10.4081/btvb.2023.51
Adenosine diphosphate (ADP) receptors and protein-kinase C (PKC) isoforms play different role in platelet activity. In the present study, whole blood platelet adhesion at 200 - 1800 s-1 shear rates was investigated by Impact-R system, measuring percent of surface coverage (SC) by platelets. Gradual heightened shear rate par-alleled increase of platelet adhesion. At relatively low shear (200 and 1000 s-1) blockade of neither P2Y1 receptor nor P2Y12 receptor (by A2P5P and 2MeSAMP, respectively) affected SC. At high shear rate (1800 s-1) reduction of SC was observed by 2MeSAMP. Treatment of blood with PKCδ inhibitor (rottlerin) but not PKCα,β inhibitor (Gö6976) diminished platelet adhe-sion. Among all the agents, only combination of 2MeSAMP and rottlerin used at subthreshold concentrations was able to inhibit platelet adhesion under high shear condition. We suggest that platelet agonist-induced P2Y12 and PKCδ signaling essentially stimulates platelet adhesion under flow condition, the important initiating step of thrombin formation.
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2785-5309
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en
Yulia Einav
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Bleeding, Thrombosis and Vascular Biology
Platelet adhesion; ADP-receptors; PKC isoforms.
Interaction between adenosine diphosphate receptors and protein-kinase C isoforms in platelet adhesion under flow condition 
Text.Serial.Journal
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Brief Reports
2023-02-16
2022-08-24
2023-01-27
2023-03-21
Bleed Thromb Vascul Biol
Yulia Einav
2023/02/16
10.4081/btvb.2023.51
Faculty of Engineering, Holon Institute of Technology, Holon
2785-5309
1
Platelet adhesion; ADP-receptors; PKC isoforms.
en
Yacoub D, Theoret JF, Villneuve L, et al. Essential role of pro-tein kinase C delta in platelet signaling, alpha IIB beta 3 acti-vation, and thromboxane A2 release. J Biol Chem 2006;281: 30024-35.
Interaction between adenosine diphosphate receptors and protein-kinase C isoforms in platelet adhesion under flow condition 
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2
                                                                                                                  BRIEF REPORT

Interaction between adenosine diphosphate receptors and protein-kinase C isoforms
in platelet adhesion under flow condition
Boris Shenkman,1 Ivan Budnik,2 Yulia Einav3
1
  National Hemophilia Center, Sheba Medical Center, Ramat Gan, Israel; 2Department of Pathophysiology, Sechenov First Moscow
State Medical University, Moscow, Russian Federation; 3Faculty of Engineering, Holon Institute of Technology, Holon, Israel



ABSTRACT
     Adenosine diphosphate (ADP) receptors and protein-kinase C (PKC) isoforms play different role in platelet activity. In the present
study, whole blood platelet adhesion at 200 - 1800 s-1 shear rates was investigated by Impact-R system, measuring percent of surface
                                                                        coverage (SC) by platelets. Gradual heightened shear rate par-
                                                                        alleled increase of platelet adhesion. At relatively low shear (200
                                                                        and 1000 s-1) blockade of neither P2Y1 receptor nor P2Y12 re-
   Correspondence: Yulia Einav, Faculty of Engineering, Holon In-       ceptor  (by A2P5P and 2MeSAMP, respectively) affected SC. At
   stitute of Technology, Holon, Israel.                                high shear rate (1800 s-1) reduction of SC was observed by




                                                                                              ly
   Tel./Fax: +972.35026769.                                             2MeSAMP. Treatment of blood with PKCδ inhibitor (rottlerin)
   E-mail: yulia_e@hit.ac.il                                            but not PKCα,β inhibitor (Gö6976) diminished platelet adhe-




                                                                                      on
                                                                        sion. Among all the agents, only combination of 2MeSAMP and
   Citation: Shenkman B, Budnik I, Einav Y. Interaction between         rottlerin used at subthreshold concentrations was able to inhibit
   adenosine diphosphate receptors and protein-kinase C isoforms in     platelet adhesion under high shear condition. We suggest that




                                                                                 e
   platelet adhesion under flow condition. Bleeding, Thrombosis, and
                                                                        platelet agonist-induced P2Y12 and PKCδ signaling essentially
   Vascular Biology 2023;2:51.

