Differential Involvement of Tyrosine and Serine/Threonine Kinases in Platelet Integrin _IIbß₃ Exposure

From the Department of Haematology, University Hospital Utrecht, and the Institute for Biomembranes, Utrecht University, Utrecht, the Netherlands.

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

	Abstract

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Abstract—The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin _IIbß₃ (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on ¹²⁵I-fibrinogen binding to _IIbß₃ and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of _IIbß₃ exposure by 30 µmol/L (ADP stimulation) and 35 to 40 µmol/L (-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to 80% (for -thrombin). Studies with a submaximal dose of herbimycin A (50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as _IIbß₃ exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in _IIbß₃ exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP- and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and -thrombin.

Key Words: integrin _IIbß₃ • fibrinogen binding • aggregation • human platelets • protein phosphorylation

	Introduction

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Platelet aggregation is mediated by the coupling of fibrinogen to integrin _IIbß₃ (ie, glycoprotein IIb/IIIa) via binding sites that are exposed when these cells are activated. Our understanding of the intracellular mechanisms that control the exposure of ligand-binding sites on platelet integrin _IIbß₃ is far from complete. A major stimulating pathway in platelets involves tyrosine kinases, which may signal to the processes mediating platelet-platelet contact and formation of focal adhesions.¹ A second stimulating route involves hydrolysis of polyphosphoinositides and formation of diacylglycerol, inositol 1,4,5-tris-phosphate, and phosphatidic acid. Diacylglycerol activates PKC, whereas inositol 1,4,5-tris-phosphate mobilizes Ca²⁺, thereby inducing rearrangements of the plasma membrane and cytoskeleton that facilitate anchorage of _IIbß₃ and possibly exposure of the binding sites.^{1 2}

There is uncertainty regarding the role of protein kinases in inside-out signaling to _IIbß₃. Platelet activation is accompanied by tyrosine phosphorylation of 60-, 64-, 75-, and 130-kDa proteins, a step that precedes ligand binding to _IIbß₃ and subsequent outside-in signaling through this integrin.^{3 4} One or more of these tyrosine phosphorylations may therefore function in the intracellular control of _IIbß₃, which accords with the effect of certain PTK inhibitors. For instance, genistein, erbstatin, and tyrphostins inhibit aggregation and secretion induced by -thrombin, collagen, and PAF.^{5 6 7 8 9 10} The binding of fibrinogen and the activation-dependent antibody PAC-1 induced by -thrombin and ADP is also abolished.¹¹ In contrast, other studies could not confirm these observations and emphasized the poor specificity of these inhibitors.^{12 13 14}

Serine/threonine phosphorylation might also contribute to the regulation of _IIbß₃. Activation of PKCs with phorbol esters, such as phorbol myristate acetate, exposes binding sites for fibrinogen and the activation-dependent antibody PAC-1 and induces aggregation.^{15 16} Bisindolylmaleimide derivatives, which block PKC activity, strongly reduce -thrombin– and collagen-induced aggregation^{17 18 19} and -thrombin–induced fibrinogen binding.²⁰ The target of PKC appears to be the ß₃ subunit of the integrin, as phorbol myristate acetate–induced binding and ³² P incorporation correlate linearly²⁰ and stimulation with -thrombin increases the stoichiometry of ß₃ phosphorylation from 5±2% to 80±10%.²⁰

In the course of our studies on _IIbß₃ control, we found that ADP and -thrombin differed greatly in their sensitivity to inhibitors of protein kinases. Thus, we explored the relative contributions of tyrosine and serine/threonine phosphorylation in further detail, giving special attention to indirect modulation of _IIbß₃ by secreted ADP, formation of TxA₂, and the correlation between _IIbß₃ exposure and optical aggregation responses.

	Methods

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Materials
Human -thrombin, epinephrine, imipramine, indomethacin, BSA (RIA grade), and protein A–peroxidase were purchased from Sigma Chemical Co; staurosporine, ADP, PAF, PEP, PK, and the PKC inhibitor bisindolylmaleimide I (GF 109203X) were from Boehringer. Bisindolylmaleimide II and V, calphostin C, geldanamycin, tyrphostin A47, and PPACK were from Calbiochem. Sepharose 2B was obtained from Pharmacia Biotech and BSA (demineralized) from Organon Technika. Na[¹²⁵I], with a specific activity of 629 GBq/mg, and 5-hydroxy[side chain-2-¹⁴C]tryptamine creatinine sulfate ([¹⁴C]-serotonin), with a specific activity of 1.85 to 2.29 GBq/mmol, were from Amersham International. [³²P]Orthophosphate with a specific activity of 314 TBq/mmol was from New England Nuclear. FITC was obtained from Pierce Chemical Co; dPPA and herbimycin A were from Biomol Research Laboratories, and the anti-phosphotyrosine antibodies 4G10 and PY20 were from Upstate Biotechnology Inc. Fibrinogen (grade L) was from Chromogenix. FITC-labeled monoclonal mouse antibody to the platelet glycoprotein IIIa (ß₃) subunit was obtained from Dakopatts. FITC calibration beads (Quantum 24 series) were obtained from Flow Cytometry Standards Corp.

Platelet Isolation
Freshly drawn venous blood from healthy volunteers (with informed consent) who claimed not to have taken any medication in the previous 14 days was anticoagulated with 0.1 volume of 130 mmol/L trisodium citrate. Platelet-rich plasma was obtained by centrifugation (200g, 5 minutes, 22°C), and the 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 (demineralized). The platelet count was adjusted to 200x10³/µL.

