Key Points

  • First comprehensive and time-resolved characterization of platelet cAMP/PKA signaling upon iloprost treatment.

  • More than 2700 phosphorylation sites quantified between 4 time points and from 3 individual healthy donors.

Abstract

One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)–signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of platelet inhibition and activation in 3 independent biological replicates. We quantified over 2700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multipronged process involving different kinases and phosphatases as well as many previously unanticipated proteins and pathways.

Introduction

In the blood flow, a delicate equilibrium between activating and inhibiting stimuli prevents spontaneous platelet aggregation and occlusion of blood vessels while allowing steady plug formation in case of blood vessel lesions. One of the compounds keeping platelets in their resting state is prostacyclin I2 (PGI2), derived from endothelial cells through conversion of prostaglandin H2 by prostacyclin synthase.1  PGI2 binds specifically to the Gs-coupled prostacyclin receptor on the platelet plasma membrane,2  thus stimulating the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)–signaling cascade. This leads to downstream phosphorylation events resulting in the inactivation of small G proteins of the Ras and Rho families, inhibition of Ca2+ release, and modulation of actin cytoskeleton dynamics.3  Due to their central role in platelet inhibition, defects in cAMP and guanosine 3′,5′-cyclic monophosphate (cGMP) pathways might contribute to cardiovascular diseases,4  whereas prostacyclin and modulators of platelet cyclic nucleotide levels are highly interesting pharmacologic targets.5  In the clinic, drugs modulating cAMP or cAMP-regulating pathways, such as the phosphodiesterase 3 (PDE3) inhibitor cilostazol or the P2Y12 receptor inhibitors clopidogrel or prasugrel, are used for antiplatelet treatment.5,6 

Due to the short half-life of prostacyclin, more stable analogs such as iloprost with a half-life time of over 20 minutes were developed.7  In this study, we analyzed platelets prepared from fresh blood donations to investigate molecular mechanisms underlying platelet inhibition upon stimulation of the PGI2 receptor. Using quantitative mass spectrometry (MS), we analyzed thousands of phosphorylation events after stimulation with 2 or 5nM iloprost (3 biological replicates each), respectively, for 0, 10, 30 or 60 seconds.

This is the first study to elucidate time-resolved changes in phosphorylation patterns of human platelets upon stimulation of the cAMP/PKA pathway. We quantified a total of 2738 phosphorylated peptides, corresponding to 2598 and 1457 phosphorylation sites of high (probability of >0.9 for correct phosphorylation site localization within the peptide sequence) and lower (<0.9) confidence from 1223 unique proteins (see supplemental Tables 3-5, available on the Blood Web site). Among those are almost 300 proteins with differential phosphorylation patterns of which 137 are potential PKA targets, compared with the 15 PKA targets established in platelets so far.3  Our data provide unprecedented insight into the crosstalk between the inhibiting cAMP/PKA pathway and downstream targets such as ubiquitin, Rho-Rac, and Ca2+/inositol phosphate–signaling pathways.

In addition, we assessed the biological variance of the basal phosphoproteome in human platelets, relatively quantifying 700 phosphopeptides between 4 different healthy donors. As we previously demonstrated for the complete human platelet proteome,8  the variance of basal phosphorylation levels in healthy donors seems to be surprisingly low as well–with a median coefficient of variation (CV) of 16% from the expected 1:1:1:1 ratios.

In summary, our novel quantitative data on platelet phosphorylation provide a rich source for further functional research and represent another step toward a deeper understanding of the mechanisms which contribute to platelet physiology as well as pathophysiology. Some of the signaling proteins detected in this study are so-far unknown key players of platelet activation and inhibition; thus, they might be novel candidates for monitoring functional defects or impaired responsiveness of human platelets to antiplatelet treatment, imposing an increased risk for severe cardiovascular or other adverse secondary effects.

Materials and methods

Platelet preparation

Blood was obtained from healthy volunteers according to our Institute’s guidelines and the Declaration of Helsinki. Our studies with human platelets were approved by the ethics committee of the University of Würzburg (study numbers: 67/92 and 114/04).

Full blood (40 mL) was immediately collected into sample tubes containing 10 mL of citrate buffer (CCD, 100mM Na2HPO4 × 2 H2O, 7mM citric acid, 140mM glucose; pH 6.5) and 10 U/mL apyrase. Platelet-rich plasma (PRP) was obtained by centrifugation at 300g for 20 minutes at room temperature (RT). The PRP layer was removed and diluted 1:1 with HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (145mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES, and 10mM d-glucose; pH 7.4). The diluted PRP was centrifuged at 200g for 10 minutes at RT to reduce leukocyte contamination. Subsequently, the platelets were pelleted from the supernatant by centrifugation at 400g for 20 minutes at RT. Each pellet was finally diluted in Tyrode's buffer (2.2mM Na2HPO4 × 2 H2O, 7.8mM NaH2PO4 × H2O, 10mM NaHCO3, 150mM NaCl, 5mM KCl, 5mM glucose, pH 6.8) and incubated for 20 minutes at 37°C. Meanwhile, a small aliquot of each pellet was used for cell counting on a cell-sorting machine (Sysmex; Norderstedt, Germany). Aliquots of the platelet suspension were either incubated for 10, 30, or 60 seconds with 2 or 5nM iloprost or vehicle at 37°C. For western blot analysis, 50 µL of platelet suspension was mixed with 100 µL of 3× sodium dodecyl sulfate (SDS) gel-loading buffer. For proteome analysis, the remaining samples were stopped by adding the lysis buffer (50mM Tris, 1% SDS, 150mM NaCl, 1 tablet PhosStop/10 mL, pH 7.8) in a ratio of 1:2 to each platelet sample. Lysed samples were immediately shock-frozen in liquid nitrogen and stored at −80°C until further usage.

Sample preparation and liquid chromatography-mass spectrometry analysis

All steps of the analytical workflow, including sample preparation, proteolytic digestion, quality control, iTRAQ labeling, global proteome analysis, phosphopeptide enrichment, mass spectrometry, spectrum processing, database searching, and quantification are detailed in the supplemental Materials and methods. Lysed samples (200 µg each) were processed separately and digested using trypsin. Digest efficiency and reproducibility were quality controlled9  prior to labeling samples with 4 different isobaric iTRAQ 4plex labels. Samples were pooled and 3 µg each were used for global proteome quantification. Afterward, samples were subjected to TiO2-based phosphopeptide enrichment, as described previously.10  Data interpretation was conducted using the Proteome Discoverer software (Thermo Scientific). A false discovery rate of <1% was applied and phosphorylation site localization probabilities were determined by using the phosphoRS11  algorithm. Raw data and search results can be accessed using the ProteomeXchange accession: PXD000242.

Western blot analysis

For western blot analysis the samples were added directly to SDS gel loading buffer, then analyzed by SDS polyacrylamide gel electrophoresis (PAGE). Separated proteins were transferred to nitrocellulose membrane, blocked with 3% milk in Tris-buffered saline (TBS, 150mM NaCl, 50mM Tris) containing 0.1% Tween and incubated with primary antibodies against phosphorylated GRP2Ser587 (CalDAG-GEF1), SrcTyr530, VASPSer157, VASPSer239,12  GSK3αSer21, GSK3βSer9, ZyxinSer142/143, Filamin-ASer2152, and LASPSer146, as well as total GRP2, Src, Filamin-A, Zyxin and PKA substrate antibody overnight at 4°C (see supplemental Methods). For visualization of the signal, goat anti-rabbit or anti-mouse immunoglobulin G (IgG)–conjugated with horseradish peroxidase was used as a secondary antibody followed by ECL detection (Amersham; Pharmacia Biotech). Blots were scanned using SilverFast software and analyzed densitometrically by NIH Image J software for uncalibrated optical density.

