (NEN Life Science Products, Boston, MA; NEX-330) was estimated by the percent of the total I-125 which could be bound to a high concentration of GP IIb/IIIa coated on SPA fluomicrospheres. A series of concentrations of GP IIb/IIIa-coated fluomicrospheres (233, 116, 58, 29, 45, 7, 3.6, 0 mg fluomicrospheres/ml) were incubated for 1 hr with 1 nM [125I]L-692,884 in HN buffer containing 1 mM CaCl2. After centrifugation (5000 ×g for 30 s), an aliquot of the supernatant was counted in a gamma counter. The ratio of the unbound counts to the total counts in the absence of added fluomicrospheres were fitted to the equation: Ratio = Ratiomax [GP IIb/IIa]/([GP IIb/IIa] + Kd), whereKd is the dissociation constant and Ratiomax is the maximum percentage of the total radioactivity which can be bound. Typically, this ratio suggests the radiochemical purity is >95%. When the ratio was less than 90%, the batch of [125I]L-692,884 was not used.

    Because the specific activity of the [125I]-L-692,884 is close to theoretical for carrier-free I-125, millicurie amounts of the material do not produce a reliable UV spectral signal even given the ∼20,000 M−1 cm−1extinction coefficient for iodobenzene moiety. The concentration and also the specific activity of [125I]L-692,884 was determined by nonlinear least-squares analysis of saturation binding curves done at five different fixed ratios of radioactive and nonradioactive L-692,884. GP IIb/IIIa-coated fluomicrospheres (0.6 mg/ml; 0.2 nM receptors) were incubated with 12 serial 2-fold dilutions of [125I]L-692,884 starting at 4 nM (assuming a theoretical specific activity of 2200 Ci/mmol) in the presence and absence of EDTA (10 mM) in a Packard Optiplate. The above-mentioned serial dilutions were repeated for [125I]L-692,884 which was diluted 2-, 4-, 12- and 256-fold with unlabeled L-692,884. The plate was heat-sealed and shaken overnight before counting in a Packard Top Count. The observed CPM at each given added concentration of labeled (H) and unlabeled (C) L-692,884 were fitted to the equation: CPM = (CPMmax αH)/(C + α H +Kd) + α H NSB, where α multiplied by H is the true concentration of [125I]L-692,884 and the other fitted parameters CPMmax, Kd and NSB represent the maximum binding for undiluted [125I]L-692,884, the dissociation constant and nonspecific binding, respectively. This nonlinear least-squares fit was performed by SigmaPlot (SPSS, Chicago, IL) with weights equal to CPM−2. The specific activity of [125I]L-692,884 could be estimated by dividing 2200 Ci/mmol by α and adjusting if necessary for the radiochemical purity estimated above. The actual concentration of [125I]L-692,884 in the stock solution was determined directly as the product of α and the concentration calculated assuming theoretical specific activity of 2200 Ci/mmol.

    SPA assay for binding of nonradiolabeled antagonists to purified forms of GP IIb/IIIa.

    The binding affinity of nonradiolabeled GP IIb/IIIa antagonists was determined by incubating GP IIb/IIIa antagonists at room temperature in a final volume of 200 μl with 0.2 nM [125I]L-692,884 and 0.2 nM receptors (form A or form B) coated onto SPA fluomicrospheres in the HN buffer (fig. 5B). For very potent GP IIb/IIIa antagonists the receptor concentration was lowered to 0.02 nM to increase the dynamic range of the assay. Controls containing no GP IIb/IIIa antagonist or EDTA were included to determine the maximal binding capacity (Bmax) and nonspecific binding, respectively. The serial dilutions were performed in a Packard Optiplate followed by the addition of radioactivity and fluomicrosphere by a Packard Multiprobe Robot. The fluomicrospheres were stirred at 200 rpm in a 100-ml beaker to produce a homogeneous suspension. The plates were heat-sealed and shaken overnight on a Titer Tek plate shaker before measurement of bound CPM in a Packard Topcount microplate scintillation counter.

