DOI 10.1007/s10517-015-2923-8 Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
205
Global Tests in Evaluation of the Function of Proand Anticoagulant Systems: Present and Future V. V. Udut*,**, I. I. Tyutrin***, M. A. Solov’ev*, V. F. Klimenkova***, E. F. Malyugin***, O. S. Karchagina*, E. V. Borodulina*, and A. V. Turenko*** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 162-165, February, 2015 Original article submitted April 13, 2014 Comparative analysis of the effectiveness of global tests for evaluation of the blood coagulation systems demonstrated the possibility of obtaining express data on hemostasis stages I-II and III when working with native substrate (whole blood). Changes in viscous characteristics of the whole blood recorded ex vivo during hemocoagulation allowed us to propose the concept of permanency of fibrinogenesis as the obligatory in vivo process determining the hemostatic potential, an integral characteristic of the hemocoagulation cycle providing sufficient blood fluidity and limiting extravasation of blood components under conditions of permeability disturbances and damage to the vascular wall. Key Words: hemocoagulation; hemostatic potential; whole blood; global tests; low-frequency piezoelectric thromboelastography Fatal outcomes of different diseases directly or indirectly associated with thrombosis and hemorrhages suggest that these complications are the leading cause of death. Discovery of genetic defects in the system of regulation of aggregation state of the blood (RASB) such as Leiden mutation (F5), prothrombin, plasminogen activator/inhibitor gene polymorphism (PAI-1), and platelet receptor mutations extended our knowledge on the ethiopathogenesis of primary (genetically determined) and secondary (acquired) thrombophilias. New data on the role of endothelium in RASB functioning and its effect on thromboresistance, vascular tone and permeability, blood cells adhesion, and angiogenesis allow us considering endothelial dysfunction as the key element of the pathogenesis of atherosclerosis, coronary heart disease, hypertension, cancer, diabetes mellitus, gestosis, and many other diseases. The status of RASB system is usually assessed by using clotting, amidolytic, and ELISA assays [4,5,7]. *E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine; **Tomsk National Research State University; ***Mednord-Tekhnika, Tomsk, Russia. Address for correspondence: [email protected]. M. A. Solov’ev
These methods have several basic imperfections such as low sensitivity, absence of standardization, longterm samples preparation, and the use of citrate plasma
Fig. 1. LPTEG diagram of aggregate state of the blood from a healthy individual. Ordinate: amplitude of the studied process (rel. units); abscissa: time of the observation (min). A0: zero point of amplitude in t0 on the time scale; t1: reaction period; time passed from zero point to maximum amplitude decrease (A1); t2: time of attaining A2 amplitude; A2: amplitude increase by 100 rel. units (A2A1); t3: time of coagulation (gel point) determined automatically by attaining 50% change in curve slope; A3: gel point amplitude; A4: amplitude in 10 min after gel point; t5: time of attaining maximum amplitude (A5) (time of fibrin-platelet clot formation); A6: amplitude in 10 min after attaining maximum amplitude.
0007-4888/15/15920205 © 2015 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
[1,2,10,11]. Despite the appearance of new technologies for detection of genetic thromboplilias, endothelial dysfunction markers, and antiphospholipid antibodies, the diagnostics and prevention of thrombosis and thrombohemorrhagic complications remain largely unsolved problems [12]. Nowadays, there are some innovations in clinical and laboratory practice, such as new personification
technology (point-of-care testing) with global tests to evaluate RASB system: thrombin generation test (TGT), thromboelastography (TEG), turbidimetric fibrinogenesis assay (TFA), Fourier transform mechanical spectroscopy (FTMS), and low-frequency piezoelectric thromboelastography (LPTEG) [6,13,14,15]. Here we compared the effectiveness of these methods in RASB system evaluation.
Fig. 2. Hemocoagulation curves. MA: maximum clot amplitude.