Preparation of ¹²⁵I-Labeled Fibrinogen and FITC-Fibrinogen
Fibrinogen was made fibrin- and fibronectin-free by separation on a gelatin–Sepharose 4B column and radiolabeled with Na[¹²⁵I] by a modified Iodo-Gen method. Details of these procedures have been described elsewhere.²¹ The labeling with FITC was performed as described.²²

Platelet Aggregation
Gel-filtered platelets (200x10³/µL) were preincubated with vehicle (DMSO), herbimycin A (5 minutes, 15 µmol/L), or bisindolylmaleimide I (1 minute, 5 µmol/L) with or without the ADP scavenger PEP/PK (14 mmol/L PEP, 150 U/mL PK). Suspensions were stirred (900 rpm) in the presence of fibrinogen (1 µmol/L) at 37°C. Subsequently, platelets were stimulated with 10 µmol/L ADP, 10 µmol/L epinephrine, 200 nmol/L PAF, or 0.2 U/mL -thrombin, and aggregation was measured in a Chrono-Log Lumiaggregometer (Chrono-Log Corp) at 37°C. The agonist concentrations used in these experiments induced maximal aggregation of gel-filtered platelets. When -thrombin was used as an agonist, 30 nmol/L PPACK was added 30 seconds after stimulation to prevent coagulation. Aggregation was measured as the change in light transmission. Quantitative data reflect the extent of aggregation at 5 minutes after stimulation.

Binding of ¹²⁵I-Labeled Fibrinogen
Gel-filtered platelets (200x10³/µL) were preincubated with different concentrations of the kinase inhibitors. Preincubation times and concentrations were as follows: for herbimycin A, 5 minutes, 15 and 30 µmol/L; geldanamycin, 10 minutes, 5 and 10 µmol/L; tyrphostin A47, 10 minutes, 300 and 500 µmol/L; bisindolylmaleimide I, II, and V, 1 minute, 5 µmol/L; and calphostin C, 30 minutes, 3 µmol/L at 22°C without stirring. Under these conditions the inhibitors completely blocked protein phosphorylation as established in dose-response studies on tyrosine phosphorylation and pleckstrin phosphorylation (data not shown). Platelets were stimulated with 200 nmol/L PAF, 10 µmol/L ADP, or 10 µmol/L epinephrine in the presence of 1 µmol/L ¹²⁵I-fibrinogen or with 0.2 U/mL -thrombin in the absence of ¹²⁵I-fibrinogen. PPACK was added 3 minutes after stimulation with -thrombin to prevent coagulation, whereas ¹²⁵I-fibrinogen was added 2 minutes thereafter.

After a 15-minute incubation at 22°C with ¹²⁵I-fibrinogen, each sample (in triplicate) of 200 µL was drawn, 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, 4 minutes, 22°C) in a Beckman Microfuge E. 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 compared with the total radioactivity in the pellet plus supernatant. The data were corrected for nonspecific binding, defined as the binding of ¹²⁵I-fibrinogen to unstimulated platelets (7232±3021 fibrinogen molecules per platelet), which was in the same range as in stimulated platelets in the presence of a 100-fold molar excess of nonradiolabeled fibrinogen.²¹

Binding of FITC-Labeled PAC-1, Anti-ß₃ Antibody, and Fibrinogen
Gel-filtered platelets (200x10³/µL) were preincubated with vehicle (DMSO), herbimycin A (5 minutes, 15 or 30 µmol/L), or bisindolylmaleimide I or V (1 minute, 5 µmol/L) at 22°C. Platelets were stimulated with 0.2 U/mL -thrombin in the presence of 40 µmol/L FITC-labeled-PAC-1 or 10 µg/mL FITC-labeled monoclonal antibody against the ß₃ subunit. After a 15-minute incubation in the dark, the platelets were diluted to 20x10³/µL, fixed in Tyrode's buffer containing 1% paraformaldehyde, and analyzed by flow cytometry (FACScan, Becton Dickinson). The data obtained with FITC–PAC-1 were corrected for nonspecific binding, defined as the binding of FITC–PAC-1 to unstimulated platelets (mean fluorescence intensity of 8±4).

For the fibrinogen-binding experiments, platelets were stimulated with 0.2 U/mL -thrombin in the absence of FITC-fibrinogen. PPACK was added 3 minutes after stimulation to prevent coagulation, and 30 µmol/L FITC-fibrinogen (final concentration) was added 2 minutes thereafter. After a 15-minute incubation with FITC-labeled-fibrinogen in the dark at 22°C, platelets were fixed and analyzed as described. Fibrinogen binding was quantified by using FITC calibration beads. These beads emit a fluorescence intensity equivalent to a fixed number of FITC molecules in suspension, from which a standard curve is prepared that relates the median channel number of beads to the number of fluorescein equivalents per bead. The number of fibrinogen molecules bound per platelet was calculated from the median channel number obtained from each platelet sample divided by the known fluorescence-to-fibrinogen ratio (see Reference 22²² for details). The data were corrected for nonspecific binding, defined as the binding of FITC-fibrinogen to unstimulated platelets (mean fluorescence intensity of 35±9).

