Anesth Pain Med 2009; 4: 133～137 ￭실험연구￭
The effects of red blood cells on coagulation: a thromboelastographic study
Department of Anesthesiology and Pain Medicine, Sungkunkwan University School of Medicine, *The Catholic University of Korea College of Medicine, Seoul, Korea
Sangmin Maria Lee, M.D., Joo Yeon Lee, M.D., Daemyoung Jeong, M.D., and Keon Hee Ryu, M.D.*
Background: There are reports suggesting the effect of red blood platelet-rich plasma (PRP).9) In patients with sickle cell disease, cells (RBCs) on blood coagulation. The effects of red blood cells the change in RBC membrane that occurs in deoxygenated (RBCs) on coagulation were investigated in vitro while maintaining sickle RBCs was shown to release platelet factor 3, and, sub- other coagulation elements constant. Methods: Twenty-five healthy male volunteers were enrolled. sequently, to activate platelets to cause vasoocclusive crisis in Citrated fresh whole blood was drawn from each subjects and hypoxic condition.10) The RBC membrane normally has two processed into washed RBCs and platelet-rich plasma (PRP). To aminophospholipids (phosphatidyl serine and phosphatidyl etha- make six different hematocrit groups with each blood, PRP was mixed with the same volume of serially diluted washed RBCs. nolamine) in inner leaflet of the membrane bilayer, and they Reaction time, coagulation time, clot formation rate, and maximum are exposed to outer membrane leaflet of deoxygenated and ir- amplitude were measured using recalcified TEG. reversibly sickled RBCs and senescent normal cells.8,11) Results: The mean ± SD of six different hematocrit was 38.0 ± Phosphatidyl serine provides a catalytic surface for coagulation 2.3% (group 1), 28.9 ± 2.2% (group 2), 21.3 ± 1.9% (group 3), 12) 13.8% ± 1.6% (group 4), 7.1 ± 1.0% (group 5), and 0 ± 0% (group factors and thus can exhibit the procoagulant activity. Others, 6). The platelet count ranged from 141,000 to 292,000/mm3. however, proposed that hemodilution may increase blood Maximum amplitude (r = −0.4213, P ＜ 0.001) and alpha angle coagulability.13) Tocantins et al14) demonstrated hypercoagulable (r = −0.216, P ＜ 0.05) showed statistically significant negative changes after hemodilution of whole blood up to 40% with linear relationship with hematocrit. Conclusions: A gradual reduction in hematocrit was associated crystalloids. Additionally, hemodilution of blood up to 20% with a shortened coagulation time, no changes in reaction time. This with either crystalloid or colloid improved coagulation profile study results suggest that a gradual reduction in the RBC mass in an in vivo study.15) The precise mechanism of the effects of in vitro accelerates coagulation and forms stronger fibrin strands. hemodilution is unclear, but a relative decrease in antithrombin (Anesth Pain Med 2009; 4: 133∼137) concentration was speculated to be the cause, and the proper- Key Words: coagulation, red blood cell, thromboelastography. ties of diluent itself do not appear to play an important role. This discrepancy may be stemming from the fact that in INTRODUCTION most studies the effects of RBCs on coagulation have been in- vestigated by comparing a limited number of blood test results, A number of reports suggested the red blood cells (RBCs) although homeostasis of coagulation is achieved by an appro- 1-8) affect blood coagulation via various mechanisms. The pro- priate balance between procoagulants and anticoagulants with coagulant activity of RBCs was shown by a shortened onset interactions of endothelium, blood cellular components (platelets time of coagulation in whole blood of patients suffering from and RBCs), and coagulation proteins in plasma. For example, deep vein thrombosis or preeclampsia, compared with that of the role of RBCs in uremia and in severe anemia was tested using bleeding time and/or platelet adhesiveness.16,17) There was 논문접수일：2009년 3월 12일 a negative correlation between the log value of bleeding time 책임저자：이상민, 서울시 강남구 일원동 50번지 and the hematocrit in uremic patients, and transfusion of RBCs 삼성서울병원 마취통증의학과 우편번호: 135-230 increased the platelet adhesiveness and returned the bleeding Tel: 02-3410-0319, Fax: 02-3410-0369 time to normal in spite of a reduction of the total platelet E-mail: [email protected] count by 20%.