Results

Quality control of samples

To ensure that the detected phosphopeptide regulations do not originate from variations of protein levels (eg, due to technical variation in sample processing, digestion, etc9 ), we additionally compared protein levels between the different time points for each biological replicate, using 1% of the labeled samples prior to phosphopeptide enrichment. The CV was below 12% for all replicates over all quantified proteins (∼1000 per replicate), which is close to the expected technical error of iTRAQ experiments. In addition, we subjected a single 5nM iloprost-stimulated time course to label-free quantitation, as detailed in the supplemental Material and methods, as an alternative and unbiased strategy to also identify phosphorylation-independent regulation events.

As we have demonstrated recently, intrasubject as well as intersubject variances of the global protein expression patterns are low in human platelets, as 85% of the relatively quantified proteins showed no significant variation.8  After purification of platelets of the required quality without preactivation was established,8  we addressed the intersubject variance of basal phosphorylation patterns to determine whether reproducible phosphorylation time courses can be obtained from different donors. Quantifying 700 phosphopeptides between “resting” platelets from 4 healthy donors, we assessed a CV of 16%. Even for low abundant membrane receptors such as P2X1 with ∼1400 copies per platelet, the CV for the phosphopeptide 382DLAApTSSTLGLQENMR397 was only 15%. These data indicate that intersubject comparisons of time-resolved phosphoproteomic profiles in human platelets are feasible.

Classification of phospho-regulated proteins

To determine which phosphopeptides are significantly regulated, for each replicate iTRAQ, ratios obtained from the Proteome Discoverer software were normalized against the corresponding global analysis, hence compensating for pipetting imprecisions and other systematic errors based on the assumption that the majority of peptides remain stable. Then, peptide-spectrum matches of each replicate were grouped according to protein accessions and the positions of phosphorylation sites in the respective protein sequence, as determined by the phosphoRS algorithm. For each of those groups, the corresponding medians of ratios and SDs were calculated. Grouping and median calculation were repeated to combine all 3 biological replicates. Subsequently, in order to evaluate the significance of regulations, the distribution of the logarithmically transformed ratios was modeled for every iTRAQ channel using a skewed normal distribution centered on the median and normalized to the upper and lower 34th percentiles. Thus, a peptide was considered as regulated with >99% confidence if the corresponding ratio had a probability >99% to deviate from the normal distribution. Finally, only those peptide groups presenting either (1) a regulation confidence >99% in at least 2 replicates or (2) more than twofold upregulation or downregulation in a single replicate were considered to generate a final set of reliably regulated phosphopeptides.

In total, 299 unique proteins show a regulation of phosphorylation upon stimulation with iloprost. Notably, these proteins are henceforth referred to as “regulated proteins.” According to our previous data on human platelets,8  this corresponds to ∼6% of the platelet proteome. Comparing this set of 299 regulated proteins with the human proteome based on GO annotation13  (see Table 1 and supplemental Table 1 for details) reveals an enrichment of proteins involved in “cytoskeleton rearrangement and organization” (26 proteins) and “vesicle-mediated transport” (54 proteins). According to the GPS 2.1 algorithm for kinase consensus sequence prediction,14  many of these regulations occur within a PKA consensus sequence. Furthermore, many of these are early responses (first 10 seconds) as well, indicating an early inhibition of degranulation and actin rearrangement by PKA phosphorylation.

Table 1

Selection of significantly enriched biological processes

Enriched biological process (P value of enrichment)All proteinsPotentially PKA regulatedEarly responders (10 s), 2nM/5 nM
Vesicle-mediated transport (1.1 × 10−2054 19 14/14 
Platelet activation (4.4 × 10−1222 5/7 
Actin cytoskeleton organization (1.5 × 10−1226 15 11/10 
Regulation of small GTPase–mediated signal transduction (5.5 × 10−1226 13 10/9 
Platelet degranulation (1.5 × 10−812 3/3 
Negative regulation of cytoskeleton organization (3.7 × 10−710 2/3 
Blood coagulation (1.5 × 10−628 7/9 
Regulation of Ras protein signal transduction (4.8 × 10−521 8/4 
Cell-surface receptor linked signaling pathway (1.9 × 10−242 24 11/12 
Enriched biological process (P value of enrichment)All proteinsPotentially PKA regulatedEarly responders (10 s), 2nM/5 nM
Vesicle-mediated transport (1.1 × 10−2054 19 14/14 
Platelet activation (4.4 × 10−1222 5/7 
Actin cytoskeleton organization (1.5 × 10−1226 15 11/10 
Regulation of small GTPase–mediated signal transduction (5.5 × 10−1226 13 10/9 
Platelet degranulation (1.5 × 10−812 3/3 
Negative regulation of cytoskeleton organization (3.7 × 10−710 2/3 
Blood coagulation (1.5 × 10−628 7/9 
Regulation of Ras protein signal transduction (4.8 × 10−521 8/4 
Cell-surface receptor linked signaling pathway (1.9 × 10−242 24 11/12 

Selection of significantly enriched biological processes, according to gene ontology (GO) annotation,13  within the regulated proteins. Given is the number of proteins which can be assigned to the respective biological process from the share of all regulated proteins, the potentially PKA-regulated proteins as well as the early responders which show significant regulation already 10 seconds after stimulation.

Among the regulated proteins are 16 kinases, which can be classified in those which show an early (10 seconds) or later (≥30 seconds) response to platelet inhibition as summarized in Table 2.

Table 2

Summary of regulated kinases

AccessionGeneSiteRegulation
Kinase early responders, 10 s    
 Q8TD19 NEK9 Ser827 ↓105nM 
 Q9UKE5 TNIK Ser1021/1028 ↑105nM 
  Ser764/Ser766/Ser769 ↑105nM 
 P07948 LYN Thr489/Tyr492/Ser495/Tyr501/Thr502/Thr504 ↑60/302/5nM 
 Q00537 CDK17 Ser9 ↑105nM 
  Ser180 ↑105nM 
  Ser182 ↑102nM 
 Q02750 MAP2K1 Ser377/Thr378/Ser385/Thr386/Thr388 ↓105nM 
  Ser385/Thr386 ↑105nM 
 Q93100 PHKB Ser27 ↑102/5nM 
  Ser29 ↑102/5nM 
 Q8N4C8 MNK1 Ser699 ↑102/5nM 
  Ser701 ↑102/5nM 
  Ser732 ↓105nM 
  Ser993 ↑105nM 
 Q9BZ23 PANK2 Ser189 ↑30/102/5nM 
 O95819 MAP4K4 Ser900/Thr907/Thr909/Ser913 ↑102/5nM 
 Q15746 MLCK Ser1438 ↓105nM 
  Ser1773 ↑30/102/5nM 
 P12931 SRC Tyr530 ↑105nM 
Kinase late responders, >10 s    
 Q12851 MAP4K2 Ser365/367 ↑305nM 
 P46019 PHKA2 Ser729 ↑302/5nM 
 P52333 JAK3 Ser17 ↑605nM 
 P49840 GSK3A Thr19/Ser20/Ser21/Ser39 ↑105nM 
 P27448 MARK3 Tyr441/Ser442 ↑305nM 
AccessionGeneSiteRegulation
Kinase early responders, 10 s    
 Q8TD19 NEK9 Ser827 ↓105nM 
 Q9UKE5 TNIK Ser1021/1028 ↑105nM 
  Ser764/Ser766/Ser769 ↑105nM 
 P07948 LYN Thr489/Tyr492/Ser495/Tyr501/Thr502/Thr504 ↑60/302/5nM 
 Q00537 CDK17 Ser9 ↑105nM 
  Ser180 ↑105nM 
  Ser182 ↑102nM 
 Q02750 MAP2K1 Ser377/Thr378/Ser385/Thr386/Thr388 ↓105nM 
  Ser385/Thr386 ↑105nM 
 Q93100 PHKB Ser27 ↑102/5nM 
  Ser29 ↑102/5nM 
 Q8N4C8 MNK1 Ser699 ↑102/5nM 
  Ser701 ↑102/5nM 
  Ser732 ↓105nM 
  Ser993 ↑105nM 
 Q9BZ23 PANK2 Ser189 ↑30/102/5nM 
 O95819 MAP4K4 Ser900/Thr907/Thr909/Ser913 ↑102/5nM 
 Q15746 MLCK Ser1438 ↓105nM 
  Ser1773 ↑30/102/5nM 
 P12931 SRC Tyr530 ↑105nM 
Kinase late responders, >10 s    
 Q12851 MAP4K2 Ser365/367 ↑305nM 
 P46019 PHKA2 Ser729 ↑302/5nM 
 P52333 JAK3 Ser17 ↑605nM 
 P49840 GSK3A Thr19/Ser20/Ser21/Ser39 ↑105nM 
 P27448 MARK3 Tyr441/Ser442 ↑305nM 

