Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation

Abstract

Background

Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed.

Methods and Results

Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca²⁺ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbβ3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation.

Conclusion

Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation.

 

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Keywords

collagen receptor - kinase inhibitors - Bruton's tyrosine kinase - protein kinase C - secretion

Introduction

Platelets have crucial roles in hemostasis, thrombo-inflammation, infection, and cancer.[1] [2] Membrane-proteins such as G-protein coupled receptors (GPCRs) and tyrosine (Y)-protein kinase-linked receptors mediate the activation of platelets in response to numerous agonists.[3] [4] GPCRs include receptors for thrombin (PAR1, PAR4), thromboxane A₂ (TP), and ADP (P2Y₁, P2Y₁₂)[2] [5]; tyrosine kinase-linked receptors are glycoprotein VI (GPVI), C-type lectin receptor-2 (CLEC-2), GPIbα, FcγRIIA, and integrin αIIbβ3.[2] [6] Since there is a growing need to control platelet hyperreactivity, new approaches to inhibit platelets are sought.[2]

GPVI, a platelet-specific tyrosine kinase-linked collagen receptor, signals via the Fc receptor γ-chain and the spleen tyrosine kinase (Syk), similar to the signaling of the B cell receptor (BCR) and related immune receptors.[7] [8] B cell studies originally established that a BCR-/immunoreceptor tyrosine-based activation motif (ITAM)-induced and membrane-associated Src/Syk/PI3K/Btk/PLCγ2 signalosome with additional signaling components (protein kinase C [PKC], Akt, calcineurin, mitogen-activated protein kinase [MAPK] nuclear factors) controls final B cell responses.[9] [10]

The BCR signalosome concept was readily transferred to platelets and the ITAM-based signaling complexes induced by GPVI or CLEC-2 activation.[8] [9] GPVI agonists including collagen, cross-linked collagen-related peptide (CRP-XL), and the snake venom toxin convulxin, activate platelets via Src family kinases (SFKs), which induce dual Y-phosphorylation of proteins with the ITAM.[9] This recruits the SH2 domain-containing Syk to the membrane followed by SFK-mediated Syk Y352 phosphorylation and kinase activation associated with autophosphorylation (Y525/526).[9] [11] Syk substrates are linkers for activation of T cells (LAT) and Bruton's tyrosine kinase (Btk), which stimulate Y-phosphorylation/activation of phospholipase Cγ 2 (PLCγ2) and platelet activation.[12]

GPVI stimulation also activates phosphoinositide 3-kinases (PI3Ks) by their recruitment to membranes via their SH2-domain binding to Y-phosphorylated proteins such as LAT or by interaction with GPCR βγ subunits.[13] Activated PI3Ks via their product phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) also recruit PH-domain containing proteins (i.e., PLCγ2, Btk, Akt) to the membranes and activate them.[13]

However, the sequential signaling events in ITAM-mediated responses, the interactions of Y- with S/T-protein kinases, the crosstalk with other pathways, and the coupling to specific functional responses are not well defined. Recent developments of selective and potent kinase inhibitors of the ITAM signaling components Syk, PI3K, and Btk provide novel approaches to study this pathway in human platelets.[2]

Syk inhibitors and first-/second-generation Btk inhibitors, ibrutinib and acalabrutinib, respectively, inhibited various functions of human platelets and tyrosine phosphorylation of downstream targets.[14] [15] [16] [17] [18] Earlier studies showed that ibrutinib strongly inhibits many tyrosine kinases including SFKs and Tec in addition to Btk, whereas acalabrutinib is Btk selective.[19] Antiplatelet effects of several novel Btk/Syk inhibitors and reversible/irreversible Btk inhibitors were compared,[20] [21] but with limited phosphorylation data. In contrast, extensive phosphoproteomic data were obtained with GPVI-activated human platelets[22] and compared with functional effects of several Syk and Btk inhibitors.[23] These studies provided substantial evidence for the important role of tyrosine kinases and especially Btk in GPVI-induced platelet activation, but the phosphorylation data had limitations. Most studies used only one time point after activation, indicating that the data reported represent a static snapshot, and not a dynamic view on platelet signaling. Most papers also primarily addressed Y-phosphorylation of Syk, Btk, and their direct substrates LAT and PLCγ2, but not additional S/T protein kinases. PKC activation was analyzed by phosphoantibodies against PKC consensus phospho-sites,[14] [22] similarly also Akt and MAPK.[22] However, these procedures do not detect specific substrates.

Many tyrosine protein kinases are known to be regulated by S/T protein kinases, but this has been rarely studied in human platelets. In our previous phosphoproteomic approaches, we detected numerous ADP- and/or prostacyclin (PGI2)-regulated phosphoproteins in human platelets and noted that ADP stimulated S/T phosphorylation of several tyrosine protein kinases such as JAK3, Btk, TNK2, and Syk.[24] Previously, we focused on the regulation of the individual platelet tyrosine protein kinases Syk and Btk. Analysis of Syk serine phosphorylation in response to GPIbα stimulation by beads coated with the toxin echicetin, GPVI stimulation by the toxin convulxin, and by ADP detected prominent Syk S297 phosphorylation preferentially dependent on PKCα/β.[25] [26] Syk S297 phosphorylation was negatively affected by PKA or the protein phosphatase 2A (PP2A) and correlated well with reduced Syk Y-phosphorylation/kinase activity.[26] [27] We also compared the differential regulation of Syk and Btk by PKC, PKA, and PP2A in human platelets and noted that PP2A does not directly affect Btk pS180.[28]

Now, we hypothesized that the analysis of GPVI-activated multisite protein phosphorylation kinetics in human platelets identifies interactions of key Y-kinases (SFK, Syk, Btk) and S/T-kinases (PKC, MEK1/2, Erk1/2, p38, Akt) within a signaling network, which is required for granule exocytosis and αIIbβ3 integrin activation. To define time-dependent dynamics of GPVI signaling in human platelets, we quantified relevant convulxin-regulated phosphoproteins (14 phospho-sites) during an extended activation time of 10 to 300 seconds and six different time points. Interactions of GPVI-stimulated Y- and S/T-protein kinases and resulting functional effects on platelets, in particular regulation of intracellular Ca²⁺ mobilization, degranulation, and αIIbβ3 integrin activation, were assessed by the effects of selective inhibition of Syk, Btk, and PKC.