133 134 Anesth Pain Med Vol. 4, No. 2, 2009
Thromboelastography (TEG) which measures the dynamic and calcium chloride (0.2 M, 0.03 ml, Haemoscope corpo- viscoelasticity during whole blood clotting process has been ration) was added for recalcification. The blood and calcium widely used in investigational and clinical field,1,3,4,18) and ap- chloride were mixed by moving the TEG pin up and down pears to have advantages over other techniques in assessing five times, and TEG was maintained at 37oC. Four drops of blood coagulability. In this technique, coagulation is activated mineral oil was added to the surface of the blood to prevent when the blood contacts with inner surface of cup of TEG evaporation. TEG recording began 4 min after recalcification, rather than vascular endothelium. Several TEG variables have and measured variables were reaction time (r, min), coagulation been used to elucidate the blood clotting process. Briefly, the time (k, min), clot formation rate (α, o ), and maximum ampli- reaction time (r) is the time taken to form the initial clot by tude (MA, mm). activation of coagulation factors together with interaction with Data were analyzed using a statistical package (SPSS 7.5 for ionized calcium and platelets. Coagulation time (k) is the time windows; SPSS, Chicago, IL). Analysis of variance of TEG taken to reach an amplitude of 20 mm and is considered the variables was performed to test the dilutional effect on function of coagulation factors and platelets. The clot formation coagulation. For comparison of groups, analysis of variance rate (α angle) represents the rate of clot formation and is the was performed based on ranks followed by Dunnetts’ method. function of fibrinogen and platelets. The maximum amplitude The relationship between hematocrit and each of four TEG (MA) mainly depends on the quantitative and qualitative func- variables were obtained by regression analysis calculating tion of platelets. correlation. All data are presented as means ± SD, and P ＜ The specific objective of the study was to investigate the 0.05 was considered significant. hypothesis that blood coagulability (the rate of clot formation and/or the strength of clot formed) is affected by the amount RESULTS of RBCs while other coagulation elements (coagulation factors and platelets) remain relatively constant. One subject with severe thrombocytopenia (platelet count, 26,000/mm3) was excluded from analysis. Average age of the − MATERIALS AND METHODS 24 subjects was 33 ± 7 years (range, 21 45 years), platelet count was 222,000 ± 40,000/mm3 (range, 141,000 − 292,000/ After the approval of the Institutional Research Review mm3) and hematocrit was 38 ± 2 % (range, 33.5−44%). The Board and obtaining written informed consent, 25 healthy vol- RBC dilutional effects on coagulation of 24 patients are shown unteers were included in this study. They were adult male in Fig. 1. Reaction time and coagulation time remained un- subjects with no history of smoking, coagulation disorder, ane- changed at different levels of RBC concentration, but clot for- mia, liver disease, and any recent medications within two mation rate (α) and maximum amplitude (MA) decreased weeks. Aseptic venopuncture was made in the antecubital vein using a 20-guage venous catheter. The first 2 ml of blood was discarded to avoid tissue contamination, and 9 ml of blood was collected in a test tube containing sodium citrate solution (1 ml, 3.8%). The citrated whole blood sample was centrifused immediately at 300 g for two minutes to separate the PRP from the packed RBCs (pRBCs). Separated pRBCs were wash- ed three times by adding the same volume of normal saline, centrifusing it at 3,000 g for two and half minutes, and re- moving supernatant at each time. The washed pRBCs were divided into six alliquotes (0.3 ml each), and normal saline was added to make pRBCs and normal saline in a ratio of 5：0, 4：1, 3：2, 2：3, 1：4, and 