V. V. Udut, I. I. Tyutrin, et al.
MATERIALS AND METHODS The study was performed at the E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine and included 38 conventionally healthy volunteers (mean age 23±6.5 years); all examinees signed informed consent for participation in the study. Whole blood was used as the model medium. Standardization of the preparation stage was provided as follows: the blood (1 ml) was taken from the cubital vein without tourniquet application; ternary siliconized syringe (2.5 ml) and disposable cuvette made of medical plastic (0.45 ml) were used; the test was started immediately after blood sampling (in 10-12 sec). The results of global RASB test (TGT, TEG, TFA, and LPTEG) were analyzed. FTMS data were used as historical control, because we do not have devices to perform this study in Russia [13]. For recording of the coagulation process by the new LPTEG method, an ARP-01M Mednord hardware and software complex was used [3,8,9]. The analyzed parameters of coagulation were calculated as shown in Figure 1.
RESULTS The point-of-care mode (ex vivo studies) and the use of the whole blood allow evaluation of the interactions between the RASB system components under conditions of inevitable stressor influence standardized contact activation of hemocoagulation (constant volume and area of cuvette). Indeed, whole blood samples
Fig. 3. LPTEG and hemocoagulation scheme.
207 containing endothelium products (some of these act within limited time intervals: from 10–3 to 30 sec), blood cells, and serum factors of hemostasis provides instant objective information on the hemostatic potential and endothelial function [11,12]. Hemocoagulation processes assessed by TEG, TFA, LPTEG, and FTMS are shown on Figure 2. Lateral assembly and cross-linking of fibrin take ≈30±5 min and can be recorded by various methods. Gel point (time of blood coagulation) is a very important parameter characterizing initiation of blot formation and reflecting blood transition from sol (liquid state) to gel (solid state). This parameter is evaluated by two methods: FTMS and LPTEG. Fibrinolytic activity is evaluated by TEG, FTMS, and LPTEG. The initial stage of coagulation can be analyzed only by LPTEG. For the rest methods, this stage corresponds to “lag time” (TGT, TFA), “reaction time” (TEG), or “establishment of an incipient clot” (FTMS) [13]. Thus, among global tests for hemostatic potential evaluation in the whole blood, LPTEG best fulfils the main requirements for hemostasis evaluation tests (efficiency, informativeness, reliability). Superposition LPTEG curve of the whole blood coagulation and the hypothetic stage-by-stage hemocoagulation model provided a novel view on the RASB system as an interaction of spatially and functionally isolated parts (vascularplatelet, coagulative, anticoagulative, and fibrinolytic) in the maintenance of adequate hemostatic potential allowing permanent in vivo fibrinogenesis under conditions of activation, suppression, and limitations (Fig. 3).
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Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
From the viewpoint of the systemic analysis, the hemostatic potential of the whole blood is the resultant function of the RASB system in vivo, while its competence is determined by temporarily non-linear process of fibrinogenesis with well-known and axiomatic stages of initiation/amplification, propagation, lateral assembly and cross-linking of fibrin, clot retraction, and lysis. Temporal determinant of fibrinogenesis nonlinearity is determined by enzymatic potential of endothelial-intravascular continuum. Without contest of the thrombine role in this process, we should understand that its concentration and, more likely, its activity regulated by anticoagulative potential provided by C and S proteins, thrombomoduline, AT-3, α2-macroglobulin, NO, and prostacyclines determines completeness or incompleteness of fibrinogenesis under conditions of suppression or limitation of fibrin formation rate by its intermediates and vascular wall products. Thus, seconds and milliseconds needed for thrombin generation in vivo are enough for fibrinogenesis initiation, while its consistency directly correlates with the intensity of formation and volume of thrombin pool together with volume blood flow in different zones of the vascular bed.
REFERENCES 1. V. P. Baluda, M. V. Baluda, I. I. Deyanov, and I. K. Tlepshukov, Physiology of Hemostasis [in Russian], Moscow (1995).
