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Vol. 33, No. 1, 2003
Issue release date: June 2003
Section title: Paper
Pathophysiol Haemost Thromb 2003;33:23–29
(DOI:10.1159/000071638)

Calibrated Automated Thrombin Generation in Frozen-Thawed Platelet-Rich Plasma to Detect Hypercoagulability

Regnault V.a · Béguin S.c · Lecompte T.a,b
aINSERM ERIT-M 0323 et EA 3452, Faculté de Médecine, Université Henri Poincaré, et bHématologie Biologique, CHU de Nancy, Nancy, France; cSynapse BV, Cardiovascular Research Institute, University of Maastricht, Maastricht, TheNetherlands
email Corresponding Author

Abstract

To enhance the practical applicability of the calibrated automated thrombogram (CAT) we investigated whether frozen-thawed platelet-rich plasma (ft-PRP) can be used to assess the function of the protein C inhibitory pathway, while preserving the natural phospholipid composition. Recalcified ft-PRP triggered with 0.5 pM recombinant human tissue factor shows a median thrombin potential of 1,779 nM·min, against 1,576 nM·min for fresh PRP. To obtain ∼70% inhibition, 6.7 nM activated protein C (APC) has to be added, instead of 25 nM in fresh PRP; so the relative APC resistance of PRP appears to depend upon the presence of intact platelets. Factor VIII, added to normal ft-PRP to obtain a concentration of 3.3 U/ml, increases the thrombin potential in the presence of APC 1.5-fold, from 524 to 808 nM·min, in keeping with previously published increases in thrombotic risk in patients with high factor VIII levels. We conclude that thrombography in ft-PRP, with and without added APC, can be used to assess known risk factors for thrombosis, which allows the design of large clinical studies aimed at proving the relationship between thrombin potential and clinical outcome.

© 2003 S. Karger AG, Basel


  

Key Words

  • Activated protein C
  • Factor VIII
  • Platelets
  • Thrombin generation

References

  1. Laffan M, Tuddenham E: Assessing thrombotic risk. BMJ 1998;317:520–523.
  2. Hemker HC, Béguin S: Phenotyping the clotting system. Thromb Haemost 2000;84:747–751.
  3. Regnault V, Béguin S, Wahl D, De Maistre E, Hemker HC, Lecompte T: Thrombinography shows acquired resistance to activated protein C in patients with lupus anticoagulants. Thromb Haemost 2003;89:208–212.
  4. Heemskerk JWM, Bevers EM, Lindhout T: Platelet activation and blood coagulation. Thromb Haemost 2002;88:186–193.
  5. Monroe DM, Hoffman M, Roberts HR: Platelets and thrombin generation. Arterioscler Thromb Vasc Biol 2002;22:1381–1389.
  6. Soria JM, Almasy L, Souto JC, Buil A, Martinez-Sanchez E, Mateo J, Borell M, Stone WH, Lathrop M, Fontcuberta J, Blangero J: A new locus on chromosome 18 that influences normal variation in activated protein C resistance phenotype and factor VIII activity and its relation to thrombosis susceptibility. Blood 2003;101:163–167.
  7. Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki O, Kanabayashi T, Yamamoto I, Mauryama I, Suzuki K: Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood 1990;75:1396–1399.
  8. Regnault V, De Maistre E, Geschier C, Briquel ME, André E, Stoltz JF, Lecompte T: A new fast one-step immunopreparation for activated protein C. Thromb Haemost 1995;73:1365.
  9. Dahlbäck B, Carlsson M, Svensson PJ: Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993;90:1004–1008.
  10. Hemker HC, Giesen PLA, Ramjee M, Wagenvoord R, Béguin S: The thrombogram: monitoring thrombin generation in platelet rich plasma. Thromb Haemost 2000;83:589–591.
  11. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Béguin S: Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15.
  12. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Béguin S: Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15.
  13. Rosing J, van Rijn JLMM, Bevers EM, van Dieijen G, Comfurius P, Zwaal RF: The role of activated human platelets in prothombin and factor X activation. Blood 1985;65:319–332.
  14. Mann KG, Butenas S, Brummel K: The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003;23:17–25.
  15. Taylor FB, Peer GT, Lockhart MS, Ferrell G, Esmon CT: Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001;97:1685–1688.
  16. Hampton JW, Matthew C: Similarities between baboon and human blood clotting. J Appl Physiol 1966;21:1713–1716.
  17. Taube J, McWilliam N, Luddington R, Byrne C, Baglin T: Activated protein C resistance: Effect of platelet activation, platelet-derived microparticles, and atherogenic lipoproteins. Blood 1999;93:3792–3797.
  18. Jane SM, Mitchell CA, Hau L, Salem HH: Inhibition of activated protein C in platelets. J Clin Invest 1989;83:222–226.
  19. Nishioka J, Ning M, Hayashi T, Suzuki K: Protein C inhibitor secreted from activated platelets efficiently inhibits activated protein C on phosphatidylethanolamine of platelet membrane and microvesicles. J Biol Chem 1998;273:11281–11287.
  20. Smirnov MD, Esmon CT: Phosphatidylethanolamine incroporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994;269:816–819.
  21. Koster T, Blann AD, Briet E, Vandenbroucke JP, Rosendaal FR: Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 1995;345:152–155.
  22. Kamphuisen PW, Eikenboom JCJ, Bertina RM: Elevated factor VIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol 2001;21:731–738.