 

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Methods

Reagents and Antibodies

Convulxin and GF109203X were obtained from Enzo Life Sciences (Lausen, Switzerland). PRT-060318 was from Selleckchem (Houston, Texas, United States). Acalabrutinib was purchased from Abcam (Cambridge, United Kingdom). AR-C669931 (AR-C) was from the Medicines Company (Parsippany, New Jersey, United States). Total Syk (4D10), total PLCγ2 (B-10), and total Akt1 (B-1) antibodies were from Santa Cruz Biotechnology (Dallas, Texas, United States). Phospho-Syk (S297, Y352, Y525/526), total Btk (D3H5), phospho-Btk (S180, Y223, Y551), phospho-PLCγ2 (Y759, Y1217), phospho-LAT Y220, phospho-Akt (T308, S473), phospho-MEK1/2 S217/S221, phospho-Erk1/2 T202/Y204, phospho-p38 T180/Y182, total MEK1/2, α-actinin, and β-actin were provided by Cell Signaling Technologies (Danvers, Massachusetts, United States). Secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibodies were obtained from BioRad Laboratories (Hercules, California, United States). FITC-conjugated mouse anti-human CD62P, CD63, and PAC-1 antibodies were from BD Biosciences (Heidelberg, Germany).

 

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Blood Donors, Ethics Approval, and Informed Consent

Blood collection was performed as previously described.[28] This study was approved in accordance with the Declaration of Helsinki by the local Ethics Committee of the University Medical Center Mainz (study no. 837.302.12; 25.07.12; 2018–13290_1; 27.07.2018).

 

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Isolation of Human Platelets and Preparation of Fura-2-Loaded Platelets

For immunoblotting and flow cytometry, human platelets were washed and isolated as previously described.[28] For the measurement of cytosolic Ca²⁺ rises, the protocol was slightly modified based on our previous publication.[29] Briefly, platelet-rich plasma (PRP) was prepared via centrifugation at 260 × g for 10 minutes at room temperature (RT), supplemented with 1:10 vol/vol acid citrate dextrose (ACD) (80 mM trisodium citrate, 183 mM glucose, 52 mM citric acid). Platelets in PRP were pelleted by centrifugation at 2,360 × g for 2 minutes and resuspended in HEPES buffer (136 mM NaCl, 2.7 mM KCl, 2 mM MgCl₂, 5.5 mM glucose, 10 mM HEPES, 0.1% BSA, pH 6.6) in the presence of apyrase (1 U/mL) and 1:15 vol/vol ACD. After a further centrifugation step, platelets were resuspended in HEPES buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM glucose, 10 mM HEPES, pH 7.5) and loaded with Fura-2 acetoxymethyl ester (3 µM) and Pluronic (0.4 µg/mL) for 30 minutes at RT. Fura-2-loaded platelets were adjusted to a platelet concentration of 2 × 10⁸/mL with HEPES buffer, pH 7.5.

 

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Light Transmission Aggregometry

Light transmission aggregometry (LTA) was performed as previously described.[28] Briefly, using an Apact4S Plus aggregometer (DiaSys, Flacht, Germany), washed human platelets (200 µL, 3 × 10⁸/mL) were preincubated with vehicle control (0.1% DMSO), 1 µM PRT-060318, 5 µM acalabrutinib or 5 µM GF109203X for 5 minutes at 37°C and then stimulated by 50 ng/mL convulxin under stirring. Samples for immunoblotting were collected at 0, 10, 30, 60, 120, and 300 seconds by adding 100 µL of 3× Lämmli buffer (200 mM Tris/HCl, 15% (v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromophenol blue, 1:10 β-mercaptoethanol), and boiled for 10 minutes at 95°C with gentle shaking.

 

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SDS-PAGE and Immunoblotting Analysis of Phosphoproteins

SDS-polyacrylamide electrophoresis (SDS-PAGE), immunoblotting, and phosphoprotein analysis were performed as previously described.[28] Briefly, proteins in prepared samples were separated by 8% gels and transferred to polyvinylidene fluoride membranes. After blocking membranes with 2% BSA in 1× TBS-T for 1 hour at RT, the membranes were incubated overnight at 4°C with specific antibodies with 2% BSA in 1× TBS-T. The incubated membranes were washed three times with 1× TBS-T and incubated with relevant HRP-conjugated secondary antibody for 2 hours at RT with 2% BSA in 1× TBS-T, and then rewashed three times with 1× TBS-T. The membranes were developed by electrochemiluminescence (ECL) detection. The antibodies used are listed above. An alternative fluorescence-based detection system [IRDye 800 CW goat anti-rabbit (LiCor); Bio-Rad ChemiDoc MP Imaging System] was also used with selected platelet samples and compared with our regular ECL detection system, and found superiority of the latter in terms of signal sizes and signal-to-noise ratios.

