Ligand Inhibition of the Platelet Glycoprotein IIb-IIIa Complex Function as a Calcium Channel in Liposomes*

Platelet glycoproteins IIb and IIIa function as a fi- brinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen 7 COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln- Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior in- cubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 PM His-12-Va1, analogous to RGDS inhibition of His-12-Val binding to platelets. His- 12-Val inhibited a minor component of transmem- brane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 4sCa flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His- 12-Val and RGDS, steric considerations, or differences

(8-lo), and a tetrapeptide (RGDS) near the carboxyl terminus of the a chain or a tripeptide (RGD) near the amino terminus of the a chain (11)(12)(13)(14)(15). The synthetic peptide analogues of these regions inhibit platelet aggregation and Fgn binding to activated platelets and are mutually inhibitory in binding studies (11)(12)(13)(14)(15)(16). The data suggest that these peptides share common or closely related binding sites on GPIIb-IIIa.
Ligand binding to GPIIb-IIIa may signal secondary events in the platelet and alter the conformation and distribution of the IIb-IIIa complex. Binding of Fgn, RGDS, or the y dodecapeptide induces clustering of platelet , and occupancy of GPIIb-IIIa by Fgn is necessary to maintain Na+/H+ exchange in epinephrine-stimulated platelets (18). Parise et al. (19) have demonstrated that binding of LGGAKQAGDV or RGDS to soluble GPIIb-IIIa alters the hydrodynamic properties of the complex and renders GPIIb susceptible to thrombin hydrolysis. This suggests that binding of either the dodecapeptide or RGDS induces an unfolding of the GPIIb-IIIa complex.
In a previous study we reported that the purified GPIIb-IIIa complex but not the dissociated glycoproteins can facilitate calcium movement across a phospholipid membrane when the complex is inserted into the surface of liposomes (20). This apparent Ca2+ channel is inhibited by a monoclonal antibody to the GPIIb-IIIa complex. In studies of calcium homeostasis in normal and thrombasthenic platelets, Brass (21) demonstrated that the GPIIb-IIIa complex may play a role in Ca2+ flux across the platelet plasma membrane. In studies using a monoclonal antibody (TM83) to the GPIIb-IIIa complex and GRGDSP with aequorin-loaded platelets, Yamaguchi et al. (22) concluded that the IIb-IIIa complex was involved in Ca2+ influx during platelet activation with thrombin and collagen but not phorbol 12-myristate 13-acetate. In the current study, we investigated whether binding of His-12-Val or RGDS alters the apparent calcium channel function of GPIIb-IIIa in this liposome model. GPZZb-ZZIa Preparation-GPIIb and -1IIa were prepared as previously reported (20,21,(22)(23)(24) from Triton X-114-solubilized platelet membranes from clinically outdated washed platelet concentrates. Membranes were suspended in 1% (v/v) precondensed Triton X-114, 10 mM Tris, 0.15 M NaC1, pH 7.4, with 0.4 mM phenylmethylsulfonyl fluoride, 100 pg/ml leupeptin, incubated overnight at 4 "C, and centrifuged at 78,000 X g for 1 h at 4 "C. The detergent phase was applied to a 6% sucrose cushion, heated a t 37 "C for 5 min, centrifuged at 1500 x g for 5 min, and the detergent micelle layer removed. Further purification of proteins was achieved by lentil-lectin Sepharose chromatography with 10% a-methyl-D-mannoside elution. Detergent was partially removed with Bio-Beads SM-2. Each GPIIb-IIIa preparation was assayed for total protein by a modified Lowry procedure (25) and for residual Triton X-114 by spectrophotometric assay (26). This preparative method yielded GPIIb-IIIa in complex as determined by GPIIb resistance to thrombin hydrolysis (28,29). Preparations maximally contained 0.27 mg of Triton/mg of protein. Previous data demonstrated that this residual detergent did not qualitatively alter the experimental results. Proteins were analyzed by SDS-PAGE, reduced and nonreduced, using 7.5% gels; only two bands with M, consistent with GPIIb and GPIIIa were detectable by combined silver and Coomassie Blue stains of gels of any protein preparation used.
