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Tissue Factor and Tissue Factor Pathway Inhibitor Levels during and after Cardiopulmonary Resuscitation
Thrombosis Research 96 (1999) 107–113
REGULAR ARTICLE
Tissue Factor and Tissue Factor Pathway Inhibitor Levels during and after Cardiopulmonary Resuscitation Satoshi Gando, Satoshi Nanzaki, Yuji Morimoto, Shigeaki Kobayashi and Osamu Kemmotsu Department of Anesthesiology and Intensive Care, Hokkaido University School of Medicine, Kita-ku, N15 W7, Japan. (Received 20 January 1999 by Editor A. Takada; revised/accepted 7 April 1999)
Abstract Disseminated intravascular coagulation frequently occurs after global ischemia and reperfusion due to cardiac arrest. The present study was performed to demonstrate the role of tissue factor for coagulation pathway activation, as well as to investigate the precise time course of tissue factor pathway inhibitor (TFPI) during and after cardiopulmonary resuscitation (CPR). Thirty-two of out-of-hospital cardiac arrest patients were classified into two groups, those who achieved return of spontaneous circulation (ROSC) (n513) and those without ROSC (n519). Ten normal healthy volunteers served as control subjects. Serial levels of tissue factor and TFPI were measured during and after cardiac arrest and CPR. In patients with ROSC, cardiac arrest and CPR led to persistent increases in the levels of tissue factor that peaked 6 hours after arrival at the Emergency Department. Tissue factor levels in patients without ROSC also showed marked elevations compared to those of the control subjects. In both groups, the levels of TFPI were significantly lower than those in the control subjects. However, we could not find differences in the levels of the two markers between the patients Abbreviations: TFPI, tissue factor pathway inhibitor; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation. Corresponding author: Satoshi Gando, MD, Department of Anesthesiology and Intensive Care, Hokkaido University School of Medicine, N15 W7, Kita-ku, Sapporo, 060 Japan. Tel: 181 (11) 716 1161; Fax: 181 (11) 716 9666; E-mail: ,[email protected] dai.ac.jp..
with ROSC and those without ROSC. In conclusion, we demonstrated persistent elevation of the tissue factor levels associated with low TFPI during and after CPR in patients with out-of-hospital cardiac arrest. These results indicate the activation of the extrinsic coagulation pathway without adequate TFPI generation, which may contribute to thrombin activation and fibrin formation after whole-body ischemia and reperfusion. 1999 Elsevier Science Ltd. All rights reserved. Key Words: Tissue factor; Tissue factor pathway inhibitor; Cardiac arrest; Cardiopulmonary resuscitation
F
or global ischemia due to cardiac arrest there is frequent evidence of disseminated intravascular coagulation [1]. We previously demonstrated that during and after out-of-hospital cardiac arrest, fibrinopeptide A—a marker of thrombin activation—markedly elevated, followed by an increase in plasminogen activator inhibitor, which provides evidence of impaired fibrinolysis [2]. These data suggest that hypoxia, ischemia, and reperfusion accelerate coagulation, then suppress fibrinolysis. The physiologic trigger for coagulation appears to be the formation of complex between tissue factor and factor VII/VIIa. The VIIa tissue factor complex initiates the extrinsic coagulation pathway by activating both factors IX and X, leading to thrombin generation and fibrin formation [3]. An important inhibitor of tissue factor-initiated coagulation is the tissue factor pathway inhibitor (TFPI) [3]. Tissue hypoxia and ischemia cause endothelial
0049-3848/99 $–see front matter 1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00073-0
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S. Gando et al./Thrombosis Research 96 (1999) 107–113
damage, increase in tissue factor activity, and production of the factor Xa activator [4,5]. Using a baboon model of focal cerebral ischemia, Thomas and colleagues [6] showed that tissue factor-mediated events contribute to the no-reflow phenomenon in noncapillary microvessels after focal cerebral ischemia and reperfusion. Recently plasma TFPI levels in various diseases such as cardiovascular disease, blood disease, thromboembolic disease, and disseminated intravascular coagulation have been reported [7]. Endothelium under inflammatory conditions may synthesize increased amounts of TFPI [3]; however, few data are available for the TFPI levels during ischemia and reperfusion. To test the hypothesis that the exposure of tissue factor to the circulation contributes to intravascular coagulation, and to investigate the precise time course of the tissue factor and TFPI levels during and after human cardiac arrest, plasma levels of tissue factor and TFPI were prospectively measured.
