PDF herunterladen
Thromb Haemost 2004; 91(05): 977-985
DOI: 10.1160/TH03-10-0638
Platelets and Blood Cells
Schattauer GmbH

The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation

Grigoris T. Gerotziafas
1   Service d’Hématologie Biologique, Hôtel Dieu, Paris, France
2   LCL, Ivry sur Seine, France
,
Tahar Chakroun
1   Service d’Hématologie Biologique, Hôtel Dieu, Paris, France
,
François Depasse
2   LCL, Ivry sur Seine, France
,
Pantelis Arzoglou
3   Laboratory of Biochemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, Greece
,
Meyer M. Samama
1   Service d’Hématologie Biologique, Hôtel Dieu, Paris, France
2   LCL, Ivry sur Seine, France
,
Ismail Elalamy
1   Service d’Hématologie Biologique, Hôtel Dieu, Paris, France
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 21. Oktober 2003

Accepted after revision 18. Februar 2004

Publikationsdatum:
01. Dezember 2017 (online)

Auch verfügbar auf
    Lizenzen und Reprints

Summary

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 ×109/l to 150 ×109/l were spiked with rFVIIa (1 µg/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope™ assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the Pselectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot’s firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%.The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets’ count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.

Keywords

Thrombin generation - thrombocytopenia - recombinant factor VIIa - novoseven - thromboelastography - platelet activation - tissue factor
 