Proteoliposome Preparation-Large unilamellar PC vesicles were prepared by reverse phase as previously described (20)(21)(22)(23)(24)(25)(26)(27). PC (25 mg in hexane, 100 mg/ml) was dried to a thin film in a 50-ml roundbottom flask in uacuo for 3 h. The lipid film was resuspended in 1.5 ml of ether, then in 0.25 ml of 10 mM HEPES pH 7.40,0.150 M NaC1, and the fluorescent Caz+ indicator, Fura-2, as pentapotassium salt (30 pM). This mixture was sonicated for 2 min in a bath sonicator. The ether was removed by rotary evaporation to form a lipidic gel. This gel was sonicated for 1 min with 1 ml of GPIIb-IIIa (200 pg/ml in M Ca") and Fura-2 (30 pM). Liposomes were washed two times by centrifugation at 14,000 X g for 10 min at 4 "C. Liposomes were relatively homogenous in size and entrapped volume with a mean diameter of 0.1 pm by laser light scattering with a Coulter N4 submicron particle sizer. On average, 70% of added protein was incorporated, with 48.6 2 0.8% of protein in an outside-out orientation as determined by neuraminidase cleavage of sialic acid residues (28). As previously reported, the GPIIb-IIIa complex incorporated onto the PC liposomes bound Fgn with approximate Kd = 10" M (20, 28, 29) and bound monoclonal antibodies against GPIIb-IIIa (20). SDS-PAGE of proteins associated with SDS-solubilized liposome preparations revealed only two bands consistent with GPIIb and GPIIIa (20). Gel filtration studies confirmed that Fura-2 remained within the liposomes (20).
Calcium Transit Studies-Liposomes were utilized within 1 h of preparation and kept on ice until use. Temperature and pH were rigorously controlled. Double-distilled deionized 18 milliohm water was utilized in all buffers. Fura-2-loaded liposomes M intravesicular Caz+) were injected into Tris buffer (0.15 M NaCl, 10-'-1O2 M Ca2+), 20 pl of liposome suspension, to 980 p1 of buffer. This approximates infinite cis-entry conditions (saturating external calcium).
The Fura-2 fluorescence was measured (Xex 345 nm, L,,, 510 nm; slit widths of 2 and 5, respectively) in a Perkin-Elmer 650-10s fluorescence spectrophotometer. These emission and excitation wavelengths minimized the contribution from intrinsic liposomal fluorescence and beam dispersion. This system measures only Ca2+ which actually enters the liposome, not surface-associated ion. Peptide Effects-Prior to Caz+ transit studies, GPIIb-IIIa liposomes were incubated with His-12-Val (0-200 pM) or RGDS (0-200 pM) for 20 min at 25 "C; liposomes were then injected into Ca2+-containing buffer as described above. The peptides were homogeneous by high pressure liquid chromatography and thin layer chromatography. Amino acid analysis of the peptides was consistent with their predicted composition. The ability of these peptides to inhibit platelet aggregation was demonstrated by standard methods. TO assess the ability of RGDS binding to alter the effect of the His-12-Val on calcium entry, the liposomes were incubated with RGDS (170 p M ) prior to the addition of dodecapeptide (10-25 pM). Parallel incubations were performed on PC (nonprotein) and GPIIb-IIIa liposomes without peptide as controls. Experiments were performed six times in quadruplicate.

Ca2+
Movement into Vesicles-Ca2+ entrance was monitored by absolute fluorescence (Xex 345 nm, X . , 510 nm) of Fura-2loaded vesicles incubated in Ca" buffers. As previously reported (19), kinetic studies performed from 5 s to 15 min demonstrated that the onset of influx was rapid, reaching 83.6% of maximum by 5 s with a minimal, but detectable, additional increase over the subsequent 10 s ( p = 0.0013, 15 s compared with 5 s). No further influx occurred over 15 min.