 

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Measurement of Calibrated Changes in Cytosolic Ca²⁺ Rises

Using a high-throughput FlexStation 3 device (Molecular Devices, San Jose, United States), the elevation of intracellular Ca²⁺ in Fura-2-loaded platelets was measured in 96-well plates as previously described.[30] Briefly, 200 µL of platelets/well (2 × 10⁸/mL) were pretreated with PRT-060318, acalabrutinib, or GF109203X for 10 minutes at 37°C. The platelet suspension was supplemented with vehicle (HEPES buffer, pH 7.5) or 0.1 mM EGTA as needed before starting the measurement. Convulxin (final concentration 50 ng/mL) was injected by automatic pipetting at a high rate of 125 µL/s for maximal platelet response. For each column, fluorescence responses were monitored for 10 minutes at 37°C, recording 510 nm emission fluorescence at two excitation wavelengths (340 and 380 nm). The Fura-2 fluorescence ratio of each well was acquired every 4 seconds. The calibration wells contained Fura-2-loaded platelets and 0.1% Triton-X-100 in the presence of either 2 mM CaCl₂ or 1 mM EGTA/Tris for determining Rmax and Rmin values, thus resulting in nanomolar changes in intracellular Ca²⁺. Duplicate time traces capturing nanomolar changes in intracellular Ca²⁺ concentration were subjected to floating point averaging via an Excel script. These traces were subsequently assessed for the area under the curve (AUC; expressed in nM × s) over a 10-minute period.[31] To facilitate comparisons across different experimental days involving various blood donors, curve parameters were normalized against the control condition, specifically when the agonist was administered with a vehicle medium and no inhibitor. This control condition was standardized at 100%. The subsequent analysis of inhibitor effects involved expressing these effects as percentage changes relative to the established control condition. Notably, the normalization process was independently conducted for experimental runs featuring either CaCl₂ or EGTA.

 

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Flow Cytometry

After incubation with PRT-060318, acalabrutinib, or GF109203X for 5 minutes or with AR-C669931 for 15 minutes at 37°C, washed human platelets (2 × 10⁸/mL) were stimulated with 50 ng/mL convulxin for 5 minutes at RT. Platelets were stained with PAC-1-FITC (recognizing activated αIIbβ3 integrin), anti-CD63-FITC, or anti-CD62P-FITC antibodies for 10 minutes at RT and then fixed with formaldehyde in HEPES buffer (final concentration 0.5%) for 30 minutes at RT, followed by adding 1 mL HEPES buffer (pH 7.4) to stop fixation. After centrifugation at 800 × g for 10 minutes, platelets were resuspended in 500 µL of HEPES buffer (pH 7.4) and analyzed by flow cytometry using a BD FACSCANTO II and FACS DIVA software (BD biosciences, Heidelberg, Germany) as previously described.[32]

 

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Statistical Analysis

Data are presented as means ± standard deviation (SD), from n ≥ 3 independent experiments with platelets from at least three healthy donors. Statistical analysis was performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, California, United States). One-way or two-way ANOVA, followed by a Sidak's multiple comparison test, was used for the comparison of more than two groups. A p-value <0.05 was considered significant.

 

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Results

Convulxin Induces Very Rapid, Reversible Y-phosphorylation, Followed by Delayed, Also Reversible Multisite S/T-phosphorylation of Syk, Btk, and Downstream Targets

For the kinetic experiments, we preferred to use the tetrameric C-type lectin convulxin, since it dimerizes and further clusters very rapidly the GPVI receptor molecules in contrast to the cross-linked CRP polypeptide. Using convulxin, we were able to distinguish between very early, intermediate and later protein phospho-sites and also to detect reversible protein phosphorylation within 5 minutes.

Convulxin induces phosphorylation of Syk (Y352, Y525/526, S297) and Btk (Y551, Y223, S180) in human platelets.[28] These earlier studies used a limited activation window time, a small phospho-site spectrum, and primarily addressed Syk and Btk. Here, we significantly extended the time courses to compare early phosphorylation changes (at 10 seconds and 30 seconds and later events up to 300 seconds). Furthermore, we enlarged the spectrum of analyzed phospho-sites to other protein kinases with suspected involvement in long-term GPVI signaling. These include the MAPK-related kinases, MEK1/2, Erk1/2 and p38, and the PI3K-dependent kinase Akt.[13] [33] [34] We analyzed phospho-sites known to be clearly linked to activation of the defined protein kinases and/or to serve as a direct substrate of these kinases ([Supplementary Table S1], available in the online version).

In human platelets stimulated with the GPVI agonist convulxin, we determined, with several time points within 300 second activation, the phosphorylation effects using a panel of 14 specific phospho-antibodies, recognizing the following phosphoproteins: Syk (S297, Y352, Y525/526), LAT (Y220), Btk (S180, Y223, Y551), PLCγ2 (Y759, Y1217), MAPKs (MEK1/2 S217/221, Erk1/2 T202/Y204, p38 T180/Y182), and Akt (T308, S473). We found an equally rapid and strong convulxin-induced Y-phosphorylation of Syk and Btk, which was maximal within 10 seconds, reversible, and preceded their S-phosphorylation ([Fig. 1A, B]). Next to PLCγ2 and LAT, also the MAPKs MEK1/2, Erk1/2, and p38 showed a reversible pattern of phosphorylation ([Fig. 1A, B]). In contrast, the Akt T308 and S473 phosphorylation was slower in onset and more persistent ([Supplementary Fig. S1], available in the online version). Overall, this kinetic analysis indicated that the Y-phosphorylation of Syk, LAT, Btk, and PLCγ2 preceded the S/T phosphorylation of Syk, Btk, MEK1/2, Erk1/2, p38, and Akt. At longer time points, all phosphorylation events, except for Akt, reversed within the 5-minute stimulation period, indicating the activity of Y/S/T protein phosphatases.