    The EC50 values for the nonradiolabeled GP IIb/IIIa antagonists were determined by a nonlinear least-squares fit of CPM = (Bmax −Bmin)/(1 + (I/EC50)n) +Bmin, where I is the concentration of the compound tested, n is the Hill slope,Bmax is the maximum binding observed without the test compound and Bmin is the nonspecific binding signal. The value ofBmin was sometimes set equal to the nonspecific binding determined by EDTA when the data set did not contain sufficiently high concentration to determine this value independently. The average standard error of the mean for EC50 determinations was ±20%.

    Direct binding of radiolabeled GP IIb/IIIa antagonist to resting and activated platelets.

    The direct binding of radiolabeled GP IIb/IIIa antagonist to platelets at room temperature was determined after separation of bound and free material by centrifugation (fig.6). Various concentrations of the GP IIb/IIIa antagonist (e.g., [125I]L-692,884 and [3H]L-734,217) were incubated with GFP (typically 2 × 108 cell/ml) in platelet buffer (138 mM NaCl, 6.0 mM KCl, 1.0 mM CaCl2, 1.7 mM NaH2PO4, 6.3 mM HEPES, containing 1% dextrose and 2% bovine serum albumin at pH 7.4) in the presence and absence of 10 μM L-738,167. After equilibration, platelets were pelleted by centrifugation (15,000 × gfor 30 s) and the supernatant removed. Pellets of bound [125I]L-692,884 were counted directly in a gamma counter. Pellets of tritiated compounds (e.g., [3H]L-734,217) were eluted first by the addition of 10 μM L-738,167 before the addition of an aliquot to 4.5 ml of Readysafe scintillation cocktail and counting in a beta counter. The specific binding (CPMBound) was determined from the difference in bound counts in the absence and presence of 10 μM L-738,167. The concentration of total radioactive compound required to produce half-maximal binding (K1/2) was determined by a nonlinear least-squares fit of the binding isotherm to CPMBound = Bmax/(1 + (K1/2/L)n), where L is the concentration of total radiolabeled compound added, n is the Hill slope andBmax is the maximum specific binding capacity. The concentration of GP IIb/IIIa receptors used in the assay was determined by titration with [3H]L-738,167. The concentration of free radiolabeled compound required to produce half-maximal binding (Kd) was calculated from the observed K1/2 after correction by subtraction of 50% of the receptor concentration.

    Figure 6
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    Figure 6

    Binding of [3H]L-734,217 to resting and thrombin-activated platelets containing 18 nM GP IIb/IIIa receptors. GFP (1 or 2 × 108 cells/ml) were incubated at room temperature with a range of concentrations of [3H]L-734,217 and either 10 nM thrombin (triangles) or no agonist (circles) for 4.5 min before centrifugation to separate bound and free ligands. Analysis of the binding isotherms, as described under “Methods,” suggests dissociation constants of 5 nM and 600 nM for thrombin-activated and resting platelets, respectively.

    The measurement of the binding of [3H]L-734,217 to thrombin-activated platelets was performed 4.5 min after the addition of 10 nM thrombin to 1 × 108platelets/ml containing various concentration of [3H]L-734,217.

    Competition of nonradiolabeled GP IIb/IIIa antagonists with the binding of [125I]L-692,884 to GFP.

    The binding of a nonradiolabeled GP IIb/IIIa antagonist to GFP was determined by competition with the binding of [125I]L-692,884. GFP (typically 2 × 108 cell/ml, ∼30 nM receptors) were incubated at room temperature with 15 nM [125I]L-692,884 and a wide range of concentrations of the nonradiolabeled GP IIb/IIIa antagonist. After equilibration, platelets were centrifuged (15,000 × g for 1 min) and pellets were counted directly in a gamma counter. The concentration of total added GP IIb/IIIa antagonist (IC50) which prevents 50% of the maximum binding of [125I]L-692,884 was determined by a nonlinear least-squares fit of the displacement curve to the equation: CPMBound = (Bmax −Bmin)/(1 + (I/IC50)n) +Bmin, where I is the concentration of GP IIb/IIIa antagonist added, n is the Hill slope, Bmax is the binding observed in the absence of GP IIb/IIIa antagonist and Bminis the nonspecific binding signal. The concentration of GP IIb/IIIa receptors in the GFP was determined by titration with [3H]L-738,167. In this assay, the concentration of GP IIb/IIIa receptors and the concentration of [125I]L-692,884 are much less than theKd of [125I]L-692,884 for resting platelets. The concentration of free nonradiolabeled compound required to produce half-maximal displacement (Kd) of [125I]L-692,884 from resting platelets was obtained directly from the IC50 after subtraction of 50% of the GP IIb/IIIa receptor concentration to account for depletion of the added ligand by binding to the receptors.

