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

Tomasz Brzoska; Yuko Suzuki; Hideto Sano; Seiichirou Suzuki; Martyna Tomczyk; Hiroki Tanaka; Tetsumei Urano

doi:10.1160/TH16-09-0722

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2017; 117(04): 682-690
DOI: 10.1160/TH16-09-0722

Coagulation and Fibrinolysis

Schattauer GmbH

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

Authors

Tomasz Brzoska

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Yuko Suzuki

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Hideto Sano

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Seiichirou Suzuki

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Martyna Tomczyk

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Hiroki Tanaka

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan
Tetsumei Urano

¹Department of Medical Physiology, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Japan

Abstract

Summary

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-de- pendent 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).

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Fibrinolysis - thrombin-activatable fibrinolysis inhibitor - coagulation initiation region - plasminogen - thrombomodulin

Volltext

Referenzen

References
1 Gaffney PJ, Edgell TA, Whitton CM. The haemostatic balance - Astrup revisited. Haemostasis 1999; 29: 58-71
2 Hoffman M, Monroe DM. 3rd A cell-based model of hemostasis. Thromb Haemost 2001; 85: 958-965
3 Hayashi T, Mogami H, Murakami Y. et al. Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo. Pflugers Arch 2008; 456: 1239-1251
4 Morrissey JH, Tajkhorshid E, Rienstra CM. Nanoscale studies of protein-membrane interactions in blood clotting. J Thromb Haemost 2011; 9 (01) 162-167
5 Brzoska T, Tanaka-Murakami A, Suzuki Y. et al. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi. PLoS One 2015; 10: e0122196
6 Collen D, Lijnen HR. Tissue-type plasminogen activator: a historical perspective and personal account. J Thromb Haemost 2004; 2: 541-546
7 Ichinose A, Takio K, Fujikawa K. Localization of the binding site of tissue-type plasminogen activator to fibrin. J Clin Invest 1986; 78: 163-169
8 Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost 2005; 93: 647-654
9 Law RH, Caradoc-Davies T, Cowieson N. et al. The X-ray crystal structure of full-length human plasminogen. Cell Rep 2012; 1: 185-190
10 Rijken DC, Lijnen HR. New insights into the molecular mechanisms of the fibrinolytic system. J Thromb Haemost 2009; 7: 4-13
11 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271: 16603-16608
12 Wang YF, Tsirka SE, Strickland S. et al. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nature Med 1998; 4: 228-231
13 Mosnier LO, Buijtenhuijs P, Marx PF. et al. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844-4846
14 Samson AL, Borg RJ, Niego B. et al. A nonfibrin macromolecular cofactor for tPA-mediated plasmin generation following cellular injury. Blood 2009; 114: 1937-1946
15 Brzoska T, Suzuki Y, Mogami H. et al. Binding of thrombin-activated platelets to a fibrin scaffold through alpha(IIb)beta(3) evokes phosphatidylserine exposure on their cell surface. PLoS One 2013; 8: e55466
16 Reutelingsperger CP. Annexins: key regulators of haemostasis, thrombosis, and apoptosis. Thromb Haemost 2001; 86: 413-419
17 Iwasaki A, Suda M, Nakao H. et al. Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein. J Biochem 1987; 102: 1261-73
18 Tait JF, Gibson D, Fujikawa K. Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem 1989; 264: 7944-7949
19 Thiagarajan P, Tait JF. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990; 265: 17420-17423
20 Dachary-Prigent J, Freyssinet JM, Pasquet JM. et al. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood 1993; 81: 2554-2565
21 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
22 Wang W, Nagashima M, Schneider M. et al. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem 2000; 275: 22942-22947
23 Zwaal RF, Comfurius P, Bevers EM. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids. Biochim Biophys Acta 2004; 1636: 119-128
24 Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-1132
25 Collet JP, Montalescot G, Lesty C. et al. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circulation Res 2002; 90: 428-434
26 Campbell RA, Overmyer KA, Selzman CH. et al. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood 2009; 114: 4886-4896
27 Suzuki Y, Yasui H, Brzoska T. et al. Surface-retained tPA is essential for effective fibrinolysis on vascular endothelial cells. Blood 2011; 118: 3182-3185
28 Bouma BN, Marx PF, Mosnier LO. et al. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). Thromb Res 2001; 101: 329-354
29 Mao SS, Cooper CM, Wood T. et al. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem 1999; 274: 35046-35052
30 Mosnier LO, Meijers JC, Bouma BN. Regulation of fibrinolysis in plasma by TAFI and protein C is dependent on the concentration of thrombomodulin. Thromb Haemost 2001; 85: 5-11
31 Wang W, Boffa MB, Bajzar L. et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273: 27176-27181
32 Sakharov DV, Plow EF, Rijken DC. On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B. J Biol Chem 1997; 272: 14477-14482
33 Mosnier LO, Bouma BN. Regulation of fibrinolysis by thrombin activatable fibrinolysis inhibitor, an unstable carboxypeptidase B that unites the pathways of coagulation and fibrinolysis. Arterioscler Thromb Vasc Biol 2006; 26: 2445-2453
34 Schadinger SL, Lin JH, Garand M. et al. Secretion and antifibrinolytic function of thrombin-activatable fibrinolysis inhibitor from human platelets. J Thromb Haemost 2010; 8: 2523-2529
35 Carrieri C, Galasso R, Semeraro F. et al. The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet-mediated fibrinolysis resistance: a thromboelastographic study in whole blood. J Thromb Haemost 2011; 9: 154-162
36 Suzuki K, Nishioka J, Hayashi T. et al. Functionally active thrombomodulin is present in human platelets. J Biochem 1988; 104: 628-632

Zusatzmaterial

Zusatzmaterial (PDF)