Fig. 1 Time-dependent phosphorylation of multiple protein kinases and substrates in human platelets induced by convulxin. Washed human platelets were stimulated with 50 ng/mL convulxin (cvx) at 37°C under stirring, and activation was stopped after 0, 10, 30, 60, 120, or 300 seconds with Lämmli buffer. (A) Representative blots showing convulxin-stimulated protein phosphorylation of: Syk S297, Syk Y352, Syk Y525/526, LAT Y220, Btk S180, Btk Y223, Btk Y551, PLCγ2 Y759, PLCγ2 Y1217, MEK1/2 S217/221, Erk1/2 T202/Y204, and p38 T180/Y182. Antibodies against total Syk, total Btk, total PLCγ2, total MEK1/2, and α-actinin were used as loading controls. (B) The phosphorylation of Syk (i), Btk (ii), LAT (iii), PLCγ2 (iv), MEK1/2 S217/221 (v), Erk1/2 T202/Y204, and p38 (vi) was analyzed, and compared with staining for total Syk, total Btk, α-actinin, total PLCγ2, total MEK1/2, and α-actinin, respectively. Quantitative data are represented as mean ± SD from three independent experiments with platelets from three healthy donors (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, 0 second versus other time points of convulxin-treated platelets. SD, standard deviation.

 

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Inhibition of Syk, Btk, or PKC Differentially Affects the Convulxin-Induced Multisite Protein Phosphorylation

To further investigate the hierarchy of protein kinases in the GPVI signaling pathway, we then systematically studied the effects of Syk, Btk, or PKC inhibitors on protein phosphorylation events in the GPVI signalosome.

In human platelets, the compound PRT-060318 (1 µM) has been characterized as a selective Syk inhibitor.[25] [35] [36] We first confirmed that PRT-060318 blocked the convulxin-induced Syk Y525/526 phosphorylation, as an indicator of Syk activity ([Fig. 2]). However, it did not inhibit, even increased Syk Y352 phosphorylation, an established SFK substrate sequence. Syk inhibition also strongly suppressed Syk S297 phosphorylation and other Y/S/T phospho-sites, i.e., LAT, Btk, PLCγ2, MEK1/2, Erk1/2, p38, and Akt ([Fig. 2], [Supplementary Fig. S1], available in the online version). Overall, this indicated that these phosphorylation events are downstream of the activated Syk kinase.

Fig. 2 General suppression of convulxin-induced phosphorylation by Syk inhibitor PRT-060318 (PRT) except for Syk Y352. Washed human platelets were treated with 0.1% DMSO or 1 µM PRT-060318 (PRT) for 5 minutes, prior to stimulation with 50 ng/mL convulxin (cvx) under stirring, and activation was stopped after 0, 10, 30, 60, 120, or 300 seconds with Lämmli buffer. (A) Representative blots show convulxin-stimulated protein phosphorylation with or without PRT-060318 preincubation including Syk S297, Syk Y352, Syk Y525/526, LAT Y220, Btk S180, Btk Y223, Btk Y551, PLCγ2 Y759, PLCγ2 Y1217, MEK1/2 S217/221, Erk1/2 T202/Y204, and p38 T180/Y182. Antibodies against total Syk, total Btk, total PLCγ2, total MEK1/2, and α-actinin were used as loading controls. (B) Phosphorylation of Syk (i–iii), Btk (iv–vi), LAT (vii), PLCγ2 (viii–ix), MEK1/2 S217/221 (x), Erk1/2 T202/Y204 and p38 T180/Y182 (xi–xii) was analyzed, and compared with total Syk, total Btk, α-actinin, total PLCγ2, total MEK1/2, and α-actinin, respectively. Quantified data are mean ± SD from three independent experiments with platelets from three healthy donors (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, DMSO versus PRT-060318-treated platelets in response to convulxin at the same time points. Note, original blots of convulxin-induced protein phosphorylation are identical to those of [Fig 1]. SD, standard deviation.

Second, we compared the roles of Syk and Btk in the GPVI-signalosome by using the Btk inhibitor, acalabrutinib. Platelet treatment with acalabrutinib abolished the phosphorylation of Btk Y223 ([Fig. 3]), a well-known Btk autophosphorylation site representing Btk activity ([Supplementary Table S1], available in the online version), under basal and convulxin-stimulated conditions. Acalabrutinib also strongly inhibited the convulxin-stimulated phosphorylation of PLCγ2 Y759/Y1217, MEK1/2 S217/221, Erk1/2 T202/Y204, p38 T180/Y182, Syk S297, Btk S180 ([Fig. 3]), and Akt T308/S473 ([Supplementary Fig. S2], available in the online version), but not of Syk Y352, Y525/526, LAT Y220, and Btk Y551 ([Fig. 3]). Interestingly, acalabrutinib, similar to PRT, moderately increased Syk Y352 phosphorylation, which was statistically not significant. These data hence indicate that Btk, but not Syk, controls PLCγ2 Y-phosphorylation/activation, Syk S297/Btk S180 phosphorylation, and MEK1/2, Erk1/2, p38, Akt activation. However, also Syk may regulate these functions indirectly via Btk.

Fig. 3 Partial suppression of convulxin-induced phosphorylation by Btk inhibitor acalabrutinib, but not Syk Y352, Y525/526, LAT Y220, and Btk Y551. Washed platelets were treated with 0.1% DMSO or 5 µM acalabrutinib for 5 minutes, prior to stimulation with 50 ng/mL convulxin (cvx) under stirring, and activation was stopped after 0, 10, 30, 60, 120, or 300 seconds with Lämmli buffer. (A) Representative blots showing convulxin-stimulated protein phosphorylation with or without acalabrutinib preincubation: Syk S297, Syk Y352, Syk Y525/526, LAT Y220, Btk S180, Btk Y223, Btk Y551, PLCγ2 Y759, PLCγ2 Y1217, MEK1/2 S217/221, Erk1/2 T202/Y204, and p38 T180/Y182. Antibodies against total Syk, total Btk, total PLCγ2, total MEK1/2, and α-actinin were used as loading controls. (B) Phosphorylation of Syk (i–iii), Btk (iv–vi), LAT (vii), PLCγ2 (viii–ix), MEK1/2 S217/221 (x), Erk1/2 T202/Y204, and p38 T180/Y182 (xi–xii) was analyzed and compared with total Syk, total Btk, α-actinin, total PLCγ2, total MEK1/2, and α-actinin, respectively. Quantitative data are represented as the mean ± SD from three independent experiments with platelets from three healthy donors (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, DMSO versus acalabrutinib-treated platelets in response to convulxin at the same time points. SD, standard deviation.