Measurement of Protein Tyrosine Phosphorylation
Gel-filtered platelets (200x10³/µL) were preincubated with vehicle (DMSO), different concentrations of herbimycin A (5-minute preincubation), or bisindolylmaleimide I (1 minute, 5 µmol/L) at 22°C. Subsequently platelets were stimulated with 0.2 U/mL -thrombin, 10 µmol/L ADP, or 10 µmol/L dPPA in the presence of 1 µmol/L fibrinogen for 15 minutes. Aliquots of the stimulated platelets were lysed in 10x lysis buffer (10% Nonidet P40, 5% n-octylglucoside, 1% SDS, 10 mmol/L orthovanadate, 10 mmol/L PMSF, 200 µg/mL soybean trypsin inhibitor, 50 mmol/L N-ethylmaleimide, and 100 mmol/L benzamidine). The lysate was incubated for 3 hours at 4°C with the monoclonal antibody PY20. Subsequently the antigen-antibody complex was precipitated with 5 µL of protein A–Sepharose beads (45 minutes, 22°C). After extensive washing in 1x lysis buffer (1% Nonidet P40, 0.5% n-octylglucoside, 0.1% SDS, 137 mmol/L NaCl, 2.68 mmol/L KCl, 0.42 mmol/L NaH₂PO₄, 1.7 mmol/L MgCl₂, 11.9 mmol/L NaHCO₃, and 0.2% BSA), the bound protein was extracted from the Sepharose beads by boiling for 5 minutes in a twofold-concentrated Laemmli electrophoresis sample buffer. Proteins were separated by SDS–7.5% PAGE and electrophoretically transferred (1 hour, 100 V) to nitrocellulose membranes in 25 mmol/L Tris–192 mmol/L glycine (pH 8.3) and 20% methanol (vol/vol) using a Bio-Rad mini trans-blot system (Bio-Rad). The blots were blocked with PBS containing 4% BSA (RIA grade) for 1 hour at room temperature and subsequently incubated (16 hours, 4°C) with the anti-phosphotyrosine antibody 4G10. After extensive washing in PBS containing 1% Tween and 1% BSA (RIA grade), the blots were incubated with peroxidase-labeled protein A (2 µg/mL, 2 hours, 4°C). Subsequently the blots were treated with Renaissance chemiluminescence Western blot reagent and exposed to Renaissance autoradiography film (Dupont NEN Research Products).

Measurement of PKC Activity
Platelets were labeled with 3.7 MBq of carrier-free [³²P]Pi/mL of platelet-rich plasma for 1 hour at 37°C. Platelet suspensions were acidified to pH 6.5, centrifuged, and resuspended in HEPES-Tyrode's buffer (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 were incubated for 1 minute with bisindolylmaleimide I (5 µmol/L) or for 5 minutes with herbimycin A (15 and 40 µmol/L) before stimulation with 0.2 U/mL -thrombin. Samples were collected 15 minutes after stimulation, transferred into threefold-concentrated Laemmli sample buffer, and boiled for 5 minutes prior to SDS-PAGE (11%). Gels were stained with Coomassie Brilliant Blue, and the radioactive bands were visualized by autoradiography.

[¹⁴C]Serotonin Secretion
Platelets were labeled in platelet-rich plasma with 1 µmol/L [¹⁴C]serotonin for 30 minutes at 37°C. Subsequently the platelet suspension was acidified to pH 6.5 and washed on a Sepharose 2B column equilibrated in Ca²⁺-free Tyrode's solution. The experiments were performed at 22°C in the presence of 2.5 µmol/L imipramine to prevent reuptake of secreted serotonin by the platelets. Samples were collected 15 minutes after addition of the agonists in 0.15 vol of 1.035 mol/L formaldehyde in saline (4°C) to stop serotonin secretion. After centrifugation (10 000g, 2 minutes, 22°C), the supernatants were counted according to standard procedures. The data are expressed as the percentage of maximal secretable [¹⁴C]serotonin (-thrombin, 5 U/mL, 15 minutes, 22°C).

Presentation of Data
Data are expressed as mean±SD from 3 to 6 (aggregation), 4 to 6 (¹²⁵I-fibrinogen binding), and 3 (FITC-labeled anti-ß₃ monoclonal antibody–, FITC-fibrinogen–, and FITC–PAC-1 binding) experiments. Statistical significance was determined by Student's t test for unpaired data and considered significant at P<.05.

	Results

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Role of PTKs and PKC in Fibrinogen Binding to _IIbß₃
Fig 1A shows that herbimycin A dose-dependently inhibited ¹²⁵I-fibrinogen binding to _IIbß₃ induced by ADP (10 µmol/L) and -thrombin (0.2 U/mL). Half-maximal inhibition was reached at 17 µmol/L herbimycin A for ADP and 28 µmol/L herbimycin A for -thrombin; at respective concentrations of 30 and 40 µmol/L, complete blockade was observed. Similar inhibition was found with the PTK inhibitors geldanamycin, a benzoquinoid antibiotic related to herbimycin A, and tyrphostin A47, a structurally different PTK inhibitor (Table 1). Geldanamycin reduced both ADP- and -thrombin–induced ligand binding at 5 µmol/L and completely inhibited ligand binding at 10 µmol/L (Table 1). Tyrphostin A47 dose-dependently inhibited both ADP- and -thrombin–induced fibrinogen binding, with maximal inhibition at 350 µmol/L.

