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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1651-1660

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1651.)
© 2000 American Heart Association, Inc.

Thrombosis

Inhibition of Platelet Integrin {alpha}IIbß3 by Peptides That Interfere With Protein Kinases and the ß3 Tail

Ingeborg Hers; José Donath; Pieter E. M. H. Litjens; Gijsbert van Willigen; Jan-Willem N. Akkerman

From the Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, Utrecht, the Netherlands. Dr Hers is now at the Department of Biochemistry, School of Medical Sciences, Bristol, UK.

Correspondence to Prof Dr J.W.N. Akkerman, Department of Haematology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail j.w.n.akkerman{at}laboratory.azu.nl


*    Abstract
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Abstract—{alpha}-Thrombin stimulation of human platelets initiates inside-out signaling to integrin {alpha}IIbß3 (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of {alpha}IIbß3 via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the ß3 subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and {alpha}IIbß3 ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited {alpha}-thrombin–induced ligand binding to {alpha}IIbß3, but a reversed peptide with Y->F substitutions (KFSGFIF) had no effect. Ligand binding to {alpha}IIbß3 was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and ß2-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the ß3 subunit. The LLITIHDR peptide, matching the membrane-proximal domain of ß3 (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of ß3, reduced {alpha}-thrombin–induced ligand binding by 60±4%, 97±1%, and 97±2% (n=3) at 500 µmol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin {alpha}IIbß3 and identify target domains in ß3 that contribute to the regulation of this integrin.


Key Words: integrin {alpha}IIbß3 • glycoprotein IIb/IIIa • protein kinase C • protein tyrosine kinase • platelets


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Integrins are heterodimeric cell surface receptors consisting of noncovalently associated {alpha} and ß subunits.1 Each subunit contains a large extracellular domain, a transmembrane domain, and a relatively short cytoplasmic tail (typically <70 residues for each subunit). Integrins function in a variety of biological processes, such as the differentiation, growth, and migration of cells, and in inflammation and wound healing. Many integrins are subject to intracellular modulation of their activation state for ligands, a process known as inside-out signaling.2 3 4 An increase in binding affinity and subsequent ligand binding generates signals into the cell, a process known as outside-in signaling.5 6 7 8

The regulation of platelet integrin {alpha}IIbß3 is a good illustration of the significance of the rapid affinity regulation of integrins. Unstimulated platelets express {alpha}IIbß3 in a conformation inaccessible to ligands, thereby preventing platelet-platelet interaction. On activation, inside-out signaling converts the integrin to a functional receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin.9 Fibrinogen binding to {alpha}IIbß3 couples the platelets together, forming an aggregate, which is a vital step in the cessation of bleeding. Patients with Glanzmann’s thrombasthenia, a severe bleeding disorder, have platelets that either lack {alpha}IIbß3 (type I) or contain a defective {alpha}IIbß3 that is unable to expose ligand binding sites (type III).10 The Ser752->Pro mutation in the ß3 subunit of type III patients is of particular interest because it results in the loss of ligand binding.11

Platelet agonists trigger inside-out signaling to {alpha}IIbß3 via protein tyrosine kinases (PTKs) as herbimycin A, tyrphostin A47, and geldanamycin inhibit ligand binding.12 The tyrosine phosphorylation of proteins with molecular masses of 54, 60, 64, 75, and 130 kDa during binding site exposure suggests that they may contribute to the affinity regulation of {alpha}IIbß3.13 14 15 Only a few agonists signal to {alpha}IIbß3 via PTK and protein kinase C (PKC, a Ser/Thr kinase), as illustrated by the inhibition by bisindolylmaleimide I and II and calphostin C and also by the fact that phorbol 12-myrisate 13-acetate (an activator of the PKC family) and 12-deoxyphorbol-13-phenylacetate-20-acetate (an activator of the PKCß subtype) trigger the sustained exposure of ligand binding sites. Activated PKC contributes to PTK activation and preserves the high-affinity state of the integrin, probably via stoichiometric phosphorylation of the ß3 subunit.16 Although kinase inhibitors have provided a first insight into the signaling pathways that control platelet {alpha}IIbß3, their broad specificity and the concern about their effects on other steps in platelet activation are obvious limitations. In the present study, we have applied specific peptides designed to interfere with the activity of these kinases and the accessibility of their possible target domains in {alpha}IIbß3. These peptides were (1) YIYGSFK, a substrate peptide for the Src-family; (2) RKRCLRRL, a peptide that binds irreversibly to the catalytic domain of PKC; and (3) 4 peptides mimicking domains in the cytoplasmic tail of the ß3 subunit containing potential phosphorylation sites for PTKs and PKCs.


