10.4081/btvb.2023.93
The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.
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Ángel García
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Bleeding, Thrombosis and Vascular Biology
hemostasis
Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2
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Original Articles
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2023-07-18
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Bleed Thromb Vascul Biol
Ángel García
2023/10/11
10.4081/btvb.2023.93
Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, and Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela
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Bye AP, Unsworth AJ, Vaiyapuri S, et al. Ibrutinib inhibits platelet integrin αIIbβ3 outside-in signaling and thrombus stability but not adhesion to collagen. Arterioscler Thromb Vasc Biol. 2015;35:2326-635.
Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2
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                                                                                                               ORIGINAL ARTICLE

Restrained glycoprotein VI-induced platelet signaling by tyrosine
protein phosphatases independent of phospholipase Cγ2
Jingnan Huang,1,2 Delia I. Fernández,1,2 Jinmi Zou,2,3 Xueqing Wang,1 Johan W.M. Heemskerk,3 Ángel García1
1
 Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela,
and Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain; 2Cardiovascular Research Institute Maastricht,
Department of Biochemistry, Maastricht University, The Netherlands; 3Synapse Research Institute, Maastricht, The Netherlands



ABSTRACT
    The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide
3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate
GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the
                                                                       Shp1/2 inhibitor NSC87877 on platelet activation via GPVI
                                                                       using light transmission aggregometry, Ca2+ flux assay, western
                                                                       blotting and flow cytometry. Effect measurements of selective




                                                                                                         ly
  Correspondence: Johan W.M. Heemskerk, Synapse Research In-           PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877




                                                                                                 on
  stitute, Kon. Emmaplein 7, 6217 KD Maastricht , The Netherlands.     enhanced platelet aggregation induced by the GPVI agonist,
  E-mail: jwmheem722@outlook.com                                       collagen-related peptide (CRP). Furthermore, NSC87877 an-
  Citation: Huang J, Fernández DI, Zou J, et al. Restrained glyco-     tagonized the effects of PI3Kb inhibition, but not of Btk inhi-




                                                                                           e
  protein VI-induced platelet signaling by tyrosine protein phos-      bition. Both NSC87877 and TGX221 suppressed the
  phatases independent of phospholipase Cγ2. Bleeding,                 CRP-induced phosphorylation of PLCγ2 at activation site
  Thrombosis, and Vascular Biology 2023;2:93.

  Key words: glycoprotein VI, tyrosine kinase, platelet signaling, ty-
                                                                                   us
                                                                       Tyr759. These findings indicate that drug interference of the
                                                                       two phosphatases Shp1/2 subtly enhances GPVI-induced
  rosine phosphatase, hemostasis.                                      platelet responses via a mechanism not involving PLCγ2 acti-
                                                                              al
                                                                       vation, even upon PI3K inhibition.
    Acknowledgments: JH and DIF were enrolled in a joint PhD proj-
                                                                    ci

    ect of the universities of Maastricht (the Netherlands) and Santiago
    de Compostela (Spain).
                                                                  er


    Contributions: JH, DIF, designed and performed experiments and
                                                                 m



    analyzed the data; JZ, XW, performed experiments and interpreted
    data; JWMH, AG, designed the study and provided funding and
                                                                              Introduction
    supervision; JH, JWMH, AG, conceptualized and wrote the man-
                                                      om




    uscript. All authors contributed to the drafting of the manuscript,            Platelet stimulation by the collagen receptor, glycoprotein
    are accountable for all aspects of the work, and approved the final       VI (GPVI), induces granule secretion and platelet aggregation
    version to be published.                                                  through a tyrosine kinase signaling cascade, involving Src-fam-
                                               -c




