Introduction

Top Abstract Introduction Methods Results Discussion References

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; Hamm et 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 (ReoPro^TM) (Epic Investigators, 1994; Jordan et al., 1996), which blocks several integrins, and the specific reversible GP IIb/IIIa blocker tirofiban (AGGRASTAT^TM) (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.

	Methods

Top Abstract Introduction Methods Results Discussion References

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 of Kouns 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 µM transepoxysuccinyl-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--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).

View larger version (32K): [in this window] [in a new window]   Fig. 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 figure 2A. 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 = F_max (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; J is ([Ligand] + [Receptor] + K_d); and K_d is the apparent dissociation constant of the fluorescent ligand (Hulme and Birdsall, 1992). The accuracy of these K_d values depends on the accuracy of the measured GP IIb/IIIa concentrations, because a GP IIb/IIIa concentration in excess of the K_d 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.

View larger version (25K): [in this window] [in a new window]   Fig. 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  e^{kobs t}) + b, where t is time in seconds, a is an amplitude, k_obs is a first-order rate constant in seconds¹ and b is the intercept with axis y. These values of k_obs were plotted against the concentration of L-736,622 (fig. 3A) and fitted to the equation k_obs = k_ass [L-736,622] + k_diss to determine the values of k_ass (association rate constant) and k_diss (dissociation rate constant).

View larger version (22K): [in this window] [in a new window]   Fig. 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 equation kobs = kass[L-736,622] + kdiss, where kass and 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  ekdiss t) + b, where t is time in seconds, a is an amplitude, kdiss is a dissociation rate constant in seconds1 and b is the intercept with axis y, providing kdiss of 0.0329 s1 (t1/2 = 21 s). A plot of the residuals from the fitted line is shown in the lower part of the panel.

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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 = (F_max  F_min)/(1 + (I/IC₅₀)ⁿ) + F_min, were I is the concentration of the compound tested, n is the Hill slope, F_max is the maximum binding observed without the test compound and F_min is the nonspecific binding signal. The K_d for the nonfluorescent compound was calculated from the concentration needed to displace 50% of the fluorescent label (IC₅₀), the K_d 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 (R_TOTAL) according to the equation: K_d = (IC₅₀  R_TOTAL/2)/(1 + ([L-736,622]_TOTAL  R_TOTAL/2)/K_{d L-736,622}). K_d values into the low nanomolar range can be determined by this method.

View larger version (22K): [in this window] [in a new window]   Fig. 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.

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.

View larger version (25K): [in this window] [in a new window]   Fig. 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.

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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 (B_max) 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.

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 × g for 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 (CPM_Bound) 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 (K_1/2) was determined by a nonlinear least-squares fit of the binding isotherm to CPM_Bound = B_max/(1 + (K_1/2/L)ⁿ), where L is the concentration of total radiolabeled compound added, n is the Hill slope and B_max 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 (K_d) was calculated from the observed K_1/2 after correction by subtraction of 50% of the receptor concentration.

View larger version (19K): [in this window] [in a new window]   Fig. 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.

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: CPM_Bound = (B_max  B_min)/(1 + (I/IC₅₀)ⁿ) + B_min, where I is the concentration of GP IIb/IIIa antagonist added, n is the Hill slope, B_max is the binding observed in the absence of GP IIb/IIIa antagonist and B_min is 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 the K_d of [¹²⁵I]L-692,884 for resting platelets. The concentration of free nonradiolabeled compound required to produce half-maximal displacement (K_d) 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.

	Results

Top Abstract Introduction Methods Results Discussion References

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 K_d. 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 a K_d of 3.8 ± 1.5 nM for form A (fig. 2B) and 4.5 ± 1.1 nM for form B (data not shown).

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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 determine K_d values for the nonfluorescent GP IIb/IIIa antagonist into the low nanomolar range.

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 a K_d 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.

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 K_d of 480 nM was obtained for both resting human or dog platelets. When platelets were stimulated with 20 µM ADP, the apparent K_d 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 K_d values calculated for L-734,217 are based on an assumed K_d 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 K_d 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 a K_d 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 a K_d of 620 nM for the binding of L-734,217 to resting platelets (data not shown). This value is in excellent agreement with the K_d 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.

	Discussion

Top Abstract Introduction Methods Results Discussion References

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 K_d 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 (Gan et 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; Cook et 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 (K_d ~5 nM) for activated receptors. Consequently, at least 30 nM L-734,217 {1/2 [Receptors] + K_d^L-734,217 (1 + [Fibrinogen]/K_d^Fibronogen)} 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 (K_d ~ 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 K_D of L-734,217 with resting platelets and demonstrates that occupancy of receptors on resting platelets is not necessary to observe in vivo efficacy 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 a K_D 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-induced ex 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 K_d values for resting platelets much greater than the physiological receptor concentration {K_d^Resting  [Receptors]}, and also much greater than the IC₅₀ for inhibition of platelet aggregation in platelet-rich plasma (K_d^Resting  IC₅₀^PRP), 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.