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    Results

    Isolation of Form A and Form B from Human Platelets

    Human GP IIb/IIIa was purified by passing platelet lysates sequentially over a Concanavalin A affinity column and a Sepharose 4B-hexyl-RGDS affinity column (see fig. 1). Approximately 5 to 10% of the total GP IIb/IIIa was retained by an RGDS-affinity column and is designated as “form A.” The remainder of the GP IIb/IIIa was purified over a Sephacryl S-300 HR size exclusion column and is designated as “form B.” Structurally, there is no detectable difference between form A and form B on reduced and nonreduced SDS-PAGE gels (fig. 1). Rechromatography of form B on the RGDS-affinity column did not lead to any additional binding, which suggests that form B was not converted into form A in solution or on the affinity column. However, these two solubilized forms of GP IIb/IIIa are functionally distinguishable. Form A is analogous to activated GP IIb/IIIa receptor on platelets because it binds fibrinogen, whereas form B does not and is therefore functionally analogous to a resting receptor on platelets (Kouns et al., 1992).

    Binding Affinity of Antagonists to Both Forms of Purified GP IIb/IIIa

    Binding of a fluorescent ligand to purified forms of GP IIb/IIIa.

    The binding of the dansyl-containing GP IIb/IIIa antagonist L-736,622 to purified GP IIb/IIIa can be detected by a 3- to 4-fold enhancement in the fluorescence emission of L-736,622 in the bound complex relative to the unbound ligand (fig. 2A). Figure 2B shows a binding isotherm measured at a concentration of GP IIb/IIIa in excess of the Kd. This concentration is necessary to get a detectable fluorescent signal for the bound ligand when compared with the background fluorescence and light scattering. The equilibrium binding is saturable and yields aKd of 3.8 ± 1.5 nM for form A (fig.2B) and 4.5 ± 1.1 nM for form B (data not shown).

    The association and dissociation rate constants were measured by stopped-flow (fig. 3). Measured association rate constants of 9.14 × 106 M−1s−1 and 9.66 × 106 M−1s−1 were obtained for form A (data not shown) and form B (fig. 3A), respectively. The magnitude of these numbers suggests that the association of L-736,622 to the purified forms of GP IIb/IIIa may be a diffusion-controlled process. Dissociation rate constants of 0.0329 s−1and 0.0355 s−1 were obtained for form A (data not shown) and form B (fig. 3B), respectively. The measured rate constants were shown to be independent of the nonfluorescent ligand used to prevent rebinding in the stopped-flow measurements (data not shown). Based on these kinetically determined rate constants, equilibrium dissociation constants (kdiss/kass) of 3.6 nM and 3.7 nM were calculated for form A and form B of GP IIb/IIIa, respectively. These values are in agreement with the values obtained by equilibrium binding and indicate that the dansyl-containing GP IIb/IIIa antagonist L-736,622 binds to both forms of GP IIb/IIIa with equal affinity.

    Binding affinity of nonfluorescent antagonists to purified forms of GP IIb/IIIa.

    Displacement of the dansyl-containing L-736,622 from either form of GP IIb/IIIa by a nonfluorescent GP IIb/IIIa antagonists provides a convenient assay for quantifying the affinity of compounds for these two physically separable forms (fig. 4). This assay, as described in detail under “Methods,” can determineKd values for the nonfluorescent GP IIb/IIIa antagonist into the low nanomolar range.