Kinases showing an early and late response upon iloprost stimulation. ↓105nM corresponds to downregulation of phosphorylation after 10 seconds in the 5nM iloprost samples. If the phosphorylation site localization was not confident (eg, Lyn, MAPKK1, MEKKK4, and GSK3α), all potentially phosphorylated residues are listed according to phosphoRS.

In addition, 3 phosphatases, namely PTPRJ (Ser1311: ↑30/102/5nM), CTDSPL (Ser32: ↓302nM), and PTPN12 (Ser332: ↓602nM; 351EEILQPPEPHPVPPILp(TPSPPSAFPT)VTTVWQDNDR386: ↑30/105nM; 565TVSLp(TPSPTTQVET)PDLVDHDNTSPLFR591: ↓605nM) and 3 phosphatase regulatory subunits, namely PPP2R5D (Ser573: ↑105nM), PPP1R14A (Ser26: ↓302nM; Ser128 and Ser130: ↓605nM), PPP1R3D (Ser77 and Ser78: ↓302nM) were phospho-regulated upon platelet inhibition.

Analyzing all regulated phosphopeptides for the potential presence of consensus motifs of all classes of kinases using GPS 2.1 clearly suggests the additional involvement of CK1/2 and Akt1 kinases in the processes of platelet inhibition and activation. GO-term enrichment analysis with regard to protein function (see Table 3, supplemental Table 2 for details) reveals 6 proteins with Src-homology (SH)3/SH2 adaptor activity. Furthermore, among the regulated proteins, 16 are calmodulin binding and 22 small GTPase regulator proteins. Of the latter, many are connected to Rho-Rac and Rab pathways. In principle, GTPase regulators represent interesting key players in platelet regulation, as they are essential switches of cellular changes and responses upon stimulation.15 

Table 3

Protein functions which are significantly enriched within the 299 regulated proteins

Enriched protein function (P value of enrichment)All proteinsPotentially PKA regulatedEarly responders, 10 s, 2nM/5 nM
Actin binding (1.6 × 10−1126 17 7/8 
Small GTPase regulator activity (1.7 × 10−1022 10 12/7 
Calmodulin binding (8.6 × 10−916 3/3 
SH3/SH2 adaptor activity (1.9 × 10−41/1 
Ubiquitin ligase binding (1.8 × 10−30/2 
Serine/threonine kinase activity (4.4 × 10−312 2/4 
Ubiquitin protein ligase activity (8.2 × 10−23/2 
Tyrosine kinase activity (9.0 × 10−20/2 
Enriched protein function (P value of enrichment)All proteinsPotentially PKA regulatedEarly responders, 10 s, 2nM/5 nM
Actin binding (1.6 × 10−1126 17 7/8 
Small GTPase regulator activity (1.7 × 10−1022 10 12/7 
Calmodulin binding (8.6 × 10−916 3/3 
SH3/SH2 adaptor activity (1.9 × 10−41/1 
Ubiquitin ligase binding (1.8 × 10−30/2 
Serine/threonine kinase activity (4.4 × 10−312 2/4 
Ubiquitin protein ligase activity (8.2 × 10−23/2 
Tyrosine kinase activity (9.0 × 10−20/2 

Protein functions which are significantly enriched within the 299 regulated proteins, according to GO annotation.13  Given is the number of (1) proteins which can be assigned to the respective function from all regulated proteins, (2) potentially PKA-phosphorylated proteins (according to GPS 2.1 consensus motif prediction) as well as (3) early responders which show significant regulation already 10 seconds after stimulation.

Validation using western blot

We used site-specific antibodies against phosphorylated GRP2Ser587, SrcTyr530, VASPSer157, VASPSer239, GSK3αSer21, GSK3βSer9, ZyxinSer142/143, Filamin-ASer2152, LASPSer146, and against total GRP2, Src, Filamin-A, Zyxin to verify some of our results by immunoblotting. As depicted in Figure 1 (and supplemental Figure 1) our MS-based findings could be validated using platelets obtained from additional healthy donors. Indeed, in some cases, the western blot data indicate an even higher increase in phosphorylation, whereas in accordance with the quantitative MS data, phosphorylation of Filamin-ASer2152 remains unchanged upon iloprost treatment, however, increases upon platelet activation with thrombin (supplemental Figure 1).

Figure 1

Time course of PKA substrate phosphorylation in iloprost-stimulated human platelets. Washed human platelets (3 × 108/mL) were incubated with iloprost (2nM) for the indicated time and processed for western blot analysis with antibodies directed against (A) Phospho-PKA substrates (RRXpS/T), (B) P-GRP2Ser587, P-GSK3αSer21, P-GSK3βSer9, P-LASPSer146, P-VASPSer157, P-VASPSer239, or total GRP2 (CalDAG-GEFI) antibodies. (C) Immunoblots were scanned and intensities of the phospho-specific antibodies were normalized to the total GRP2 signal. All images were quantified by the Image J program and expressed as fold changes, where control samples were taken as 1. Results are mean ± SEM, n = 4. +P < .05 compared with the control; *P < .05 compared with 15-second samples.

Figure 1

Time course of PKA substrate phosphorylation in iloprost-stimulated human platelets. Washed human platelets (3 × 108/mL) were incubated with iloprost (2nM) for the indicated time and processed for western blot analysis with antibodies directed against (A) Phospho-PKA substrates (RRXpS/T), (B) P-GRP2Ser587, P-GSK3αSer21, P-GSK3βSer9, P-LASPSer146, P-VASPSer157, P-VASPSer239, or total GRP2 (CalDAG-GEFI) antibodies. (C) Immunoblots were scanned and intensities of the phospho-specific antibodies were normalized to the total GRP2 signal. All images were quantified by the Image J program and expressed as fold changes, where control samples were taken as 1. Results are mean ± SEM, n = 4. +P < .05 compared with the control; *P < .05 compared with 15-second samples.