0：5. Each reconstituted pRBC samples (0.3 ml) were mixed with autologous PRP (0.3 Fig. 1. The relationship between hematocrit and four TEG variables. ml) to maintain the same amount of autologous PRP. Each ci- Maximum amplitude (MA, r=−0.4213, P ＜ 0.001) and alpha angle (r= trated blood specimen (0.33 ml) was placed into the TEG cup, −0.216, P ＜ 0.05) showed negative linear relationship with hematocrit. Sangmin Maria Lee, et al：RBC effects on coagulation 135 steadily with hemoconcentration (P ＜ 0.05). Hematologic and respectively), indicating stronger fibrin strands. TEG variables of 24 subjects at six different dilutions are shown in Table 1. Hematocrit decreased gradually by the study DISCUSSION design as blood was diluted, while platelet count was similar in all six dilutions. Except for blood specimens containing no In this study, a gradual hemodilution increased the rate of RBCs, hemodilution decreased the reaction time (at dilution clot formation and the strength of clot formed, when the 1:4) and coagulation time (at all dilutions) and increased the amount of platelets and coagulation factors remained unchang- clot formation rate (at all dilutions) and maximum amplitude ed. However, the procoagulant activity of hemodilution was not (at dilutions 3:2, 2:3, and 1:4), suggesting increased coagula- observed in platelets and PRP without RBCs. bility with hemodilution compared with the blood with no This observation is different from results of other studies dilution. that proposed the procoagulant effects of RBCs, and several Because of the difference in the baseline hematocrit of all mechanisms are proposed. First, in the biochemical mechanism, subjects, the 144 data points were divided into six groups RBCs appear to stimulate platelet activation and aggregation. based on the hematocrit value: Group 1, 35% ＜ Hct ≤ 44%; For example, platelets mixed with RBCs, after stimulated by Group 2, 25% ＜ Hct ≤ 35%; Group 3, 17% ＜ Hct ≤ collagen, increased thromboxane B2 (TXB2) production and ad- 25%; Group 4, 9% ＜ Hct ≤ 17%; Group 5, 0% ＜ Hct ≤ enosine diphosphate (ADP) release as compared with platelet 9%; and Group 6, 0% (Table 2). The relationship between alone.19) Additionally, stimulation of platelets with collagen in hematocrit and four TEG variables were analyzed using the vitro in the presence of metabolically active RBCs increased Spearman’s correlation. The reaction time remained unchanged the serotonin release by platelets indicating platelet activation.20) at all RBC concentrations (Fig. 1). Similar to the data in Second, RBC membranes appear to affect the coagulation Table 1, the coagulation time (K) decreased gradually in process. Phospholipid, especially phosphatidyl serine, exerts cat- Group 2 through Group 5, indicating accelerated coagulation. alytic surface for activation of plasma coagulation factors. However, the coagulation time of Group 6 with no RBCs was Although phosphatidyl serine is normally provided by activated essentially similar to that of Group 1. Clot formation rate (α) platelets (platelet factor 3), RBC membrane has twice as much and maximum amplitude increased gradually as blood is di- phosphatidyl serine. This phenomenon explains the clot-promot- luted (r = −0.4213, P ＜ 0.001 and r = −0.216, P ＜ 0.05, ing effect of damaged or hemolyzed RBCs. Morphological
Table 1. Hematologic and TEG Variables of 24 Subjects with Six Different Dilutions
Dilution 5:0 Dilution 4:1 Dilution 3:2 Dilution 2:3 Dilution 1:4 Dilution 0:5
Hematocrit 37.5 ± 2.2 28.8 ± 2.2 21.5 ± 1.7 13.6 ± 1.4 7.2 ± 1.0 0 ± 0 Reaction time 10.7 ± 2.3 10.2 ± 2.6 10.3 ± 2.8 9.8 ± 1.9 9.6 ± 2.4* 11.3 ± 2.9 Coagulation time 17.1 ± 2.7 15.6 ± 6.2* 14.0 ± 3.2* 13.1 ± 2.4* 13.5 ± 3.2* 16.4 ± 3.9 Clot formation rate 42 ± 7 49 ± 6* 54 ± 7* 57 ± 7* 54 ± 7* 47 ± 9 Maximum amplitude 43 ± 5 45 ± 6 48 ± 5* 51 ± 6* 49 ± 6* 48 ± 6*
Values are expressed as mean ± SD. *P ＜ 0.05 as compared with dilution 5:0.
