TY - JOUR
T1 - Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear
AU - Jooss, Natalie J.
AU - Smith, Christopher W.
AU - Pike, Jeremy A.
AU - Farndale, Richard W.
AU - Henskens, Yvonne M.C.
AU - Watson, Steve P.
AU - Heemskerk, Johan W.M.
AU - Poulter, Natalie S.
N1 - Funding Information:
Funding information European Union’s Horizon 2020 research and innovation program; Marie Sklodowska-Curie Grant Number: 766118.
Funding Information:
Funding informationEuropean Union's Horizon 2020 research and innovation program; Marie Sklodowska-Curie Grant Number: 766118.N.J.J. was funded by a European Union's Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant (766118). N.J.J. is registered as a joint PhD student at Maastricht University (the Netherlands) and the University of Birmingham (United Kingdom). S.P.W. is a British Heart Foundation Professor (CH 03/003). The authors thank Barry Coller for providing the a2ß1 antibody 6F1, Arandeep Dosanjh for labeling Nb28, and Dr Samantha Montague and Prof Neil Morgan for helpful discussions. N.J.J. designed and performed the experiments, analyzed data, prepared figures, and wrote the manuscript. C.W.S. performed the experiments and wrote the manuscript. J.A.P. developed the clustering analysis pipeline. R.W.F. supplied collagen-like peptides. Y.H. J.W.M.H. and S.P.W. acquired funding and supervised the study. N.S.P. designed the experiments, analyzed data, provided supervision and funding, and wrote the manuscript. All authors have read and approved the final paper. S.P.W. and N.S.P. have a patent for the anti-GPVI nanobodies (WO2022/136457). R.W.F. is the Chief Scientific Officer of CambCol Laboratories Ltd. The other authors have no competing interests to disclose.
Funding Information:
N.J.J. was funded by a European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant (766118). N.J.J. is registered as a joint PhD student at Maastricht University (the Netherlands) and the University of Birmingham (United Kingdom) . S.P.W. is a British Heart Foundation Professor (CH 03/003). The authors thank Barry Coller for providing the a2ß1 antibody 6F1, Arandeep Dosanjh for labeling Nb28, and Dr Samantha Montague and Prof Neil Morgan for helpful discussions.
Publisher Copyright:
© 2023 The Author(s)
PY - 2022/8
Y1 - 2022/8
N2 - Background: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. Objectives: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. Methods: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. Results: Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. Conclusion: Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.
AB - Background: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. Objectives: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. Methods: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. Results: Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. Conclusion: Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.
KW - glycoprotein VI
KW - nanobody
KW - platelet activation
KW - receptor clustering
KW - thrombus formation
U2 - 10.1016/j.jtha.2023.04.028
DO - 10.1016/j.jtha.2023.04.028
M3 - Article
C2 - 37150294
SN - 1538-7933
VL - 21
SP - 2260
EP - 2267
JO - Journal of Thrombosis and Haemostasis
JF - Journal of Thrombosis and Haemostasis
IS - 8
ER -