2. Z. S. Barkagan and A. P. Momot, Diagnostics and Controlled Therapy of Hemostasis Disorders [in Russian], Moscow (2008). 3. Diagnosis and Prevention of Thromboses and Thromboembolism [in Russian], Ed. G. T. Kairov, Tomsk (2011). 4. V. V. Dolgov and P. V. Svirin, Laboratory Diagnosis of Hemostasis Disorders [in Russian], Moscow; Tver (2005). 5. A. A. Kishkun, Clinical Laboratory Diagnosis [in Russian], Moscow (2010). 6. A. P. Momot, Pathology of Hemostasis. Principles and Algorithms of Clinical Laboratory Diagnosis [in Russian], St. Petersburg (2006). 7. V. F. Klimenkova, Yu. A. Bochkov, M. N. Shpisman, and I. I. Tyutrin, Fundament. Issled., No. 8-2, 330-333 (2012). 8. Yu. A. Ovsyannikov and I. I. Tyutrin, Patent RF No. 107929, A Cuvette for Blood Tests, Byull., No. 25, September 10, 2011. 9. I. I. Tyutrin, V. V. Udut, and M. N. Shpisman, Low-Frequency Piezothromboelastography in the Diagnosis of Hemostasis Disturbances [in Russian], Tomsk (2013). 10. I. I. Tyutrin, V. F. Klimenkova, and V. V. Udut, Eksp. Klin. Farmakol., 77, No. 2, 21-25 (2014). 11. I. I. Tyutrin, V. V. Udut, and V. F. Klimenkova, Patogenez, 11, No. 4, 54-58 (2013). 12. M. A. Solov’ev, I. I. Tyutrin, V. V. Udut, and V. F. Klimenkova, Med.-Biol., Soc.-Psikhol. Problemy Bezopasnosti Chrezvych. Situatsiyakh, No. 4, 55-60 (2013). 13. P. A. Evans, K. Hawkins, M. Lawrence, et al., Med. Eng. Phys., 30, No. 6, 671-679 (2008). 14. H. C. Hemker, S. Wielders, H. Kessels, and S. Beguin, Thromb. Haemost., 70, No. 4, 617-624 (1993). 15. A. S. Wolberg, Blood Rev., 21, No. 3, 131-142 (2007).
205
Global Tests in Evaluation of the Function of Proand Anticoagulant Systems: Present and Future V. V. Udut*,**, I. I. Tyutrin***, M. A. Solov’ev*, V. F. Klimenkova***, E. F. Malyugin***, O. S. Karchagina*, E. V. Borodulina*, and A. V. Turenko*** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 162-165, February, 2015 Original article submitted April 13, 2014 Comparative analysis of the effectiveness of global tests for evaluation of the blood coagulation systems demonstrated the possibility of obtaining express data on hemostasis stages I-II and III when working with native substrate (whole blood). Changes in viscous characteristics of the whole blood recorded ex vivo during hemocoagulation allowed us to propose the concept of permanency of fibrinogenesis as the obligatory in vivo process determining the hemostatic potential, an integral characteristic of the hemocoagulation cycle providing sufficient blood fluidity and limiting extravasation of blood components under conditions of permeability disturbances and damage to the vascular wall. Key Words: hemocoagulation; hemostatic potential; whole blood; global tests; low-frequency piezoelectric thromboelastography Fatal outcomes of different diseases directly or indirectly associated with thrombosis and hemorrhages suggest that these complications are the leading cause of death. Discovery of genetic defects in the system of regulation of aggregation state of the blood (RASB) such as Leiden mutation (F5), prothrombin, plasminogen activator/inhibitor gene polymorphism (PAI-1), and platelet receptor mutations extended our knowledge on the ethiopathogenesis of primary (genetically determined) and secondary (acquired) thrombophilias. New data on the role of endothelium in RASB functioning and its effect on thromboresistance, vascular tone and permeability, blood cells adhesion, and angiogenesis allow us considering endothelial dysfunction as the key element of the pathogenesis of atherosclerosis, coronary heart disease, hypertension, cancer, diabetes mellitus, gestosis, and many other diseases. The status of RASB system is usually assessed by using clotting, amidolytic, and ELISA assays [4,5,7]. *E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine; **Tomsk National Research State University; ***Mednord-Tekhnika, Tomsk, Russia. Address for correspondence: [email protected]. M. A. Solov’ev
These methods have several basic imperfections such as low sensitivity, absence of standardization, longterm samples preparation, and the use of citrate plasma
Fig. 1. LPTEG diagram of aggregate state of the blood from a healthy individual. Ordinate: amplitude of the studied process (rel. units); abscissa: time of the observation (min). A0: zero point of amplitude in t0 on the time scale; t1: reaction period; time passed from zero point to maximum amplitude decrease (A1); t2: time of attaining A2 amplitude; A2: amplitude increase by 100 rel. units (A2A1); t3: time of coagulation (gel point) determined automatically by attaining 50% change in curve slope; A3: gel point amplitude; A4: amplitude in 10 min after gel point; t5: time of attaining maximum amplitude (A5) (time of fibrin-platelet clot formation); A6: amplitude in 10 min after attaining maximum amplitude.