Novel putative targets of PKA

In his comprehensive review article about cAMP/cGMP-dependent signaling in platelets, Smolenski recently published a list of 17 PKA/PKG targets.3  Our data not only confirm these findings, but substantially expand our knowledge about putative PKA targets in human platelets. From the 12 PKA substrates described by Smolenski, we could detect 9 with putative PKA sites; in addition, we could confirm phosphorylation of the established PKG substrate IRAG at Ser657 and Ser670, both upregulated 10 seconds after iloprost stimulation. However, direct crosstalk between cAMP and cGMP are unlikely.16  From that list, 2 PKA substrates are missing in our data due to the peptide sequence which is rather too short or too long for confident identification after tryptic digestion.

In total about half of all regulated proteins (137) show a regulation of potential PKA sites. Moreover, 22 proteins have >1 regulated peptide containing a potential PKA site, for example, PDE3A and MRVI1/IRAG. Interestingly, phosphorylation of PDE3A (Ser311 and 312: ↑102/5nM; Ser428: ↑30/102/5nM) is upregulated 10 seconds after iloprost treatment, suggesting an immediate negative feedback loop.17  In contrast, AMP deaminase 2 is ↑305nM on Ser76 whereas another potential PKA target Ser168 is ↓102nM, suggesting a positive feedback loop to maintain cAMP levels.18  In total, 30 proteins are potential early PKA targets (changes after 10 seconds) after both 2nM and 5nM iloprost stimulation (see Table 4). This list includes known mediators of platelet activation/inhibition but also indicates an important role for so-far unknown proteins such as the endosulfine family (ENSA, ARPP19) or BIN2.

Table 4

Potential PKA targets showing early responses 10 seconds after both 2nM and 5nM iloprost treatment

AccessionGeneProtein namePutative PKA siteAccessionGeneProtein namePutative PKA site
O14828 SCAMP3 Secretory carrier–associated membrane protein 3 S32 Q8N4C8 MNK1 Misshapen-like kinase 1 S699 and S701 
O00501 CLDN5 Claudin-5 T207 Q8N5J2 FAM63A Protein FAM63A S441 
O43312 MTSS1 Metastasis suppressor protein 1 S272 Q92974 ARHGEF2 Rho guanine nucleotide exchange factor 2 S886 
O43768 ENSA α-endosulfine S109 Q93100 PHKB Phosphorylase b kinase regulatory subunit β S27 
O94929 ABLIM3 Actin-binding LIM protein 3 S277 Q96MK2 FAM65C Protein FAM65C S340* 
O95644 NFATC1 Nuclear factor of activated T cells, cytoplasmic 1 S245 Q99490 AGAP2 Arf-GAP with GTPase, ANK repeat and PH domain–containing protein 2 T647 
O95819 MAP4K4 Mitogen-activated protein kinase kinase kinase kinase 4 S900* Q9BZ72 PTTPNM2 Membrane-associated phosphatidylinositol transfer protein 2 S1277 
P16150 SPN Leukosialin T341 Q9C0C9 UBE2O Ubiquitin-conjugating enzyme E2 O S515 
P56211 ARPP19 cAMP-regulated phosphoprotein 19 S104 Q9P270 SLAIN2 SLAIN motif-containing protein 2 S391 
Q14162 SCARF1 Endothelial cells scavenger receptor S589 and S605 Q9UBW5 BIN2 Bridging integrator 2 S259 
Q14432 PDE3A cGMP-inhibited 3′,5′-cyclic phosphodiesterase A S312 Q9UJU6 DBNL Drebrin-like protein S269* 
Q15149 PLEC Plectin S4386 Q9UKE5 TNIK TRAF2 and NCK-interacting protein kinase S1021* 
Q684P5 RAP1GAP2 Rap1 GTPase-activathg protein 2 S7 Q9Y2J2 EPB41L3 Band 4.1-like protein 3 S420 
Q7LDG7 RASGRP2 RAS guanyl-releasing protein 2 S116, 117, and S587 Q9Y385 UBE2J1 Ubiquitin-conjugating enzyme E2 J1 S266 
Q8IWB9 TEX2 Testis-expressed sequence 2 protein S295 Q9Y6F6 MRVI1 Protein MRVI1 S657 and S670 
AccessionGeneProtein namePutative PKA siteAccessionGeneProtein namePutative PKA site
O14828 SCAMP3 Secretory carrier–associated membrane protein 3 S32 Q8N4C8 MNK1 Misshapen-like kinase 1 S699 and S701 
O00501 CLDN5 Claudin-5 T207 Q8N5J2 FAM63A Protein FAM63A S441 
O43312 MTSS1 Metastasis suppressor protein 1 S272 Q92974 ARHGEF2 Rho guanine nucleotide exchange factor 2 S886 
O43768 ENSA α-endosulfine S109 Q93100 PHKB Phosphorylase b kinase regulatory subunit β S27 
O94929 ABLIM3 Actin-binding LIM protein 3 S277 Q96MK2 FAM65C Protein FAM65C S340* 
O95644 NFATC1 Nuclear factor of activated T cells, cytoplasmic 1 S245 Q99490 AGAP2 Arf-GAP with GTPase, ANK repeat and PH domain–containing protein 2 T647 
O95819 MAP4K4 Mitogen-activated protein kinase kinase kinase kinase 4 S900* Q9BZ72 PTTPNM2 Membrane-associated phosphatidylinositol transfer protein 2 S1277 
P16150 SPN Leukosialin T341 Q9C0C9 UBE2O Ubiquitin-conjugating enzyme E2 O S515 
P56211 ARPP19 cAMP-regulated phosphoprotein 19 S104 Q9P270 SLAIN2 SLAIN motif-containing protein 2 S391 
Q14162 SCARF1 Endothelial cells scavenger receptor S589 and S605 Q9UBW5 BIN2 Bridging integrator 2 S259 
Q14432 PDE3A cGMP-inhibited 3′,5′-cyclic phosphodiesterase A S312 Q9UJU6 DBNL Drebrin-like protein S269* 
Q15149 PLEC Plectin S4386 Q9UKE5 TNIK TRAF2 and NCK-interacting protein kinase S1021* 
Q684P5 RAP1GAP2 Rap1 GTPase-activathg protein 2 S7 Q9Y2J2 EPB41L3 Band 4.1-like protein 3 S420 
Q7LDG7 RASGRP2 RAS guanyl-releasing protein 2 S116, 117, and S587 Q9Y385 UBE2J1 Ubiquitin-conjugating enzyme E2 J1 S266 
Q8IWB9 TEX2 Testis-expressed sequence 2 protein S295 Q9Y6F6 MRVI1 Protein MRVI1 S657 and S670 
*

Phosphorylation could not be confidently localized within the peptide sequence.

Discussion

For the first time, our data provide a time-resolved and comprehensive insight into phosphorylation-dependent signaling upon cAMP-mediated inhibition of human platelets stimulated with the stable prostacyclin analog iloprost. It is obvious that platelet inhibition is a highly complex and concerted process, involving a variety of kinases and phosphatases with substantial crosstalk to activatory signaling pathways. All 3 major steps of platelet inhibition, (1) inactivation of small G proteins of the Ras/Rho family, (2) inhibition of Ca2+ release from intracellular stores, and (3) modulation of actin cytoskeleton dynamics,3  are represented by a variety of regulated proteins in the present study. Our quantitative phosphoproteomic data not only confirm known and expected time courses of proteins known to be involved in platelet inhibition, such as VASP, LASP, CalDAG-GEF119  (Figure 1), and Rap1GAP2,20  but also provide a detailed overview of many more phosphopeptides 10, 30, and 60 seconds after iloprost treatment, compared with untreated controls. Besides activation of PKA, platelet inhibition also seems to directly interfere with a variety of signaling pathways, such as Wnt,21  MEK/ERK, p38 MAPK, JNK,22  Src. We could detect a phosphorylation-dependent regulation of many proteins involved in known Gq-, Gi-, and G12/13-signaling pathways in platelets,23  such as phospholipase C (PLC)β, MLCK, Myosin, cytosolic phospholipase A2 (cPLA2), CalDAG-GEF1, Talin, IP3R, LIM-K. For assessing and summarizing the global data, we selected 3 GO terms which are important for platelets, namely (1) platelet activation, (2) platelet degranulation, (3) cytoskeleton organization, and created protein interaction networks based on STRING24  (Figures 2-4).