Third, we examined the role of PKC activity in the GPVI-signalosome. Previously, we characterized GF109203X (5 µM) as a potent and selective human platelet pan-PKC inhibitor and reported that PKC isoforms (most likely PKC α/β) regulate Syk S297 and Btk S180 phosphorylation as a negative feedback mechanism.[27] [28] With the extended time courses and the broader spectrum of analyzed phosphoproteins, we now monitored effects of GF109203X on convulxin-induced protein phosphorylation of Syk, LAT, Btk, PLCγ2, Akt, and MAPKs. GF109203X clearly enhanced the convulxin-induced Y-phosphorylation of Syk Y525/526, LAT Y220, and PLCγ2 Y759/1217, moderately increased Syk pY352, while it strongly reduced S-phosphorylation of Syk S297 and Btk S180 ([Fig. 4]). Importantly, GF109203X also abolished the phosphorylation of MEK1/2 S217/221 and Erk1/2 T202/Y204, but not of p38 T180/Y182 ([Fig. 4]). GF109203X inhibited convulxin-induced Akt S473 phosphorylation, especially at 5 minutes, but not of Akt T308 ([Supplementary Fig. S3], available in the online version).

Fig. 4 Differential suppression of convulxin-induced S/T phosphorylation by PKC inhibitor GF109203X, leaving p38 T180/Y182 unchanged. Washed human platelets were treated with 0.1% DMSO or 5 µM GF109203X (GFX) for 5 minutes at 37°C prior to stimulation with 50 ng/mL convulxin (cvx) under stirring, and stopped after 0, 10, 30, 60, 120, or 300 seconds with Lämmli buffer. (A) Representative blots showing convulxin-stimulated protein phosphorylation with(out) GF109203X preincubation for: Syk S297, Syk Y352, Syk Y525/526, LAT Y220, Btk S180, Btk Y223, Btk Y551, PLCγ2 Y759, PLCγ2 Y1217, MEK1/2 S217/221, Erk1/2 T202/Y204, and p38 T180/Y182. Antibodies against total Syk, total Btk, total PLCγ2, total MEK1/2, or α-actinin were used as loading controls. (B) Phosphorylation of Syk (i–iii), Btk (iv–vi), LAT (vii), PLCγ2 (viii–ix), MEK1/2 S217/221 (x), Erk1/2 T202/Y204 and p38 T180/Y182 (xi–xii) was analyzed and compared with total Syk, total Btk, α-actinin, total PLCγ2, total MEK1/2 and α-actinin, respectively. Quantitative data are represented as mean ± SD from three independent experiments with platelets from three donors (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, DMSO versus GF109203X-treated platelets in response to convulxin at the same time points. SD, standard deviation.

At the functional level of platelet activation, we confirmed our results[25] [26] [28] that both 1 µM PRT-060318 and 5 µM acalabrutinib abolished the convulxin-induced platelet aggregation, while 5 µM GF109203X had an only slightly inhibitory effect (data not shown). We then extended these studies to the analysis of convulxin-stimulated intracellular Ca²⁺ levels, granule secretion, and αIIbβ3 integrin activation.

 

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Inhibition of Btk but Not PKC Suppresses Convulxin-Induced Intracellular Ca²⁺ Rises

The GPVI-dependent activation of PLCγ2 requires multisite Y-phosphorylation and recruitment to the membrane via its SH2- and/or PH domain.[13] Activated PLCγ2 catalyzes the conversion of phosphatidylinositol 4,5-trisphosphate (PIP₂) to IP₃ resulting in the mobilization of Ca²⁺ from the dense tubular system to the cytoplasm with concomitant platelet activation.[12] The data so far indicated that the convulxin-induced PLCγ2 Y-phosphorylation (Y759, Y1217) was inhibited by PRT-060318 and acalabrutinib but was increased by GF109203X. Strong Inhibition of GPVI-activated PKC (likely α/β isoforms) by GF109203X was demonstrated by the downregulation of Syk S297 and Btk S180 phosphorylation.

To further clarify this phenomenon, regulation of cytosolic Ca²⁺ in Fura-2-loaded platelets was monitored using a high-throughput FlexStation 3 robot system. Experiments were performed in the absence and presence of EGTA to elucidate the effects on intracellular Ca²⁺ mobilization. Acalabrutinib (0.3–10 µM) dose-dependently inhibited the Ca²⁺ rise upon convulxin stimulation in either condition, as visualized by AUC analysis ([Fig. 5A]). Even 1 µM acalabrutinib had strong inhibitory effects. In contrast, GF109203X caused a different pattern of agonist response. At low doses, GF109203X (0.3–1 µM) slightly increased, whereas at high doses it did not reduce the Ca²⁺ mobilization induced by convulxin ([Fig. 5B]).

Fig. 5 Acalabrutinib, not GFX109203X, suppresses convulxin-induced Ca²⁺ mobilization in platelets. Fura-2-loaded human platelets were stimulated by 50 ng/mL convulxin (cvx), after preincubation with acalabrutinib or GF109203X. Calibrated intracellular Ca²⁺ rises were recorded for 10 minutes in 96-well plates at 37°C. Area under the curve (AUC) within 10 minutes of convulxin-stimulated intracellular Ca²⁺ rises was normalized to 100%. Shown are normalized AUC of intracellular Ca²⁺ rises in response to convulxin in the presence of acalabrutinib (A) or GF109203X (B). Mean ± SD from at least three independent experiments with platelets from healthy donors (n ≥ 3). ****p < 0.0001, ***p < 0.001, DMSO versus acalabrutinib or GF109203X-treated platelets in response to convulxin. SD, standard deviation.