    Figure 4A shows the displacement of the fluorescent ligand by the GP IIb/IIIa antagonist L-734,217. The measured binding affinity of L-734,217 is much greater for form A (Kd = 6 nM) than for form B (Kd = 620 nM). Unlike L-736,622, which binds to both forms of GP IIb/IIIa with equal affinity, L-734,217 shows selectivity in binding and binds with a 100-fold higher affinity to the same form of GP IIb/IIIa that exhibits high affinity for fibrinogen. As seen in figure 4B, tirofiban shows a more modest selectivity. Table 1summarizes the binding affinity for selected GP IIb/IIIa antagonists to both forms of purified GP IIb/IIIa.

    View this table:
    Table 1

    Binding constants for GP IIb/IIIa antagonists to activated (form A) and resting (form B) forms of purified GP IIb/IIIa

    Binding affinity of radiolabeled antagonists to purified GP IIb/IIIa coated on SPA fluomicrospheres.

    A high-throughput assay to determine the affinity of GP IIb/IIIa antagonists for purified receptor was developed and characterized, using SPA technology. We used a novel approach of precipitating the Triton X-100-solubilized GP IIb/IIIa onto commercially available yttrium silicate SPA fluomicrospheres (Bosworth and Towers, 1989; Cook et al., 1992; Takeuchi, 1992). Binding of radiolabeled RGD-containing heptapeptide [125I]L-692,884 to the GP IIb/IIIa-coated on SPA fluomicrospheres was measured conveniently, without the need for separation of bound and free ligand, with a Top Count scintillation counter (see “Methods”). Figure 5A shows saturable binding of [125I]L-692,884 to GP IIb/IIIa attached to SPA fluomicrospheres with aKd of 0.6 nM.

    Binding affinity of nonradiolabeled antagonists to purified forms of GP IIb/IIIa coated on SPA fluomicrospheres.

    Competition of a nonradiolabeled GP IIb/IIIa antagonist with [125I]L-692,884 provides a convenient assay for quantifying the affinity of compounds to GP IIb/IIIa coated on fluomicrospheres (fig. 5B). The affinity of the nonradiolabeled GP IIb/IIIa antagonist, expressed as an EC50 value, is obtained by a nonlinear least-squares fit of the data in figure 5B as described under “Methods.” EC50 values down to 10 pM can be measured by this method.

    Table 2 summarizes the binding affinities of fibrinogen and selected GP IIb/IIIa antagonists to both forms of GP IIb/IIIa coated onto SPA fluomicrospheres. Essentially the same EC50 values were obtained when either form A or form B were coated onto SPA beads, even for L-734,217 and L-692,884, which had shown a high degree of selectivity for form A in solution (see table 1). Additionally, fibrinogen binds with equally high affinity to both forms of GP IIb/IIIa when coated onto fluomicrospheres. The SPA assay has several advantages over the fluorescence-based form A assay for determining the affinity of antagonists for the activated form of GP IIb/IIIa. The SPA assay requires a much lower concentration of receptor (0.02 nM vs. 600 nM) and it can use the more plentiful form B of the receptor, whereas the fluorescence assay requires form A which represents only 5 to 10% of the total isolated receptor. Further, in the rational design of GP IIb/IIIa antagonists, it is not uncommon to have compounds that bind to the active form with subnanomolar dissociation constants. The high affinity of L-738,167 for activated GP IIb/IIIa (fig. 5B and table2) illustrates that the SPA assay is capable of measuring EC50 values into the 0.1 nM range. The fluorescence assay is limited to determiningKd values greater than 1 nM, whereas the SPA assay can measure EC50 values down to 10 pM. The SPA assay overcomes limitations in the fluorescence form A assay and provides a system for probing the intrinsic binding affinity of antagonists to an activated form of GP IIb/IIIa.