Figure 2

Protein interaction network summarizing all regulated proteins belonging to the GO-term “platelet activation”. From all 299 regulated proteins, only members of the GO-term “platelet activation” were searched for interactions using STRING,24  using a medium threshold confidence. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

Figure 2

Protein interaction network summarizing all regulated proteins belonging to the GO-term “platelet activation”. From all 299 regulated proteins, only members of the GO-term “platelet activation” were searched for interactions using STRING,24  using a medium threshold confidence. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

Figure 3

Protein interaction network summarizing all regulated proteins belonging to the GO-term “platelet degranulation.” From all 299 regulated proteins, only members of the GO-term “platelet degranulation” were searched for interactions using STRING,24  using a medium threshold. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

Figure 3

Protein interaction network summarizing all regulated proteins belonging to the GO-term “platelet degranulation.” From all 299 regulated proteins, only members of the GO-term “platelet degranulation” were searched for interactions using STRING,24  using a medium threshold. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

Figure 4

Protein interaction network summarizing all regulated proteins belonging to the GO-term “cytoskeleton organization.” From all 299 regulated proteins, only members of the GO-term “platelet degranulation” were searched for interactions using STRING,24  using a medium threshold. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

Figure 4

Protein interaction network summarizing all regulated proteins belonging to the GO-term “cytoskeleton organization.” From all 299 regulated proteins, only members of the GO-term “platelet degranulation” were searched for interactions using STRING,24  using a medium threshold. Potential PKA sites are highlighted with arrows and the corresponding colors indicate the time points which are regulated compared with 0-second iloprost treatment (dashed lines for downregulation). Upregulated phosphorylation sites are labeled in red and downregulated in blue. Estimated protein copy numbers are taken from Burkhart et al.8 

The Rho/Rac pathway and cytoskeletal reorganization

According to our data, the inhibitory cAMP/PKA pathway affects proteins connected to Rho-Rac pathways. During platelet activation, RhoA leads to various downstream effects such as an increase of actin polymerization, stress fiber, or focal adhesion formation.15  PKA phosphorylation could be involved in modulating the Rho-signaling pathway by phosphorylation (see Figure 4). For instance, several RhoGAP proteins which influence the hydrolysis of GTP to GDP show a regulation upon iloprost treatment, such as RhoGAP1 (site not confident –44p(SDDSKSSS)PELVTHLK58: ↓605nM), RhoGAP6 (Ser710: ↑605nM; Ser927 or 928: ↑305nM; Thr737: ↑602nM), and RhoGAP18 (Ser66: ↓105nM; site not confident –44p(SISQDSLDELS)MEDYWIELENIK66: ↑102nM). Furthermore, our results are in accordance with Nishikawa et al who have reported that MLCK is phosphorylated by PKA in vitro25  because we could identify 2 regulated phosphorylation sites at Ser1759 (↑305nM) and Ser1773 (↑302nM, ↑105nM) of which the latter is a potential PKA target. Moreover, we identified RhoGEF2 (Ser886: ↑102/5nM) and RhoGEF6 (Ser640: ↑302/5nM and Ser684: ↑305nM) as potential PKA substrates. RhoGEF2 mediates crosstalk between microtubules and activates RhoA by exchanging GDP to GTP, indicating a potentially similar mechanism as recently demonstrated for CalDAG-GEF1.20  According to Zenke et al, Ser886 is within a potential inhibiting domain of RhoGEF2 phosphorylated by PAK1,26  however, the sequence PRRRSL represents a classical PKA consensus sequence. RhoGEF6 is a nucleotide exchange factor for Rac1, which is involved in actin cytoskeleton reorganization27  and also interacts with BIN2. Indeed, BIN2 seems to be among the most regulated proteins with several phosphorylation sites responding after 10 seconds: Ser259 (↑102/5nM), Ser263 (↓105nM), 288SESEp(SVSAT)EDLAPDAAQGEDNSEIK313 (↑102nM), and 483ENENIHNQNPEELCp(TSPT)LMTSQVASEPGEAK514 (↓102nM). It contains a Bin–Amphiphysin–Rvs (BAR) domain (Val28-Asn244) which can mediate interactions with phosphatidylinositide-enriched membranes and seems to be involved in cell motility and migration.28 

Further downstream, our data elucidate a change in phosphorylation of Talin at Ser429 (↑60/302/5nM) and Tyr1116 (↓102nM; ↑105nM). Filamin A is another cytoskeletal protein strongly regulated at several phosphorylation sites: Ser1976 or Thr1978 (↓10/302/5nM), Ser2163 (↓302nM) and Ser2158 (↓305nM). Although we could not detect changes at Ser2152 (confirmed by western blot, supplemental Figure 1), a known PKA site preventing Filamin from degradation,29  we identified a novel potential PKA site at Thr2336 (↑302/5nM). Further structural proteins such as Titin (Ser21720: ↑305nM) and Fibrinogen α chain (Thr484 or Ser485 or Ser489: ↑30/102/5nM; S485: ↓605nM) are also regulated upon iloprost treatment, as illustrated in Figures 2-4.

Different Myosin isoforms are also clearly regulated upon iloprost stimulation: we identified Myosin 9 regulated at Ser1943 (↓102nM) and Myosin 9B at 5 confident phosphorylation sites (Thr1346: ↓602nM; Ser1993: ↑102nM; Ser1353: ↑105nM; Ser1354: ↑102nM; Ser1356: ↑102/5nM). Finally, Myosin 18A phosphorylation is found to be regulated at Ser987 (↓105nM), Ser1942 (↑102/5nM), and 1065RVp(SSSSELDLPS)GDHCEAGLLQLDVPLLR1093 (↑302/5nM).

Kinases and phosphatases involved in platelet inhibition

Regarding platelet activation (Figure 2) and aggregation, SH2 domain–containing proteins play an important role, serving as kinases as well as adaptor proteins.30  After iloprost treatment, 3 important SH2 domain–containing, nonreceptor tyrosine kinases involved in various downstream signaling cascades such as actin dynamics (Src),31  regulation of Ca2+ (Fyn),32  and degranulation (Lyn)33  are regulated as illustrated in Figures 2 and 4. The first, Src, is increasingly phosphorylated at Tyr530 (↑105nM), a site known to inhibit kinase activity,34  indicating a negative regulation of Src upon platelet inhibition. We confirmed these interesting MS-based findings by western blot analysis using a site-directed antibody against P-SrcTyr530, as depicted in Figure 5. The second, Lyn, is increasingly phosphorylated on the peptide 484AEERPp(TFDYLQSVLDDFYTAT)EGQYQQQP512 (↑60/302/5nM) and it has been reported that megakaryocyte-associated tyrosine-protein kinase inhibits Lyn by phosphorylation at Tyr508.35  Whether phosphorylation of any of these Ser/Thr/Tyr residues has a negative regulatory effect on kinase activity and subsequent platelet glycoprotein VI (GPVI) signaling in platelets requires further validation. At last, Fyn phosphorylation at Ser21 is decreased (↓302nM). While Fyn is involved in GPVI/integrin signaling and interacts with the phosphatidylinositol pathway,36  the function of Ser21 in Fyn is still unknown.