 

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Inhibition of Syk, Btk, and PKC Affects Convulxin-Induced Degranulation and αIIbβ3 Integrin Activation

Platelet granule secretion is tightly regulated by multiple platelet agonists.[37] Since both elevated Ca²⁺ level and PKC activity are essential for platelet degranulation,[37] [38] we determined effects of the same Syk, Btk, and PKC inhibitors on the convulxin-induced degranulation and, in comparison, αIIbβ3 integrin activation. At 1 µM PRT-060318, 1 to 5 µM acalabrutinib and 5 µM GF109203X strongly inhibited, while at 1 µM GF109203X only partially reduced the expression of CD63 ([Fig. 6A]) and CD62P ([Fig. 6B]), surface expression markers for δ- and α-granule exocytosis, respectively. Similar inhibitory effects were observed for the convulxin-stimulated PAC-1 antibody binding, as a marker for αIIbβ3 integrin activation ([Fig. 6C]). These data indicate that both GPVI-induced platelet granule secretion and αIIbβ3 integrin activation require all three protein kinases investigated, Syk, Btk, and PKC.

Fig. 6 Inhibitors of Syk, Btk, and PKC strongly down-regulate convulxin-induced platelet granule secretion and αIIbβ3 integrin activation. Washed platelets were pretreated with 0.1% DMSO, 1 µM PRT-060318 (PRT), 1–5 µM acalabrutinib, 1–5 µM GF109203X (GFX) for 5 minutes, before stimulation with 50 ng/mL convulxin (cvx) for 5 minutes. Surface expression of CD63 (A) and CD62P (B), and FITC PAC-1 antibody binding (C) were assessed by flow cytometry. The mean fluorescence intensity in control condition, established when the convulxin was administered with a vehicle medium and no inhibitor, served as 100%. Subsequent analysis of inhibitor effects entailed expressing these effects as percentage changes relative to this established control condition. Percentage data are shown, with convulxin condition normalized to 100%. Mean ± SD from four donors (n = 4). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, indicated inhibitor versus DMSO. SD, standard deviation.

 

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P2Y₁₂ Receptor Blockade Partially Impairs Convulxin-Stimulated Granule Secretion

The release of ADP from the δ-granules and subsequent activation of the platelet ADP receptors P2Y₁ and especially P2Y₁₂ are positive feedback mechanism to enhance the functional effects of GPVI agonists.[33] [39] We confirmed that the P2Y₁₂ receptor antagonist AR-C669931 dose-dependently (2.5–1,000 nM) reduced the convulxin-stimulated CD63 and CD62P surface expression, and the PAC-1 antibody binding ([Fig. 7]). Interestingly, treatment of platelets with 500 nM AR-C669931 resulted in partial reduction of δ-granule (CD63) ([Fig. 7A]) and α-granule (CD62P) ([Fig. 7B]) release of approximately 50%, whereas αIIbβ3 integrin activation was nearly abolished ([Fig. 7C]). The data indicate a differential regulation, at least quantitatively, of α/δ-granule secretion and integrin activation by secreted ADP in response to GPVI stimulation.

Fig. 7 The P2Y₁₂ receptor blocker AR-C669931 suppresses convulxin-induced platelet granule secretion and αIIbβ3 integrin activation. Washed platelets were pretreated with 0.1% DMSO, 2.5–1,000 nM AR-C669931 (AR-C) for 15 minutes before stimulation with 50 ng/mL convulxin (cvx) for 5 minutes. Surface expression of CD63 (A) and CD62P (B), and FITC PAC-1 antibody binding (C) were analyzed by flow cytometry. The control condition, established when the convulxin was administered with a vehicle medium and no inhibitor, served as 100%. Subsequent analysis of inhibitor effects entailed expressing these effects as percentage changes relative to this established control condition. Results are shown as %; stimulation by convulxin was normalized to 100%. Mean ± SD from three healthy donors (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, DMSO versus AR-C669931-treated platelets in response to convulxin. SD, standard deviation.

 

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Discussion

In this study, GPVI-stimulated multisite protein phosphorylation in human platelets demonstrates a multiple-phased kinetic pattern and interactions between tyrosine- and serine/threonine protein kinases with reversible activation and feedback regulation, thereby controlling degranulation and αIIbβ3 integrin activation.

GPVI Stimulation by Convulxin Induces Reversible Phosphorylation of S/T-kinases after Initial Y-phosphorylation of the GPVI-LAT Signalosome in a Partly PKC-Dependent Manner

The revealed transient, reversible nature of convulxin-induced Y/S/T-phosphorylation indicates a powerful role of both Y- and /S/T-protein phosphatases in human platelets.[40] [41] [42] For instance, the prominent tyrosine phosphatase TULA-2 dephosphorylates Y-phosphorylated Syk and antagonizes GPVI-signaling,[43] whereas the ST protein phosphatase PP2A dephosphorylates Syk pS297 and components of MAPK signaling.[27] [42] Interestingly, platelet tyrosine phosphorylation induced by GPVI and CLEC-2 activation was sustained for 50 minutes, when aggregation was prevented by eptifibatide,[18] suggesting that dephosphorylation can also be controlled.

Most phospho-sites studied here are located within the kinase domains and are indicators for their activation, namely Syk Y525/526, Btk Y551, MEK1/2 S217/221, Erk1/2 T202/Y204, p38 T180/Y182, and Akt T308/S473. Other sites are located within regulatory domains, which are essential for kinase activation (Syk Y352) or have regulatory effects (Syk S297, Btk S180, Btk Y223). The Syk and Btk phospho-sites are well studied at the kinase level,[9] [10] [11] and documented in the PhosphoSitePlus database.

Understanding the hierarchy, interactions, and functional impact of the protein kinases requires information on the human platelet proteome and kinome,[41] [44] properties of the protein kinase inhibitors and on their effects on platelets.[2] Because of the important role of Syk (abundant in human platelets, ∼0.78 µM) in inflammation and immune cell diseases, several potent Syk inhibitors have been developed for clinical use. The Syk inhibitor PRT-060318 strongly inhibited purified Syk (IC₅₀ 4 nM), and the activation and function of Syk in murine and human platelets.[25] [35] [36] It was reported that Syk, Btk, and PKC inhibitors did not block convulxin-induced phosphorylation of Syk Y352, a SFK-specific phospho-site essential for Syk activation,[11] indicating that SFKs are still operative under these conditions. We also found that PRT-060318 (1 µM) even prolongs the GPVI-induced Syk Y352 phosphorylation, suggesting that Syk inhibition enhances SFK activation, and Syk activation down-regulates the SFK-increased Syk pY352. It was reported that acalabrutinib-induced Btk inhibition caused Src potentiation in human platelets,[14] but this was less apparent in our experiments. On the other hand, PRT-060318 abolished Syk Y525/526 and other phospho-sites studied, indicating that Syk acts upstream of LAT, Btk, PLCγ2, PKC, and MAPKs. In immune cells, LAT is phosphorylated primarily by Syk at four conserved Y-sites (Y161, Y200, Y220, Y255), which serve as docking sites for SH2-domain-containing proteins (e.g., PLCγ2/1, PI3K, Btk).[45]

Another important tyrosine kinase within platelet GPVI signaling is Btk, a member of the Tec family.[12] Btk deficiency or dysfunction causes X-linked agammaglobulinemia (XLA), characterized by a severe impairment of B cell development and function.[10] XLA platelets showed an only moderate impairment of GPVI signaling, likely due to a redundant role of Tec.[12] [15] Since Btk is crucially involved in B cell differentiation and malignancies, several inhibitors of human Btk have been developed and clinically validated. Ibrutinib, a first-generation Btk inhibitor, strongly and irreversibly inhibited Btk (targeting Cys 481), but it also showed off-target effects on other tyrosine kinases including SFKs. The second-generation inhibitor acalabrutinib is more specific for Btk and Tec (Btk IC₅₀ 5 nM, Tec IC₅₀ 83 nM, no effect on SFKs) and also irreversibly binds to Btk Cys 481.[19] In this landmark clinical study, acalabrutinib plasma levels of 1.2 to 1.4 µM were detected in individuals taking 100 mg of acalabrutinib twice daily. This was accompanied by high Btk target occupancy in peripheral blood monocytes (>90%) with concomitant reduction of Btk Y223 autophosphorylation.[19] Based on this and the considerable expression levels in human platelets (Btk ∼1.76 µM, Tec ∼0.21 µM), we analyzed the effects of acalabrutinib on platelet protein phosphorylation. Acalabrutinib (5 µM) strongly and specifically inhibited phosphorylation of Btk Y223, PLCγ2, and MAPKs. An earlier report on B cells showed for PLCγ2 that phosphorylation of four conserved Y-sites (Y753, Y759, Y1197, Y1217) is essential for full PLCγ2 activation, mediated primarily by Btk.[46] In the present case, acalabrutinib abolished the GPVI-induced phosphorylation of PLCγ2 Y759 and Y1217, but not of Syk Y352 (SFKs site), Syk Y525/526 (Syk autophosphorylation), LAT Y220 and Btk Y551 (Syk site). Thus, the GPVI-induced PLCγ2 Y759/Y1217 phosphorylation is directly mediated by Btk, and indirectly controlled by Syk and SFKs, which is consistent with another report.[15]

Our results on a GPVI-induced sequential phosphorylation in platelets are summarized in [Supplementary Table S1] (available in the online version) and compared with database information. Useful protein kinase activity markers appear to be for: (1) SFK activity: Syk pY352; (2) Syk activity: Syk pY525/526, LAT pY220, Btk pY551, and (3) Btk activity: Btk pY223, PLCγ2 pY759/Y1217. It appears that the other S/T phospho-sites studied, inhibited by acalabrutinib or GF109203X, are downstream of both Btk and PKC. Furthermore, p38 pT180/Y182 and Akt pT308 events are downstream of Btk, but not of PKC.

 

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Markers for Tyrosine and Serine/Threonine Protein Kinase Activities in the Platelet Btk-PLCγ2-PKC Signalosome

In B cells, Btk activates PLCγ2 followed by the activation of PKCβ, MAPKs, calmodulin/calcineurin, Akt, and several transcription factors, which are all important for B cell development and functions.[10] Much less is known about related pathways in platelets. In platelets, PLCγ2 activation induces IP₃-mediated intracellular Ca²⁺ mobilization from the dense tubular system with subsequent Ca²⁺-dependent responses and also increases diacylglycerol formation with activation of PKC isoforms.[3] [4] In agreement with this, acalabrutinib (5 µM) abolished the GPVI-induced intracellular Ca²⁺ elevation and the Syk S297 and Btk S180 phosphorylation. Previously, we reported that the GPVI-induced Syk S297 and Btk S180 phosphorylation is mediated by one of the conventional PKC isoforms, and that this represents a possible inhibitory feedback mechanism of GPVI signaling.[26] [28] Our present data show that the PKC inhibitor GF109203X (likely via PKCα/β) abolishes GPVI-induced phosphorylation of Syk pS297 and Btk pS180, but enhances the Y-phosphorylation of Syk, LAT, and PLCγ2, as well the intracellular Ca²⁺ rise. These data are consistent with B cell studies showing that the PKCβ-mediated phosphorylation of Syk S297 and Btk S180 attenuates the membrane localization and activation of these protein kinases.[47] [48]

Interestingly, Akt decreased the activity of several BCR signaling targets, including Btk, Blink, and Syk.[49] In platelets, the convulxin-induced and PKC-mediated phosphorylation of Syk S297 and Btk S180 is rapid (maximal within 30 seconds and closely follows Btk/PLCγ2 activation), and is therefore a useful marker for GPVI-induced PKC activity. Inhibition of PKC with GF109203X strongly inhibited the phosphorylation of more distal PKC targets such as MEK1/2 S217/221, Erk1/2 T202/Y204, and Akt S473, but not p38 T180/Y182 or Akt pT308.

