High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro

Rohini Sekar; Angelina Mimoun; Melissa Bou-Jaoudeh; Stéphane Loyau; Sandrine Delignat; Victoria Daventure; Perrine Bonilla; Aishwarya Sudam Bahle; Krishnan Venkataraman; Julie Rayes; Yacine Boulaftali; Martine Jandrot-Perrus; Valérie Proulle; Sébastien Lacroix-Desmazes

doi:10.1016/j.jtha.2024.01.021

High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro

Rohini Sekar, Angelina Mimoun, Melissa Bou-Jaoudeh, Stéphane Loyau, Sandrine Delignat, Victoria Daventure, Perrine Bonilla, Aishwarya Sudam Bahle, Krishnan Venkataraman, Julie Rayes, Yacine Boulaftali, Martine Jandrot-Perrus, Valérie Proulle^*, Sébastien Lacroix-Desmazes^*

^*Corresponding author for this work

Cardiovascular Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Background: The recruitment of activated FVIII at the surface of activated platelets is a key step towards the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidyl-serine (PS) and, possibly, to fibrin bound to αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance.

Objectives: To characterize the FVIII-platelet interaction and its potential modulation on platelet function.

Methods: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (western blot). The FVIII-GPVI interaction was investigated by ELISA, confocal microscopy and proximity ligation assay.

Results: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supra-physiological concentrations did not induce platelet activation but rather specifically inhibit collagen-induced platelet aggregation and altered GPVI-dependent phosphorylation. FVIII rid of its chaperon protein, von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface.

Conclusions: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent a yet uncharacterized negative feedback loop to control overt platelet activation. Whether local activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site vascular injury in vivo are compatible with such a function remains to be determined.

Original language	English
Journal	Journal of Thrombosis and Haemostasis
Early online date	5 Feb 2024
DOIs	https://doi.org/10.1016/j.jtha.2024.01.021
Publication status	E-pub ahead of print - 5 Feb 2024

Bibliographical note

Funding:
This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, Université de Paris, Assistance Publique des Hôpitaux de Paris and by grants from the Bayer Hemophilia Award Program (BHAP 2019), from the Indo-French Center for Promotion of Advanced Research (CEFIPRA, Reference No. IFC//7126/ Hemophilia) and from Agence National de la Recherche (ANR-18-CE17-0010-02-Exfiltrins and ANR-21-CE17-0043- Persia). The work at VIT Vellore was supported by the VIT International research fund (Ref No. VIN/2022-23/011 dated 9th Feb. 2023). RS, AM and MBJ were recipients of fellowships from CEFIPRA, Fondation de la Recherche Médicale (FRM, Paris), Agence National de la Recherche (ANR-18-CE17-0010-02) and Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation.

Keywords

Factor VIII
platelets
GPVI

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Sekar, R., Mimoun, A., Bou-Jaoudeh, M., Loyau, S., Delignat, S., Daventure, V., Bonilla, P., Bahle, A. S., Venkataraman, K., Rayes, J., Boulaftali, Y., Jandrot-Perrus, M., Proulle, V., & Lacroix-Desmazes, S. (2024). High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro. Journal of Thrombosis and Haemostasis. Advance online publication. https://doi.org/10.1016/j.jtha.2024.01.021

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title = "High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro",

abstract = "Background: The recruitment of activated FVIII at the surface of activated platelets is a key step towards the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidyl-serine (PS) and, possibly, to fibrin bound to αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. Objectives: To characterize the FVIII-platelet interaction and its potential modulation on platelet function. Methods: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (western blot). The FVIII-GPVI interaction was investigated by ELISA, confocal microscopy and proximity ligation assay. Results: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supra-physiological concentrations did not induce platelet activation but rather specifically inhibit collagen-induced platelet aggregation and altered GPVI-dependent phosphorylation. FVIII rid of its chaperon protein, von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. Conclusions: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent a yet uncharacterized negative feedback loop to control overt platelet activation. Whether local activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site vascular injury in vivo are compatible with such a function remains to be determined.",

keywords = "Factor VIII, platelets, GPVI",

author = "Rohini Sekar and Angelina Mimoun and Melissa Bou-Jaoudeh and St{\'e}phane Loyau and Sandrine Delignat and Victoria Daventure and Perrine Bonilla and Bahle, {Aishwarya Sudam} and Krishnan Venkataraman and Julie Rayes and Yacine Boulaftali and Martine Jandrot-Perrus and Val{\'e}rie Proulle and S{\'e}bastien Lacroix-Desmazes",