1. Materials and Methods 1.1. Patients Approval for this study was obtained from the Ethics Committee of our institution. Informed consent was not obtained, because of the emergency situation with rare opportunity for family consultation. Cardiopulmonary arrest was defined as the absence of spontaneous respiration or palpable pulse and unresponsiveness. Patients over 16 years of age with cardiopulmonary arrest outside the hospital were included in this study. Patients were excluded if they were over 90 years of age, had terminal illness, hypothermia, trauma, or if they had clear signs of irreversible arrest (such as rigor mortis or dependent lividity). Ten normal volunteers (seven males/three females, age 3162 years) served as control subjects.
suscitation (CPR) procedure followed the 1992 Standards and Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiac Care [8]. ECG was monitored and blood pressure was continuously measured using a femoral arterial catheter. Patients showing a recovery of blood pressure and pulse for more than 1 hour were defined as return of spontaneous circulation (ROSC), whether or not catecholamines were used. The patients who died during CPR without ROSC were defined as death. All patients were administered about 700– 2000 IU heparin/day for the anticoagulation of the arterial catheter. Characteristics of the Emergency Medical Service system and Emergency Department were described according to the Utstein Style [9]. Patient outcome at the time of hospital discharge was evaluated based on cerebral and overall performance category [9].
1.3. Study Protocol Blood samples were collected using an arterial catheter immediately after arrival at the Emergency Department (time point 1). The initial 5 mL of every blood were discarded. The second blood samples were drawn when CPR was discontinued due to death (in cases of death) or at 30 minutes after arrival (in cases of ROSC) (time point 2). In the cases of ROSC, additional blood samples were taken 60 minutes after (time point 3), and 6 hours (time point 4) and 24 hours after (time point 5) the ROSC had been achieved. The measured parameters are as follows. Tissue factor antigen concentration (tissue factor) was measured by ELISA (Tissue Factor ELISA kit, American Diagnostica, Greenwich, CT, USA). Reference values for normal healthy control subjects were 135.468.1 pg/mL (n510). Total TFPI antigen concentration were measured by ELISA (TFPI ELISA kit, American Diagnostica, Greenwich, CT, USA). Reference values for normal healthy control subjects were 118.0612.3 ng/mL (n510).
1.2. Treatment Protocol 1.4. Statistical Analysis All patients were given artificial ventilation using a bag-valve-mask unit or esophageal tracheal double lumen airway (Combitube, Sheridan, NY, USA) with 100% oxygen and cardiac massage by the Emergency Medical Service. Cardiopulmonary re-
All data were expressed as mean6SEM. A statistical software package (Stat View 4.5, Abacus Concepts, Berkeley, CA, USA) was used for all statistical calculation analysis. Differences between more
109
S. Gando et al./Thrombosis Research 96 (1999) 107–113
Table 1. Clinical characteristics of the patients and Emergency Medical Service system
Age (years) Male/female Witnessed arrest (yes/no) Bystander CPR (yes/no) Initial rhythm at ED (vf/asystole/other) Outcome (survived/died) Time interval* 1 2 3 4 5
ROSC (n513)
Death (n519)
p value
6863 9/4 8/5 2/11
6063 16/3 16/3 1/18
0.0949 0.5677 0.2988 0.7284
5/3/5 3/10
3/11/5 0/19
0.3370 –
6.360.5 26.162.3 14.163.1 – 36.164.8
6.260.6 27.661.8 – 30.462.9 58.263.5
0.5387 0.4887 – – 0.001
ROSC indicates return of spontaneous circulation; CPR, cardiopulmonary resuscitation; ED, Emergency Department. Values are mean6SEM. *1, interval between the call receipt and the vehicle stops; 2, interval between the vehicle stops and the arrival at the ED; 3, interval between the arrival at the ED and the ROSC; 4, interval between the arrival at the ED and the cessation of CPR; 5, total CPR time.
than two matched samples were tested by Friedman’s test, followed by Wilcoxon signed-rank test. When two groups were being compared, the MannWhitney U test or chi-square test were used. A p,0.05 was considered statistically significant.
mance category54.0) and the third patient had good overall and cerebral performance categories (overall performance category5cerebral performance category51.0).