Dodecapeptide Inhibition of Ca2+ Entrance into Vesicles-The dodecapeptide, His-12-Va1, inhibited Ca" entrance into the vesicles in a dose-dependent manner (Fig. 1). The apparent ICbo was 15 WM for inhibition of influx for 10-3-10-2 M external Ca". At concentrations of dodecapeptide >30 p~, inhibition of Ca2+ influx was so complete as to permit no net detectable Ca2+ influx into vesicles after 5 min. His-12-Val had no effect on the intrinsic fluorescence of Fura-2 within liposomes. This was determined by increasing intraliposomal Ca2+ with 1 PM ionomycin in Caz+ buffers in the presence and absence of His-12-Val. In contrast to His-12-Va1, RGDS had no effect on Caz+ entrance into vesicles; the data for RGDS were identical to GPIIb-IIIa liposomes incubated with buffer ( Fig. 1). PAC-1 antibody also demonstrated no inhibition of Ca2+ influx. When GPIIb-IIIa liposomes were incubated with 75 pg/ml RGDS for 20 min prior to incubation with 10-45 p~ His-12-Va1, no inhibition of Ca2+ movement across the phospholipid membrane by His-12-Val was observed ( Table  I) or buffer (-) prior to incubation in lo-' M ca2+.
Change in intravesicular Fura-2 fluorescence (Xex 345 nM, X. , 510 nM) as an indicator of Ca2+ influx was followed over time. Data points shown are mean values and one standard deviation from six experiments. Change in fluorescence in Fura-2-loaded nonprotein PC liposomes was minimal and is subtracted from the data shown. This is detailed under "Experimental Procedures."
Calcium Transit into Intact Platelets-His-12-Val and RGDS in concentrations up to 100 p~ had no effect on steadystate Ca2+ transit across the platelet membrane as determined by 45Ca exchange. His-12-Val inhibited the entrance of 45Ca into ADP-activated gel-filtered platelets. Results from a 10min incubation with 45Ca and ADP at 25 "C after incubation with His-12-Val are shown in Table 11. Similar results are obtained with a 1-min incubation. This inhibition represented only a 10.06 k 0.06% decrease in total platelet Ca2+ influx in this system. RGDS had no effect on 45Ca influx at 1 or 10 min. To determine if Ca2+ influx inhibition was a function of peptide specificity and size (i.e. dodecapeptide versus tetrapeptide), the effect of HHLGGARQAGDV, a dodecapeptide which has limited inhibitory activity against platelet aggregation = 150 p M ) , was assessed in activated platelets.
This peptide had no effect on 45Ca2+ entrance at concentrations up to 100 pM. With Fura-2-loaded thrombin-stimulated platelets, His-12-Val effected a dose-dependent decrease in the change in Fura-2 fluorescence 30 s following activation; 50 pM His-12-Val caused a 25% decrease and 200 p~ His-12-Val a 50% decrease. The final increase in Fura-2 fluorescence at 5 min was not changed. In contrast, preincubation with La3+ caused an 83% decrease in change in Fura-2 fluorescence. RGDS (25-125 p~) had no effect on the thrombin-induced Ca2+ increase.

DISCUSSION
The present study demonstrates that binding of an analogue of the Fgn y carboxyl-terminal dodecapeptide, His-12-Va1, to GPIIb-IIIa liposomes decreases the GPIIb-IIIa-mediated Ca2+ entry into these liposomes. At concentrations of His-12-Val 230 p~, Ca2+ entry was profoundly inhibited, with no Ca2+ influx after 5 min of incubation. A related dodecapeptide with an arginine substitution at position 7, which is relatively ineffective in inhibition of platelet aggregation, failed to inhibit Ca2+ transit. The synthetic peptide, RGDS, in concentrations up to 125 p~ and a monoclonal antibody to the RGDS binding site had no effect on Ca2+ influx into liposomes. Binding of RGDS to GPIIb-IIIa liposomes prevented the His-12-Val inhibition of Ca2+ transit across the liposome membrane; therefore, the difference in inhibitory capacity between the peptides is not due to failure of RGDS to bind to GPIIb-IIIa molecules on the surface of the liposomes. The ability of Fgn as well as His-12-Val to inhibit this Ca2+ channel function suggests that the GPIIb-IIIa complex on the surface of the liposome in the activated configuration facilitates Ca2+ influx.