0007-4888/15/15920205 © 2015 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
[1,2,10,11]. Despite the appearance of new technologies for detection of genetic thromboplilias, endothelial dysfunction markers, and antiphospholipid antibodies, the diagnostics and prevention of thrombosis and thrombohemorrhagic complications remain largely unsolved problems [12]. Nowadays, there are some innovations in clinical and laboratory practice, such as new personification
technology (point-of-care testing) with global tests to evaluate RASB system: thrombin generation test (TGT), thromboelastography (TEG), turbidimetric fibrinogenesis assay (TFA), Fourier transform mechanical spectroscopy (FTMS), and low-frequency piezoelectric thromboelastography (LPTEG) [6,13,14,15]. Here we compared the effectiveness of these methods in RASB system evaluation.
Fig. 2. Hemocoagulation curves. MA: maximum clot amplitude.
V. V. Udut, I. I. Tyutrin, et al.
MATERIALS AND METHODS The study was performed at the E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine and included 38 conventionally healthy volunteers (mean age 23±6.5 years); all examinees signed informed consent for participation in the study. Whole blood was used as the model medium. Standardization of the preparation stage was provided as follows: the blood (1 ml) was taken from the cubital vein without tourniquet application; ternary siliconized syringe (2.5 ml) and disposable cuvette made of medical plastic (0.45 ml) were used; the test was started immediately after blood sampling (in 10-12 sec). The results of global RASB test (TGT, TEG, TFA, and LPTEG) were analyzed. FTMS data were used as historical control, because we do not have devices to perform this study in Russia [13]. For recording of the coagulation process by the new LPTEG method, an ARP-01M Mednord hardware and software complex was used [3,8,9]. The analyzed parameters of coagulation were calculated as shown in Figure 1.
RESULTS The point-of-care mode (ex vivo studies) and the use of the whole blood allow evaluation of the interactions between the RASB system components under conditions of inevitable stressor influence standardized contact activation of hemocoagulation (constant volume and area of cuvette). Indeed, whole blood samples
Fig. 3. LPTEG and hemocoagulation scheme.
207 containing endothelium products (some of these act within limited time intervals: from 10–3 to 30 sec), blood cells, and serum factors of hemostasis provides instant objective information on the hemostatic potential and endothelial function [11,12]. Hemocoagulation processes assessed by TEG, TFA, LPTEG, and FTMS are shown on Figure 2. Lateral assembly and cross-linking of fibrin take ≈30±5 min and can be recorded by various methods. Gel point (time of blood coagulation) is a very important parameter characterizing initiation of blot formation and reflecting blood transition from sol (liquid state) to gel (solid state). This parameter is evaluated by two methods: FTMS and LPTEG. Fibrinolytic activity is evaluated by TEG, FTMS, and LPTEG. The initial stage of coagulation can be analyzed only by LPTEG. For the rest methods, this stage corresponds to “lag time” (TGT, TFA), “reaction time” (TEG), or “establishment of an incipient clot” (FTMS) [13]. Thus, among global tests for hemostatic potential evaluation in the whole blood, LPTEG best fulfils the main requirements for hemostasis evaluation tests (efficiency, informativeness, reliability). Superposition LPTEG curve of the whole blood coagulation and the hypothetic stage-by-stage hemocoagulation model provided a novel view on the RASB system as an interaction of spatially and functionally isolated parts (vascularplatelet, coagulative, anticoagulative, and fibrinolytic) in the maintenance of adequate hemostatic potential allowing permanent in vivo fibrinogenesis under conditions of activation, suppression, and limitations (Fig. 3).