*    Methods
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 *Methods
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Materials
Streptolysin O (SLO) was obtained from Murex diagnostics. GDPßS, GTP{gamma}S, D-myo-inositol 1,4,5-tris-phosphate (IP3), bisindolylmaleimide I (GF 109203X), and ADP were purchased from Roche Molecular Biochemicals. {alpha}-Thrombin, imipramine, and peroxidase-labeled protein A were obtained from Sigma Chemical Co, and Sepharose 2B and gelatin Sepharose 4B were from Pharmacia. Sodium iodide (specific activity 629 GBq/mg) and [14C]serotonin (specific activity 1.8 to 2.2 GBq/mmol) were purchased from Amersham International. [32P]Orthophosphate with a specific activity of 314 TBq/mmol was from New England Nuclear. D-Phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK) was from Calbiochem. The anti-phosphotyrosine antibodies PY-20 and 4G10 were from Upstate Biotechnology Inc. Sulforhodamine B was from Molecular Probes Inc. Fibrinogen (grade L) was from Chromogenix, and the blocking anti–integrin ß1 subunit antibody AIIB2 was a kind gift from Dr C Damsky, University of California, San Francisco.17 The fibrinogen-derived peptide GRGDS was kindly provided by Dr Bekkers, Department of Enzymology and Protein Engineering, Utrecht University, Utrecht, the Netherlands. The polyclonal anti-Syk antibody SC-573 was from Santa Cruz Biotechnology Inc, and the swine anti-rabbit horseradish peroxidase–labeled antibody was from DAKO. Other chemicals were of analytical grade.

Peptides
Peptides were synthesized by a stepwise solid-phase peptide synthesis method and purified by C18 reverse-phase high-performance liquid chromatography (HPLC, Biosynthesis). The purity of the peptides was >99%. The molecular weights of the peptides were verified by matrix-assisted laser desorption mass spectrometry by the manufacturer. The synthesized peptides were an Src substrate peptide (Src peptide, YIYGSFK, 7 amino acids [AAs], molecular weight [MW] 877), the reversed Src peptide with Y->F substitutions (Src-R peptide, KFSGFIF, 7 AAs, MW 845), the inhibitory peptide for PKC (PKC peptide, RKRCLRRL, 8 AAs, MW 1100), and 4 peptides corresponding to different partially overlapping domains of the ß3 cytoplasmic tail (shown in Figure 1Down). These 4 peptides were as follows: ß3-1 (corresponding to residues 717 to 724, 8 AAs, MW 980), ß3-2 (residues 743 to 750, 8 AAs, MW 918), ß3-3 (residues 749 to 756, 8 AAs, MW 870), and ß3-4 (residues 755 to 762, 8 AAs, MW 925). In addition, 2 modifications of the ß3-3 peptide were synthesized with an S->A (EATATFTN) and an S->P replacement (EATPTFTN). For uptake studies, the ß3-3 and ß3-4 peptides were labeled with FITC by dissolving 1 mg peptide in 300 µL of 0.25 mol/L sodium carbonate buffer (pH 9.0) and incubated with 1.3 mmol/L FITC for 16 hours at 4°C. The FITC-labeled peptide was purified on a C8 reverse-phase HPLC column. The molecular mass of the purified peptide was verified by mass spectrometry. To assess the aspecific effects after the introduction of peptides, platelets were treated with protein C inhibitor (PCI)-1 peptide (HRHHPREHKERVEDLH, 16 AAs, MW 2112), PCI-2 peptide (HRHHPREHKEEVEDLH, 16 AAs, MW 2085), and ß2-glycoprotein I (ß2-gpI) peptide (FKEHSSLAFWK, 11 AAs, MW 1402), which were >96% pure and kindly provided by Drs M.G.L.M. Elisen and D.A. Horbach, Department of Hematology, University Medical Center Utrecht, Utrecht, the Netherlands. The PCI peptides correspond to a domain in the heparin binding site of PCI and contain individual substitutions of a basic residue in the consensus binding domain that blocks heparin binding. The ß2-gpI peptide corresponds to a sequence in the fifth domain of ß2-gpI.



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  		Figure 1. AA sequence of the ß3 cytoplasmic tail and corresponding peptides. The boxes underneath the ß3 cytoplasmic tail denote the position, AA sequence, and abbreviation (ß3-1 to ß3-4) of the ß3 peptides used in the present study. A single-letter AA code is used. The start and end positions of the peptides are indicated by arrows. Stippled boxes refer to the highly conserved membrane-proximal sequences of the {alpha}IIb and ß3 cytoplasmic tails. Note that the model is not drawn to scale.

Platelet Isolation
Freshly drawn venous blood from healthy volunteers, who claimed not have taken any medication in the previous 14 days, was anticoagulated with 0.1 vol of 13 mmol/L trisodium citrate. Platelet-rich plasma was obtained by centrifugation (200g for 15 minutes at 22°C), and platelets were isolated by gel filtration on a Sepharose 2B column equilibrated in Ca2+-free Tyrode’s solution (137 mmol/L NaCl, 2.68 mmol/L KCl, 0.42 mmol/L NaH2PO4, 1.7 mmol/L MgCl2, and 11.9 mmol/L NaHCO3, pH 7.2) containing 0.1% glucose and 0.2% BSA. The platelet count was adjusted to 2x1011 platelets per liter.