View larger version (43K): [in this window] [in a new window]   Figure 1. Effect of herbimycin A and bisindolylmaleimide I on ligand binding to IIbß3 and protein phosphorylation. For fibrinogen binding, gel-filtered platelets (200x103/µL) were preincubated for 5 minutes with various concentrations of herbimycin A (A) or for 1 minute with various concentrations of bisindolylmaleimide I (B) and subsequently stimulated with 10 µmol/L ADP (–) in the presence of 125I-fibrinogen or with 0.2 U/mL -thrombin (–) in the absence of 125I-fibrinogen. PPACK and 125I-fibrinogen were added at 3 and 5 minutes, respectively, after -thrombin. Specific binding was measured after a 15-minute incubation in the presence of 125I-fibrinogen at 22°C. Data (mean±SD, n=4) are expressed as the percentage of maximal binding in the absence of inhibitors. For protein phosphorylation, unlabeled platelets (C) or 32P-labeled platelets (D) (200x103/µL) were treated as described above. Protein tyrosine phosphorylation (C) and pleckstrin phosphorylation (D) were analyzed after 15 minutes in the presence of fibrinogen (1 µmol/L). bis I indicates bisindolylmaleimide I, 5 µmol/L; herb A, herbimycin A.

View this table: [in this window] [in a new window]   Table 1. Effect of Different PTK and PKC Inhibitors on ADP- and -Thrombin–Induced Fibrinogen Binding

The PKC inhibitor bisindolylmaleimide I, which interacts with the ATP binding site of PKC, had no effect on ADP-induced binding up to a concentration of 5 µmol/L (Fig 1B). In contrast, -thrombin–induced binding was dose-dependently reduced by bisindolylmaleimide I and was maximal at 5 µmol/L (Fig 1B). Similar results were found with bisindolylmaleimide II and the structurally different PKC inhibitor calphostin C (Table 1). Bisindolylmaleimide II differs from bisindolylmaleimide I in that the dimethylaminopropyl side group has been replaced by methylpyrrolidinoethyl. At 5 µmol/L it had no effect on ADP-induced fibrinogen binding but strongly reduced -thrombin–induced binding. Bisindolylmaleimide V, the inactive bisindolylmaleimide in which the side group is absent, had no effect. Calphostin C, which interacts with the regulatory domain of PKC, only slightly reduced ADP-induced binding, whereas it strongly interfered with -thrombin–induced binding. Noteworthy is the fact that all PKC inhibitors left 20% to 30% of the ligand binding intact, whereas the PTK inhibitors induced complete inhibition. Together these data suggest that PTKs play a role in both ADP- and -thrombin–induced _IIbß₃ exposure. In contrast, a role for PKCs appears restricted to -thrombin–induced _IIbß₃ control.

To assess whether herbimycin A and bisindolylmaleimide I efficiently blocked PTKs and PKCs under the conditions of the binding experiments, tyrosine-phosphorylated proteins (Fig 1C) and phosphorylated pleckstrin (Fig 1D) were analyzed. -Thrombin increased the phosphorylation of proteins of 75 and 130 kDa. Bands representing 60- and 64-kDa proteins were already phosphorylated in resting platelets and showed no further increase. In addition, some tyrosine phosphorylation was observed in proteins of 84 and 90 to 100 kDa. Herbimycin A strongly reduced phosphorylation of the 75-kDa band, whereas phosphorylation of the 84-, 90- to 100-, and 130-kDa bands was inhibited to a lesser extent. Bisindolylmaleimide I (5 µmol/L) slightly inhibited -thrombin–induced tyrosine phosphorylation. ADP induced a weaker increase in tyrosine phosphorylation, mainly in proteins of 60, 64, 75, and 130 kDa. Again the phosphorylation was dose-dependently reduced by herbimycin A. Bisindolylmaleimide I failed to affect the tyrosine phosphorylation of these proteins. In contrast to ADP, -thrombin (0.2 U/mL) induced phosphorylation of the 47-kDa protein pleckstrin, which is a major substrate for PKCs. Bisindolylmaleimide I (5 µmol/L) completely blocked this phosphorylation, whereas 15 to 40 µmol/L herbimycin A had no effect.

Effect of PTK and PKC Inhibition on Aggregation and Fibrinogen Binding
Because the dose-response studies suggested that the relative contributions of PTKs and PKCs in _IIbß₃ control differed among different agonists, ¹²⁵I-fibrinogen binding studies were repeated with ADP and -thrombin and compared with those evoked by epinephrine and PAF. To assess whether differences in _IIbß₃ regulation also affected platelet aggregation, the binding data (established at 22°C without stirring) were compared with concurrently run aggregation curves (established at 37°C with stirring) with platelets from the same donor. Herbimycin A was used at a submaximal concentration of 15 µmol/L, since differences in sensitivity among agonists were most apparent at low concentrations of the inhibitor (Fig 1A). This concentration reduced ADP-induced fibrinogen binding to 50% of that of untreated suspensions. The sensitivity to bisindolylmaleimide I was measured at an optimal concentration of 5 µmol/L. In addition, the effect of staurosporine (1 µmol/L) was measured, which is known to block both PTKs and PKCs.²³ Some studies were performed in the presence of the ADP scavenger mixture PEP/PK to evaluate the role of secreted ADP, as exocytosis is greatly impaired by inhibitors of PKC.^{17 18} As expected, all agonists induced secretion of [¹⁴C]serotonin in platelet suspensions stirred at 37°C (data not shown). In contrast, under ligand-binding conditions serotonin secretion was only 3±1% (n=4) of maximal in the presence of ADP, epinephrine, and PAF but 82±3% (n=7) of maximal in -thrombin–treated platelets.

