Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

Claire S Whyte, Frauke Swieringa, Tom G Mastenbroek, Ausra S Lionikiene, Marcus D Lancé, Paola E J van der Meijden, Johan W M Heemskerk, Nicola J Mutch

Research output: Contribution to journalArticle

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Abstract

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation; with tPA-mediated lysis more efficient than uPA-mediated lysis. Fluorescently-labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative and bound plasminogen and fibrinogen in a protruding 'cap'. These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

Original languageEnglish
Pages (from-to)2568 - 2578
Number of pages11
JournalBlood
Volume125
Issue number16
Early online date23 Feb 2015
DOIs
Publication statusPublished - 16 Apr 2015

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Plasminogen
Phosphatidylserines
Platelets
Thrombosis
Blood Platelets
Fibrinolysis
Fibrin
Fibrinogen
tirofiban
Hirudins
Flow cytometry
Confocal microscopy
Fluorescence microscopy
Ports and harbors
Fluorescence Microscopy
Confocal Microscopy
Scaffolds
Amides
Thrombin
Flow Cytometry

Cite this

Whyte, C. S., Swieringa, F., Mastenbroek, T. G., Lionikiene, A. S., Lancé, M. D., van der Meijden, P. E. J., ... Mutch, N. J. (2015). Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. Blood, 125(16), 2568 - 2578. https://doi.org/10.1182/blood-2014-09-599480

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. / Whyte, Claire S; Swieringa, Frauke; Mastenbroek, Tom G; Lionikiene, Ausra S; Lancé, Marcus D; van der Meijden, Paola E J; Heemskerk, Johan W M; Mutch, Nicola J.

In: Blood, Vol. 125, No. 16, 16.04.2015, p. 2568 - 2578.

Research output: Contribution to journalArticle

Whyte, CS, Swieringa, F, Mastenbroek, TG, Lionikiene, AS, Lancé, MD, van der Meijden, PEJ, Heemskerk, JWM & Mutch, NJ 2015, 'Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow', Blood, vol. 125, no. 16, pp. 2568 - 2578. https://doi.org/10.1182/blood-2014-09-599480
Whyte CS, Swieringa F, Mastenbroek TG, Lionikiene AS, Lancé MD, van der Meijden PEJ et al. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. Blood. 2015 Apr 16;125(16):2568 - 2578. https://doi.org/10.1182/blood-2014-09-599480
Whyte, Claire S ; Swieringa, Frauke ; Mastenbroek, Tom G ; Lionikiene, Ausra S ; Lancé, Marcus D ; van der Meijden, Paola E J ; Heemskerk, Johan W M ; Mutch, Nicola J. / Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. In: Blood. 2015 ; Vol. 125, No. 16. pp. 2568 - 2578.
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AU - Lancé, Marcus D

AU - van der Meijden, Paola E J

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AU - Mutch, Nicola J

N1 - Copyright © 2015 American Society of Hematology. The authors thank the Microscopy and Histology Core Facility and the Iain Fraser Cytometry Centre at the University of Aberdeen for excellent advice and use of the facilities. This work was supported by grants from the British Heart Foundation (FS/11/2/28579) (N.J.M., A.S.L.) and (PG/11/1/28461) (N.J.M., C.S.W.), the National Health Service Grampian Endowment (grant 14/43) (C.S.W., N.J.M.), Friends of Anchor (N.J.M.), the Landsteiner Foundation for Blood Transfusion Research (1006) (F.S., P.E.J.v.d.M., J.W.M.H.), and the Cardiovascular Centre Maastricht (F.S., P.E.J.v.d.M., J.W.M.H.). Travel for this project was supported by a grant from the British Society for Haemostasis and Thrombosis (N.J.M., C.S.W.).

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N2 - The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation; with tPA-mediated lysis more efficient than uPA-mediated lysis. Fluorescently-labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative and bound plasminogen and fibrinogen in a protruding 'cap'. These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

AB - The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation; with tPA-mediated lysis more efficient than uPA-mediated lysis. Fluorescently-labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative and bound plasminogen and fibrinogen in a protruding 'cap'. These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

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DO - 10.1182/blood-2014-09-599480

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JF - Blood

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