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Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo

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Abstract

The exact mechanism of blood vessel thrombus formation remains to be defined. Here, we introduce a new approach to probe thrombus formation in blood vessels of living animals using intravital microscopy in green fluorescent protein (GFP)-transgenic mice to simultaneously monitor platelet aggregation and procoagulant activity. To this end, GFP-expressing platelets and annexin A5 labeled with a fluorescent dye were employed to visualize and analyze platelet aggregation and markers of procoagulant activity (platelet surface phosphatidylserine (PS)). Laser-induced thrombi increased and then decreased in size with time in vessels of living animals, whereas platelet surface PS initiated at the site of injury and then penetrated into the thrombus. PS-positive platelets were predominantly localized in the center of the thrombus, as was fibrin generation. The experimental system proposed here is a valuable tool not only for investigating mechanisms of thrombus formation but also to assess the efficacy of antithrombotic drugs within the vasculature.

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Abbreviations

PS:

phosphatidylserine

ANX:

annexin A5

IMC:

ionomycin

References

  1. Born GV (1962) Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194:927–929

    Article  PubMed  CAS  Google Scholar 

  2. Rosado JA, Sage SO (2000) Phosphoinositides are required for store-mediated calcium entry in human platelets. J Biol Chem 275:9110–9113

    Article  PubMed  CAS  Google Scholar 

  3. Mazzucato M, Pradella P, Cozzi MR, De Marco L, Ruggeri ZM (2002) Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ib alpha mechanoreceptor. Blood 100:2793–2800

    Article  PubMed  CAS  Google Scholar 

  4. Nesbitt WS, Giuliano S, Kulkarni S, Dopheide SM, Harper IS, Jackson SP (2003) Intercellular calcium communication regulates platelet aggregation and thrombus growth. J Cell Biol 160:1151–1161

    Article  PubMed  CAS  Google Scholar 

  5. Jackson SP (2007) The growing complexity of platelet aggregation. Blood 109:5087–5095

    Article  PubMed  CAS  Google Scholar 

  6. Zwaal RF, Schroit AJ (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121–1132

    PubMed  CAS  Google Scholar 

  7. Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD (2000) Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 106:385–392

    Article  PubMed  CAS  Google Scholar 

  8. Rosen ED, Raymond S, Zollman A, Noria F, Sandoval-Cooper M, Shulman A, Merz JL, Castellino FJ (2001) Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi. Am J Pathol 158:1613–1622

    PubMed  CAS  Google Scholar 

  9. Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B (2002) Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 8:1175–1181

    Article  PubMed  CAS  Google Scholar 

  10. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) 'Green mice' as a source of ubiquitous green cells. FEBS Lett 407:313–319

    Article  PubMed  CAS  Google Scholar 

  11. Tait JF, Gibson D, Fujikawa K (1989) Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem 264:7944–7949

    PubMed  CAS  Google Scholar 

  12. Reutelingsperger CP (2001) Annexins: key regulators of haemostasis, thrombosis, and apoptosis. Thromb Haemost 86:413–419

    PubMed  CAS  Google Scholar 

  13. Dachary-Prigent J, Freyssinet JM, Pasquet JM, Carron JC, Nurden AT (1993) Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood 81:2554–2565

    PubMed  CAS  Google Scholar 

  14. Heemskerk JW, Farndale RW, Sage SO (1997) Effects of U73122 and U73343 on human platelet calcium signalling and protein tyrosine phosphorylation. Biochim Biophys Acta 1355:81–88

    Article  PubMed  CAS  Google Scholar 

  15. Mira JP, Dubois T, Oudinet JP, Lukowski S, Russo-Marie F, Geny B (1997) Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. Evidence of crucial importance of domain I type II Ca2+-binding site in the mechanism of inhibition. J Biol Chem 272:10474–10482

    Article  PubMed  CAS  Google Scholar 

  16. Heemskerk JW, Vuist WM, Feijge MA, Reutelingsperger CP, Lindhout T (1997) Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood 90:2615–2625

    PubMed  CAS  Google Scholar 

  17. Smeets EF, Heemskerk JW, Comfurius P, Bevers EM, Zwaal RF (1993) Thapsigargin amplifies the platelet procoagulant response caused by thrombin. Thromb Haemost 70:1024–1029

    PubMed  CAS  Google Scholar 

  18. Thiagarajan P, Tait JF (1990) Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 265:17420–17423

    PubMed  CAS  Google Scholar 

  19. Thiagarajan P, Tait JF (1991) Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles. J Biol Chem 266:24302–24307

    PubMed  CAS  Google Scholar 

  20. Wolf BB, Goldstein JC, Stennicke HR, Beere H, Amarante-Mendes GP, Salvesen GS, Green DR (1999) Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. Blood 94:1683–1692