  

Author Contacts

Véronique Regnault
INSERM ERIT-M 0323 ‘Nouvelles approches anti-thrombotiques’ EA 3452
Faculté de Médecine, Université Henri Poincaré, Boite Postale 184
FR–54505 Vandoeuvre-lès-Nancy (France)
Tel. +33 3 83 68 34 70, Fax +33 3 83 38 34 79, E-mail regnault@hemato.u-nancy.fr

  

Article Information

Received: March 8, 2003
Accepted: April 1, 2003
Number of Print Pages : 7
Number of Figures : 6, Number of Tables : 0, Number of References : 22

  

Publication Details

Pathophysiology of Haemostasis and Thrombosis
(Formely: Haemostasis)
Official Journal of the ‘Mediterranean League against Thromboembolic Diseases’

Vol. 33, No. 1, Year 2003 (Cover Date: Released June 2003)

Journal Editor: J. Rosing, Maastricht
ISSN: 1424–8832 (print), 1424–8840 (Online)

For additional information: http://www.karger.com/pht


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References

  1. Laffan M, Tuddenham E: Assessing thrombotic risk. BMJ 1998;317:520–523.
  2. Hemker HC, Béguin S: Phenotyping the clotting system. Thromb Haemost 2000;84:747–751.
  3. Regnault V, Béguin S, Wahl D, De Maistre E, Hemker HC, Lecompte T: Thrombinography shows acquired resistance to activated protein C in patients with lupus anticoagulants. Thromb Haemost 2003;89:208–212.
  4. Heemskerk JWM, Bevers EM, Lindhout T: Platelet activation and blood coagulation. Thromb Haemost 2002;88:186–193.
  5. Monroe DM, Hoffman M, Roberts HR: Platelets and thrombin generation. Arterioscler Thromb Vasc Biol 2002;22:1381–1389.
  6. Soria JM, Almasy L, Souto JC, Buil A, Martinez-Sanchez E, Mateo J, Borell M, Stone WH, Lathrop M, Fontcuberta J, Blangero J: A new locus on chromosome 18 that influences normal variation in activated protein C resistance phenotype and factor VIII activity and its relation to thrombosis susceptibility. Blood 2003;101:163–167.
  7. Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki O, Kanabayashi T, Yamamoto I, Mauryama I, Suzuki K: Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood 1990;75:1396–1399.
  8. Regnault V, De Maistre E, Geschier C, Briquel ME, André E, Stoltz JF, Lecompte T: A new fast one-step immunopreparation for activated protein C. Thromb Haemost 1995;73:1365.
  9. Dahlbäck B, Carlsson M, Svensson PJ: Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993;90:1004–1008.
  10. Hemker HC, Giesen PLA, Ramjee M, Wagenvoord R, Béguin S: The thrombogram: monitoring thrombin generation in platelet rich plasma. Thromb Haemost 2000;83:589–591.
  11. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Béguin S: Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15.
  12. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Béguin S: Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15.
  13. Rosing J, van Rijn JLMM, Bevers EM, van Dieijen G, Comfurius P, Zwaal RF: The role of activated human platelets in prothombin and factor X activation. Blood 1985;65:319–332.
  14. Mann KG, Butenas S, Brummel K: The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003;23:17–25.
  15. Taylor FB, Peer GT, Lockhart MS, Ferrell G, Esmon CT: Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001;97:1685–1688.
  16. Hampton JW, Matthew C: Similarities between baboon and human blood clotting. J Appl Physiol 1966;21:1713–1716.
  17. Taube J, McWilliam N, Luddington R, Byrne C, Baglin T: Activated protein C resistance: Effect of platelet activation, platelet-derived microparticles, and atherogenic lipoproteins. Blood 1999;93:3792–3797.
  18. Jane SM, Mitchell CA, Hau L, Salem HH: Inhibition of activated protein C in platelets. J Clin Invest 1989;83:222–226.
  19. Nishioka J, Ning M, Hayashi T, Suzuki K: Protein C inhibitor secreted from activated platelets efficiently inhibits activated protein C on phosphatidylethanolamine of platelet membrane and microvesicles. J Biol Chem 1998;273:11281–11287.
  20. Smirnov MD, Esmon CT: Phosphatidylethanolamine incroporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994;269:816–819.
  21. Koster T, Blann AD, Briet E, Vandenbroucke JP, Rosendaal FR: Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 1995;345:152–155.
  22. Kamphuisen PW, Eikenboom JCJ, Bertina RM: Elevated factor VIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol 2001;21:731–738.