note = "Funding: This work was supported by Institut National de la Sant{\'e} et de la Recherche M{\'e}dicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Universit{\'e}, Universit{\'e} de Paris, Assistance Publique des H{\^o}pitaux de Paris and by grants from the Bayer Hemophilia Award Program (BHAP 2019), from the Indo-French Center for Promotion of Advanced Research (CEFIPRA, Reference No. IFC//7126/ Hemophilia) and from Agence National de la Recherche (ANR-18-CE17-0010-02-Exfiltrins and ANR-21-CE17-0043- Persia). The work at VIT Vellore was supported by the VIT International research fund (Ref No. VIN/2022-23/011 dated 9th Feb. 2023). RS, AM and MBJ were recipients of fellowships from CEFIPRA, Fondation de la Recherche M{\'e}dicale (FRM, Paris), Agence National de la Recherche (ANR-18-CE17-0010-02) and Minist{\`e}re de l{\textquoteright}Enseignement sup{\'e}rieur, de la Recherche et de l{\textquoteright}Innovation.",

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doi = "10.1016/j.jtha.2024.01.021",

language = "English",

journal = "Journal of Thrombosis and Haemostasis",

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TY - JOUR

T1 - High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro

AU - Sekar, Rohini

AU - Mimoun, Angelina

AU - Bou-Jaoudeh, Melissa

AU - Loyau, Stéphane

AU - Delignat, Sandrine

AU - Daventure, Victoria

AU - Bonilla, Perrine

AU - Bahle, Aishwarya Sudam

AU - Venkataraman, Krishnan

AU - Rayes, Julie

AU - Boulaftali, Yacine

AU - Jandrot-Perrus, Martine

AU - Proulle, Valérie

AU - Lacroix-Desmazes, Sébastien

N1 - Funding: This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, Université de Paris, Assistance Publique des Hôpitaux de Paris and by grants from the Bayer Hemophilia Award Program (BHAP 2019), from the Indo-French Center for Promotion of Advanced Research (CEFIPRA, Reference No. IFC//7126/ Hemophilia) and from Agence National de la Recherche (ANR-18-CE17-0010-02-Exfiltrins and ANR-21-CE17-0043- Persia). The work at VIT Vellore was supported by the VIT International research fund (Ref No. VIN/2022-23/011 dated 9th Feb. 2023). RS, AM and MBJ were recipients of fellowships from CEFIPRA, Fondation de la Recherche Médicale (FRM, Paris), Agence National de la Recherche (ANR-18-CE17-0010-02) and Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation.

PY - 2024/2/5

Y1 - 2024/2/5

N2 - Background: The recruitment of activated FVIII at the surface of activated platelets is a key step towards the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidyl-serine (PS) and, possibly, to fibrin bound to αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. Objectives: To characterize the FVIII-platelet interaction and its potential modulation on platelet function. Methods: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (western blot). The FVIII-GPVI interaction was investigated by ELISA, confocal microscopy and proximity ligation assay. Results: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supra-physiological concentrations did not induce platelet activation but rather specifically inhibit collagen-induced platelet aggregation and altered GPVI-dependent phosphorylation. FVIII rid of its chaperon protein, von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. Conclusions: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent a yet uncharacterized negative feedback loop to control overt platelet activation. Whether local activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site vascular injury in vivo are compatible with such a function remains to be determined.

AB - Background: The recruitment of activated FVIII at the surface of activated platelets is a key step towards the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidyl-serine (PS) and, possibly, to fibrin bound to αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. Objectives: To characterize the FVIII-platelet interaction and its potential modulation on platelet function. Methods: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (western blot). The FVIII-GPVI interaction was investigated by ELISA, confocal microscopy and proximity ligation assay. Results: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supra-physiological concentrations did not induce platelet activation but rather specifically inhibit collagen-induced platelet aggregation and altered GPVI-dependent phosphorylation. FVIII rid of its chaperon protein, von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. Conclusions: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent a yet uncharacterized negative feedback loop to control overt platelet activation. Whether local activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site vascular injury in vivo are compatible with such a function remains to be determined.

KW - Factor VIII

KW - platelets

KW - GPVI

U2 - 10.1016/j.jtha.2024.01.021

DO - 10.1016/j.jtha.2024.01.021

M3 - Article

SN - 1538-7933

JO - Journal of Thrombosis and Haemostasis

JF - Journal of Thrombosis and Haemostasis

ER -

High FVIII concentrations interfere with GPVI-mediated platelet activation in vitro

Abstract

Bibliographical note

Keywords

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