Preparation of 125I-Labeled Fibronectin and 125I-Labeled Fibrinogen
Fibronectin was isolated from fresh frozen plasma by affinity chromatography on gelatin–Sepharose 4B as described elsewhere.18 Electrophoresis on a 5% polyacrylamide gel, as described by Laemmli19 after reduction with ß-mercaptoethanol, showed >99% homogeneity. The fibronectin preparation contained <10 ng von Willebrand factor per milligram of fibronectin, as determined with a von Willebrand ELISA. Fibrinogen was made fibrin and fibronectin free by passage through a gelatin–Sepharose 4B column. The fibrinogen and fibronectin were radiolabeled with sodium iodide by a modified Iodo-Gen method, as described in detail for fibrinogen.20

Isolation and Permeabilization With SLO
A 15 U/mL solution of SLO was freshly prepared in Ca2+-free Tyrode’s solution (137 mmol/L NaCl, 2.68 mmol/L KCl, 0.42 mmol/L NaH2PO4, 1.7 mmol/L MgCl2, and 11.9 mmol/L NaHCO3, pH 7.2) containing 0.1% glucose and 0.2% BSA. Gel-filtered platelets were permeabilized with 0.15 U/mL SLO for 3 minutes at 22°C before addition of the agonist, unless indicated otherwise. In each experiment, permeabilization was verified by a parallel incubation with the membrane-impermeant mediator IP3 (15 µmol/L). Suspensions that showed an IP3-induced ligand binding of {approx}22 500 fibronectin molecules per platelet were considered permeable and used for further analysis. IP3-induced phosphorylation of pleckstrin, a substrate for PKC, was >20% of the phosphorylation seen in the presence of 0.2 U/mL {alpha}-thrombin, as assessed by the program ImageQuant (Molecular Dynamics). Tyrosine phosphorylation initiated by IP3 was in the same range as that induced by the weak agonist ADP. Experiments in which IP3 induced lower responses were discarded. Permeabilization was confirmed by fluorescence-activated cell sorter (FACS) analysis in the FL-2 channel on a FACScan (Becton-Dickinson) in the presence of sulforhodamine B. The permeabilization procedure induced 16±4% (n=4) release of maximal secretable [14C]serotonin (5 U/mL {alpha}-thrombin, 20 minutes at 22°C) in the course of the experiment. [14C]Serotonin secretion was determined as described.21

Binding of 125I-Labeled Fibronectin and 125I-Labeled Fibrinogen
Gel-filtered platelets were permeabilized in the presence of different concentrations of the Src, PKC, and ß3 peptides at 22°C. 125I-labeled ligand (1 µmol/L) was added 2 minutes after the addition of SLO, which was followed by the addition of {alpha}-thrombin (0.2 U/mL) or IP3 (15 µmol/L, all final concentrations) 3 minutes later. Samples (200 µL, in triplicate) were drawn and layered on top of 100 µL of 20% (wt/vol) sucrose in Ca2+-free Tyrode’s solution in microsedimentation tubes (Sarstedt) and centrifuged (12 000g for 4 minutes at 22°C) in a Beckman Microfuge E 15 minutes after stimulation. The pellet was separated from the supernatant and counted for radioactivity in a gamma counter. The number of molecules bound per platelet was calculated from the radioactivity in the pellet fraction and compared with the total activity in the pellet plus supernatant. The data were corrected for nonspecific binding, defined as the binding of 125I-labeled ligand to unstimulated intact platelets.18 22

Measurement of Pleckstrin Phosphorylation
The activity of PKC was deduced from the phosphorylation of the 47-kDa protein pleckstrin, a major substrate for this enzyme. Platelets were labeled with 3.7 MBq carrier-free [32P]Pi per milliliter of platelet-rich plasma for 1 hour at 37°C. Platelet suspensions were acidified to pH 6.5, centrifuged, and resuspended in HEPES-Tyrode (145 mmol/L NaCl, 5 mmol/L KCl, 0.5 mmol/L Na2HPO4, 1 mmol/L MgSO4, and 10 mmol/L HEPES, pH 7.2) containing 0.1% glucose. Labeled platelets (2x1011 platelets per liter) were permeabilized in the presence of different concentrations of the Src, PKC, and ß3 peptides at 22°C and incubated with {alpha}-thrombin (0.2 U/mL) or IP3 (15 µmol/L) for 2 minutes. Samples were collected and transferred into 3x concentrated Laemmli sample buffer19 and boiled for 5 minutes before SDS-PAGE (11%). Gels were stained with Coomassie brilliant blue, and radioactive bands were visualized by autoradiography and exposure to a phosphoimager screen. For determination of the radioactivity of pleckstrin, the density of the 47-kDa band was determined by the program OptiQuant (Packard Instruments).

Measurement of Protein Tyrosine Phosphorylation
Gel-filtered platelets (2x1011 platelets per liter) were permeabilized in the presence of different concentrations of the Src, PKC, and ß3 peptides at 22°C. Subsequently, platelets were incubated with {alpha}-thrombin (0.2 U/mL) or IP3 (15 µmol/L) for 2 minutes. Samples were withdrawn, and 3x concentrated Laemmli sample buffer was added. The samples were heated at 95°C for 5 minutes, and proteins were separated on SDS/7.5% PAGE and electrophoretically transferred (1 hour at 100 V) to nitrocellulose membrane in 25 mmol/L Tris/192 mmol/L glycine (pH 8.3) and 20% methanol (vol/vol) by using a minitransblot system (Bio-Rad). The blots were blocked with Tris-buffered saline (TBS) containing 4% BSA for 1 hour at room temperature and subsequently incubated (16 hours at 4°C) with the anti-phosphotyrosine antibody 4G10. Subsequently, the blots were incubated with peroxidase-labeled protein A (2 µg/mL for 2 hours at 4°C), treated with Renaissance chemiluminescence Western blot reagent, and exposed to Kodak X-Omat Blue autoradiography film (Eastman Kodak Co). For the reprobing with an anti-Syk antibody, the blots were stripped in TBS containing 0.1% Tween 20, 2% SDS, and 2% ß-mercaptoethanol. After extensive washing, the blots were blocked with 10% BSA in TBS-Tween and incubated for 16 hours with a polyclonal antibody against Syk. Subsequently, the blots were incubated with swine anti-rabbit horseradish peroxidase–labeled antibody, and the densities of the spots were quantified with OptiQuant (Packard Instruments).