Suspensions stimulated with ADP (Fig 2A) showed rapid first- and second-wave aggregation and binding of 51 243±4760 (n=5) fibrinogen molecules per platelet (control). Herbimycin A reduced aggregation to 26% and fibrinogen binding to 58% of controls. Bisindolylmaleimide I inhibited aggregation (P<.05), but ligand binding remained unchanged. A combination of bisindolylmaleimide I and herbimycin A did not further reduce aggregation and fibrinogen binding compared with herbimycin A alone (P>.05). Finally, staurosporine suppressed aggregation to 27% and ligand binding to 10% of controls.

View larger version (20K): [in this window] [in a new window]   Figure 2. Role of protein kinases in aggregation and ligand binding to IIbß3 induced by different agonists. For aggregations (all panels, tracings and bar graphs; open bars), gel-filtered platelets were stirred (900 rpm) at 37°C in the presence of 1 µmol/L fibrinogen and 10 µmol/L ADP (A), 10 µmol/L epinephrine (B), 200 nmol/L PAF (C), or 0.2 U/mL -thrombin (D) (arrows) after preincubation with (a) DMSO (control), (b) PEP/PK, (c) herbimycin A (5 minutes, 15 µmol/L), (d) bisindolylmaleimide I (1 minute, 5 µmol/L), (e) bisindolylmaleimide I and PEP/PK, (f) bisindolylmaleimide I and herbimycin A, or (g) staurosporine (1 minute, 1 µmol/L). PPACK was added 30 seconds after -thrombin stimulation to prevent coagulation. Data (mean±SD, n3) are expressed as the percentage of maximal light transmission 5 minutes after stimulation in the absence of inhibitors. For fibrinogen binding (all panels, bar graphs; open bars), gel-filtered platelets (200x103/µL) were incubated with the same inhibitors and subsequently stimulated with ADP (A), epinephrine (B), or PAF (C) in the presence of 125I-fibrinogen or with 0.2 U/mL -thrombin (D) in the absence of 125I-fibrinogen. PPACK was added 3 minutes and 125I-fibrinogen 5 minutes after -thrombin stimulation. Specific binding was measured after a 15-minute incubation in the presence of 125I-fibrinogen at 22°C. Data (mean±SD, n3) are expressed as the percentage of maximal binding in the absence of inhibitors (100% values: ADP, 51 243±4760; epinephrine, 31 067±4230; PAF, 56 852±4430; and -thrombin, 94 430±8993 molecules fibrinogen per platelet).

Epinephrine-induced responses (Fig 2B) showed a sensitivity similar to the two inhibitors as seen with ADP. This agonist induced biphasic aggregation and binding of 31 067±4230 fibrinogen molecules per platelet (control). Removal of ADP by PEP/PK inhibited aggregation (P<.05) but had no effect on ligand binding. This finding is in agreement with the absence of serotonin secretion under ligand-binding conditions. Herbimycin A reduced aggregation to 33% and ligand binding to 52% of controls. In contrast, both responses were insensitive to bisindolylmaleimide I (P>.05). A combination of bisindolylmaleimide I and PEP/PK further reduced aggregation (P<.05), although no significant additive effect was seen on fibrinogen binding. Bisindolylmaleimide I had no effect on aggregation or ligand binding of herbimycin A–treated platelets (P>.05). Again, staurosporine strongly suppressed both aggregation (to 26% of control) and ligand binding (to 9%).

PAF induced aggregation and fibrinogen binding (56 852±4430 molecules per platelet, control) as shown in Fig 2C. Again, the effect of the inhibitors was roughly similar to that observed for ADP and epinephrine, but an important difference was the effect of bisindolylmaleimide I, which reduced aggregation to 42% and ligand binding to 68% of control. A combination of this PKC inhibitor with PEP/PK reduced aggregation further (P<.05) without affecting ligand binding. A combination of herbimycin A and bisindolylmaleimide I inhibited ligand binding (to 47%), which is stronger inhibition than that seen with herbimycin A alone. Staurosporine reduced aggregation and binding to 50% and 18%, respectively.

A further increase in sensitivity to bisindolylmaleimide I was seen with stimulation by -thrombin (Fig 2D), which was accompanied by a decrease in sensitivity to herbimycin A. This agonist induced strong and rapid aggregation and binding of 93 430±8993 molecules of fibrinogen per platelet (control). PEP/PK reduced the aggregation response to 83% (P<.05), whereas ligand binding was not affected. Thus, although -thrombin induced secretion in the binding experiments, the liberated ADP hardly interfered with ligand binding. Suspensions treated with herbimycin A showed no decrease in aggregation, but ligand binding was slightly inhibited (to 79%, P<.05). In contrast, bisindolylmaleimide I suppressed aggregation almost completely (to 18%) and inhibited ligand binding to 26%. A combination of this PKC inhibitor with PEP/PK did not further reduce aggregation and ligand binding. Herbimycin A had no significant effect on PKC-independent aggregation (P=.26) but further reduced ligand binding (P<.05). Almost complete inhibition was also seen with staurosporine. Together these data reveal that ADP- and epinephrine-induced ligand binding to _IIbß₃ depends on PTKs but not on PKCs, whereas PAF- and -thrombin–induced binding depends on both types of kinases. The aggregation curves reflect the inhibitor sensitivity for ligand binding; in addition, they illustrate the contribution of PKC-mediated secretion and further enhancement by released ADP.