Figure 5

Western blot validation of increased Src Tyr530 phosphorylation. Washed human platelets (3 × 108/mL) were incubated with iloprost (5nM) for the indicated time and processed for western blot analysis with antibodies directed against P-SrcTyr530 and total Src. Signals were quantified as described in the Figure 1B legend. Shown are representatives of 3 independent experiments.

Figure 5

Western blot validation of increased Src Tyr530 phosphorylation. Washed human platelets (3 × 108/mL) were incubated with iloprost (5nM) for the indicated time and processed for western blot analysis with antibodies directed against P-SrcTyr530 and total Src. Signals were quantified as described in the Figure 1B legend. Shown are representatives of 3 independent experiments.

Ca2+ and phosphatidylinositol signaling

During platelet activation, phosphatidylinositol and mainly Ca2+ signaling regulate most activating pathways in platelets such as integrin activation, thrombus formation, vesicle degranulation, and cytoskeletal rearrangement. Stimulation of the Thromboxane A2 receptor (TPα) leads to activation of PLC, producing PIP2 and PIP3.37  We found TPα (Ser329/Ser331: ↑105nM) regulated on a potential PKA site which could lead to receptor desensitization.37-39  Additionally, we found PITPNM2 (Ser399 or Ser400: ↑102nM; Ser1277: ↑102/5nM; Ser1326: ↑305nM) as phosphatidylinositol transporter and PIKFYVE (Ser307: ↑102nM) as a PIP2-converting kinase to be potentially phosphorylated by PKA. Further downstream on the level of effector proteins, we identified several PIP3-signaling–dependent proteins to be directly regulated by (de)phosphorylation, among those IRS-2 (Ser560: ↑305nM), GSK3α (site not confident, 19p(TSSFAEPGGGGGGGGGGPGGS)ASGPGGTGGGK50: ↑305nM), GSK3β (Thr8/Ser9/Ser13: ↑302nM), centraurin-delta2 (Ser1419: ↑605nM; Ser1435: ↑305nM) and centaurin-gamma1 (Thr647: ↑102/5nM). We could confirm an increase in phosphorylation of GSK3αSer21 and GSK3βSer9 by immunoblotting (Figure 1). For GSK3α, it was demonstrated that phosphorylation of Ser21 by PKA can lead to its enzymatic inhibition.40 

With regard to Ca2+ signaling, pleckstrin phosphorylation (Thr123: ↑305nM; Ser117: ↑605nM, a major PKC target in platelets41 ) is regulated, whereas CalDAG-GEF1 phosphorylation is regulated at Ser576 (↑102/5nM) and potentially PKA phosphorylated at Ser116 and Ser117 (both: ↑102/5nM) as well as Ser587 (↑30/102/5nM), which is in accordance with our own recent data20  demonstrating that phosphorylation of CalDAG-GEF1 inhibits Rap1b activation in human platelets. Moreover, cPLA2 phosphorylation is upregulated at Ser727 (↑302/5nM). Phosphorylation at this site might promote the dissociation of a heterotetramer of cPLA2, Annexin A2, and p11 and increases cPLA2 enzymatic activity.42  As direct regulators of intracellular calcium, we found the calcium release–activated calcium channel Orai1 being dephosphorylated (Thr295: ↓602nM) and the inositol phosphate receptor regulator MRVI1 (IRAG) increasingly phosphorylated at Ser657 and Ser670 (↑102/5nM). Both sites are known to be PKG targets preventing the release of Ca2+ from inositol phosphate sensitive stores and subsequent platelet aggregation.43  The role of Orai1 phosphorylation though is yet unclear.

In addition, we found novel putative PKA phosphorylation sites in TRPC63  at Ser839/840 and Ser92/Thr93/94, however, not regulated under the observed conditions.

A potential role for protein ubiquitination in platelet regulation

Our data also reveal differential phosphorylation upon iloprost stimulation in 7 proteins which are related to protein ubiquitination. Rad23B (Thr155/Thr159/Ser160: ↓102nM) is assumed to deliver polyubiquitinated substrates to the 26S proteasome and to participate in endoplasmic reticulum–associated degradation (ERAD) of misfolded glycoproteins, whereas Cullin4A and DCAF8 are part of the cullin-RING–based E3 ubiquitin-protein ligase.44  Cullin4A is potentially PKA phosphorylated at Ser10 (↑302/5nM) and DCAF8 phosphorylation is significantly downregulated at Ser99 (↓605nM). CYLD, a ubiquitin thiolesterase, is dephosphorylated at Ser418 (↓605nM) as well. MARCH-2 and NEDD-4 are E3 ubiquitin-protein ligases which are potentially PKA phosphorylated at Ser49 (↑302/5nM) and Ser747 (↑305nM), respectively. The latter regulates membrane-bound receptors by ubiquitination.45  Finally, TOM-like protein 1 phosphorylation is significantly regulated at Ser314/Ser321/Ser323 (↑102nM).

The reasons for phosphoregulation of components of the ubiquitin pathway after platelet inhibition–dependent stimulation remain unclear. However, there is evidence that Syk is ubiquitinated upon platelet activation by collagen, leading to a substantial increase of its enzymatic activity.46  Furthermore, cAMP/PKA-mediated ubiquitination leads to an increased proteasome and ubiquitin ligase activity in rat neurons47  and PKA phosphorylation of β-catenin prevents its ubiquitination and degradation in mouse fibroblasts, thus retaining Wnt-signaling activity.48 

Additional phosphorylation-independent regulation

To identify further iloprost-mediated processes with an alternative approach which is not solely focused on phosphopeptides, we analyzed a time course of 1 donor using label-free quantitative proteomics, as described in supplemental Material and methods. Hereby, we quantified global changes within the complete samples to identify regulation events which are not primarily based on phosphorylation, but could be mediated by further posttranslational modifications such as (de)acetylation, (de)palmitoylation,49  degradation/shedding, and formation or cleavage of disulfide bonds. To improve the robustness and reduce the number of candidates considered, we filtered all proteins for the occurrence of at least 3 significantly regulated peptides, leading to a final list of 44 proteins of which 29 were identified as regulated on the phosphopeptide level too (see supplemental Table 6). Among the 15 additional candidates are proteins which are known to be involved in platelet activation, such as APP, Calpain-1 catalytic subunit, coagulation factor V, coagulation factor XIII A chain, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β-1, Hic-5, integrin αIIb, protein disulfide-isomerase A3, glycogen phosphorylase, kindlin-3, and vWF. These results indicate that, besides phosphorylation, further post-translational modification (PTM) such as degradation/processing, ubiquitination, and disulfide bond rearrangement/formation might play an important role in platelet activation and inhibition.

Novel key regulators of platelet activity?

ENSA (Ser109) and ARPP19 (Ser104) are low-molecular-weight proteins of the endosulfine family which have been neglected in platelets so far; however, both show a striking increase in phosphorylation already 10 seconds after stimulation with iloprost. It is known that these proteins are involved in the regulation of phosphatase PP2A which in turn regulates cPLA2 and thus the release of arachidonate.21  Moreover, the early responding ENSA phosphorylation Ser109 disrupts ENSA interaction with α-synuclein (SNCA),23  which was reported as potential negative regulator of α-granule release.50  Thus, these 2 proteins represent novel substrates which might have an important role in platelet activation and inhibition. We speculate that the present data set contains many more interesting targets for functional research, of which many have been completely unknown to the field.

