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Reducing state attenuates ectodomain shedding of GPVI while restoring adhesion capacities of stored platelets: evidence addressing the controversy around the effects of redox condition on thrombosis

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Abstract

Thrombosis involves different stages including platelet adhesion to the site of injury, aggregatory events governed by integrin activation, pro-inflammatory responses recruiting leukocytes and finally, pro-coagulant activity which results in fibrin generation and clot formation. As important signaling agents, reactive oxygen species (ROS) reduce thrombus volume and growth, however given such a multistage mechanism, it is not well-elucidated how ROS inhibition modulates thrombosis. PRP-platelet concentrates (PCs) were either treated with ROS-reducing agents (1 mM NAC or 30 μM NOX inhibitor, VAS2870) or kept untreated during storage. Shedding and expression of platelet adhesion receptors in presence of inhibitors, agonists and CCCP (as controls) were analyzed by flow cytometery and western blot respectively. Besides above parameters, platelet adhesion to collagen in stored platelets was examined in presence of ROS inhibitors using fluorescence-microscopy. Highest levels of adhesion receptors shedding were achieved by ionophore and CCCP while collagen induces much more GPVI shedding than that of GPIbα. ROS inhibition reduced receptors shedding from day 3 of storage while enhanced their expressions. ROS inhibition not only did not reduce platelet adhesion capacity but it also enhanced platelets adhesion (in presence of NAC) or spreading (in presence of VAS2870) in 5 days-stored PCs. While reducing state significantly inhibits platelet aggregation and thrombus growth, our results indicated that as a first stage of thrombosis, platelet adhesion is resistance to such inhibitory effects. These findings highlight the fact that integrin-dependent platelet activation is much more vulnerable to the inhibition of ROS generation than GPVI-dependent platelet adhesion. Presumably, inhibition of platelet activating signals by ROS inhibitors preserves platelet adhesiveness to collagen due to lessening GPVI shedding.

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Abbreviations

ADAM:

A disintegrin and metalloproteinase

CaM:

Calmodulin

Ca2 +:

Calcium

Cys-residue:

Cysteine residue

DAG:

Diacylglycerol

DTS:

Dense tubular system

FcRγ:

Fc receptor gamma

GPIbα:

Glycoprotein Ibα

GPV:

Glycoprotein V

GPVI:

Glycoprotein VI

IP3:

Inositol triphosphate

ITAM:

Immunoreceptor Tyrosine-based Activation Motif

NAC:

N-acetylcysteine

NOX:

NADPH oxidase

PC:

Platelet concentrate

PI3K:

Phosphatidylinositol 3-kinase

PIP2:

Phosphatidylinositol 4, 5-bisphosphate

PKC:

Protein kinases C

PLCγ2:

Phospholipase C gamma 2

PTP:

Protein tyrosine phosphatase

P38-MAPK:

P38-mitogen-activated protein kinase

P47phox:

P47 phagocyte oxidase

ROS:

Reactive Oxygen Species

SFK:

Sarcoma family kinase

SyK:

Spleen tyrosine kinase

TRAF:

Tumor necrosis factor receptor associated factor

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Acknowledegment

This work was part of Dr. Ghasemzadeh’s approved projects (No.1394-01-33-1862 and No.1396-06-33-2099) supported by High Institute for Research and Education in Transfusion Medicine. Parts of this work were presented as thesis programs for Master students Zahra roudsari and Amin Soluki. The authors declare no conflict of interests.

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Authors and Affiliations

  1. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, 14665-1157, Tehran, Iran

    Ehteramolsadat Hosseini, Amin Solouki, Zahra Oushyani Roudsari & Mehran Ghasemzadeh

  2. Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Science, Tehran, Iran

    Faranak Kargar

Authors
  1. Ehteramolsadat Hosseini
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  2. Amin Solouki
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  3. Zahra Oushyani Roudsari
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  4. Faranak Kargar
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  5. Mehran Ghasemzadeh
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Contributions

MG provided conceptual input, supervised and designed the study, did the experiments, analyzed the data and wrote the paper. EH, provided conceptual input, co-supervised the study, did the experiments and co-wrote the paper. AS did some experiments and helped with paper preparation. ZR did some experiments. FK critically reviewed the paper.

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Correspondence to Mehran Ghasemzadeh.

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Hosseini, E., Solouki, A., Roudsari, Z.O. et al. Reducing state attenuates ectodomain shedding of GPVI while restoring adhesion capacities of stored platelets: evidence addressing the controversy around the effects of redox condition on thrombosis. J Thromb Thrombolysis 50, 123–134 (2020). https://doi.org/10.1007/s11239-020-02137-0

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