    Ethical approval and consent to participate: all blood donors gave
                                                                              ily kinases (SFK), Syk and Btk kinases.1-3 Commonly, two
    full informed consent according to the declaration of Helsinki. Ap-       GPVI-induced signaling pathways are distinguished, one via
                                      on




    proval for the studies was obtained from the medical ethics com-          phospholipase Cγ2 (PLCγ2) triggering a rise in cytosolic [Ca2+]i
    mittees of the University of Santiago de Compostela (Galician             and one via phosphoinositide 3-kinases (PI3K) that is more di-
    Investigation Ethics Committee, code 2009-270) and Maastricht
                                                                              rectly linked to integrin αIIbβ3 activation.4 Collagen and colla-
                                N




    University Medical Centre+ (MUMC+, code: 2017-0285). The ap-
    proval did not allow revealing donor characteristics other than in-       gen-related peptide (CRP) are bona fide ligands of GPVI, as
    dicated.                                                                  platelets from patients with a GPVI deficiency are greatly im-
    Availability of data and material: all generated data are included
                                                                              paired in activation and aggregation with these agonists.5-7
    in the article.                                                                Platelet GPVI signals with its co-receptor FcR g-chain, con-
                                                                              taining an immunoreceptor tyrosine-based activation motif
    Received: 18 July 2023.                                                   (ITAM). In platelets and other blood cells, the ITAM-linked re-
    Accepted: 23 September 2023.
                                                                              ceptors are modified in signaling action by tyrosine phosphatases
    Publisher’s note: all claims expressed in this article are solely those   containing an immunoreceptor tyrosine-based inhibition motifs.
    of the authors and do not necessarily represent those of their affili-    Two such phosphatases are Shp1 and Shp2 (short for SH2 do-
    ated organizations, or those of the publisher, the editors and the re-
    viewers. Any product that may be evaluated in this article or claim
                                                                              main-containing protein tyrosine phosphatases), both of which
    that may be made by its manufacturer is not guaranteed or endorsed        dephosphorylate signaling proteins upon GPVI activation.8-10 The
    by the publisher.                                                         isoform Shp1 encoded by the gene PTPTN6 is highly expressed
                                                                              in human platelets with an estimated copy number of 8,916 per
    ©
     Copyright: the Author(s), 2023
    Licensee PAGEPress, Italy                                                 cell.11 The other isoform Shp2 (gene PTPN11) has a lower ex-
    Bleeding, Thrombosis and Vascular Biology 2023; 2:93                      pression with a copy number of 3,666.
    doi:10.4081/btvb.2023.93                                                       In spite of their structural similarities, the roles of Shp1 and
    This work is licensed under a Creative Commons Attribution-
                                                                              Shp2 are known to be partly different.12 In mouse platelets, the
    NonCommercial 4.0 International License (CC BY-NC 4.0).                   isoform Shp2 suppresses GPVI- and integrin αIIbβ3-induced re-
                                                                              sponses.9 On the other hand, mouse platelet Shp1 can act as a


                                                Bleeding, Thrombosis and Vascular Biology 2023; 2:93
      Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2      41



positive regulator of GPVI-induced signaling.9,13,14 Both iso-       Cytosolic Ca2+ measurements
forms are inactivated by the compound NSC87877, which binds
to the catalytic cleft of either phosphatase.15 This compound is         Washed platelets (200×109/L) were loaded with a mixture
of current interest for tumor-targeted therapies.16 When applied     of Fura-2 acetoxymethyl ester (3 μM) and pluronic (0.4 µg/mL)
as such a drug, NSC87877 will likely affect platelet functions,      for 40 min at room temperature. After centrifugation in the pres-
although to which extent and in which way is unclear.                ence of 1:10 ACD and apyrase (1 U/mL), the dye-loaded
    In the present study with human platelets, we studied the ef-    platelets were resuspended at the same concentration into Hepes
fects of NSC87877 on signaling and activation responses in-          buffer pH 7.45.18 Samples of 200 µL in 96-wells plates were pre-
duced by the GPVI agonist CRP. This allowed us to check for          incubated with DMSO vehicle or inhibitor for 10 min, after
off-target effects of the drug, and simultaneously to define the     which 1 mM CaCl2 was added, and the temperature was adapted
combined roles of both tyrosine phosphatases Shp1 and Shp2.          to 37°C. Agonist-induced ratiometric changes in fluorescence
Our data point to a partly suppressive role on platelet aggrega-     were measured per well with a FlexStation 3 (Molecular De-
tion, not directly involving the Btk and PLCγ2 pathway.              vices, San Jose, CA, USA).21 Calibrated changes in cytosolic
                                                                     [Ca2+]i were calculated from ratiometric values.22 Measurements
                                                                     were performed in triplicate wells.
Materials and Methods
                                                                     Western blotting
Materials