Table 2. Hematologic and TEG Variables of 24 Subjects Divided into 6 Groups Based on Hematocrit
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Hematocrit 38.0 ± 2.3 28.9 ± 2.2 21.3 ± 1.9 13.8 ± 1.6 7.1 ± 1.0 0 ± 0 Reaction time 10.5 ± 2.5 9.9 ± 2.7 9.8 ± 2.8 10.0 ± 1.9 9.6 ± 2.5 11.7 ± 3.1 Coagulation time 16.7 ± 3.2 14.2 ± 3.0* 13.4 ± 3.1* 13.4 ± 2.6* 13.1 ± 2.9* 16.8 ± 4.1 Clot formation rate 40 ± 6 49 ± 5* 55 ± 5 * 57 ± 7* 56 ± 6* 46 ± 9 Maximum amplitude 41 ± 5 45 ± 6 47 ± 6* 49 ± 6* 49 ± 5* 47 ± 6*
Values are expressed as mean ± SD. *P ＜ 0.05 as compared with group 1. 136 Anesth Pain Med Vol. 4, No. 2, 2009 changes in RBC membrane also affect coagulation. Phosphati- clot formation and the strength of clot formed, when the dyl serine, normally located in inner membrane layer of RBCs, amount of platelets and coagulation factors remained unchang- is exposed itself on outer layer in sickled, deoxygenated RBCs ed. And the role of red blood cells on coagulation in the pres- or in senescent normal RBCs to cause activation of pro- ence of normal platelets and PRP in the clinical setting need thrombin to thrombin.8,11) Third, the physical role of RBCs on to be solved. coagulation also has been suggested,7,21,22) based on the fact that RBCs affect platelet aggregation by altering blood vis- REFERENCES cosity and shear rate.22,23) This is indirectly observed in red clots, a mixture of platelets, RBCs, white blood cells, and fi- 1. Zuckerman L, Cohen E, Vagher JP, Woodward E, Caprini JA: brin, formed in the venous side, in contrast to white thrombi Comparison of thromboelastography with common coagulation composed of platelets in the arterial side. tests. Thromb Haemost 1981; 46: 752-6. The mechanism of the procoagulant acitivity of hemodilution 2. Shaper AG, Kyobe J, Mwanje DK: Fibrinolytic activity and clot observed in this study is unclear. All blood samples of this strength in anemia. Am J Clin Pathol 1967; 47: 344-6. 3. Kim HW, Stubdal H, and Greenburg AG: Coagulation dynamics study contained the same volume of PRP, suggesting that low after hemodilution with polyhemoglobin. Surg Gynecol Obstet concentration of antithrombin-III should not be the cause of 1992; 175: 219-26. the procoagulants activity. The next logical question is whether 4. Holloway DS, Zuckerman L, Vagher JP, Mockros L: The effects RBC has certain inhibitory role on coagulation to prevent ex- of prostacyclin on the coagulation of whole blood. Thromb cessive clot formation. Another possible explanation may come Haemost 1983; 50: 671-5. from the complexity of coagulation process. Coagulation is the 5. Kawakami S, Kaibara M, Kawamoto Y, Yamanaka K: Rheological approach to the analysis of blood coagulation in endothelial net result of dynamic interactions among the vascular endothe- cell-coated tubes: activation of the intrinsic reaction on the lium, humoral, and cellular components in flowing blood, and erythrocyte surface. Biorheology 1995; 32: 521-36. alteration of one component may affect others with a positive 6. Stadler I, Toumbis CA, Ambrus AL: Influence of cold storage or negative feedback. altered red cell surface on the function of platelets. J med 1994; Another reason for the difference in results may be found in 25: 353-61. 7. Goldsmith HL, Kaufer ES, McIntosh FA: Effect of hematocrit on the test methodology. Several studies showing the procoagulant adenosine diphosphate-induced aggregation of human platelets in effects of RBCs were tested in the endothelial cell-coated tub- tube flow. Biorheology 1995; 32: 537-52. 