   Key words: Platelet adhesion; ADP-receptors; PKC isoforms.
                                                                            us
                                                                        stimulates platelet adhesion under flow condition, the important
                                                                        initiating step of thrombin formation.
                                                                            al
  Contributions: BS, YE, conceptualization and writing the original
  draft; IB, study design; YE, IB, review and editing; BS, IB, per-
                                                         ci

  formed experiments; BS, IB, YE, data interpretation; IB, statistical       INTRODUCTION
  analysis and preparation of figures; YE, supervision. All authors
                                                       er


  have read and agreed to the published version of the manuscript.               Adenosine diphosphate (ADP) activates human
                                                                             platelets via concomitant engagement of G-protein-coupled
                                                      m



  Conflict of interest: The authors declare no potential conflict of in-     P2Y1 and P2Y12 receptors.1,2 The P2Y1 receptor triggers the
  terest.
                                                                             mobilization of calcium from internal stores, which results
                                          om




  Ethical approval: The study has been approved by the local Ethics          in platelet shape change and weak, transient aggregation in
  Committee of the Sheba Medical Center, Israel, and each volunteer          response to ADP.3 The P2Y12 receptor plays a central role
  signed a written informed consent in accordance with the Decla-
                                    -c




  ration of Helsinki.
                                                                             in platelet activation, agonist-induced dense granule release
                                                                             and pro-coagulant activity.4 Accordingly, P2Y12 receptor
                           on




  Informed consent: Informed consent was obtained from all subjects          antagonists were reported to be clinically effective in the
  involved in the study.                                                     prevention of myocardial infarction, ischemic stroke, and
                     N




  Availability of data and material: Data and materials are available        vascular death,5,6
  by the authors.                                                                Downstream of these receptors activation, various sig-
                                                                             nal transduction pathways are involved, among them an
  Received for publication: 24 August 2022.
  Accepted for publication: 18 January 2023.
                                                                             important role play protein kinases C (PKC). PKC isoforms
                                                                             are divided to classic and novel groups.7 Human platelets
  Publisher’s note: All claims expressed in this article are solely those    possess both classic (PKCα, PKCβ) and novel PKCδ iso-
  of the authors and do not necessarily represent those of their affili-     forms.8 PKC isoforms play different roles in ADP signal-
  ated organizations, or those of the publisher, the editors and the re-
  viewers. Any product that may be evaluated in this article or claim
                                                                             ing.9,10 The role of PKC isoforms in ADP-induced platelet
  that may be made by its manufacturer is not guaranteed or endorsed         function and vice versa is not fully understood.
  by the publisher.                                                              Many studies regarding platelet agonists and inhibitors
                                                                             were performed with washed platelets or platelet-rich
  ©
   Copyright: the Author(s), 2023
  Licensee PAGEPress, Italy                                                  plasma (PRP) under static conditions. The known limita-
  Bleeding, Thrombosis and Vascular Biology 2023; 2:51                       tions of these methods can be overcome by using whole
  doi:10.4081/btvb.2023.51                                                   blood and engagement of flow.
                                                                                 In the present study, we investigated the interaction be-
  This work is licensed under a Creative Commons Attribution-
  NonCommercial 4.0 International License (CC BY-NC 4.0).                    tween ADP receptors and different PKC isoforms in
                                                                             platelet deposition under flow conditions.

                                              Bleeding, Thrombosis and Vascular Biology 2023; 2:51
16                                                        B. Shenkman et al.