    View this table:
    Table 2

    Affinity of ligands to both forms of purified GP IIb/IIIa-coated SPA fluomicrospheres

    Binding Affinity of Antagonists to Resting and Activated Platelets

    To verify the selectivities seen with purified forms of GP IIb/IIIa, studies on the binding of selected GP IIb/IIIa antagonists to resting and activated platelets were undertaken. The affinity of L-734,217 for GFP was measured by displacement of the dansyl-containing L-736,622 analogous to that shown in figure 4 for solubilized GP IIb/IIIa. The estimated Kd of 480 nM was obtained for both resting human or dog platelets. When platelets were stimulated with 20 μM ADP, the apparentKd for L-734,217 shifted to 18 nM for human or dog platelets (data not shown), which indicates selective binding of L-734,217 on activated platelets. These Kdvalues calculated for L-734,217 are based on an assumedKd of 3.7 nM for L-736,622 on resting and activated platelets analogous to the values obtained by stopped-flow measurements on purified form A and form B of GP IIb/IIIa.

    The direct binding of [3H]-L-734,217 to platelets also was determined after separation of bound and free material by centrifugation. The filled circles in figure 6 show a typical binding experiment with resting GFP and radiolabeled [3H]L-734,217. The average of seven such experiments yields a Kd of 620 ± 20 nM for binding of L-734,217 to resting human GFP. The open triangles in figure 6 show the binding of [3H]L-734,217 to GFP activated with 10 nM thrombin. This binding isotherm suggests aKd of 5 nM for the binding of L-734,217 to activated platelet receptors.

    The dissociation constants for nonradiolabeled GP IIb/IIIa antagonists to resting GFP also were determined from their competition with the binding of [125I]L-692,884, as described under “Methods.” Competition of unlabeled L-734,217 with [125I]L-692,884 yields aKd of 620 nM for the binding of L-734,217 to resting platelets (data not shown). This value is in excellent agreement with the Kd value of 620 nM directly determined with [3H]L-734,217 and illustrates the utility of this method to measure affinities to resting GFP when radiolabeled compound is not available.

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    Discussion

    Platelet activation in vivo is of pathophysiologic significance in several vascular disorders, including unstable angina, peripheral vascular disease, stroke and after angioplasty or coronary thrombolysis (Abrams and Shattil, 1991; Hamm et al., 1987;Fitzgerald et al., 1986, 1989). Methods for detection of activated platelets in the circulation are being developed and used to detect increased levels of activated platelets in certain disease states (Scharf et al., 1992; Abrams and Shattil, 1991;Abrams et al., 1990; George and Shattil, 1991). Blocking activated receptors on circulating platelets may lead to effective treatment for the prevention of acute and long-term adverse cardiovascular events in high-risk populations.

    Selective binding of fibrinogen to activated platelets, but not resting platelets, is essential in maintaining normal hemostasis. Selective binding to activated platelets also may be a desirable feature in the rational design of GP IIb/IIIa antagonists. Although the basis for the selective binding of the multivalent ligand fibrinogen to GP IIb/IIIa on activated platelets is not known (Kunicki et al., 1996), we demonstrate in this paper that selectivity can be observed with low molecular weight univalent GP IIb/IIIa antagonists.

    Historically, inhibition of platelet aggregation (Hartman et al., 1992; Egbertson et al., 1994a, 1994b) or a solid-phase assay with plates coated with GP IIb/IIIa or fibrinogen (Alig et al., 1992) have been used to measure the potency of GP IIb/IIIa antagonists. However, neither assay offers information on the affinity of a GP IIb/IIIa antagonist for resting platelets. Further, ex vivo inhibition of platelet aggregation does not yield a true measure of the affinity for highly potent compounds, because the observed IC50 in a receptor ligand binding assay can never be less than 50% of the concentration of receptors used in the assay. Because ex vivo platelet aggregation assays typically are conducted with ≥ 2 × 108 platelets/ml of platelet-rich plasma containing ∼100,000 receptors per platelet, at least 16 nM high-affinity compound will be necessary to occupy 50% of the GP IIb/IIIa receptors. Therefore, we might see little difference in the IC50 for inhibition of platelet aggregation by a GP IIb/IIIa antagonist with a dissociation constant for activated platelets of 1 nM vs. 0.01 nM.