  • References

  • 1 Schmidt ML, Gamerman S, Smith HE. et al. Recombinant activated factor VII (rFVIIa) therapy for intracranial hemorrhage in hemophilia A patients with inhibitors. Am J Hematol 1994; 47: 36-40.
    CrossrefPubMedSuche in Google Scholar
  • 2 Roberts HR. Clinical experience with activated factor VII: focus on safety aspects. Blood Coagul Fibrinolysis 1998; 09 (Suppl. 01) S115-S118.
    PubMedSuche in Google Scholar
  • 3 Revesz T, Arets B, Bierings M. et al. Recombinant factor VIIa in severe uremic bleeding. Thromb Haemost 1998; 80: 353.
    Thieme ConnectPubMedSuche in Google Scholar
  • 4 Poon MC, d’Oiron R. Recombinant activated factor VII (NovoSeven) treatment of plateletrelated bleeding disorders. International Registry on Recombinant Factor VIIa and Congenital Platelet Disorders Group. Blood Coagul Fibrinolysis 2000; 11 (Suppl. 01) S55-68.
    CrossrefPubMedSuche in Google Scholar
  • 5 Kristensen J, Killander A, Hippe E. et al. Clinical experience with recombinant factor VIIa in patients with thrombocytopenia. Haemostasis 1996; 26 (Suppl. 01) 159-64.
    PubMedSuche in Google Scholar
  • 6 Gerotziafas GT, Zervas C, Gavrielidis G. et al. Effective hemostasis with rFVIIa treatment in two patients with severe thrombocytopenia and life-threatening hemorrhage. Am J Hematol 2002; 69: 219-22.
    CrossrefPubMedSuche in Google Scholar
  • 7 Vidarsson B, Onundarson PT. Recombinant factor VIIa for bleeding in refractory thrombocytopenia. Thromb Haemost 2000; 83: 634-5.
    Thieme ConnectPubMedSuche in Google Scholar
  • 8 Waddington DP, McAuley FT, Hanley JP. et al. The use of recombinant factor viia in a jehovah’s witness with auto-immune thrombocytopenia and post-splenectomy haemorrhage. Br J Haematol 2002; 119: 286-8.
    CrossrefPubMedSuche in Google Scholar
  • 9 Culic S. Recombinant factor VIIa for refractive haemorrhage in autoimmune idiopathic thrombocytopenic purpura. Br J Haematol 2003; 120: 909-10.
    CrossrefPubMedSuche in Google Scholar
  • 10 Tranholm M, Rojkjaer R, Pyke C. et al. Recombinant factor VIIa reduces bleeding in severely thrombocytopenic rabbits. Thromb Res 2003; 109: 217-23.
    CrossrefPubMedSuche in Google Scholar
  • 11 Gerotziafas GT, Zervas K, Arzoglou P. et al. On the mechanism of action of rFVIIa administered to patients with severe thrombocytopenia and life-threatening haemorrhage. Focus on prothrombin activation. Br J Haematol 2002; 117: 705-8.
    CrossrefPubMedSuche in Google Scholar
  • 12 Hemker HC, Giesen PLA, Ramjee M. et al. The thrombogram: monitoring thrombin generation in platelet rich plasma. Thromb Haemost. 2000 83. 589-91.
    PubMedSuche in Google Scholar
  • 13 Hemker HC, Wielders S, Kessels H. et al. Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb Haemost 1993; 70: 617-24.
    Thieme ConnectPubMedSuche in Google Scholar
  • 14 Hemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb Haemost 1995; 74: 134-8.
    Thieme ConnectPubMedSuche in Google Scholar
  • 15 Mustard JF, Perry DW, Kinlough-Rathbone RL. et al. Factors responsible for ADPinduced release reaction of human platelets. Am J Physiol 1975; 228: 1757-65.
    PubMedSuche in Google Scholar
  • 16 Potevin F, Lecompte T, Favier R. et al. Rapid aequorin loading into platelets in the presence of DMSO: characteristics of the responses (changes in light transmission and in calcium) to various agonists. Thromb Haemost 1991; 66: 334-42.
    Thieme ConnectPubMedSuche in Google Scholar
  • 17 Vig S, Chitolie A, Bevan DH. et al. Thromboelastography: a reliable test?. Blood Coagul Fibrinolysis. 2001 12. 555-61.
    PubMedSuche in Google Scholar
  • 18 Billy D, Briede J, Heemskerk JW. et al. Prothrombin conversion under flow conditions by prothrombinase assembled on adherent platelets. Blood Coagul Fibrinolysis 1997; 08: 168-74.
    CrossrefPubMedSuche in Google Scholar
  • 19 Butenas S, Branda RF, van t’Veer C. et al. Platelets and phospholipids in tissue factor initiated thrombin generation. Thromb Haemost 2001; 86: 660-7.
    Thieme ConnectPubMedSuche in Google Scholar
  • 20 Hemker HC, Beguin S. Phenotyping the clotting system. Thromb Haemost 2000; 84: 741-51.
    PubMedSuche in Google Scholar
  • 21 Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23: 17-25.
    CrossrefPubMedSuche in Google Scholar
  • 22 Khurana S, Mattson JC, Westley S. et al. Monitoring platelet glycoprotein IIb/IIIafibrin interaction with tissue factor-activated thromboelastography. J Lab Clin Med 1997; 130: 401-11.
    CrossrefPubMedSuche in Google Scholar