                                                                      thereby serve as platform for subsequent platelet adhesion
MATERIALS AND METHODS                                                 onto polystyrene.17 Reconstituted blood samples (130 ml)
Study population and blood preparation                                were placed into polystyrene wells for two min and sub-
                                                                      jected to flow at defined shear rates. The wells were then
    Our study included 46 healthy volunteers who had not              thoroughly washed, stained with May-Grünwald stain, and
taken medications known to affect platelet function for at least      analyzed by the Impact-R image analysis system.
10 days before blood sampling. Peripheral vein blood was
collected in polypropylene tubes using a 21G butterfly needle         Statistical analysis
and a vacutainer system. The first 2 ml of blood were dis-
carded, and the second portion of 4.5 ml blood was drawn                   Statistical analysis was performed with IBM SPSS Sta-
into tubes containing 3.2% sodium citrate in an anticoagu-            tistics (version 23.0; IBM Corp., USA). The data of SC by
lant/blood ratio of 1:9. PRP was separated from pack cells            adhered platelets were presented as median and inter-quar-
following centrifugation of native blood for 10 min at 160g.          tile range (IQR). Multiple group comparisons were per-
The following ADP receptor antagonists were used: A2P5P               formed by Kruskal–Wallis ANOVA followed by post hoc
(20-200 μM) against P2Y1 and 2MeSAMP (20-160 μM)                      Dunn’s Q test. Two-tailed P values of less than 0.05 were
against P2Y12.3,11 PBS served as a vehicle in experiments with        considered statistically significant.




                                                                                               ly
ADP inhibitors. The following PKC inhibitors were used: 0.2-
1 μM Gö6976, 1-5 μM rottlerin and 2-10 μM D-erythro-




                                                                                       on
sphingosine against PKCα,β, PKCδ, and pan-PKC,                        RESULTS
respectively.12-15 DMSO was the vehicle for PKC isoform an-
                                                                      Dose-dependent effect of A2P5P and 2MeSAMP
tagonists, as they do not dissolve in PBS. PRP samples were



                                                                                  e
                                                                      on platelet adhesion
pre-incubated with all inhibitors for 10 min. Whole blood re-
constitution was performed adding PRP to pack cells in a ratio
equal to their volumes following blood centrifugation.
                                                                           us
                                                                          Preliminary experiments in this study showed that
                                                                      platelet adhesion intensity was just the same using native
                                                                     al
                                                                      and reconstituted blood, suggesting that even if red blood
Impact-R test                                                         cells release some amount of ADP during blood centrifu-
                                                        ci

                                                                      gation and reconstitution of whole blood, this does not af-
    This method is based on the use of polystyrene surface
                                                      er


for platelet deposition under shear stress (DiaMed, Cressier,         fect platelet adhesion. Platelet adhesion at shear rate of
Switzerland).16 Recently, it has been shown that fibrinogen           1800 s−1 was chosen according to previously found as op-
                                                     m




and von Willebrand factor are precipitated from blood and             timal reflecting arterial blood flow in our model.15 Incu-
                                                                      bation of platelet-rich plasma (PRP) with A2P5P (P2Y1
                                            om




                                                                      antagonist) at concentrations from 20 mM up to 200 mM
                                                                      did not change the rate of platelet deposition in reconsti-
                                      -c




                                                                      tuted blood (Figure 1). In contrast, 2MeSAMP signifi-
                                                                      cantly decreased SC compared to blood without inhibitors
                               on




                                                                      [from 11.5% (10.0-13.8%) to 8.0 (7.9-8.7%, P<0.5) at 40
                                                                      mM and 7.2% (6.8-7.7%, P<0.01) at 80 mM 2MeSAMP].
                          N




                                                                      This effect did not change at 160 mM compared to 80
                                                                      mM. A2P5P at 100 mM and 2MeSAMP at 80 mM were
                                                                      chosen for subsequent experiments.