    To measure the intrinsic differences in affinity between compounds, we isolated two forms of GP IIb/IIIa from human platelets and developed assays based on purified receptors. In the first assay, EC50 values are calculated from competitive binding between compounds of interest and the fibrinogen receptor antagonist [125I]L-692,884 to purified GP IIb/IIIa activated by coating onto yttrium silicate SPA fluomicrospheres (fig. 5B). Binding measurements with fibrinogen (EC50 ∼ 10 nM) and GP IIb/IIIa antagonists indicate that the SPA assay provides a binding affinity for an activated form of GP IIb/IIIa, regardless of which form of GP IIb/IIIa is coated onto the fluomicrospheres (see table 2). These results demonstrate that both forms of purified GP IIb/IIIa can exhibit identical binding profiles when attached to a fluomicrosphere. Form A and form B in solution may represent different stable “conformations” of the same molecular complex which are converted to pharmacologically indistinguishable structures when coated onto the surface of the fluomicrospheres. From a practical point of view, these results suggest that either form of GP IIb/IIIa attached to SPA fluomicrospheres exhibits the binding profile expected for an activated receptor. However, in the second assay, displacement of the fluorescent fibrinogen receptor antagonist L-736,622 by compounds of interest from form A or form B of GP IIb/IIIa solubilized in Triton X-100 micelles gave Kd values that apparently provide the binding affinity for an activated or resting form of GP IIb/IIIa, respectively (see table 1, fig. 4 and below). Thus, with these assays one can evaluate intrinsic binding affinity of ligands to both forms of purified GP IIb/IIIa, which may be critical parameters in selecting the optimal antagonists of platelet aggregation.

    The equilibrium dissociation constants for the fluorescent GP IIb/IIIa antagonist L-736,622 of 3.7 nM obtained from stopped-flow kinetic measurements (fig. 3) were the same for both forms of purified GP IIb/IIIa (fig. 1), which suggests that the fluorescent GP IIb/IIIa antagonist L-736,622 has no selectivity for activated compared with resting receptors. The naturally occurring disintegrin, echistatin (Ganet al., 1988), also shows no selectivity for form A over form B (table 1). However, as shown in figure 4A, the orally active GP IIb/IIIa antagonist L-734,217 (Duggan et al., 1995; Cooket al., 1996) shows a 100-fold selectivity for form A compared with form B. This result demonstrates that selectivity is possible with low molecular weight GP IIb/IIIa antagonists and is not a property exclusive to multivalent protein ligands like fibrinogen, Fab fragments of antibodies or some disintegrins.

    The high degree of selectivity observed with L-734,217 on purified forms of GP IIb/IIIa also was seen in its binding to resting and activated platelets. The selective binding of L-734,217 to platelets was observed directly by radiolabeled compound (fig. 6) and by competition with the binding of fluorescent L-736,622 to platelets (see “Results”). The agreement for L-734,217 of the dissociation constants observed on resting and activated platelets (fig. 6) with the dissociation constant obtained with resting and activated purified GP IIb/IIIa receptors (table 1) suggests that the form A and form B assays provide a good estimate of the binding affinity of GP IIb/IIIa antagonists for activated and resting platelets.

    Because only the activated form of GP IIb/IIIa receptors binds fibrinogen (Phillips et al., 1988), a GP IIb/IIIa antagonist would need only to bind to the activated form to interfere with fibrinogen-mediated aggregation. The plasma concentration of L-734,217 needed to produce inhibition of ex vivo platelet aggregation is dictated primarily by the high concentration of GP IIb/IIIa receptors (∼50 nM in the plasma volume of blood) rather than by the high affinity of L-734,217 (Kd ∼5 nM) for activated receptors. Consequently, at least 30 nM L-734,217 {1/2 [Receptors] +KdL-734,217 (1 + [Fibrinogen]/KdFibronogen)} would be necessary to occupy 50% of the GP IIb/IIIa receptors after platelet activation. Further increase in the affinity of a GP IIb/IIIa antagonist for activated receptors could reduce only modestly the concentration required for 50% occupancy down toward 25 nM. In agreement with this analysis, the plasma concentration of L-734,217 that is needed to produce 50% inhibition of platelet aggregation in human platelet-rich plasma activated with ADP or thrombin was determined to be 23 ± 3 nM and 27 ± 5 nM, respectively (Duggan et al., 1995). At this concentration of circulating drug, only about 5% of the receptors on resting platelets are occupied by the drug, because L-734,217 has a low affinity (Kd ∼ 600 nM) for resting platelets (fig.6). The in vivo efficacy of L-734,217 in a conscious dog model of left circumflex coronary artery electrolytic lesion has been documented (Cook et al., 1996). These authors reported that administration of 3.0 mg/kg p.o. reduced thrombus mass, prevented occlusive coronary artery thrombosis and reduced or prevented myocardial infarction and ventricular ectopy. This dose of L-734,217 results in ≥90% inhibition of ADP-induced platelet aggregation and measured blood levels of 100 to 300 nM. This concentration is lower than the value of the KD of L-734,217 with resting platelets and demonstrates that occupancy of receptors on resting platelets is not necessary to observe in vivoefficacy with the selective GP IIb/IIIa antagonist L-734,217.