To evaluate a possible role for PTKs downstream of PKC activation, platelets were stimulated with the PKC ß–activator dPPA (Fig 3). dPPA-induced fibrinogen binding (40 700±8644, n=8) was unaffected by PEP/PK but completely blocked by bisindolylmaleimide I (7±3%, n=7). Surprisingly, a submaximal concentration of herbimycin A reduced fibrinogen binding to 47±16% of control (n=4) and a maximal concentration reduced fibrinogen binding to 6±3% of control (n=4), suggesting involvement of PTKs downstream of PKC activation. In agreement with this observation, dPPA induced tyrosine phosphorylation of proteins with apparent molecular weights of 60, 64, 72, 84, and 130 kDa, an event that was completely blocked by bisindolylmaleimide I and reduced by increasing concentrations of herbimycin A (Fig 3A and B). As expected, bisindolylmaleimide I completely blocked dPPA-induced pleckstrin phosphorylation, whereas herbimycin A had no effect (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Role of protein kinases in dPPA-induced ligand binding to IIbß3. Gel-filtered platelets (200x103/µL) were incubated with inhibitors as noted in Fig 2 and subsequently stimulated with 10 µmol/L dPPA in the presence of 125I-fibrinogen. Specific binding was measured after a 15-minute incubation in the presence of 125I-fibrinogen at 22°C. Data (mean±SD, n3) are expressed as the percentage of maximal binding in the absence of inhibitors (100% value: 40 700±8644). bis I indicates bisindolylmaleimide I; herb A, herbimycin A; and stauro, staurosporine. A: Gel-filtered platelets (200x103/µL) were treated as described above. Protein tyrosine phosphorylation was analyzed after 15 minutes in the presence of fibrinogen (1 µmol/L).

Role of Tx Formation
The fact that bisindolylmaleimide I inhibited ADP-induced aggregation but not ligand binding agrees with the concept that aggregating platelets produce TxA₂, which further enhances platelet responses via activation of PKC. To assess the sensitivity of the inhibitors in the absence of this pathway, aggregation studies were repeated with indomethacin-treated platelets. Table 2 illustrates that the difference in sensitivity of ADP- and -thrombin–stimulated platelets to inhibitors of PTKs and PKCs was preserved. ADP-induced aggregation was strongly reduced by herbimycin A (P<.05), whereas bisindolylmaleimide I had no effect (P>.05). A combination of both inhibitors did not further reduce aggregation (P>.05). In contrast, bisindolylmaleimide I strongly inhibited -thrombin–induced aggregation (P<.05). Also, herbimycin A reduced aggregation, although the effect was less pronounced. The combination of inhibitors completely blocked -thrombin–induced aggregation.

Role of PTKs and PKCs in Expression of Internal _IIbß₃
Unlike ADP, epinephrine, and PAF, which activate only surface-bound _IIbß₃, -thrombin is known to express a second pool of _IIbß₃ complexes located in the open canalicular system and -granules.²⁴ This difference is illustrated by the almost-twofold higher fibrinogen binding induced by -thrombin compared with that induced by ADP, PAF, or epinephrine (Fig 2). To investigate the role of PTKs and PKCs in the surface expression of internal _IIbß₃, the accessibility of _IIbß₃ for an anti-ß₃ antibody was compared with the number of exposed _IIbß₃. Fig 4 shows that -thrombin induced an almost-twofold increase in the binding of an FITC–anti-ß₃, revealing the surface expression of the internal pool. This expression was almost completely blocked by 5 µmol/L bisindolylmaleimide I, whereas the inactive bisindolylmaleimide V and herbimycin A had no significant effect.

View larger version (31K): [in this window] [in a new window]   Figure 4. Surface expression of the internal pool of IIbß3. Gel-filtered platelets (200x103/µL) were preincubated with DMSO (control), 5 µmol/L bisindolylmaleimide I or V (respectively bis I and bis V), or 15 or 30 µmol/L herbimycin A (herb A) and stimulated with 0.2 U/mL -thrombin in the presence of an FITC-labeled antibody against the ß3-subunit or FITC–PAC-1 or in the absence of FITC-fibrinogen. After a 15-minute incubation with antibody, platelets were fixed and the fluorescence analyzed by flow cytometry. For fibrinogen binding, PPACK was added 3 minutes after stimulation and FITC-fibrinogen was added 5 minutes after stimulation. After a 15-minute incubation with FITC-fibrinogen, platelets were fixed and analyzed as described. Hatched bar represents binding to unstimulated platelets. Data are expressed as percentage of maximal median fluorescence with -thrombin (mean±SD, n=4). (100% FITC-fibrinogen binding=mean fluorescence intensity of 155±20=88 570±11 428 molecules fibrinogen per platelet, 100% FITC–anti-ß3 binding=mean fluorescence intensity of 196±14, 100% FITC–PAC-1 binding=130±12).

-Thrombin–induced binding of FITC-fibrinogen was almost completely prevented by bisindolylmaleimide I, in line with findings for the binding of ¹²⁵I-fibrinogen (Fig 2D). Again, the inactive bisindolylmaleimide V had no effect. Herbimycin A had little effect on FITC-fibrinogen binding at 15 µmol/L but strongly interfered with binding at 30 µmol/L, which also accords with the binding data for ¹²⁵I-fibrinogen. Similar results were found with PAC-1, an antibody against the activated conformation of _IIbß₃. Bisindolylmaleimide I strongly inhibited -thrombin–induced binding of FITC–PAC-1, whereas bisindolylmaleimide V had no effect. Again, a dose-dependent reduction was seen in the presence of herbimycin A.