Potential mechanisms for His-12-Val inhibition of Ca2+ transit include His-12-Val-induced conformational change in the GPIIb-IIIa complex, change in topographical distribution of GPIIb-IIIa on the surface of the liposomes, or His-12-Valinduced changes in the four potential Ca2+ binding regions on the GPIIb molecule (32, 33). Tsein et al. (34) have proposed that such Ca2+ binding domains provide selectivity to Ca2+ "trigger proteins." The difference in inhibitory capacity between dodecapeptide and RGDS may be related to nonidentity of binding sites of these two peptides or differences in conformational changes associated with ligand-receptor interaction. While both peptides induce conformational changes in the soluble GPIIb-IIIa complex (19), subtle differences in these changes may account for differences in Ca2+ channel inhibition. While data from a number of studies suggest that His-12-Val and RGDS bind to related sites on the GPIIb-IIIa complex, these sites may not be identical. Despite the mutually inhibitory nature of RGDS and LGGAKQAGDV binding, Santoro and Lawing (35) with affinity labeling, Williams and Gralnick (15) with binding competition, and Bennett et al. (16) have hypothesized that the binding sites for these peptides are not identical but that ligand-induced conformational changes may be responsible for mutually exclusive binding.
Our studies with His-12-Val and the intact platelet extend and are in general accord with those of Brass (21),Yamaguchi et al. (22), and Sinigaglia et al. (36). Their data suggest that GPIIb-IIIa may play a role in calcium flux in the intact platelet. The lack of inhibition with RGDS in the current study is in contrast to the inhibition of Ca2+ influx by GRGDSP observed by Yamaguchi et al. (22) and by GRGDS observed by Sinigaglia et al. (36) in stimulated platelets. This difference may be due to differences in methodology, i.e. dimethyl sulfoxide-treated platelets in the Yamaguchi study following thrombin and collagen activation versus intact, ADP, and thrombin-activated platelets, or from different time points chosen for analysis. The 10-min time point in the current 45Ca study follows the completion of transmembrane Ca2+ influx (37, 38), and both control and peptide-treated platelet data may include a component of efflux. This efflux would actually result in an underestimate of His-12-Val inhibition. Differences among these three studies should not arise from differences among RGDS, GRGDSP, and GRGDS since the ICso values of these peptides for Fgn y chain binding to activated platelets are similar (16). The Sinigaglia study (36) measured the total increase in platelet Ca2+, while the current 45Ca study examines transmembrane Ca2+ movement. The Fura-2 platelet data include a significant component of calcium release from intracellular sites; however, the La3+ inhibition suggests a significant contribution from extracellular Ca2+ to [CaZ+li at 30 s. The complex nature of changes in Ca2+ concentration in the activated platelet with its multiple sources of Ca2+ in contrast to the liposome model render the former a more difficult system to interpret. The observation that ADP-induced activation and secretion by human platelets proceeds normally in the absence of external calcium indicates that the Caz+ influx mediated by GPIIb-IIIa may play only a subsidiary role in platelet activation. The current data indicate that His-12-Val inhibits only a minor component of Ca2+ movement into the platelets. This may explain the apparent discordance between these data and those of Powling and Hardisty (39) who were able to demonstrate normal calcium influx into ADP-activated quin-2-loaded thrombasthenic platelets. Our platelet data, in concert with the liposome model data, suggest that GPIIb-IIIa may play a minor but significant role in transmembrane calcium influx into activated platelets. This calcium could potentially alter local perimembrane Ca2+ concentration or affect GPIIb-IIIa directly. Specific ligand binding may terminate GPIIb-IIIamediated influx. Further studies will determine the precise nature of this channel and mechanism of inhibition and define its role in platelet activation.