    PubMed  CAS  Google Scholar 

  21. Wolfs JL, Comfurius P, Rasmussen JT, Keuren JF, Lindhout T, Zwaal RF, Bevers EM (2005) Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction. Cell Mol Life Sci 62:1514–1525

    Article  PubMed  CAS  Google Scholar 

  22. Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4:552–565

    Article  PubMed  CAS  Google Scholar 

  23. Furie B, Furie BC (2005) Thrombus formation in vivo. J Clin Invest 115:3355–3362

    Article  PubMed  CAS  Google Scholar 

  24. Goto S, Tamura N, Ishida H, Ruggeri ZM (2006) Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5′-diphosphate receptor stimulation and cyclic calcium signaling. J Am Coll Cardiol 47:155–162

    Article  PubMed  CAS  Google Scholar 

  25. Matsui H, Sugimoto M, Mizuno T, Tsuji S, Miyata S, Matsuda M, Yoshioka A (2002) Distinct and concerted functions of von Willebrand factor and fibrinogen in mural thrombus growth under high shear flow. Blood 100:3604–3610

    Article  PubMed  CAS  Google Scholar 

  26. Andre P, Denis CV, Ware J, Saffaripour S, Hynes RO, Ruggeri ZM, Wagner DD (2000) Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins. Blood 96:3322–3328

    PubMed  CAS  Google Scholar 

  27. Gross PL, Furie BC, Merrill-Skoloff G, Chou J, Furie B (2005) Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development. J Leukoc Biol 78:1318–1326

    Article  PubMed  CAS  Google Scholar 

  28. Dubois C, Panicot-Dubois L, Gainor JF, Furie BC, Furie B (2007) Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model. J Clin Invest 117:953–960

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

We greatly appreciate the gift of four calcium-binding site mutant of annexin 5A from Dr. Francoise Russo-Marie (Institut Cochin de Ge´ne´tiqueMole´culaire, Unite´ INSERM U332, 75014 Paris, France). We thank Dr. Satoshi Nakamura (Director for Finance and Hospital, Hamamatsu University School of Medicine) for critical discussion and Dr. Yuko Suzuki (Department of Physiology) for technical advice. This work was supported by Grant-in-Aid for Young Scientists (B: 17790885) to T.H., Grant-in-Aid for Scientific Research (C: 18590204) to T.U. from the Japan Society for the Promotion of Science (JSPS), a grant from the Smoking Research Foundation to T.U., and a grants from Takeda Science Foundation to H.M.

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Authors and Affiliations

  1. Second Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Tadataka Hayashi, Toshio Nakamura & Hiroyuki Konno

  2. Department of Physiology, Hamamatsu University School of Medicine, Handa-yama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka, 431-3192, Japan

    Tadataka Hayashi, Hideo Mogami, Yusuke Murakami & Tetsumei Urano

  3. Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Yusuke Murakami & Naohiro Kanayama

  4. Bioenvironmental Science, Okazaki Institute for Integrative Bioscience, Okazaki, Japan

    Hideo Mogami

Authors
  1. Tadataka Hayashi
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  2. Hideo Mogami
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  3. Yusuke Murakami
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  4. Toshio Nakamura
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  5. Naohiro Kanayama
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  6. Hiroyuki Konno
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  7. Tetsumei Urano
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Corresponding author

Correspondence to Hideo Mogami.

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Figure S1; Movie 1

Dual monitoring of [Ca2+]i and surface-exposed PS. Fluo-4-loaded platelets on fibrinogen-coated glass-bottomed dishes were perfused with solution containing 100 nM ANX. Application of 10 μM ionomycin resulted in a rapid increase in the fluorescence intensity of fluo-4 followed by that of ANX. The fluo-4-loaded platelets developed many filopodial projections and shrank into rounded shapes with sudden loss of fluo-4 dye. The movie was made from merged images of fluo-4 and ANX captured every 5 s for 10 min (MOV 1.34 MB)

Figure S2; Movie 2

A laser-induced thrombus in the mesenteric venule of a GFP mouse. Platelets are accumulating at the site of injury. Images were taken every 27 ms for 11 s at the same focal plane (MOV 2.44 MB)

Figure S3

Optical slices of a laser-induced thrombus in the mesenteric venule of a GFP mouse. Focal plane images along the z axis from the vessel wall to the luminal face of a thrombus were taken at 33 ms per frame. Nine representative images were selected from 28 z-sections at 1.4-μm optical slice thickness. Scale bar, 10 μm (GIF 175 KB)

High-resolution image (TIFF 245 KB)

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Hayashi, T., Mogami, H., Murakami, Y. et al. Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo. Pflugers Arch - Eur J Physiol 456, 1239–1251 (2008). https://doi.org/10.1007/s00424-008-0466-9

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  • DOI: https://doi.org/10.1007/s00424-008-0466-9

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