Analysis of [¹⁴C]serotonin secretion was in line with these observations. Bisindolylmaleimide I reduced dense granule secretion from 82±3% to 3±1%. Herbimycin A had no significant effect at 15 µmol/L (83±10%, P>.05) and only slightly inhibited secretion at 30 µmol/L (67±5%, n=5, P<.05). Together these data indicate that the surface expression of internal _IIbß₃ requires activation of PKCs but is independent of PTK activity. Instead, both PTKs and PKCs are required for the exposure of ligand-binding sites on both pools of _IIbß₃ complexes, as demonstrated by the lower binding of FITC-fibrinogen and FITC–PAC-1 compared with the binding of FITC–anti-ß₃.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study applied inhibitors of PTKs and PKCs to define the roles of these kinases in the exposure of ligand-binding sites on integrin _IIbß₃ by different agonists. Together the data show that there is a dominant role for PTKs and little involvement of PKCs in sites exposed by ADP and epinephrine, a role for both types of kinases in sites exposed by PAF, and a dominant role for PKCs in sites exposed by -thrombin, with a smaller but still significant contribution of PTKs.

It was important to select an appropriate PTK inhibitor, since different inhibitors show diverse effects on signal transduction mechanisms^{6 12} and nonspecific effects cannot always be ruled out. Genistein is a potent antagonist of the Tx receptor in addition to its PTK-inhibitory effect.^{13 25} Erbstatin inhibits phosphoinositide metabolism, PKC, and serotonin secretion.⁶ Tyrphostin AG213 inhibits virtually all steps in platelet signal transduction, eg, Ca²⁺ mobilization, phosphoinositide metabolism,^{5 26} and activation of phospholipases C and D^{5 9 27} and PKC,⁹ as well as serotonin secretion,^{5 9} and inhibits the enzymatic activity of GTP-utilizing proteins.²⁸ At present, herbimycin A might be the inhibitor of choice, as it blocks kinases of the src family²⁹ and pp72^{syk 30} but leaves PKC activation (Reference 31³¹ and this study) and TxA₂ formation³¹ unchanged. Fukazawa et al³² demonstrated that herbimycin A selectively binds to the kinase domain of PTKs, thereby inhibiting access to ATP.

Nonreceptor tyrosine kinases in platelets include the src family members pp60^src, pp61^fyn, pp62^yes, pp61,^hck and pp54/58^{lyn 33,34}; pp72^syk; and pp125^FAK. Only pp60^src and pp72^syk are activated in the absence of fibrinogen binding and are therefore candidates for control of _IIbß₃ under the conditions of our binding experiments. pp60^src is activated within seconds,^{35 36 37 38} while pp72^syk is rapidly phosphorylated and activated by -thrombin,^{4 39} collagen,⁴ ADP,⁴ and PAF.⁴⁰ pp72^syk is further activated on ligand binding to _IIbß₃.³⁸ Herbimycin A inhibited ligand binding induced by all agonists studied, although -thrombin–induced binding was less sensitive than that induced by ADP, epinephrine, and PAF. Because the binding of ¹²⁵I-fibrinogen was analyzed after 15 minutes (22°C, nonstirring), it is possible that outside-in signaling after fibrinogen binding affects PTK activation at later stages of incubation. However, essentially similar results were obtained with PAC-1, an antibody directed against the exposed conformation that binds to _IIbß₃ without inducing signal generation. pp72^syk is the only platelet tyrosine kinase identified at present whose activity is directly stimulated by _IIbß₃ occupancy.^{4 41} Fibrinogen binding also leads to integrin-dependent phosphorylation of proteins such as p140 and p50–68, which is most likely initiated by receptor cross-linking.⁴² At present it is uncertain whether phosphorylation of these proteins signals back to _IIbß₃. Phosphorylation of pp125^FAK and the recently reported tyrosine phosphorylation of the ß₃ subunit only occur after platelet aggregation⁴³ and therefore might take part in postaggregatory events such as cytoskeleton attachment and clot retraction.

In general, the sensitivity to herbimycin A seen in the binding studies is also observed in optical aggregation experiments, although the latter is more strongly reduced with weak agonists. This may reflect an effect on postoccupancy signaling events that take part in the stabilization of aggregation. As expected there is a slight inhibition by PEP/PK, illustrating the contribution of secreted ADP, but the sensitivity to herbimycin A seen in the fibrinogen-binding studies remains preserved. Thus, herbimycin A inhibited the platelet aggregation induced by all agonists studied, but the -thrombin–induced response was less sensitive than the responses induced by ADP, epinephrine, and PAF. This finding is in contrast to those of Schoenwaelder et al,³¹ who found no effect of herbimycin A on -thrombin–induced aggregation even after a 24-hour incubation with the inhibitor.

Bisindolylmaleimide I (GF 109203X) is an inhibitor of the different PKC isozymes , ß₁, ß₂, and in both purified enzyme systems and intact cells.¹⁷ Its potency and specificity make it a suitable PKC inhibitor for the present studies. Although it slightly inhibited ADP-induced platelet aggregation via an inhibitory effect on TxA₂-mediated responses, PAF- and -thrombin–induced responses were more strongly reduced. This difference in sensitivity for the PKC inhibitor was even more evident in the ligand-binding studies, wherein ADP- and epinephrine-induced fibrinogen binding was unaffected by the inhibitor. PKC activation by ADP or epinephrine has been the subject of much controversy. ADP induces aggregation without the formation of inositol trisphosphate⁴⁴ or an increase in cytosolic Ca²⁺,^{45 46} and pleckstrin is only slightly phosphorylated.²⁰ Pulcinelli et al⁴⁷ found no effect of the PKC inhibitor Ro 31–8220 on ADP-induced fibrinogen binding, in agreement with the present study, but their conclusion that the same is true for other agonists is refuted by the present data. Other studies confirmed the role of PKC in -thrombin–induced aggregation and found inhibition by bisindolylmaleimide derivatives Ro 31–7549⁴⁸ and Ro 31–8220.¹⁸ However, a much greater reduction was found with bisindolylmaleimide I (Reference 17¹⁷ and this study), possibly because Ro 31–8220 stimulates other kinases independent of its ability to inhibit PKC.⁴⁹ In support of a role for PKCs in _IIbß₃ exposure, Gabbeta et al⁵⁰ reported on a patient with normal numbers of _IIbß₃ integrins but diminished pleckstrin phosphorylation after platelet activation, suggesting lowered PKC activity. Platelets from this patient showed reduced binding of the activation-dependent antibody PAC-1 after stimulation with -thrombin and PAF but not with ADP.⁵⁰