Statistics
Data are expressed as mean±SD, with n indicating the number of observations, and were analyzed by Student t test for paired observations. Differences were considered significant at P<0.05.


*    Results
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Integrin {alpha}IIbß3 Is Preserved in Human Platelets Permeabilized by SLO
Platelets were permeabilized by treatment with SLO. The optimal SLO concentration was determined by incubation in the presence of the fluorescent indicator sulforhodamine B and by monitoring the uptake of the dye by flow cytometry. Uptake was half maximal at {approx}0.13 U/mL and maximal at 0.20 U/mL SLO (Figure 2ADown). The cell-impermeable activator IP3, which releases Ca2+ from intracellular stores, and GTP{gamma}S, which activates trimeric G proteins, induced ligand binding of 22 500±4100 and 17 230±3280 fibronectin molecules per platelet, respectively, at 0.15 U/mL SLO, illustrating that inside-out signaling mechanisms to {alpha}IIbß3 were at least partly preserved. Above this SLO concentration, the functional properties of {alpha}IIbß3 were lost. At the optimal concentration of SLO (0.15 U/mL), the platelet population showed a normal distribution of sulforhodamine B fluorescence, indicating that all platelets were permeabilized (Figure 2BDown). This concentration was used in subsequent experiments.



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  		Figure 2. Platelet permeabilization with SLO. A, Effect of membrane-impermeable activators of {alpha}IIbß3 at different concentrations of SLO. Gel-filtered platelets (2x1011 cells per liter) were incubated with the indicated concentrations of SLO for 3 minutes before addition of Tyrode’s solution (buffer [b]), 200 µmol/L GTP{gamma}S, or 15 µmol/L IP3 in the presence of 125I-labeled fibronectin. The specific ligand binding to {alpha}IIbß3 was measured 15 minutes after addition of GTP{gamma}S or IP3 and expressed as the number of fibronectin molecules bound per platelet (left y-axis). Under the same conditions, the platelets were incubated with sulforhodamine B, subsequently diluted to 2x1010 cells per liter, and analyzed by FACS analysis (open squares, right y-axis, fluorescence, in arbitrary units). B, Gel-filtered platelets incubated for 3 minutes in the presence of the cell-impermeable fluorescent dye sulforhodamine B without (-SLO) and with (+SLO) 0.15 U/mL SLO, diluted to 20x103 platelets per microliter, and subsequently analyzed by FACS.

Because permeabilization procedures are likely to affect cell function, platelet aggregation and secretion were compared in intact and SLO-treated platelets. Compared with intact platelets, permeabilized platelets aggregated normally after stimulation with {alpha}-thrombin but slightly slower after stimulation with ADP (Figure 3ADown). [14C]Serotonin secretion was also slower after permeabilization and amounted to 87±7% (n=7) of intact platelets after 5 minutes of stimulation with 0.2 U/mL {alpha}-thrombin (Figure 3BDown). To assess whether permeabilized platelets were capable of activating {alpha}IIbß3, the specific binding of 125I-labeled fibronectin to {alpha}-thrombin–stimulated platelets was measured. SLO-treated platelets showed 85±6% (n=8) of the binding of intact platelets (Figure 3CDown). These data illustrate that despite a slight decrease in aggregation, secretion, and ligand binding to {alpha}IIbß3, permeabilized platelets preserved >=80% of their functional responses.



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  		Figure 3. Effect of SLO permeabilization on platelet functions. A, Aggregation tracings of intact (-SLO curves) and SLO-treated (+SLO curves) platelets stimulated with 0.2 U/mL {alpha}-thrombin and 10 µmol/l ADP (in the presence of 1 µmol/L fibrinogen). B, Secretion of [14C]serotonin by intact (open symbols) and SLO-treated (closed symbols) platelets incubated with 0.1 (squares) and 0.2 U/mL (triangles) {alpha}-thrombin or with buffer (circles). Data are expressed as percentage of total secretable serotonin, as defined in Methods. C, Binding of 125I-labeled fibronectin to intact (open symbols) and SLO-permeabilized (closed symbols) platelets. Platelets were stimulated with 0.2 U/mL {alpha}-thrombin (squares) or buffer (circles) in the presence of 1 µmol/L 125I-labeled fibronectin, and at different times, samples were collected. The specific ligand binding was measured and expressed as molecules fibronectin bound per platelet (mean±SD, n=3).

Although fibrinogen is the natural ligand for {alpha}IIbß3 in stirred platelet suspensions, binding studies with {alpha}-thrombin–stimulated platelets are easily disturbed by fibrin formation. To confirm previous findings that 125I-labeled fibronectin and fibrinogen are bound to {alpha}IIbß3 with the same kinetics,18 the specific binding of 125I-labeled fibrinogen to {alpha}IIbß3 was measured under conditions that prevented {alpha}-thrombin–induced fibrin formation. Platelets were first incubated with {alpha}-thrombin for 2 minutes and then with the thrombin-neutralizing agent PPACK (30 nmol/L) for another 2 minutes. This was followed by 15 minutes in the presence of 125I-labeled ligand. As shown in Table 1Down, there was no significant difference between the binding of fibronectin and fibrinogen. Also, the use of PPACK did not interfere if the platelets were stimulated with an optimal {alpha}-thrombin concentration (0.2 U/mL). The presence of the {alpha}IIbß3 antagonist GRGDS abolished fibronectin and fibrinogen binding by >95%, confirming that both ligands are bound to {alpha}IIbß3. A possible binding of fibronectin to ß1 integrins on the platelet was investigated in experiments with the ß1 antagonist AIIB2. This antibody changed neither fibronectin nor fibrinogen binding, indicating that the binding data reflected primarily ligand binding to {alpha}IIbß3.


