Low-dose Btk inhibitors selectively block platelet activation by CLEC-2

Pip Nicolson; Sophie H Nock; Joshua Hinds; Lourdes Garcia Quintanilla; Christopher Smith; Joana Campos; Alexander Brill; Jeremy Pike; Abdullah Khan; Natalie Poulter; Deirdre Kavanagh; Stephanie Watson; Callum N Watson; Hayley Clifford; Aarnoud Huissoon; Alice Pollitt; Johannes A Eble; Dickens Pratt; Steve Watson; Craig Hughes

doi:10.3324/haematol.2019.218545

Low-dose Btk inhibitors selectively block platelet activation by CLEC-2

Pip Nicolson, Sophie H Nock, Joshua Hinds, Lourdes Garcia Quintanilla, Christopher Smith, Joana Campos, Alexander Brill, Jeremy Pike, Abdullah Khan, Natalie Poulter, Deirdre Kavanagh, Stephanie Watson, Callum N Watson, Hayley Clifford, Aarnoud Huissoon, Alice Pollitt, Johannes A Eble, Dickens Pratt, Steve Watson, Craig Hughes

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Abstract

Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

Original language	English
Pages (from-to)	208-219
Number of pages	12
Journal	Haematologica
Volume	106
Issue number	1
DOIs	https://doi.org/10.3324/haematol.2019.218545
Publication status	Published - 16 Jan 2021

Bibliographical note

Funding Information: This work was supported by British Heart Foundation (BHF) Programme grant (RG/13/18/30563), a BHF Clinical Fellowship to PLRN (FS/17/20/32738), a BHF Senior Basic Science Research Fellowship to AB (FS/19/30/34173), an AMS springboard grant to AYP (SBF002\1099) and the University of Birmingham’s Institute of Translation Medicine and Institute of Cardiovascular Sciences; SPW holds a BHF Chair (CH03/003). JAE is supported by the Deutsche Forschungsgemeinschaft (DFG grant: Eb177/13-1). Btk deficient DT40 cells, plasmid constructs and rabbit anti-Btk antibody were a kind gift from Dr Mike Tomlinson (University of Birmingham, UK). PLR Nicolson has received research grants from Rigel Pharmaceuticals, Novartis Pharmaceuticals and materials from Principia Biopharma.

Keywords

platelets
disorders of platelet function
venous thrombosis
molecular pharmacology
Animals
Platelet Membrane Glycoproteins
Platelet Activation
Humans
Lectins, C-Type
Blood Platelets
Mice
Protein Kinase Inhibitors/pharmacology

ASJC Scopus subject areas

Hematology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3324/haematol.2019.218545Licence: Other (please provide link to licence statement

NicholsonP2020Low
Phillip L.R. Nicolson, Sophie H. Nock, Joshua Hinds, Lourdes Garcia-Quintanilla, Christopher W. Smith, Joana Campos, Alexander Brill, Jeremy A. Pike, Abdullah O. Khan, Natalie S. Poulter, Diedre M. Kavanagh, Stephanie Watson, Callum N. Watson, Hayley Clifford, Aarnoud P. Huissoon, Alice Y. Pollitt, Johannes A. Eble, Guy Pratt, Steve P. Watson, and Craig E. Hughes. Low dose Btk inhibitors selectively block platelet activation by CLEC-2 Haematologica. 2020; 105:xxx doi:10.3324/haematol.2019.218545
Final published version, 1.64 MBLicence: Creative Commons: Attribution-NonCommercial (CC BY-NC)

Cite this

Nicolson, P., Nock, S. H., Hinds, J., Garcia Quintanilla, L., Smith, C., Campos, J., Brill, A., Pike, J., Khan, A., Poulter, N., Kavanagh, D., Watson, S., Watson, C. N., Clifford, H., Huissoon, A., Pollitt, A., Eble, J. A., Pratt, D., Watson, S., & Hughes, C. (2021). Low-dose Btk inhibitors selectively block platelet activation by CLEC-2. Haematologica, 106(1), 208-219. https://doi.org/10.3324/haematol.2019.218545

