Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation

Shuchi Gupta; Christoph Konradt; Adam Corken; Jerry Ware; Bernhard Nieswandt; Jorge Di Paola; Mei Yu; Demin Wang; Marvin T. Nieman; Sidney W. Whiteheart; Lawrence F. Brass

doi:10.1073/pnas.2007642117

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation

Shuchi Gupta, Christoph Konradt, Adam Corken, Jerry Ware, Bernhard Nieswandt, Jorge Di Paola, Mei Yu, Demin Wang, Marvin T. Nieman, Sidney W. Whiteheart, Lawrence F. Brass

Molecular and Cellular Biochemistry

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

25 Scopus citations

Abstract

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

Original language	English
Pages (from-to)	24316-24325
Number of pages	10
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	117
Issue number	39
DOIs	https://doi.org/10.1073/pnas.2007642117
State	Published - Sep 29 2020

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing Bi-Karchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1−/− mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLCγ2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.)

Funding Information:
We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing BiKarchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1?/? mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLC?2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.)

Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.

Keywords

Dense granules
GPVI signaling
Homeostasis
Platelets
Vascular integrity

ASJC Scopus subject areas

General

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10.1073/pnas.2007642117

Cite this

Gupta, S., Konradt, C., Corken, A., Ware, J., Nieswandt, B., Di Paola, J., Yu, M., Wang, D., Nieman, M. T., Whiteheart, S. W., & Brass, L. F. (2020). Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation. Proceedings of the National Academy of Sciences of the United States of America, 117(39), 24316-24325. https://doi.org/10.1073/pnas.2007642117

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title = "Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation",

abstract = "Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.",

keywords = "Dense granules, GPVI signaling, Homeostasis, Platelets, Vascular integrity",

author = "Shuchi Gupta and Christoph Konradt and Adam Corken and Jerry Ware and Bernhard Nieswandt and {Di Paola}, Jorge and Mei Yu and Demin Wang and Nieman, {Marvin T.} and Whiteheart, {Sidney W.} and Brass, {Lawrence F.}",

note = "Funding Information: ACKNOWLEDGMENTS. We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing Bi-Karchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1−/− mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLCγ2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.) Funding Information: We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing BiKarchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1?/? mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLC?2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.) Publisher Copyright: {\textcopyright} 2020 National Academy of Sciences. All rights reserved.",

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doi = "10.1073/pnas.2007642117",

language = "English",

volume = "117",

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Gupta, S, Konradt, C, Corken, A, Ware, J, Nieswandt, B, Di Paola, J, Yu, M, Wang, D, Nieman, MT, Whiteheart, SW & Brass, LF 2020, 'Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation', Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 39, pp. 24316-24325. https://doi.org/10.1073/pnas.2007642117

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation. / Gupta, Shuchi; Konradt, Christoph; Corken, Adam et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, No. 39, 29.09.2020, p. 24316-24325.

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

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AU - Nieman, Marvin T.

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AU - Brass, Lawrence F.

N1 - Funding Information: ACKNOWLEDGMENTS. We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing Bi-Karchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1−/− mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLCγ2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.) Funding Information: We thank Gordon Ruthel from The Penn Vet Imaging Core (PVIC) for his technical assistance with the imaging studies. Imaging experiments were performed in the Penn Vet Imaging Core Facility (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). The flow cytometry experiments were performed at the Flow Cytometry and Cell Sorting Resource Laboratory (University of Pennsylvania). We thank Jing BiKarchin and Michael S. Marks (Department of Pathology and Laboratory Medicine, Department of Physiology, University of Pennsylvania) for providing BLOC1?/? mice. This work was supported by NIH, National Heart, Lung, and Blood Institute Grants P01 HL40387 (to L.F.B.) and P01 HL120846 (to L.F.B.), and by a University of Pennsylvania Research Foundation grant (to L.F.B.). Imaging experiments were carried out on instrumentation supported by NIH Grant S10 OD021633-01 (to the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). PLC?2 mutant mice were generated using NIH Grants AI079087 (to D.W.) and HL130724 (to D.W.) Publisher Copyright: © 2020 National Academy of Sciences. All rights reserved.

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N2 - Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

AB - Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

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Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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