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  		Table 1. Comparison Between {alpha}-Thrombin–Induced Fibronectin and Fibrinogen Binding

To assess whether the activation state of integrin {alpha}IIbß3 was sensitive to agents that do not penetrate intact cells, platelets were stimulated with ADP (10 µmol/L) and {alpha}-thrombin (0.2 U/mL), which induced the binding of 22 537±6261 (n=4) and 59 045±9250 (n=8) fibronectin molecules per platelet, respectively (Figure 4Down). Although the cell-impermeable GDP analogue GDPßS slightly reduced ligand binding to intact platelets, the inhibition in permeabilized platelets was much stronger, resulting in {approx}80% ({alpha}-thrombin) to 90% (ADP) inhibition by 400 µmol/L GDPßS. Together, these results illustrate that permeabilized platelets preserve sensitivity to inside-out regulation and at the same time are accessible to membrane-impermeable agents.



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  		Figure 4. The G-protein blocker GDPßS inhibits agonist-induced ligand binding to integrin {alpha}IIbß3. Gel-filtered platelets were incubated for 1 minute with different concentrations of GDPßS before addition of Tyrode’s solution (A) or 0.15 U/mL SLO (B) for 3 minutes. Subsequently, platelets were stimulated with 10 µmol/L ADP and 0.1 U/mL {alpha}-thrombin in the presence of 125I-labeled fibronectin. Specific binding was measured 15 minutes after stimulation. Data are expressed as percentage of control (100%: for ADP, 22 537±6261 [mean±SD] fibronectin molecules per platelet, n=4; for {alpha}-thrombin, 59 045±9250 [mean±SD] fibronectin molecules per platelet, n=8).

Because the permeabilization procedure was designed to introduce peptides that possibly interfere with {alpha}IIbß3 regulation, a peptide mimicking residues 749 to 756 in the ß3 tail, designated ß3-3, and a peptide mimicking residues 755 to 7762, designated ß3-4 (Figure 1Up), were labeled with FITC and added to the platelet suspension before permeabilization. Subsequent FACS analysis revealed a complete shift of the platelet suspension, indicating that all platelets had accumulated a certain amount of the FITC-labeled ß3-3 (Figure 5Down) and ß3-4 (not shown). To further address the specificity of {alpha}IIbß3 inhibition by the peptides, secretion studies were performed in the presence of ß3-4 and in the presence of a peptide related to an inhibitory peptide of Src kinases, named Src-R (see below). None of these peptides interfered significantly with the secretion of [14C]serotonin induced by 0.2 U/mL {alpha}-thrombin, illustrating that a major platelet function was undisturbed (Table 2Down).



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  		Figure 5. Introduction of peptides into SLO-treated platelets. Gel-filtered platelets were incubated with 25 µmol/L FITC-labeled ß3-3 for 3 minutes, treated with buffer or SLO, as indicated, and analyzed by FACS. FL1-H indicates fluorescence 1-height.


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  		Table 2. Peptide Introduction Into SLO-Treated Platelets: Effect on Secretion

Effect of Src and PKC Peptides on {alpha}IIbß3 Exposure
Earlier studies revealed that ligand binding to {alpha}IIbß3 was greatly impaired in the presence of bisindolylmaleimide I and herbimycin A, which are potent inhibitors of the PKC family and tyrosine kinases, respectively. Because such a pharmacological approach might be biased by aspecific side effects, the role of these protein kinases was evaluated by introducing peptides that interfere with these enzyme superfamilies. Permeabilized platelets stimulated with {alpha}-thrombin bound at 59 771±9775 125I-labeled fibronectin molecules per platelet. To evaluate the role of Src kinases in affinity regulation of {alpha}IIbß3, the Src peptide YIYGSFK was introduced. This is a substrate analogue of Src kinases and competes with the natural substrate for phosphorylation.23 The peptide dose-dependently inhibited {alpha}-thrombin–induced ligand binding, with almost complete inhibition at 1000 µmol/L. In contrast, ligand binding to intact platelets was unaffected (Figure 6ADown). The PKC inhibitory peptide RKRCLRRL binds irreversibly to a conserved Cys residue in the catalytic domain of PKC and inhibits the activity of PKC isoforms {alpha}, ß, and {gamma} in vitro.24 This peptide dose-dependently reduced ligand binding, showing a maximal inhibition of {approx}75%. No effect of the PKC peptide was found in intact platelets. The reversed peptide of the Src peptide with Y->F substitutions did not change {alpha}-thrombin–induced ligand binding (Src-R in Table 3Down) or [14C]serotonin secretion (Table 2Up). To assess possible aspecific effects of these treatments, peptides were introduced that were not related to signaling elements in platelets. These were the peptides corresponding to domains of PCI, named PCI-1 and PCI-2, and of ß2-gpI. None of these peptides interfered significantly with {alpha}-thrombin–induced ligand binding up to a concentration of 1000 µmol/L (Table 3Down). Thus, these data confirm the earlier findings based on pharmacological inhibitors of PKC and tyrosine kinases and indicate more specifically that Src kinases take part in the regulation of the activation state of {alpha}IIbß3.