5,9) 7) ing or in tube flow. In this study, blood coagulability was 8. Test ST, Mitsuyoshi J: Activation of the alternative pathway of tested by TEG. This technique measures shear elasticity of fi- complement by calcium-loaded erythrocytes resulting from loss of brin strands formed in a real time fashion and is known to re- membrane phospholipids asymmetry. J Lab Clin Med 1997; 130: semble clinical coagulation on the surgical field.1,3,4,18) In TEG, 169-82. 8. Kaibara M: Rheology of blood coagulation. Biorheology 1996; 33: surface activation of coagulation is initiated by metallic surface 101-17. of cup instead of vascular endothelium. It is possible that sur- 9. Toumbis CA, Stadler I, Ambrus JL: Effect of red blood cells from face activation of coagulation process by the metallic cup of patients with sickle cell disease on platelet factor 3 release. Am TEG might have affected or masked the role of RBCs in ini- J Hematol 1992; 40: 149-50. tiating blood coagulation. Another limitation of TEG is that 10. Chiu D, Lubin B, Roelofsen B, Van Deene LLM: Sickled erythrocytes accelerate clotting in vitro: an effect of abnormal coagulability is tested in a stagnant blood instead of circulating membrane lipid asymmetry. Blood 1981; 58: 398-401. blood. 11. Marcus AJ: The role of lipid in blood coagulation. Adv Lipid Res The next logical question is what is the role of hemodilu- 1966; 4: 1-37. tion on coagulation in the clinical arena, in the presence of 12. Tocantins LM, Carroll RT, Holburn RH: The clot accelerating normal platelets and PRP. This study clearly demonstrated that effect of dilution on blood and plasma. Relation to the mechanism blood coagulability is not reduced by decreasing RBC concen- of coagulation of normal and hemophilic blood. Blood 1959; 6: 720-39. tration. Does hemodilution activate coagulation to an excessive 13. Lee KM: Perioperative hemodilution. Korean J Anesthesiol 2005; degree to cause thrombosis? TEG variables of all studied 49: 1-10. group were within the normal range, suggesting no excessive 14. Ruttmann TG, James MFM, Viljoen JF: Hemodilution induces a activation is anticipated. hypercoagulable state. Br J Anaesth 1996; 76: 412-4. In conclusion, a gradual hemodilution increased the rate of 15. Fernandez F, Goudable C, Sie P, Ton-That H, Durand D, Suc JM, Sangmin Maria Lee, et al：RBC effects on coagulation 137
et al: Low hematocrit and prolonged bleeding time in uremic N, et al: Enhancement of platelet reactivity and modulation of patients: effect of red cell transfusions. Br J Hematol 1985; 59: eicosanoid production by intact erythrocytes. A new approach to 139-148. platelet activation and recruitment. J Clin Invest 1991; 87: 571-80. 16. Hellem AJ, Borchgrevink CF, Ames SB: The role of red cells in 20. Bell DN, Spain S, Goldsmith HL: The effect of red blood cells hemostasis: the relation between hematocrit, bleeding time and on the ADP-induced aggregation of human platelets in flow platelet adhesiveness. Br J Hematol 1961; 7: 42-50. through tubes. Thromb Haemost 1990; 63: 112-21. 17. Kang YG: Thromboelastography in liver transplantation. Semin 21. Karino T, Goldsmith HL: Adhesion of human platelets to collagen Thromb Hemost 1995; 21(suppl 4): 34-44. on the walls distal to tubular expansion. Microvasc Res 1979; 17: 18. Valles J, Santos MT, Aznar J, Marcus AJ, Martinez-Sales V, 238-62. Portoles M, et al: Erythrocytes metabolically enhance collagen- 22. Goldsmith HL: Red cell motions and wall interactions in tube induced platelet responsiveness via increased thromboxane produc- flow. Fed Proc 1971; 30: 1578-88. tion, adenosine diphosphate release, and recruitment. Blood 1991; 23. Turitto VT, Weiss H: Platelet and red cell involvement in mural 78: 154-62. thrombogenesis. Ann NY Acad Sci 1982; 416: 363-76. 19. Santos MT, Valles J, Marcus AJ, Safier LB, Broekman MJ, Islam