                                                                                       ly
                                                                          For western blotting according to established procedures,19
     Cross-linked CRP was purchased from CambCol Laborato-           washed platelets (800×109/L) were preincubated with DMSO ve-




                                                                               on
ries (Cambridge, UK). NSC87877 came from Cayman Chemical             hicle, TGX221 and/or NSC87877. Also added was 9 μM integrilin
(Ann Arbor, MI, USA). TGX221 was from sources described              to prevent aggregation. Platelet samples (300 μL) were stimulated
before,17 while ibrutinib was from Toronto Research Chemicals        with 0.5 μg/mL CRP for different times, after which reactions




                                                                          e
(North York, Canada). Used as fluorescent stains were Alexa          were stopped by addition of 300 μL 2× lysis buffer. The process-
Fluor (AF) 647-conjugated anti-human CD62P mAb (Biole-
gend, London, UK) and FITC-PAC1 mAb (BD Bioscience,
                                                                    us
                                                                     ing of lysed samples by reduction, boiling, and loading onto Nu-
                                                                     PAGE 4-12% Bis-Tris gels was as before,19 as were the
Franklin Lakes, NJ, USA). Other materials for functional assays      electrophoresis and blotting conditions. Primary antibodies used
                                                               al
and for Western blotting are as described elsewhere.18,19            were anti human phospho-PLCγ2 Y759 mAb (dilution 1:300), rab-
                                                                     bit anti-phospho-Syk Y525+Y526 mAb (dilution 1:1,000), and rabbit
                                                 ci

Preparation of blood and platelets                                   anti-phospho-Src Y418 mAb (dilution 1:1,000). In addition, lanes
                                               er


                                                                     were re-probed for rabbit anti-GAPDH (G9545, Sigma) as a load-
    Blood was drawn into 3.2% trisodium citrate from healthy         ing control. The probed blotting membranes were incubated with
                                              m



volunteers through venipuncture. Donors had not received anti-       horseradish peroxidase-labeled goat anti-mouse IgG or goat anti-
platelet medication for at least two weeks and gave full informed    rabbit IgG (dilution 1:5,000), and then processed using an en-
                                     om




consent according to the declaration of Helsinki. Studies were       hanced chemiluminescence system and Image Lab.19
approved by the local Medical Ethics Committees.
    Platelet-rich plasma (PRP) and washed platelet preparations
                                                                     Flow cytometry
                               -c




were isolated following an established procedure to remove
other blood cells.20 In brief, PRP was obtained by centrifugation        Platelets suspended at 50×109/L were pre-incubated with
                       on




at 240 g for 15 min. After the addition of 10 vol% acid citrate      DMSO vehicle, NSC87877 (20 µM), TGX221 (3 µM), and/or
dextrose (ACD) (80 mM trisodium citrate, 183 mM glucose, 52          ibrutinib (1 µM) for 10 min at room temperature. The cells were
mM citric acid), platelets were isolated by centrifugation at
                  N