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  		Figure 6. {alpha}-Thrombin–induced fibronectin binding is inhibited by the Src and PKC peptides. A, Gel-filtered platelets incubated for 3 minutes with Tyrode’s solution (open squares, similar for both peptides) or 0.15 U/mL SLO in the presence of different concentrations of Src peptide (closed triangles) or PKC peptide (open circles). Subsequently, platelets were stimulated with 0.2 U/mL {alpha}-thrombin in the presence of 125I-labeled fibronectin. Specific binding was measured at 15 minutes after stimulation and expressed as percentage of control (mean±SD, n=3; 100%: in permeabilized platelets, 59 771±9775 fibronectin molecules per platelet; in intact platelets, 91 253±12 025 fibronectin molecules per platelet). B and C, Effect of Src and PKC peptide on {alpha}-thrombin–induced tyrosine and pleckstrin phosphorylation. Unlabeled platelets (B) or 32P-labeled platelets (C) were permeabilized with 0.15 U/mL for 3 minutes in the presence of different concentrations of Src or PKC peptide, as indicated, before stimulation with 0.2 U/mL {alpha}-thrombin. Two minutes after stimulation, samples were collected for protein separation on PAGE and immunoblotting with antibody 4G10 to detect tyrosine-phosphorylated proteins (B) and for analysis of pleckstrin phosphorylation (C). D, Quantification of the density of the 100-kDa band (hatched bars) and 55-kDa band (open bars) of panel B with the program OptiQuant (Packard Instruments), with the density of the Syk bands used as a standard for protein quantification. A similar decrease in density at increasing concentrations of Src peptide was found for the 60-kDa band and to a lesser extent for the 125-kDa band. The data are representative of 3 similar results.


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  		Table 3. Effect of Different Peptides on {alpha}-Thrombin–Induced Ligand Binding to Permeabilized Platelets

To confirm that the peptides indeed interfered with their target proteins, tyrosine-phosphorylated proteins (Figure 6BUp) and phosphorylated pleckstrin (Figure 6CUp) were analyzed. Unstimulated permeabilized platelets showed tyrosine phosphorylation of proteins with molecular masses of {approx}50, 55, 60, 100, and 125 kDa, in accordance with earlier reports on intact platelets.25 26 In addition, a weak tyrosine phosphorylation was found in proteins of 84/87 kDa. {alpha}-Thrombin stimulation further increased the tyrosine phosphorylation of these proteins and, in addition, triggered phosphorylation of a protein with a molecular mass of 70 kDa. The Src peptide inhibited these tyrosine phosphorylations dose-dependently with almost complete inhibition at 1000 µmol/L, which is in line with the expected competition with the natural substrates (Figure 6DUp). In contrast, neither the PKC peptide nor the reversed Src-R peptide interfered with these tyrosine phosphorylations. As expected, the PKC peptide strongly interfered with the phosphorylation of pleckstrin, which in platelets is a major PKC substrate. There was a dose-dependent reduction in {alpha}-thrombin–induced pleckstrin phosphorylation with a maximum at {approx}200 µmol/L. This phosphorylation was unchanged in the presence of maximal concentrations (1000 µmol/L) of Src peptide and Src-R. Thus, the Src peptide and the PKC peptide interfered with their respective target enzymes without showing cross-reactivity. Therefore, their effect on the affinity regulation of {alpha}IIbß3 might result from direct interference with the role of these kinases in inside-out signaling to {alpha}IIbß3.

Identification of Cytoplasmic Domains in ß3 Involved in Affinity Regulation of {alpha}IIbß3
In Chinese hamster ovary cells, mutations and deletions in the ß3 cytoplasmic tail interfere with the affinity state of chimeric {alpha}IIbß3.4 11 27 28 These observations indicate that ß3 might be a target for protein kinases that take part in inside-out signaling to {alpha}IIbß3, either via a direct effect or via phosphorylation of 1 or more proteins that control the affinity state of the integrin. To identify these residues, 4 peptides were constructed that mimic different domains of the ß3 cytoplasmic tail, as illustrated in Figure 1Up. The peptide that resembled the membrane proximal part of the ß3 tail, named ß3-1, failed to interfere with the {alpha}-thrombin–induced fibronectin binding. In contrast, peptides ß3-2, ß3-3, and ß3-4 dose-dependently reduced {alpha}-thrombin–induced ligand binding (Figure 7ADown). At 500 µmol/L peptide, ß3-2 reduced the binding by 60±4%, ß3-3 reduced it by 97±1%, and ß3-4 reduced it by 97±2% compared with ligand binding to permeabilized platelets in the absence of these peptides. The modified ß3-3 peptides with an S->A and an S->P replacement showed the same inhibition as ß3-3 (data not shown). No effect of these peptides was seen on intact platelets (data not shown). These results indicate that sequences at the carboxy-terminal end between Asn743 and Thr762 of ß3 take part in the regulation of the affinity state of {alpha}IIbß3.