                                                                      Effect of A2P5P and 2MeSAMP on platelet adhesion
                                                                      at different shear rate
                                                                          At static conditions limited number of platelets adhered
                                                                      (Figure 2). Gradual increase of platelet adhesion was ob-
                                                                      served at shear rates of 200, 1000 and 1800 s−1. However,
Figure 1. Dose-depending effect of A2P5P and 2MeSAMP on               no inhibitor was able to affect platelet deposition at low
platelet deposition under flow conditions PRP was pre-incubated       (200 s-1) and intermediate (1000 s-1) shear rate. In contrast,
with PBS (control, white columns) and increasing                      substantial reduction of platelet deposition was observed
concentrations of A2P5P (solid lines) and 2MeSAMP (dashed             by 2MeSAMP at1800 s−1 (SC 7.2% (6.8-7.7%). Despite the
lines) for 10 min, then mixed with autologous packed cells to         lack of inhibitory effect of A2P5P alone, the combination
reconstitute whole blood and subjected to Impact-R test for 2         of both drugs had an additive effect on platelet adhesion,
min at 1800 s–1 shear rate. The median surface coverage (SC)          further decreasing SC to 5.5% (5.0-6.0% compared to
(25th-75th percentile) is presented. *P<0.01 vs. control (n=8).
                                                                      2MeSAMP alone, P<0.01).

                                       Bleeding, Thrombosis and Vascular Biology 2023; 2:51
    Interaction between adenosine diphosphate receptors and protein-kinase C isoforms in platelet adhesion under flow condition 17




Role of PKC isotypes in platelet adhesion
    At high shear (1800-1) among the PKC inhibitors, D-
erythro-sphingosine and rottlerin reduced SC from 11.0%
(10.0-13.0%) in control to 4.2% (3.7-7.1%, P<0.001) and
7.3% (5.7-7.6%, P<0.01), respectively, whereas Gö6976
did not affect platelet adhesion (Figure 3A). In the follow-
ing experiments, interaction between the ADP and PKC an-
tagonists was assayed. For this purpose, the agents were
used at subthreshold concentrations, just at 20% of the con-
centrations used above. As expected, the inhibitors used
separately did not affect platelet adhesion (Figure 3B). In
contrast, the combination of 2MeSAMP with D-erythro-
sphingosine reduced SC to 6.0% vs. D-erythro-sphingosine
alone (9.6%, P<0.01), and that of 2MeSAMP with rottlerin
reduced SC to 7.4%, vs. rottlerin alone (10.2%, P<0.05).
Platelet adhesion was not changed by concomitant blood




                                                                                       ly
treatment with 2MeSAMP and Gö6976.




                                                                               on
                                                                     Figure 2. Shear rate-dependent effect of A2P5P and 2MeSAMP
                                                                     on platelet adhesion PRP was pre-incubated with PBS (control,
                                                                     white columns), 100 mM A2P5P, 80 mM 2MeSAMP or both
DISCUSSION AND CONCLUSIONS




                                                                         e
                                                                     inhibitors in combination for 10 min, then mixed with
                                                                     autologous packed cells to reconstitute whole blood and
    The aim of this study was to evaluate the role of P2Y1
and P2Y12 ADP receptors as well as PKCαβ1 and PKCδ iso-
forms in platelet adhesion under shear flow conditions. The
                                                                   ussubjected to the Impact-R test at the indicated shear rates for 2
                                                                     min. The median surface coverage (SC) (25th-75th percentile) is
                                                                     presented. *P<0.01, **P<0.001 represents difference vs. PBS-
                                                            al
study employed the Impact-R device measuring whole
                                                                     treated samples (n=8).
blood platelet adhesion to a polystyrene surface, where fib-
                                               ci
                                             er
                                            m
                                   om
                             -c
                      on
                 N




Figure 3. Role of PKC isotypes in platelet adhesion under flow. A) Modulation of platelet deposition by inhibitors of PKC isotypes.
PRP samples were pre-incubated for 10 min with a vehicle (0.33% DMSO), 10mM D-erythro-sphingosine (D-Sphing.), 5 μM rottlerin
and 1 μM Gö6976.14-18 Whole blood reconstitution was performed, and the samples were subjected to the Impact-R test for 2 min at
1800 s−1 shear rate. The median surface coverage (SC) is presented. *P<0.05 and **P<0.01 vs. control (n=10). B) Combined effect of
subthreshold concentrations (20% of those used above) of P2Y12 and of PKC isotype antagonists on platelet deposition. PRP was pre-
incubated for 10 min with 16 μM 2MeSAMP in the presence or absence of 2 μM D-erythro-sphingosine, 1μM rottlerin, and 0.2 μM
Gö6976. Whole blood reconstitution was performed, and samples were subjected to the Impact-R test for 2 min at 1800 s-1 shear rate.
The SC median (25th-75th percentile) is presented. *P<0.05, **P<0.01 vs. control, #P<0.01 and ##P<0.001 vs. 2MeSAMP (n=10).