                                                                     then stimulated with CRP at 1 mM CaCl2. Measurements of
5,500 g for 2 min. The pelleted platelets were resuspended into      platelet activation markers with AF-647 anti-CD62P mAb (2.5
Hepes buffer pH 6.6 (10 mM Hepes, 136 mM NaCl, 2.7 mM                µg/mL) and FITC PAC1 mAb (1.25 µg/mL) were performed
KCl, 2 mM MgCl2, 5.5 mM glucose, and 0.1% bovine serum al-           using an Accuri C6 flow cytometer (10,000 events). Data were
bumin). After the addition of apyrase (1 U/mL) and 6.6 vol%          analyzed with FlowJo software.21
ACD, another centrifugation step was performed to obtain
washed platelets. For functional assays, these were resuspended      Statistics and data processing
in Hepes-Tyrode’s buffer (134 mM NaCl, 0.34 mM Na2HPO4,
2.9 mM KCl, 12 mM NaHCO3, 20 mM Hepes, 5 mM glucose,                     Statistical analysis was performed with GraphPad Prism 8
and 1 mM MgCl2, pH 7.3) at requested platelet count.                 software (San Diego, CA, USA). A paired Student t-test was used
                                                                     with significance set at p<0.05.
Light transmission aggregometry
    Aggregation of washed platelets (250×109/L) was measured         Results
at 37°C under stirring with a Chronolog aggregometer (Havertown,
PA, USA). No fibrinogen was added, implicating that the aggre-       Shp1/2 inhibition by NSC87877 enhances
gation response relied on fibrinogen secretion. Samples of 300 µL    glycoprotein VI-induced platelet aggregation
were pre-incubated with either dimethylsulfoxide (DMSO),
NSC87877 (10-20 µM) and/or TGX221 (1-3 µM) for 5 min at                  We first established how inhibition of Shp1/2 by NSC87877
room temperature, followed by a temperature adjustment to 37°C.      affected GPVI-induced platelet aggregation, as a standard


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:93
42                                                           J. Huang et al.




platelet function assay. The compound was used at a concentra-          Shp1/2 inhibition by NSC87877 reduces
tion of 10 µM, which was previously shown to block the cat-             glycoprotein VI-induced platelet PLCγ2 tyrosine
alytic activity of both Shp1 and Shp2.15 In response to
sub-maximal doses of CRP (0.2-1.0 µg/mL), we found a signif-
                                                                        phosphorylation and Ca2+ responses
icant enhancing effect of NSC87877 on the aggregation re-                    Given the ability of the Shp1/2 isoforms to alter the GPVI-
sponse, when comparison to the vehicle control condition                induced tyrosine phosphorylation of signalosome components
(Figure 1A and B). This agreed with the GPVI-restraining role           Syk, Src and PLCγ2,10 we assessed the phosphorylation of crit-
of Shp2, previously observed in mouse platelets.10 At the highest       ical tyrosine residues in these proteins.19 Western blot analysis
CRP dose of 5.0 µg/mL, the aggregation was already maximal,             of platelets stimulated with sub-maximal CRP (0.5 µg/mL),
precluding an effect of NSC87877 (Figure 1B).                           using the broad anti-phospho-tyrosine mAb 4G10, indicated a
     For assessment of the contribution of GPVI-induced PI3K            high near-optimal phosphorylation of most protein bands after
activation, we used the PI3K inhibitor, TGX221, which was pre-          45 s and 90 s of stimulation (Figure 2A). Using selective phos-
viously shown to nearly completely block GPVI-induced throm-            pho-tyrosine mAbs, we confirmed this for phospho-Syk
bus formation.17 Initial experiments using 1 or 3 µM TGX221             Tyr525+526, phospho-Src Tyr419 and phospho-PLCγ2 Tyr759 (Fig-
indicated a consistent, donor-independent aggregation response          ure 2B). Quantitative blot analysis of 90-s samples indicated
at 3 µM (data not shown), which concentration was then chosen.          that preincubation with NSC87877 did not influence the three
In TGX221-treated platelets, the CRP-induced aggregation was            tyrosine phosphorylation sites (Figure 2C-E). By itself,
reduced by ~50% (Figure 1C). Importantly, the reduction was




                                                                                                   ly
                                                                        TGX221 slightly reduced only the Tyr759 phosphorylation of
rescued by the simultaneous treatment with 10 µM NSC87877               PLCγ2. Strikingly, the combination of NSC87877 and