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  		Figure 7. Effect of ß3 peptides on {alpha}-thrombin–induced ligand binding to {alpha}IIbß3. A, Gel-filtered platelets incubated for 3 minutes with 0.15 U/mL SLO in the presence of different concentrations of peptides ß3-1 (open squares), ß3-2 (closed squares), ß3-3 (triangles), or ß3-4 (circles). Subsequently, platelets were stimulated with 0.2 U/mL {alpha}-thrombin in the presence of 125I-labeled fibronectin. Specific binding was measured 15 minutes after stimulation and expressed as percentage of control (100%: 59 771±9775 [mean±SD] fibronectin molecules per platelet, n=3). Fibronectin binding to intact platelets in the presence of the peptides was 102±9% of the binding in the absence of peptides. B and C, {alpha}-Thrombin–induced tyrosine and pleckstrin phosphorylation in the presence of ß3 peptides. Unlabeled platelets (B) or 32P-labeled platelets (C) were permeabilized with 0.15 U/mL for 3 minutes in the presence of buffer or 500 µmol/L peptide ß3-2, ß3-3, or ß3-4 before addition of 0.2 U/mL {alpha}-thrombin or 15 µmol/L IP3. Two minutes after stimulation with {alpha}-thrombin, samples were collected for protein separation on PAGE and immunoblotting with antibody 4G10 to detect tyrosine-phosphorylated proteins (B) and for analysis of pleckstrin phosphorylation (C). D, Quantification of the density of the 125-kDa band (left-hatched bars), the 55-kDa band (open bars), and the 20-kDa band (right-hatched bars) of panel B. The constant density seen for the 125-kDa band was also observed for the 60- and 42-kDa bands. The increase in density found for the 20-kDa band was also seen for the 100-kDa band. The data are representative of 3 similar results.

Because these peptides contain Ser, Thr, and Tyr residues and might therefore be targets for protein kinases, a possible interference with the natural substrates for PTKs and PKC was investigated by analyzing {alpha}-thrombin–induced tyrosine phosphorylation (Figure 7BUp) and pleckstrin phosphorylation (Figure 7CUp) in the presence of ß3-2, ß3-3, and ß3-4. Incubation with 500 µmol/L peptide did not change most of these protein phosphorylations. An exception was the tyrosine phosphorylation of the 20- and 100-kDa bands that increased in the presence of the peptides (Figure 7DUp).


*    Discussion
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 *Discussion
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The major findings of the present study are as follows: (1) {alpha}-Thrombin initiates inside-out signaling to {alpha}IIbß3 via 1 or more Src kinases and via PKC. (2) Residues 743 to 762 in the carboxy-terminal end of the ß3 subunit contain domains involved in the regulation of the activation state of {alpha}IIbß3. PTKs known to be activated before ligand binding to {alpha}IIbß3 include Syk and Src kinases.14 Because aggregation by {alpha}-thrombin is normal in Syk-deficient mouse platelets, this kinase is probably not involved in inside-out signaling to {alpha}IIbß3.29 Platelets contain the Src kinases Src, Fyn, Yes, Lyn, and Hck, with Src showing the greatest expression. Activation of Src follows Gi-coupled receptor stimulation and is therefore a very early step during platelet activation.30 The YIYGSFK peptide is a specific substrate for all these Src kinases and is phosphorylated at the N-terminus on Tyr-3 by Src, Fyn, and Lyn with a Km of 55 µmol/L.23 It is a very poor substrate for membrane receptor–linked PTKs and non–receptor-linked cytosolic PTKs other than Src. The present results reveal complete inhibition of ligand binding to {alpha}IIbß3 at 1000 µmol/L YIYGSFK, indicating that 1 or more members of the Src-family play a critical role in inside-out signaling to this integrin. At present, it is uncertain which effectors are controlled by Src. Because {alpha}IIbß3 is not tyrosine-phosphorylated during the initial stages of platelet activation and tyrosine phosphorylation of the ß3-subunit critically depends on aggregation,31 32 it is unlikely that the ß3 subunit is a direct target of Src. Alternatively, the inhibition of ligand binding by YIYGSFK might reflect interference with the phosphorylation of an integrin regulatory protein. A candidate is ß3-endonexin, which contains a Tyr residue and in Chinese hamster ovary cells binds to the ß3 cytoplasmic tail, thereby increasing ligand binding to {alpha}IIbß3.33 Another candidate is a calcium and integrin binding protein (molecular mass 25 kDa), which contains 2 Tyr residues and binds to {alpha}IIb.34