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title = "Low-dose Btk inhibitors selectively block platelet activation by CLEC-2",

abstract = "Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.",

keywords = "platelets, disorders of platelet function, venous thrombosis, molecular pharmacology, Animals, Platelet Membrane Glycoproteins, Platelet Activation, Humans, Lectins, C-Type, Blood Platelets, Mice, Protein Kinase Inhibitors/pharmacology",

author = "Pip Nicolson and Nock, {Sophie H} and Joshua Hinds and {Garcia Quintanilla}, Lourdes and Christopher Smith and Joana Campos and Alexander Brill and Jeremy Pike and Abdullah Khan and Natalie Poulter and Deirdre Kavanagh and Stephanie Watson and Watson, {Callum N} and Hayley Clifford and Aarnoud Huissoon and Alice Pollitt and Eble, {Johannes A} and Dickens Pratt and Steve Watson and Craig Hughes",

note = "Funding Information: This work was supported by British Heart Foundation (BHF) Programme grant (RG/13/18/30563), a BHF Clinical Fellowship to PLRN (FS/17/20/32738), a BHF Senior Basic Science Research Fellowship to AB (FS/19/30/34173), an AMS springboard grant to AYP (SBF002\1099) and the University of Birmingham{\textquoteright}s Institute of Translation Medicine and Institute of Cardiovascular Sciences; SPW holds a BHF Chair (CH03/003). JAE is supported by the Deutsche Forschungsgemeinschaft (DFG grant: Eb177/13-1). Btk deficient DT40 cells, plasmid constructs and rabbit anti-Btk antibody were a kind gift from Dr Mike Tomlinson (University of Birmingham, UK). PLR Nicolson has received research grants from Rigel Pharmaceuticals, Novartis Pharmaceuticals and materials from Principia Biopharma.",

year = "2021",

month = jan,

day = "16",

doi = "10.3324/haematol.2019.218545",

language = "English",

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Nicolson, P, Nock, SH, Hinds, J, Garcia Quintanilla, L, Smith, C, Campos, J, Brill, A , Pike, J, Khan, A, Poulter, N, Kavanagh, D, Watson, S, Watson, CN, Clifford, H, Huissoon, A, Pollitt, A, Eble, JA, Pratt, D, Watson, S & Hughes, C 2021, 'Low-dose Btk inhibitors selectively block platelet activation by CLEC-2', Haematologica, vol. 106, no. 1, pp. 208-219. https://doi.org/10.3324/haematol.2019.218545

TY - JOUR

T1 - Low-dose Btk inhibitors selectively block platelet activation by CLEC-2

AU - Nicolson, Pip

AU - Nock, Sophie H

AU - Hinds, Joshua

AU - Garcia Quintanilla, Lourdes

AU - Smith, Christopher

AU - Campos, Joana

AU - Brill, Alexander

AU - Pike, Jeremy

AU - Khan, Abdullah

AU - Poulter, Natalie

AU - Kavanagh, Deirdre

AU - Watson, Stephanie

AU - Watson, Callum N

AU - Clifford, Hayley

AU - Huissoon, Aarnoud

AU - Pollitt, Alice

AU - Eble, Johannes A

AU - Pratt, Dickens

AU - Watson, Steve

AU - Hughes, Craig

N1 - Funding Information: This work was supported by British Heart Foundation (BHF) Programme grant (RG/13/18/30563), a BHF Clinical Fellowship to PLRN (FS/17/20/32738), a BHF Senior Basic Science Research Fellowship to AB (FS/19/30/34173), an AMS springboard grant to AYP (SBF002\1099) and the University of Birmingham’s Institute of Translation Medicine and Institute of Cardiovascular Sciences; SPW holds a BHF Chair (CH03/003). JAE is supported by the Deutsche Forschungsgemeinschaft (DFG grant: Eb177/13-1). Btk deficient DT40 cells, plasmid constructs and rabbit anti-Btk antibody were a kind gift from Dr Mike Tomlinson (University of Birmingham, UK). PLR Nicolson has received research grants from Rigel Pharmaceuticals, Novartis Pharmaceuticals and materials from Principia Biopharma.

PY - 2021/1/16

Y1 - 2021/1/16

N2 - Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

AB - Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

KW - platelets

KW - disorders of platelet function

KW - venous thrombosis

KW - molecular pharmacology

KW - Animals

KW - Platelet Membrane Glycoproteins

KW - Platelet Activation

KW - Humans

KW - Lectins, C-Type

KW - Blood Platelets

KW - Mice

KW - Protein Kinase Inhibitors/pharmacology

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U2 - 10.3324/haematol.2019.218545

DO - 10.3324/haematol.2019.218545

M3 - Article

C2 - 31949019

SN - 0390-6078

VL - 106

SP - 208

EP - 219

JO - Haematologica

JF - Haematologica

IS - 1

ER -

Low-dose Btk inhibitors selectively block platelet activation by CLEC-2

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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