2.2. Changes in Tissue Factor and TFPI
2. Results 2.1. Baseline Characteristics Characteristics of the patients and Emergency Medical Service system are shown in Table 1. We studied 32 patients, 13 of whom successfully achieved ROSC while the remaining 19 patients died without ROSC. Except total CPR time, baseline characteristics between the two groups were the same. None of the patients in either group were taking drugs that affect blood coagulation. Table 2 shows the etiology of the cardiac arrest. There were statistical differences in the dose of epinephrine (p50.0004) and the use of other catecholamines (p50.002) between the two groups (Table 3). With regards to the outcome of the patients with ROSC, 10 died in a period of 2.3860.9 days and three were discharged from the hospital. At the time of discharge, two of the three patients were in a vegetative state (overall performance category5cerebral perfor-
Figure 1 shows the results of the measurement of tissue factor. In patients with ROSC, tissue factor levels at time point 1 (at the arrival to the Emergency Department) through time point 5 (24 hours after arrival) were significantly higher than those in the control subjects. The levels reached the highest values at time point 4 (6 hours after arrival), which showed a significant statistical difference from time
Table 2. Etiology of the cardiac arrest ROSC
Death
6
13 (12)
3 3 0 1 0
2 0 3 0 1
Cardiac (presumed) Noncardiac Central nervous system Respiratory Vascular Other Unknown ROSC indicates return of spontaneous circulation.
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S. Gando et al./Thrombosis Research 96 (1999) 107–113
Table 3. Clinical data and therapy during cardiopulmonary resuscitation
Epinephrine (mg) Other catecholamine (yes/no) Countershock (yes/no) Mean arterial pressure (mm Hg) Heart rate (beats/min)
ROSC (n513)
Death (n519)
p value
3.360.6 10/3 4/9 8868 11268
7.260.8 3/16 9/10 – –
0.0004 0.002 0.5669 – –
ROSC indicates return of spontaneous circulation.
point 1 (p,0.05). Tissue factor levels in the dead patients also showed marked differences compared to those in the control subjects. Figure 2 shows the changes in TFPI. At time points 1 and 2, the levels of the TFPI in both groups were significantly lower than those in the control subjects. After time point 2, TFPI values in patients with ROSC increased significantly compared to those of time point 1. p values for the Friedman test in patients with ROSC were 50.011 for tissue factor and 50.0036 for TFPI. We could not find any differences in the levels of the two markers between the patients with ROSC and those without ROSC.
3. Discussion
Fig. 1. Bar graphs showing changes in tissue factor antigen concentration (tissue factor) during and after cardiopulmonary resuscitation at time points 1, 2, 3, 4, and 5. See “Methods” for definitions of the time points. Open bar, control subjects; hatched bar, patients with return of spontaneous circulation (ROSC); dark stippled bar, patients without ROSC (death). *p,0.05, **p,0.01, 1p,0.001, 11p,0.0001 vs. control. #p,0.05 vs. time point 1.
Fig. 2. Bar graphs showing changes in tissue factor pathway inhibitor (TFPI) during and after cardiopulmonary resuscitation at time points 1, 2, 3, 4, and 5. See “Methods” for definitions of the time points. Open bar, control subjects; hatched bar, patients with return of spontaneous circulation (ROSC); dark stippled bar, patients without ROSC (death). *p,0.05 vs. control, #p,0.05 and p,0.01 vs. time point 1.