Other potential substrates for Src kinases include Ras-GAP (120 kDa),35 the cytoskeleton protein cortactin (85/87 kDa), and a number of unidentified proteins termed p54, p65, and p130 to 140.36 Src peptide inhibited tyrosine phosphorylation of proteins with molecular masses of 55 and 100 kDa. Also, the tyrosine phosphorylation of a 60-kDa protein was reduced, possibly reflecting Src and interference with the autophosphorylation of the positive regulatory Tyr416 residue.14 Thus, each of these effector molecules of Src is a candidate for a role in the affinity regulation of integrin {alpha}IIbß3. In addition to activating PTKs, {alpha}-thrombin is known to activate the Ser/Thr kinase PKC. Platelets express the PKC isoforms {alpha}, ß, {delta}, {theta}, {eta}, and {zeta},37 38 39 which are translocated from the cytosol to the membrane on platelet stimulation by {alpha}-thrombin, platelet-activating factor, or phorbol 12-myrisate 13-acetate.40 The RKRCLRRL peptide is an analogue of the PKC peptide substrate RKRTLRRL, in which the Thr residue has been substituted for a Cys residue. It inactivates the activity of PKC{alpha}, PKCß, and PKC{gamma} by a covalent disulfide linkage to a conserved site in the catalytic domain.24 {alpha}-Thrombin–induced ligand binding to {alpha}IIbß3 was strongly reduced by the PKC peptide, but the maximal inhibition was never >75%, suggesting that part of the binding was insensitive to the peptide. A similar, apparently PKC-insensitive, ligand binding is observed in platelets treated with PKC inhibitors, which amounts to {approx}20%.12 These results indicate that PKC contributes to {alpha}IIbß3 regulation, in agreement with earlier work based on the PKC inhibitors bisindolylmaleimide I and II and calphostin C in intact platelets.12 Candidate substrates for PKC are the ß3 tail of {alpha}IIbß316 41 42 and the regulatory protein ß3-endonexin, with 1 and 3 consensus motifs, respectively, for PKCs.

The ß3 cytoplasmic tail is a potential target for intracellular signals generated by these kinases. The point mutation Ser752->Pro in the ß3 cytoplasmic tail was associated with an apparent {alpha}IIbß3 activation defect in a patient with Glanzmann’s thrombasthenia.10 Prolonged inhibition of Ser/Thr phosphatases by calyculin A increased the phosphorylation of Thr753, which was accompanied by a decrease in platelet adhesion and spreading on surface-coated fibrinogen.42 In an attempt to compete with the native integrin subunit for regulatory signals, peptides that mimic different, but partially overlapping, domains of the ß3 subunit were designed. Peptide ß3-1, which resembles the membrane-proximal part of the ß3 tail (residues 717 to 724), had no effect on {alpha}-thrombin–induced ligand binding. In contrast, peptides ß3-2 (residues 743 to 750), ß3-3 (residues 749 to 756), and ß3-4 (residues 755 to 762), which mimic regions at the carboxy-terminal of the ß3 tail, interfered with agonist-induced ligand binding. Also, the modified ß3-3 peptides with an S->A (EATATFTN) and an S->P replacement (EATPTFTN) inhibited ligand binding, indicating that the Ser residue (mimicking S752 in ß3) is not critical for ß3 function, in agreement with earlier findings in HEL cells.43 No effect of these inhibitory peptides was found on {alpha}-thrombin–induced tyrosine phosphorylation and phosphorylation of pleckstrin, indicating that the interaction with the major substrates of these kinases was left undisturbed.

The ß3 domains mimicked by these peptides contain Tyr, Ser, and Thr residues. The Tyr residues in NNPLYKEA (743 to 750, mimicked by peptide ß3-2) and TNITYRGT (755 to 762, peptide ß3-4) may be part of the docking site of a regulatory protein. The NPXY motif (present in peptide ß3-2) is highly conserved among many cell surface receptors, including ß subunits of integrins, the LDL receptor, and tyrosine kinase–linked receptors.41 Deletion of the NPXY motif or a Tyr747->Ala substitution blocked integrin function28 and abolished {alpha}IIbß3-mediated internalization of fibrinogen-coated particles.44 Liu et al43 found that the Tyr residues in the NPLY and NITY motifs are essential in {alpha}IIbß3 affinity regulation. A cell-permeable peptide mimicking residues 747 to 762 in the cytoplasmic tail of {alpha}IIbß3 strongly reduced adhesion of HEL and ECM304 cells to surface-coated fibrinogen.44 Substitution of Tyr747 and/or Tyr759 for Phe resulted in loss of the inhibitory function of the peptide. The regulatory protein ß3-endonexin binds to the NITY motif in the ß3 cytoplasmic tail45 (residues 756 to 760), which might explain the inhibitory effect of peptide ß3-4. A second mechanism by which peptide ß3-3 and ß3-4 may affect inside-out signaling is by direct interference with the PKC-mediated phosphorylation of the ß3 cytoplasmic tail. The ß3 tail is phosphorylated on Thr after {alpha}-thrombin stimulation.46 A potential candidate is the Thr residue in the NITY motif of ß3 (Thr758), which is in a domain (TYR) that is recognized and phosphorylated by PKC. Alternatively, Thr762 might also be a target for PKC.47 In support of this concept are findings in Chinese hamster ovary cells, in which deletion of the last 4 AAs (YRGT) of ß3 disrupted the TYR motif and blocked binding of the antibody PAC-1.27

The present results in combination with reported data support the concept that activation of Src kinases is a common step in inside-out signaling to {alpha}IIbß3 by most platelet-activating agents. In addition, PKC contributes to inside-out signaling in platelets stimulated by {alpha}-thrombin. Inhibition of ligand binding to {alpha}IIbß3 by peptides mimicking the carboxy-terminal part of the ß3 tail shows that residues 749 to 762 are likely targets for signals generated by these kinases.


*    Acknowledgments
 
This study was supported in part by a grant from the Netherlands Thrombosis Foundation (No. 92002) and a grant from the Netherlands Organization for Scientific Research/Netherlands Heart Foundation (No. 902-526-094/940-50-102). G.v.W. is a research fellow of the Catharijne Foundation.

Received July 23, 1999; accepted November 25, 1999.


*    References
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