In conclusion, our study represents the first global approach to elucidate time-resolved phosphorylation changes after cAMP-dependent PKA stimulation of human platelets using iloprost. In total, we quantified >2700 phosphopeptides thus yielding unprecedented insights into fundamental processes leading to platelet inhibition. Almost 300 proteins show regulated phosphopeptides, 137 of which are putative PKA targets. Our data clearly indicate that cAMP-dependent platelet inhibition involves the crosstalk of different signaling pathways, obviously through the concerted regulation of at least 16 kinases and 7 phosphatases, and thus is much more complex than originally expected.

Besides known players and substrates of platelet inhibition, we identified many so-far unknown players. We thus hypothesize that these data generated through a discovery-driven characterization of signaling events in human platelets will be the basis to identify and establish novel key players and target proteins in platelet signaling. Moreover, it seems obvious that besides phosphorylation, further dynamic PTM might also play an important role in regulating platelet activation, even in the early stages of cAMP/PKA signaling. Future experiments will have to characterize these novel PKA substrates on the functional level to define their roles in platelet inhibition.

This article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgment

We thank Claudia Schütz for excellent technical assistance.

This work was supported by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, by the Bundesministerium für Bildung und Forschung (MedSys Project SARA 31P5800: J.G., P.N., O.K., U.W., A.S., and R.Z., 01E010003: U.W.), and Deutsche Forschungsgemeinschaft (SFB688/TPA2 [U.W., S.G.], SPP1335 [O.K.]).

F.B. is candidate at the University of Dortmund, Germany. J.V. is candidate at the University of Tübingen, Germany, and this work is submitted in partial fulfillment of the requirement of the PhD.

Authorship

Contribution: F.B. designed and performed research, analyzed the data, and wrote the paper; J.G. analyzed data, collected the platelets from donors, and wrote the paper; S.G. performed western blots and contributed to study design and writing the paper; M.V., P.N., J.V., O.K., and L.M. analyzed the data; U.W. contributed to study design; A.S. contributed to study design and writing the paper; and R.P.Z. conceived of the study, analyzed the data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: René P. Zahedi, Leibniz-Institut für Analytische Wissenschaften–ISAS– e.V., Otto-Hahn-Strasse 6b, D-44227 Dortmund, Germany; e-mail: rene.zahedi@isas.de; and Albert Sickmann, Leibniz-Institut für Analytische Wissenschaften–ISAS–e.V., Otto-Hahn-Strasse 6b, D-44227 Dortmund, Germany; e-mail: albert.sickmann@isas.de.

References

1
Moncada
 
S
Gryglewski
 
R
Bunting
 
S
Vane
 
JR
An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation.
Nature
1976
, vol. 
263
 
5579
(pg. 
663
-
665
)
2
Dutta-Roy
 
AK
Sinha
 
AK
Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets.
J Biol Chem
1987
, vol. 
262
 
26
(pg. 
12685
-
12691
)
3
Smolenski
 
A
Novel roles of cAMP/cGMP-dependent signaling in platelets.
J Thromb Haemost
2012
, vol. 
10
 
2
(pg. 
167
-
176
)
4
Van Geet
 
C
Izzi
 
B
Labarque
 
V
Freson
 
K
Human platelet pathology related to defects in the G-protein signaling cascade.
J Thromb Haemost
2009
, vol. 
7
 
suppl 1
(pg. 
282
-
286
)
5
Dogné
 
JM
Hanson
 
J
Pratico
 
D
Thromboxane, prostacyclin and isoprostanes: therapeutic targets in atherogenesis.
Trends Pharmacol Sci
2005
, vol. 
26
 
12
(pg. 
639
-
644
)
6
Barn
 
K
Steinhubl
 
SR
A brief review of the past and future of platelet P2Y12 antagonist.
Coron Artery Dis
2012
, vol. 
23
 
6
(pg. 
368
-
374
)
7
Siess
 
W
Lapetina
 
EG
Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.
Biochem J
1990
, vol. 
271
 
3
(pg. 
815
-
819
)
8
Burkhart
 
JM
Vaudel
 
M
Gambaryan
 
S
, et al. 
The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways.
Blood
2012
, vol. 
120
 
15
(pg. 
e73
-
e82
)
9
Burkhart
 
JM
Schumbrutzki
 
C
Wortelkamp
 
S
Sickmann
 
A
Zahedi
 
RP
Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.
J Proteomics
2012
, vol. 
75
 
4
(pg. 
1454
-
1462
)
10
Beck
 
F
Lewandrowski
 
U
Wiltfang
 
M
, et al. 
The good, the bad, the ugly: validating the mass spectrometric analysis of modified peptides.
Proteomics
2011
, vol. 
11
 
6
(pg. 
1099
-
1109
)
11
Taus
 
T
Köcher
 
T
Pichler
 
P
, et al. 
Universal and confident phosphorylation site localization using phosphoRS.
J Proteome Res
2011
, vol. 
10
 
12
(pg. 
5354
-
5362
)
12
Geiger
 
J
Brandmann
 
T
Hubertus
 
K
Tjahjadi
 
B
Schinzel
 
R
Walter
 
U
A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways.
Anal Biochem
2010
, vol. 
407
 
2
(pg. 
261
-
269
)
13
Ashburner
 
M
Ball
 
CA
Blake
 
JA
, et al. 
The Gene Ontology Consortium
Gene ontology: tool for the unification of biology.
Nat Genet
2000
, vol. 
25
 
1
(pg. 
25
-
29
)
14
Xue
 
Y
Ren
 
J
Gao
 
X
Jin
 
C
Wen
 
L
Yao
 
X
GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy.
Mol Cell Proteomics
2008
, vol. 
7
 
9
(pg. 
1598
-
1608
)
15
Ridley
 
AJ
Rho GTPases and cell migration.
J Cell Sci
2001
, vol. 
114
 
Pt 15
(pg. 
2713
-
2722
)
16
Wangorsch
 
G
Butt
 
E
Mark
 
R
, et al. 
Time-resolved in silico modeling of fine-tuned cAMP signaling in platelets: feedback loops, titrated phosphorylations and pharmacological modulation.
BMC Syst Biol
2011
, vol. 
5
 pg. 
178
 
17
Hunter
 
RW
Mackintosh
 
C
Hers
 
I
Protein kinase C-mediated phosphorylation and activation of PDE3A regulate cAMP levels in human platelets.
J Biol Chem
2009
, vol. 
284
 
18
(pg. 
12339
-
12348
)
18
Fain
 
JN
Wieser
 
PB
Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.
J Biol Chem
1975
, vol. 
250
 
3
(pg. 
1027
-
1034
)
19
Subramanian
 
H
Zahedi
 
RP
Sickmann
 
A
Walter
 
U
Gambaryan
 
S
Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation.
J Thromb Haemost
2013
, vol. 
11
 
8
(pg. 
1574
-
1582
)
20
Schultess
 
J
Danielewski
 
O
Smolenski
 
AP
Rap1GAP2 is a new GTPase-activating protein of Rap1 expressed in human platelets.
Blood
2005
, vol. 
105
 
8
(pg. 
3185
-
3192
)
21
Moscardó
 
A
Vallés
 
J
Piñón
 
M
Aznar
 
J
Martínez-Sales
 
V
Santos
 
MT
Regulation of cytosolic PlA2 activity by PP1/PP2A serine/threonine phosphatases in human platelets.
Platelets
2006
, vol. 
17
 