                                                                                           on
(Figure 1C). In this setting, an increased dose of 20 µM                TGX221 caused a significant reduction in phospho-Tyr419 of
NSC87877 overruled the inhibitory effect of TGX221, and did             Src and in phospho-Tyr759 of PLCγ2, when compared to
even enhance the aggregation (Figure 1D). Together, these find-         NSC87877 alone (Figure 2C-E). In contrast, the phosphoryla-




                                                                                     e
ings suggest that NSC87877, likely by antagonizing the negative         tion extent of Syk was not affected by the combination of in-
regulation by Shp2, enhances the CRP/GPVI-induced signaling
bypassing PI3K activation.                                                     us
                                                                        hibitors. This pointed to a Shp1-like (positive regulatory)
                                                                        action mechanism on tyrosine phosphorylation events, down-
                                                                        stream of Syk, which becomes apparent under condition of
                                                                       al
                                                                        PI3Kb inhibition.
                                                                             The Tyr759 PLCγ2 phosphorylation is a known regulatory
                                                         ci

                                                                        site for phospholipase activity,23 thus implicating a role in
                                                                        downstream platelet Ca2+ responses. To investigate this further,
                                                       er


                                                                        we measured in Fura-2-loaded platelets the rises in cytosolic
                                                                        [Ca2+]i induced by GPVI agonist CRP.21 However, NSC87877
                                                      m




                                                                        treatment did not significantly affect the [Ca2+]i traces, neither
                                             om




                                                                        in the absence nor presence of TGX221 (Figure 3A and B). On
                                                                        the other hand, in agreement with our aggregation data, the
                                                                        treatment with TGX221 (with or without NSC87877) halved
                                       -c




                                                                        the rises in [Ca2+]i induced by CRP. Collectively, these data
                                                                        point to a suppressive effect of Shp1/2 inhibition on CRP-in-
                               on




                                                                        duced PLCγ2 tyrosine phosphorylation and ensuing Ca2+ re-
                                                                        sponses in platelets.
                          N




                                                                        Contribution of glycoprotein VI-mediated Btk
                                                                        activation
                                                                            Downstream of GPVI, the tyrosine kinase Btk is a known reg-
Figure 1. Enhanced collagen-related peptide-induced platelet            ulator of PLCγ2 activation. In particular, Btk catalyzes the phos-
aggregation by inhibition of Shp1/2. A-B) Washed platelets              phorylation of PLCγ2 at Tyr759, thereby enhancing its activity in
(250×109/L) were incubated with vehicle (Veh.) or Shp1/2 in-            evoking [Ca2+]i rises.24 In order to check for a role of Btk in the
hibitor NSC87877 (NSC, 10 μM) for 10 min. Platelet aggrega-             signaling modulation by Shp1/2, we used the selective, irre-
tion was monitored in response to indicated doses of                    versible inhibitor ibrutinib.25 Strikingly, we found that the CRP-
collagen-related peptide (CRP, 0.2-5 μg/mL); A) Representative
light transmission (%T) changes (axes 480 s, 100%T) of platelet         induced Ca2+ response was almost completely abrogated by
aggregation induced by 0.5 μg/mL CRP; B) Effect of NSC87877             ibrutinib alone, an effect that could not be reverted by the addi-
on maximal aggregation induced by CRP (0.2-5 μg/mL); C-D)               tional presence of NSC87877 (Figure 3C and D). In another ap-
Platelets (250×109/L) were incubated with vehicle control               proach, we examined the effect of ibrutinib alone or in
(Veh.), NSC87877 (NSC, 20 μM), TGX221 (TGX, 3 μM) or a
combination for 10 min; C) Representative light transmission            combination with NSC87877 on CRP-induced integrin αIIbβ3 ex-
changes (axes 480 s, 100%T) on maximal aggregation induced              pression (Figure 4A) and P-selectin expression (Figure 4B), using
by 0.5 μg/mL CRP; D) Effect of NSC87877 and TGX221 on                   flow cytometry. In none of these responses, the inhibitory effect
maximal aggregation, expressed as % of vehicle control. Mean            of ibrutinib was overcome by NSC87877, regardless of the CRP
values ± SD (n=3 donors); *p<0.05, **p<0.005, ***p<0.001,               dose that was used (Figure 4). Together, this pointed to absence
****p<0.0001 vs. vehicle (paired Student’s t-test).
                                                                        of Shp1/2 regulation of GPVI-induced responses acting via Btk.