  • 23 Oshita K, Az-ma T, Osawa Y. et al. Quantitative measurement of thromboelastography as a function of platelet count. Anesth Analg 1999; 89: 296-9.
    PubMedSuche in Google Scholar
  • 24 Bowbrick VA, Mikhailidis DP, Stansby G. Influence of platelet count and activity on thromboelastography parameters. Platelets 2003; 14: 219-24.
    CrossrefPubMedSuche in Google Scholar
  • 25 Gaydos LA, Freirech EJ, Mantel N. The quantitative relation between platelet count and hemorrhage in patients with acute leukemia. N Engl J Med 1962; 266: 905-9.
    CrossrefPubMedSuche in Google Scholar
  • 26 Rebulla P, Finazzi G, Marangon F. et al. The Threshold for Prophylactic Platelet Transfusions in Adults with Acute Myeloid Leukemia. N Engl J Med 1997; 337: 1870-5.
    CrossrefPubMedSuche in Google Scholar
  • 27 Wandt H, Frank M, Ehninger G. et al. Safety and cost effectiveness of a 10 × 109/l trigger for prophylactic platelet transfusions compared with the traditional 20 × 109/l trigger: a prospective comparative trial in 105 patients with acute myeloid leukemia. Blood 1998; 91: 3601-6.
    PubMedSuche in Google Scholar
  • 28 Wandt H, Ehninger G, Gallemeier WM. New strategies for prophylactic platelet transfusion in patients with hematologic diseases. Oncologist 2001; 06: 446-50.
    CrossrefPubMedSuche in Google Scholar
  • 29 Erhardtsen E. Pharmacokinetics of recombinant activated factor VII (rFVIIa). Semin Thromb Hemost 2000; 26: 385-91.
    Thieme ConnectPubMedSuche in Google Scholar
  • 30 Kjalke M, Ezban M, Monroe DM. et al. High dose factor VIIa increases initial thrombin generation and mediates platelet activationj in thrombocytopenia-like conditions in a cellbased model system. Br J Haematol 2001; 114: 114-20.
    CrossrefPubMedSuche in Google Scholar
  • 31 Hoffman M, Monroe D, Roberts HR. Platelet dependent action of high-dose factor VIIa. Blood 2002; 100: 364-5.
    CrossrefPubMedSuche in Google Scholar
  • 32 Mann KG, Butenas S. Mechanism of factor VIIa-dependent coagulation in hemophilia blood. Blood 2002; 100: 365.
    PubMedSuche in Google Scholar
  • 33 Monroe DM, Hoffman M, Oliver JA. et al. Platelet activity of high dose factor VIIa is independent of tissue factor. Br J Haematol 1997; 99: 542.
    CrossrefPubMedSuche in Google Scholar
  • 34 Lisman T, Moschatsis S, Adelmeijer J. et al. Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation. Blood 2003; 101: 1864-70.
    CrossrefPubMedSuche in Google Scholar
  • 35 Veldman A, Hoffman M, Ehrenforth S. New insights into the coagulation system and implications for new therapeutic options with recombinant factor VIIa. Curr Med Chem 2003; 10: 797-811.
    CrossrefPubMedSuche in Google Scholar
  • 36 Morrissey JH, Macik BG, Neuenschwander PF. et al. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81: 734-44.
    PubMedSuche in Google Scholar
  • 37 van’t Veer C, Golden NJ, Mann KG. Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa. Blood 2000; 95: 1330-5.
    PubMedSuche in Google Scholar
  • 38 Butenas S, Brummel KE, Paradis SG. et al. Influence of factor VIIa and phospholipids on coagulation in “acquired” haemophilia. Arterioscler Thromb Vasc Biol 2003; 23: 123-9.
    CrossrefPubMedSuche in Google Scholar
  • 39 Sorensen B, Johansen P, Christiansen K. et al. Whole blood coagulation profiles employing minimal tissue factor activation. J Thromb Haemost 2003; 01: 551-8.
    CrossrefPubMedSuche in Google Scholar
  • 40 Torbet J. The thrombin activation pathway modulates the assembly, structure and lysis of human plasma clots in vitro. Thromb Haemost 1995; 73: 785-92.
    Thieme ConnectPubMedSuche in Google Scholar
  • 41 Blomback B, Carlsson K, Fatah K. et al. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation. Thromb Res 1994; 75: 521-38.
    CrossrefPubMedSuche in Google Scholar
  • 42 Giesen PLA, Rauch U, Bohrmann B. et al. Blood borne tissue factor: another view of thrombosis. Proc Natl Acad Sci USA 1999; 96: 2311-5.
    CrossrefPubMedSuche in Google Scholar
  • 43 Morrissey JH. Tissue factor: an enzyme cofactor and a true receptor. Thromb Haemost 2001; 86: 66-74.
    Thieme ConnectPubMedSuche in Google Scholar
  • 44 Nemerson Y, Contino PB. Tissue factor, flow and the initiation of coagulation. In Hypercoagulable states. Fundamental aspects, acquired disorders and congenital thrombophilia. Seghatchian MJ, Samama MM, Hecker SP. eds. CRC Press; Boca Raton, New York, London, Tokyo: 1996: 21-8.
    Suche in Google Scholar
  • 45 Ingerslev J, Holm M, Christiansen K. et al. Levels of prothrombin activation peptide F1+2 in patients with a bleeding tendency. Blood Coagul Fibrinolysis 1998; 9 Suppl 1: S129-34.
    PubMedSuche in Google Scholar