Polyphosphate co-localizes with factor XII on plateletbound fibrin and augments its plasminogen activator activity

This work was supported by grants FS/11/2/28579 (N.J.M. & A.S.L) from the British Heart Foundation and by the University of Aberdeen Development Trust (J.L.M. & N.J.M.). P.Y.K is supported by an Early Career Award and New Investigator Fund from the Hamilton Health Sciences. We are grateful to the following students for contributions to the project, Natasha Walker & Thomas Nolan. We also thank both 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. We also thank Dr Jeffrey Weitz from McMaster University, Canada for the kind gift of HRG.Peer reviewedPostprin

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

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 tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-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 the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow