Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor

• Published in: Thrombosis and haemostasis Vol. 117; no. 4; p. 682
• Main Authors: Brzoska, Tomasz, Suzuki, Yuko, Sano, Hideto, Suzuki, Seiichirou, Tomczyk, Martyna, Tanaka, Hiroki, Urano, Tetsumei
• Format: Journal Article
• Language: English
• Published: Germany 2017
• Subjects: Blood Coagulation - drug effects; Blood Platelets - drug effects; Blood Platelets - enzymology; Carboxypeptidase B2 - antagonists & inhibitors; Carboxypeptidase B2 - pharmacology; Dose-Response Relationship, Drug; Fibrin - metabolism; Fibrinolysis - drug effects; Humans; Intravital Microscopy - methods; Microscopy, Confocal - methods; Phosphatidylserines - metabolism; Platelet Activation - drug effects; Platelet-Rich Plasma - cytology; Platelet-Rich Plasma - diagnostic imaging; Platelet-Rich Plasma - enzymology; Protease Inhibitors - pharmacology; Thrombomodulin - metabolism; Time Factors; plasminogen; thrombin-activatable fibrinolysis inhibitor; Fibrinolysis; thrombomodulin; coagulation initiation region
• ISSN: 2567-689X
• DOI: 10.1160/TH16-09-0722

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).