 

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PKC-Dependent Protein Phosphorylation Phases Induced by GPVI-Mediated Btk Activation Are Required for Granule Exocytosis and αIIbβ3 Integrin Activation

Based on the observed effects of the protein kinase inhibitors PRT-060318 (Syk), acalabrutinib (Btk), and GF109203X (likely PKCα/β isoforms) on GPVI-stimulated multisite phosphorylation, we got evidence that the interactions of Syk, Btk, and PKC control platelet granule secretion and αIIbβ3 integrin activation. However, the PKC inhibitor GF109203X at 1 µM showed less strong inhibitory functional effects. Possibly, inhibitor concentrations saturating for PKC were not reached, as the intraplatelet concentration of the conventional PKCα and PKCβ isoforms can be calculated as approximately 2.9 µM. Increasing the concentration of GF109203X to 5 µM resulted in a similarly strong inhibition of these responses as with PRT-060318 or acalabrutinib. While PKC and Ca²⁺-regulated pathways are known to regulate platelet granule release,[37] other protein kinases such as MAPKs and Akt may also contribute. In a previous phosphoproteomic study, we observed that multiple proteins of the platelet secretory machinery are regulated at the phosphorylation level by ADP.[24] This is relevant in the present context since ADP is released from the δ-granules and subsequently stimulates the P2Y₁ and P2Y₁₂ receptors.[39] We found that the P2Y₁₂ blocker AR-C669931, at 1 µM, reduced the convulxin-induced δ-granule and α-granule exocytosis up to 50%, whereas this abolished the αIIbβ3 integrin activation, suggesting that the convulxin-stimulated platelet degranulation is only partly controlled by the P2Y₁₂ pathway. Interactions of P2Y₁₂ and GPVI signaling are clinically relevant, as ADP enhances GPVI signaling.[50]

Taken together, we identified a GPVI-induced multi-phased interactive network of Y- and S/T-protein kinases in human platelets, which is characterized by regulative multisite phosphorylation patterns ([Fig. 8]). Syk down-regulates SFK activity indicated by decreased Syk pY352. PKC not only activates MAPKs, but also directly phosphorylates Syk S297 and Btk S180 as negative feedback regulation. GPVI-induced granule exocytosis and αIIbβ3 integrin activation are primarily mediated by PKC and involve autocrine ADP release which enhances this process, but additional protein kinases are likely to participate. Acalabrutinib, an approved irreversible Btk/Tec inhibitor, specifically inactivates human blood cell and platelet Btk kinase activity (chemical kinase knockout) at clinically observed concentrations. There is increasing evidence that not linear signaling pathways, but rather rapid phosphorylation–dephosphorylation cycles of multiple proteins underlie the regulation of complex and important biological functions.[51] Of clinical relevance, the human GPVI signaling network contains multiple Y/S/T protein kinases, which are already targeted by an increasing number of Y- and S/T-protein kinase inhibitors already used in patients with malignant and/or inflammatory diseases, often in combination. There is a need to enhance the understanding of the GPVI-controlled human platelet signaling network and the possible benefits of novel interventions.

Fig. 8 Model showing bi-directional links between GPVI signalosome effects and platelet functions. Central (green background area) is the signalosome network of multiple tyrosine kinases (SFK, Syk, Btk) and serine/threonine kinases (PKC, MEK1/2, Erk1/2, p38, Akt), which are (in)activated by multisite phosphorylation changes. The phosphorylation of PLCγ2, MEK1/2, and Erk1/2, similarly as platelet functional responses, is Btk- and PKC-dependent, which includes mechanisms of feedback inhibition (Syk S297 and Btk S180 phosphorylation) and feedforward enhancement (P2Y₁₂ stimulation by secreted ADP). Indicated pathways were revealed by specific inhibition of Syk, Btk, or PKC. While this model represents the data obtained, additional proteins, phospho-sites, and alternative signaling pathways can be involved as well. For further details, see text.

 

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Conflict of Interest

J.W.M.H. is a scientific advisor for Synapse Research Institute. P.Z. was supported by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement TICARDIO no. 813409. P.Z. was enrolled in a joint PhD project of the Universities of Maastricht (NL) and Mainz (DE). The other authors declare no relevant conflicts of interest.

Acknowledgment

Figures were created using GraphPad 9.5.1 and Biorender.com.

Authors' Contribution

Conceptualization: K.J. and U.W.; methodology: P.Z., S.v.U.-S., L.H.; data curation: P.Z.; formal analysis: P.Z. and U.W.; writing—original draft preparation: P.Z., U.W., and K.J.; writing—review and editing: P.Z., U.W., K.J., F.A.S., A.S., J.W.M.H., and M.J.E.K.; funding and supervision: A.S. and K.J.

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Publikationsverlauf

Eingereicht: 31. Oktober 2023

Angenommen: 16. April 2024

Accepted Manuscript online:
23. April 2024

Artikel online veröffentlicht:
15. Mai 2024