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:93
      Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2          43



                                                                       PLCγ2 tyrosine phosphorylation rather than enhancing it. Also,
Discussion                                                             the Ca2+ signal downstream of PLCγ2 was not enhanced. We
    Prior work has indicated that the tyrosine phosphatases Shp1       found that the combination of NSC87877 plus TGX221 markedly
and Shp2 play nonredundant, partly contrary roles in ITAM-me-          suppressed the phosphorylation of PLCγ2 at Tyr759, and to a lesser
diated platelet activation.9,13,14 Both Shp isoforms are known to      degree of Src at Tyr419 after CRP stimulation. Furthermore, inhi-
co-regulate the GPVI signaling pathway, by differently affecting       bition of the upstream tyrosine kinase Btk - a major regulator of
the activity of SFK, Syk and the effector protein PLCγ2.10 In the      Tyr759 PLCγ2 phosphorylation - caused a prominent reduction
present paper, we show that platelet treatment with the pan-           in the Ca2+ signal, which was not reverted nu PI3K inhibition. Al-
Shp1/2 inhibitor NSC87877, a potential anti-cancer drug, en-           together, these findings suggest that the ability of tyrosine phos-
hanced GPVI-induced platelet aggregation responses, thus               phatase Shp2 to dampen the GPVI-induced signaling in platelets
pointing to a net antagonistic effect of the combined tyrosine         concerns a pathway aside from PLCγ2 and PI3K.
phosphatases on ITAM signaling by GPVI. Interestingly, the                  In mice, megakaryocytic deletion of Shp1 resulted in platelets
blockage of Shp1/2 with NSC87877 enhanced platelet aggrega-            being less responsive to CRP, whereas deletion of Shp2 caused
tion even under conditions of PI3K β blockage. Given the rela-         hyper-responsiveness.9,13,14 Accordingly the present inhibitory ef-
tive importance of the PI3K β isoform,17 this indicated                fect of NCS87877 on GPVI PLCγ2 phosphorylation needs to be
interference with a non-PI3K pathway.                                  attributed to the Shp1 isoform. In contrast, the enhancing effect
    In contrast to the results of platelet aggregation, we noticed     of the drug on platelet aggregation points to a role of the Shp2
that Shp1/2 inhibition with NSC87877 suppressed CRP-induced            isoform. Given the role of SFK in platelet aggregation and the




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Figure 2. Unchanged collagen-related peptide-induced tyrosine phosphorylation by inhibition of Shp1/2 in contrast to inhibition of
phosphoinositide 3-kinase β. Washed platelets (800×109/L) were incubated with vehicle medium, NSC87877 (NSC, 20 μM), TGX221
(TGX, 3 μM) or combination of both for 10 min. The cells were then stimulated with collagen-related peptide (CRP, 0.5 μg/mL) for 45-
90 s. Changes of protein tyrosine phosphorylation were measured by western blotting using anti-Tyr 4G10 mAb. A) Representative
western blot of collagen-related peptide-induced tyrosine phosphorylation changes in platelets. Molecular size of separated kDa markers
is indicated on the left side; B) Blots showing collagen-related peptide-induced increased phosphorylation (45 s) of phospho-phospho-
lipase Cγ2 Tyr759, phospho-Src Tyr419 and phospho-Syk Tyr525+526, using specific antibodies; C-E) Densitometric analysis of tyrosine
phosphorylation changes after 45 s of Syk p-Tyr525+526 (C), Src p-Tyr419 (D), and phospholipase Cγ2 p-Tyr759 (E). Data are presented as
arbitrary staining units (AU) versus sample-size control (anti-nicotinamide adenine dinucleotide phosphate mAb), normalized for the
vehicle control condition. Mean values ± standard deviation (n=3 donors). *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001 vs. vehicle
control (paired Student’s t-test).


