Contribution of Erythrocytes to Thrombin Generation in Whole Blood

Summary

Thrombin generation (TG) initiated by diluted tissue-factor was investigated in whole human blood, in platelet-rich plasma (PRP), in platelet-poor plasma (PPP), and in PPP supplemented with red blood cells (RBCs). TG was characterized by the lag time preceding the thrombin burst and by the endogenous thrombin potential (ETP). RBCs at normal haematocrit were found to influence the lag time to the same extent as platelets. When TG was carried out in PRP or in PPP + RBCs, both the ETP and lag time were dependent on the platelet count or on the haematocrit, but the shapes of the dose-response curves were different. The inhibition of TG in PPP+ RBCs by two direct thrombin and factor Xa inhibitors: hirudin and DX 9065A, and two antithrombin III (AT)-dependent anticoagulants: heparin and SR 90107A was found to be similar to that previously described in PPP and in PRP: hirudin and DX 9065A only delayed TG whereas heparin and SR 90107A both delayed and decreased TG. FACscan analysis following labelling with FITC-annexin V or with phycoerythrin-labelled anti-glycophorin A of samples taken in the course of TG initiated in PPP + RBCs showed that no significant haemolysis occurred and revealed that 0.51 ± 0.075% (mean ± sem, n = 3) of RBCs steadily exposed procoagulant phospholipids on their outer surface throughout the TG course. Furthermore, incubation of factors Xa and Va with washed RBCs sampled during TG in PPP +RBCs resulted in a significant and constant prothrombinase activity.

Taken together, these data show for the first time that normal RBCs may participate in the haemostatic process through exposure of procoagulant phospholipids.

• References

• 1 Hemker HC. Thrombin generation, an essential step in haemostasis and thrombosis. In: Haemostasis and Thrombosis. (ed 3). Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. eds London, UK: Churchill Livingstone; 1994: p 477.
• 2 Mann KG, Neisheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1.
• 3 Zwaal RFA, Comfurius P, Bevers EM. Platelet procoagulant activity and microvesicle formation. Its putative role in hemostasis and thrombosis. Biochim Biophys Acta 1992; 1180: 1.
• 4 Tracy PB, Allen DH, Worfolk LA, Lawler RR. Monocytes/macrophages regulation of coagulant events. Haemostas 1996; 26 suppl 6.
• 5 Tijburg PNM, De Groot PG. Endothelial cell-mediated thrombin formation. Semin Thromb Hemostas 1992; 18: 256.
• 6 Helley D, Eldor A, Girot R, Ducrocq R, Guillin MC, Bezeaud A. Increased procoagulant activity of red blood cells from patients with homozygous sickle-cell disease and β-thalassemia. Thromb Haemost 1996; 76: 322.
• 7 Kuypers FA, Lewis RA, Hua M, Schott MA, Discher D, Ernst JD, Lubin B. Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood 1996; 87: 1179.
• 8 Wood BL, Gibson DF, Tait JF. Increased erythrocyte phosphatidylserine exposure in sickle cell disease: Flow-cytometric measurement and clinical associations. Blood 1996; 88: 1873.
• 9 Ruf A, Pick M, Deutsch V, Patscheke H, Goldfarb A, Rachmilewitz EA, Guillin MC, Eldor A. In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with β-thalassemia major. Brit J Haematol 1997; 98: 51.
• 10 Hemker BC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9.
• 11 Béguin S, Lindhout T, Hemker HC. The effect of trace amount of tissue factor on thrombin generation in platelet rich plasma, its inhibition by heparin. Thromb Haemost 1989; 61: 25.
• 12 Kessels H, Béguin S, Andree H, Hemker HC. Measurement of thrombin generation in whole blood. The effect of heparin and aspirin. Thromb Haemost 1994; 72: 78.
• 13 Hemker BC, Wielders SJH, Kessels H, Béguin S. Continuous registration of thrombin generation in plasma. Its use for the determination of thrombin potential. Thromb Haemost 1993; 70: 617.
• 14 Dachary-Prigent J, Toti F, Satta N, Pasquet JM, Uzan A, Freyssinet JM. Physiopathological significance of catalytic phospholipids in the generation of thrombin. Semin Thromb Hemostas 1996; 22: 157.
• 15 Sixma JJ. Role of blood platelets, plasma proteins and the vessel wall in haemostasis. In: Haemostasis and Thrombosis. (ed 2). Bloom AL, Thomas DP. (eds) London, UK: Churchill Livingstone; 1987: p 283.
• 16 Santos MT, Valles J, Marcus AJ, Safier LB, Broekman MJ, Islam N, Ullman HL, Eiroa AM, Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. J Clin Invest 1991; 87: 571.
• 17 Tans G, Rosing J, Thomassen MCLDG, Heeb MJ, Zwaal RFA, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641.
• 18 Lormeau JC, Herault JP. Comparative inhibition of extrinsic and intrinsic thrombin generation by standard heparin, a low molecular weight heparin and the synthetic ATIII-binding pentasaccharide. Thromb Haemost 1993; 69: 152.
• 19 Lindhout T, Blezer R, Hemker HC. The anticoagulant mechanism of action of recombinant hirudin (CGP 39393) in plasma. Thromb Haemost 1990; 64: 464.
• 20 Herbert JM, Bernat A, Dol F, Herault JP, Crepon B, Lormeau JC. DX 9065A, a novel synthetic, selective and orally active inhibitor of factor Xa: In vitro and in vivo studies. J Pharmacol Exp Ther 1996; 276: 1030.
• 21 Tait JF, Gibson D. Measurement of membrane phospholipid asymmetry in normal and sickle-cell erythrocytes by means of annexin V binding. J Lab Clin Med 1994; 123: 741.
• 22 Connor J, Pak CC, Schroit AJ. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. J Biol Chem 1994; 269: 2399.
• 23 Cadroy Y, Hanson SR. Effects of red blood cell concentration on haemostasis and thrombus formation in a primate model. Blood 1990; 75: 2185.
• 24 Francis Jr RB, Johnson CG. Vascular occlusion in sickle cell disease: Current concepts and unanswered questions. Blood 1991; 77: 1405.