Our results are consistent with a role for PKCs in PAF- and -thrombin–induced inside-out signaling, leading to exposure of ligand-binding sites on _IIbß₃ on the platelet surface. It may be argued, however, that the reduction in -thrombin–induced ligand binding and platelet aggregation is due to impaired surface expression of internal _IIbß₃ from the -granules and the open canalicular system. Indeed, bisindolylmaleimide I completely blocked the surface expression of internal _IIbß₃ and reduced the binding of an anti-ß₃ antibody to the range of that for the external pool. The binding of fibrinogen and PAC-1 was well below that range, indicating that PKC activity is required for the exposure of surface _IIbß₃ as well as for the recruitment of internal _IIbß₃.

The bisindolylmaleimide I–insensitive _IIbß₃ exposure observed with PAF and -thrombin was further reduced by herbimycin A. Also staurosporine, a potent inhibitor of both PTKs and PKCs,^{23 51} induced a further reduction than did bisindolylmaleimide I alone. This suggest that PKCs only partially contribute to _IIbß₃ exposure by these agonists and that the essential component of the response is mediated by PTKs. In favor of independent roles for PTKs and PKCs is the early wave of tyrosine phosphorylation that is similar for all agonists, whether or not they activate PKCs.^{3 4 52 53} Protein phosphorylation under ligand-binding conditions partly supports this conclusion. Herbimycin A did not interfere with pleckstrin phosphorylation, in agreement with studies by Schoenwaelder et al.³¹ However, bisindolylmaleimide I reduced the overall level of -thrombin–induced tyrosine phosphorylation, whereas ADP-induced tyrosine phosphorylation was hardly effected. Since tyrosine phosphorylation was determined under ligand-binding conditions (15 minutes after stimulation), this might reflect a role for PKC in the later stages of tyrosine phosphorylation. Indeed, early tyrosine phosphorylation has been shown to be unaffected by PKC inhibitors.⁵⁴ The PKC ß-activator dPPA induced tyrosine phosphorylation and exposed _IIbß₃, and both responses were inhibited by herbimycin A. Phorbol ester triggers tyrosine phosphorylation of several proteins similar to those phosphorylated after -thrombin stimulation.³ Phosphorylation at Ser-12 by PKC might increase the substrate affinity of pp60^src.³⁵ Thus, activated PKC is unable to expose _IIbß₃ in the absence of tyrosine kinase activity. In previous studies we showed that ligand-binding sites on _IIbß₃ exposed by -thrombin remain accessible, whereas sites exposed by ADP gradually closed. Inhibition of PKC rapidly closed the binding sites exposed by -thrombin, suggesting a role for PKC in sustained exposure of ligand-binding sites on _IIbß₃.²⁰

The present results, in combination with published data, can be best explained by assuming the following model (Fig 5). Platelet agonists activate PTKs, but the degree of PKC activation differs among different agonists. PTKs shift surface _IIbß₃ molecules from the closed conformation (_IIbß₃⁰) to the exposed conformation (_IIbß₃*), allowing ligand binding. In the absence of ligands, the integrin rapidly returns to the closed conformation.⁵⁵ PKCs play three roles in _IIbß₃ control: first, they activate PTKs; second, they make the internal pool of _IIbß₃ available from -granules and surface-connecting tubules during the secretion response; and third, they directly phosphorylate the ß₃-chain and prevent its return to the closed formation.

View larger version (12K): [in this window] [in a new window]   Figure 5. Model depicting regulation of IIbß3 exposure by protein kinases. Following stimulation by an agonist, PTKs and PKCs are activated. PTK activation (1) appears a general property of platelet agonists; PKC activation (2) differs among agonists. Direct activation by PKC (3) can replace step (1). PTKs convert the closed integrin (IIbß30) to the exposed configuration (IIbß3*), which in the absence of ligand rapidly recloses, probably due to dephosphorylation by protein tyrosine phosphatases (PTPs). Depending on the degree of PKC activation, internal IIbß3 is made available (4) and the ß3-chain is phosphorylated (5), which opposes its return to the closed configuration. Dephosphorylation by Ser/Thr protein phosphatases of the 1-type converts the integrin to the closed conformation. (s) indicates surface.

	Selected Abbreviations and Acronyms

 

dPPA = 12-deoxyphorbol-13-phenylacetate-20-acetate PAF = platelet-activating factor PEP = phospho(enol)pyruvate PK = pyruvate kinase PKC = protein kinase C PPACK = D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone PTK = protein tyrosine kinase Tx = thromboxane

	Acknowledgments

 
This study was supported in part by a grant from the Netherlands Thrombosis Foundation (No. 92002).

Received February 5, 1997; accepted November 11, 1997.

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