                                      Bleeding, Thrombosis and Vascular Biology 2023; 2:51
18                                                          B. Shenkman et al.




rinogen and von Willebrand factor were bound to poly-                         active antiplatelet agents with dual specificity for platelet
styrene preceeding platelet adhesion.17 We found that P2Y1                    P2Y1 and P2Y12 adenosine diphosphate receptors. Europ J
                                                                              Med Chem 2016;107:204-8.
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failed to affect platelet deposition at both static and rela-                 sary for adenosine 5′-diphosphate-induced platelet aggrega-
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at high shear rate (1800 s−1) inhibition of P2Y12 but not                4.   Kunapuli SP, Dorsam RT, Kim S, et al. Platelet purinergic re-
P2Y1 was followed by reduction of platelet adhesion. De-                      ceptors. Curr Opin Pharmacol 2003;3:175–80.
spite the lack of the effect of A2P5P alone, synergistic in-             5.   Oliphant CS, Doby J B, Blade CL, et al. Emerging P2Y12 re-
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                                                                                                     ly
inhibited platelet adhesion onto von Willebrand factor-col-              9.   Unsworth AJ, Smith H, Gissen P, et al. Submaximal inhibition




                                                                                             on
lagen surface under shear rates of 750 and 1500 s−1.19 We                     of protein kinase C restores ADP-induced dense granule se-
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have shown that blockade of P2Y12 was also more effective
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than P2Y1. The applied concentrations of ADP antagonists                10.   Mundell SJ, Jones ML, Hardy AR, et al. Distinct roles for pro-




                                                                                       e
in our study were higher than in other studies. However, it                   tein kinase C isoforms in regulating platelet purinergic recep-
must be taken into consideration that the effective concen-
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                                                                               us
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                                                          ci

blood, and on the extent of shear rate.                                 12.   Koivunen J, Aaltonen V, Koskela S, et al. Protein kinase C
     It is well known that protein kinase C isoforms play an                  alpha/beta inhibitor Go6976 promotes formation of cell junc-
                                                        er


important role in platelet granule secretion, activation, ag-                 tions and inhibitors invasion of urinary bladder carcinoma
gregation, and procoagulant activity. In the present study                    cells. Cancer Res 2005;64:5693-701.
                                                       m




                                                                        13.   Bong KA, Se KJ, Hee SK, et al. Rottlerin, a specific inhibitor
we used inhibitors of PKCαβ1 (Gö6976), PKCδ (rottlerin),                      of protein kinase C-delta, impedes barrier repair response by
                                             om




as well as the pan-PKC inhibitor (D-erythro-sphingosine)                      increasing intracellular free calcium. J Invest Dermatol
to explore their role in platelet adhesion under flow condi-                  2006;126:1348-55.
tions. We showed that PKCδ, but not PKCαβ is responsible                14.   Lee TH, Chen JL, Liu PS, et al. Rottlerin, a natural polyphenol
                                       -c




for the reduction of platelet deposition. This is consistent                  compound, inhibits upregulation of matrix metalloproteinase-
                                                                              9 and brain astrocytic migration by reducing PKC-δ-depen-
with the data that PKCδ signaling is required for platelet
                                on




                                                                              dent ROS signal. J Neuroinflammation 2020;17:177.
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PKC isoforms regulate P2Y12 function in experiments in                        ing protein kinase C inhibitor: D-erythro-sphingosine. Arch
                           N




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                                        Bleeding, Thrombosis and Vascular Biology 2023; 2:51