208
Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
From the viewpoint of the systemic analysis, the hemostatic potential of the whole blood is the resultant function of the RASB system in vivo, while its competence is determined by temporarily non-linear process of fibrinogenesis with well-known and axiomatic stages of initiation/amplification, propagation, lateral assembly and cross-linking of fibrin, clot retraction, and lysis. Temporal determinant of fibrinogenesis nonlinearity is determined by enzymatic potential of endothelial-intravascular continuum. Without contest of the thrombine role in this process, we should understand that its concentration and, more likely, its activity regulated by anticoagulative potential provided by C and S proteins, thrombomoduline, AT-3, α2-macroglobulin, NO, and prostacyclines determines completeness or incompleteness of fibrinogenesis under conditions of suppression or limitation of fibrin formation rate by its intermediates and vascular wall products. Thus, seconds and milliseconds needed for thrombin generation in vivo are enough for fibrinogenesis initiation, while its consistency directly correlates with the intensity of formation and volume of thrombin pool together with volume blood flow in different zones of the vascular bed.
REFERENCES 1. V. P. Baluda, M. V. Baluda, I. I. Deyanov, and I. K. Tlepshukov, Physiology of Hemostasis [in Russian], Moscow (1995).
2. Z. S. Barkagan and A. P. Momot, Diagnostics and Controlled Therapy of Hemostasis Disorders [in Russian], Moscow (2008). 3. Diagnosis and Prevention of Thromboses and Thromboembolism [in Russian], Ed. G. T. Kairov, Tomsk (2011). 4. V. V. Dolgov and P. V. Svirin, Laboratory Diagnosis of Hemostasis Disorders [in Russian], Moscow; Tver (2005). 5. A. A. Kishkun, Clinical Laboratory Diagnosis [in Russian], Moscow (2010). 6. A. P. Momot, Pathology of Hemostasis. Principles and Algorithms of Clinical Laboratory Diagnosis [in Russian], St. Petersburg (2006). 7. V. F. Klimenkova, Yu. A. Bochkov, M. N. Shpisman, and I. I. Tyutrin, Fundament. Issled., No. 8-2, 330-333 (2012). 8. Yu. A. Ovsyannikov and I. I. Tyutrin, Patent RF No. 107929, A Cuvette for Blood Tests, Byull., No. 25, September 10, 2011. 9. I. I. Tyutrin, V. V. Udut, and M. N. Shpisman, Low-Frequency Piezothromboelastography in the Diagnosis of Hemostasis Disturbances [in Russian], Tomsk (2013). 10. I. I. Tyutrin, V. F. Klimenkova, and V. V. Udut, Eksp. Klin. Farmakol., 77, No. 2, 21-25 (2014). 11. I. I. Tyutrin, V. V. Udut, and V. F. Klimenkova, Patogenez, 11, No. 4, 54-58 (2013). 12. M. A. Solov’ev, I. I. Tyutrin, V. V. Udut, and V. F. Klimenkova, Med.-Biol., Soc.-Psikhol. Problemy Bezopasnosti Chrezvych. Situatsiyakh, No. 4, 55-60 (2013). 13. P. A. Evans, K. Hawkins, M. Lawrence, et al., Med. Eng. Phys., 30, No. 6, 671-679 (2008). 14. H. C. Hemker, S. Wielders, H. Kessels, and S. Beguin, Thromb. Haemost., 70, No. 4, 617-624 (1993). 15. A. S. Wolberg, Blood Rev., 21, No. 3, 131-142 (2007).