6
(pg. 
405
-
415
)
22
Roberts
 
W
Magwenzi
 
S
Aburima
 
A
Naseem
 
KM
Thrombospondin-1 induces platelet activation through CD36-dependent inhibition of the cAMP/protein kinase A signaling cascade.
Blood
2010
, vol. 
116
 
20
(pg. 
4297
-
4306
)
23
Boettcher
 
JM
Hartman
 
KL
Ladror
 
DT
, et al. 
Membrane-induced folding of the cAMP-regulated phosphoprotein endosulfine-alpha.
Biochemistry
2008
, vol. 
47
 
47
(pg. 
12357
-
12364
)
24
Snel
 
B
Lehmann
 
G
Bork
 
P
Huynen
 
MA
STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene.
Nucleic Acids Res
2000
, vol. 
28
 
18
(pg. 
3442
-
3444
)
25
Nishikawa
 
M
de Lanerolle
 
P
Lincoln
 
TM
Adelstein
 
RS
Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase.
J Biol Chem
1984
, vol. 
259
 
13
(pg. 
8429
-
8436
)
26
Zenke
 
FT
Krendel
 
M
DerMardirossian
 
C
King
 
CC
Bohl
 
BP
Bokoch
 
GM
p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor.
J Biol Chem
2004
, vol. 
279
 
18
(pg. 
18392
-
18400
)
27
Yang
 
N
Higuchi
 
O
Ohashi
 
K
, et al. 
Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.
Nature
1998
, vol. 
393
 
6687
(pg. 
809
-
812
)
28
Sánchez-Barrena
 
MJ
Vallis
 
Y
Clatworthy
 
MR
, et al. 
Bin2 is a membrane sculpting N-BAR protein that influences leucocyte podosomes, motility and phagocytosis.
PLoS ONE
2012
, vol. 
7
 
12
pg. 
e52401
 
29
Chen
 
M
Stracher
 
A
In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain.
J Biol Chem
1989
, vol. 
264
 
24
(pg. 
14282
-
14289
)
30
Pawson
 
T
Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems.
Cell
2004
, vol. 
116
 
2
(pg. 
191
-
203
)
31
O’Brien
 
KA
Gartner
 
TK
Hay
 
N
Du
 
X
ADP-stimulated activation of Akt during integrin outside-in signaling promotes platelet spreading by inhibiting glycogen synthase kinase-3β.
Arterioscler Thromb Vasc Biol
2012
, vol. 
32
 
9
(pg. 
2232
-
2240
)
32
Kim
 
S
Kunapuli
 
SP
Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase.
J Biol Chem
2011
, vol. 
286
 
27
(pg. 
24170
-
24179
)
33
Cho
 
MJ
Pestina
 
TI
Steward
 
SA
Lowell
 
CA
Jackson
 
CW
Gartner
 
TK
Role of the Src family kinase Lyn in TxA2 production, adenosine diphosphate secretion, Akt phosphorylation, and irreversible aggregation in platelets stimulated with gamma-thrombin.
Blood
2002
, vol. 
99
 
7
(pg. 
2442
-
2447
)
34
Kaplan
 
KB
Bibbins
 
KB
Swedlow
 
JR
Arnaud
 
M
Morgan
 
DO
Varmus
 
HE
Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.
EMBO J
1994
, vol. 
13
 
20
(pg. 
4745
-
4756
)
35
Hirao
 
A
Hamaguchi
 
I
Suda
 
T
Yamaguchi
 
N
Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.
EMBO J
1997
, vol. 
16
 
9
(pg. 
2342
-
2351
)
36
Watson
 
SP
Auger
 
JM
McCarty
 
OJ
Pearce
 
AC
GPVI and integrin alphaIIb beta3 signaling in platelets.
J Thromb Haemost
2005
, vol. 
3
 
8
(pg. 
1752
-
1762
)
37
Yan
 
FX
Yamamoto
 
S
Zhou
 
HP
Tai
 
HH
Liao
 
DF
Serine 331 is major site of phosphorylation and desensitization induced by protein kinase C in thromboxane receptor alpha.
Acta Pharmacol Sin
2002
, vol. 
23
 
10
(pg. 
952
-
960
)
38
Walsh
 
MT
Foley
 
JF
Kinsella
 
BT
The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for prostacyclin-mediated desensitization.
J Biol Chem
2000
, vol. 
275
 
27
(pg. 
20412
-
20423
)
39
Kelley-Hickie
 
LP
O’Keeffe
 
MB
Reid
 
HM
Kinsella
 
BT
Homologous desensitization of signalling by the alpha (alpha) isoform of the human thromboxane A2 receptor: a specific role for nitric oxide signalling.
Biochim Biophys Acta
2007
, vol. 
1773
 
6
(pg. 
970
-
989
)
40
Bragado
 
MJ
Aparicio
 
IM
Gil
 
MC
Garcia-Marin
 
LJ
Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase-3A serine 21 phosphorylation in boar spermatozoa.
J Cell Biochem
2010
, vol. 
109
 
1
(pg. 
65
-
73
)
41
Craig
 
KL
Harley
 
CB
Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C.
Biochem J
1996
, vol. 
314
 
Pt 3
(pg. 
937
-
942
)
42
Tian
 
W
Wijewickrama
 
GT
Kim
 
JH
, et al. 
Mechanism of regulation of group IVA phospholipase A2 activity by Ser727 phosphorylation.
J Biol Chem
2008
, vol. 
283
 
7
(pg. 
3960
-
3971
)
43
Antl
 
M
von Brühl
 
ML
Eiglsperger
 
C
, et al. 
IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation.
Blood
2007
, vol. 
109
 
2
(pg. 
552
-
559
)
44
Jin
 
J
Arias
 
EE
Chen
 
J
Harper
 
JW
Walter
 
JC
A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1.
Mol Cell
2006
, vol. 
23
 
5
(pg. 
709
-
721
)
45
Hallows
 
KR
Bhalla
 
V
Oyster
 
NM
, et al. 
Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel.
J Biol Chem
2010
, vol. 
285
 
28
(pg. 
21671
-
21678
)
46
Dangelmaier
 
CA
Quinter
 
PG
Jin
 
J
Tsygankov
 
AY
Kunapuli
 
SP
Daniel
 
JL
Rapid ubiquitination of Syk following GPVI activation in platelets.
Blood
2005
, vol. 
105
 
10
(pg. 
3918
-
3924
)
47
Myeku
 
N
Wang
 
H
Figueiredo-Pereira
 
ME
cAMP stimulates the ubiquitin/proteasome pathway in rat spinal cord neurons.
Neurosci Lett
2012
, vol. 
527
 
2
(pg. 
126
-
131
)
48
Hino
 
S
Tanji
 
C
Nakayama
 
KI
Kikuchi
 
A
Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination.
Mol Cell Biol
2005
, vol. 
25
 
20
(pg. 
9063
-
9072
)
49
Dowal
 
L
Yang
 
W
Freeman
 
MR
Steen
 
H
Flaumenhaft
 
R
Proteomic analysis of palmitoylated platelet proteins.
Blood
2011
, vol. 
118
 
13
(pg. 
e62
-
e73
)
50
Park
 
SM
Jung
 
HY
Kim
 
HO
, et al. 
Evidence that alpha-synuclein functions as a negative regulator of Ca(++)-dependent alpha-granule release from human platelets.
Blood
2002
, vol. 
100
 
7
(pg. 
2506
-
2514
)