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:93
44                                                            J. Huang et al.




                                                                         Src-dependent action mechanism of Shp,9 the molecular pathway
                                                                         is likely Src-dependent. However, this hypothesis needs further
                                                                         confirmation.


                                                                         Conclusions
                                                                             Considering the current interest of Shp2 inhibitors in tumor-
                                                                         targeted therapies,16 we conclude that the drug NSC87877 mod-
                                                                         erately enhances platelet aggregation upon GPVI stimulation
                                                                         through the inhibition of Shp2. The drug hence may help to nor-
                                                                         malize platelet plug formation in hemostatic-impaired patients.




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Figure 3. Impaired collagen-related peptide-induced platelet
Ca2+ responses by inhibition of phosphoinositide 3-kinase β or
Btk, but not of Shp1/2. A-B) Samples of Fura-2-loaded platelets
(200×108/L) were pre-incubated with vehicle control (Veh.),
NSC87877 (NSC, 20 μM), TGX221 (TGX, 3 μM) or combina-
tion for 10 min, before adding to a 96-wells plate. After addition
of 1 mM CaCl2, the loaded platelets were stimulated with colla-
gen-related peptide (CRP, 10 μg/mL), while 340/380 nm fluo-
rescence ratio changes were recorded per well; A)                        Figure 4. Additional effects Shp1/2 and Btk inhibition on col-
Representative traces of calibrated nM changes in [Ca2+]i per            lagen-related peptide-induced platelet activation markers by
condition; B) Effects of NSC87877 and TGX221 on integrated               flow cytometry. Washed platelets (50×109/L) were incubated for
[Ca2+]i rises (area-under-curve over 10 min). Data are normal-           10 min with vehicle control (Veh.), NSC87877 (NSC, 20 μM),
ized to the control condition per experiment; C-D) Fura-2-               ibrutinib (Ibru., 1 μM) or combination, and subsequently acti-
loaded platelets were pre-incubated with vehicle control, NSC            vated with collagen-related peptide (0.1-5.0 μg/mL). A, B) Ex-
(20 μM), ibrutinib (Ibru., 1 μM) or combination for 10 min.              tent of integrin αIIbβ3 activation and P-selectin expression after
Stimulation of the loaded platelets was again with 1 mM CaCl2            10 min, as determined by flow cytometry. Shown are represen-
and 10 μg/mL CRP; C) Representative nM [Ca2+]i traces per con-           tative heatmaps of inhibitor effects, assessed as percentage re-
dition; D) Effects of NSC87877 and ibrutinib on integrated               sponses of positive platelets, with values indicated. Color code
[Ca2+]i rises (area-under-curve over 10 min). Data are normal-           gives scaled values between 0% (blue) to 100% (red) in com-
ized to the control condition per experiment; B, D) Mean values          parison to vehicle control per agonist dose. Means of duplicate
± SD (n=3 donors); *p<0.05, **p<0.005, ***p<0.001,                       runs per blood sample for 3 donors; *p<0.05 vs. vehicle (paired
****p<0.0001 vs. vehicle (paired Student’s t-test).                      Student’s t-test).


                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:93
      Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2         45



                                                                           Shp1 in platelet activation by the collagen receptor glycopro-
References                                                                 tein VI. J Biol Chem 2000;275:28526-31.
                                                                     14.   Ma P, Cierniewska A, Signarvic R, et al. A newly identified
 1. Rayes J, Watson SP, Nieswandt B. Functional significance of
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