    Similar in vivo efficacy also was observed by Cook et al. (1997) for the nonselective GP IIb/IIIa antagonist L-738,167 in the same animal model. L-738,167 binds to resting platelets with aKD of 0.1 to 0.2 nM (Bednar et al., 1997). Cook et al. (1997) found that administration of 0.1 mg/kg p.o. resulted in comparable efficacy, with ≥90% inhibition of ex vivo platelet aggregation and measured total blood levels of ∼80 nM. A 0.03 mg/kg p.o. dose of L-738,167 achieved sustained 40 to 70% inhibition of ADP-inducedex vivo platelet aggregation and modest 2- to 3-fold elevation in bleeding time (Cook et al., 1997). Less than 2-fold increase in template bleeding time was observed at a dose of L-734,217 that elicited ∼80% inhibition of ADP-induced platelet aggregation (Cook et al., 1996). Both the selective compound L-734,217 and the nonselective compound L-738,167 show similar efficacy in the conscious dog model of left circumflex coronary artery electrolytic lesion (J.J. Cook, personal communication). This comparable efficacy demonstrates that selective compounds, which do not bind to resting platelets, need not suffer any loss in efficacy resulting from their inability to bind to resting platelets.

    Selective GP IIb/IIIa antagonists, with Kdvalues for resting platelets much greater than the physiological receptor concentration {KdResting ≫ [Receptors]}, and also much greater than the IC50 for inhibition of platelet aggregation in platelet-rich plasma (KdResting ≫ IC50PRP), will show little binding to circulating platelets at therapeutic doses. Such selective GP IIb/IIIa antagonists may have advantages in reducing the likelihood of undesired side effects. Because GP IIb/IIIa receptors on platelets and megakaryocytes would not be chronically occupied by an antagonist, the possibility of side effects resulting from drug-induced thrombocytopenia (Bednar et al., 1996) and outside-in signaling by receptors occupied with a GP IIb/IIIa antagonist would be reduced. In general, such a selective compound tends to approach the ideal drug, which does not interact with anything except when it is needed and then only at a unique site of action. Clinical studies with selective GP IIb/IIIa antagonists will be necessary to demonstrate the putative advantages of these compounds. Selective GP IIb/IIIa antagonists, which bind preferentially to activated receptors, represent a new therapeutic approach in antithrombotic therapy.

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    Footnotes

    • Send reprint requests to: Dr. Rodney A. Bednar, Department of Pharmacology, WP 26–265, Merck Research Laboratories, West Point, PA 19486-0004.

    • Abbreviations:
      ADP
      adenosine diphosphate
      EDTA
      ethylenediaminetetraacetic acid
      GFP
      gel-filtered platelets
      GP
      glycoprotein
      HEPES
      N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
      RGD
      arginine-glycine-aspartic acid
      SDS-PAGE
      sodium dodecyl sulfate-polyacrylamide gel electrophoresis
      SPA
      scintillation proximity assay
      kass
      association rate constant
      kdiss
      dissociation rate constant
      • Received June 26, 1997.
      • Accepted February 23, 1998.
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