In the former study [2], we demonstrated that plasma fibrinopeptide A, a marker of thrombin activation, and a cross-linked fibrin degradation product (D-dimer), a marker of fibrin formation (and subsequent fibrinolysis), had persistently elevated from the arrival at the Emergency Department to 24 hours after the arrival in patients with out-of-hospital cardiac arrest. Bo¨ttiger et al. [1] also showed the activation of blood coagulation after cardiac arrest. The exposure of blood to tissue factor and the formation of factor VIIa tissue factor
S. Gando et al./Thrombosis Research 96 (1999) 107–113
complexes appear to be the primary mechanism for initiating blood coagulation during hemostasis [3,10]. Activation of factor XII initiates the contact activation reactions that result in activation of factor XI; however, these reactions are only required for blood to clot normally in a glass test tube. Therefore, the contact activation reactions are not required for hemostasis [10]. The interpretation of plasma tissue factor antigen assay used in this study may be complicated by the fact that it is not clear whether tissue factor detectable by our assay is in a functional form. However, since tissue factor requires phospholipid for its activity, free soluble tissue factor in plasma does not reflect the potential for clotting activity [11]. Further, we proved significant correlations between plasma tissue factor and various thrombin markers such as prothrombin fragment F112, thrombin antithrombin III complex, fibrinopeptide A, and D-dimer in patients with sepsis and trauma [12]. In the present study, we did not measure thrombin markers. However, this evidence suggests that the elevated tissue factor levels are closely linked to the thrombin activation and fibrin formation during and after CPR observed in the former studies [1,2]. Several mechanisms by which cardiac arrest and resuscitation cause coagulation abnormalities are proposed. The impact of interventions during CPR, the amount of endogenously released and exogenously administered catecholamines, presence of indwelling catheter, and underlying etiology such as coronary artery disease may have influences on blood coagulation; however, we believe that the profound effect of cardiac arrest and resuscitation overshadow these effects. Tissue hypoxia and ischemia cause endothelial damage with a consequent increase in tissue factor activity and production of factor Xa activator [4,5]. We recently confirmed endothelial injury manifested by liberation of soluble thrombomodulin, a novel marker of endothelial injury, into the circulation in patients with cardiac arrest (unpublished data, 1998). The endothelial injury disrupts a barrier that normally separates the circulating blood from tissue factor, with the resultant binding of plasma factor VII to tissue factor [7,10,13]. Increased vascular permeability after ischemia and reperfusion observed by Goto et al. [14] may expose perivascular tissue factor to the plasma compartment with subsequent activation of the extrin-
111
sic coagulation pathway. In addition to constitutive expression, the tissue factor gene is inducibly expressed in several vascular cell types, including monocytes, endothelial cells, and smooth muscle cells [15]. P-selectin [16], oxygen-free radicals [17,18], and thrombin [2] generated during ischemia and reperfusion may induce tissue factor in monocytes and endothelial cells. Cytokines such as tumor necrosis factor-a and interleukin 1-b also induce the tissue factor gene [15]. However, we could not find any increase in these cytokines during and after CPR (unpublished data, 1998). Thus the above-mentioned factors (except cytokines) may contribute to the elevation of tissue factor observed in our present study. The TFPI is found in at least three intravascular pools [7,19]. A major pool is bound to the endothelial surface and the second pool is plasma TFPI. A smaller pool is circulating and is to a great extent in complex form with lipoproteins. A third pool of TFPI is located in platelet. Our assay used in the present study measures all parts of the TFPI. The endothelium is the principle site of synthesis responsible for maintaining the plasma levels of TFPI and the TFPI is removed from the plasma by the liver, kidney, and the low-density lipoprotein receptor-related protein-dependent mechanism [7,19]. The mechanisms by which cardiac arrest and CPR induced TFPI decrease are not precisely known. No patients were administered intravenous drip infusion before their arrival to the Emergency Department, so the effect of the hemodilution can be denied. Low levels of TFPI are occasionally seen in tissue factor-induced disseminated intravascular coagulation, but no unanimity has been achieved with regard to TFPI plasma level in patients diagnosed as having disseminated intravascular coagulation [7,19,20]. Neutrophil elastase cleaves TFPI between Thr87 and Thr88, which is within the polypeptide that links the first and second Kunitz domain [10]. This impairs the ability of TFPI to neutralize both factor VIIa tissue factor and factor Xa [10]. We already reported increased neutrophil elastase release in patients with cardiac arrest [21]. Thus, the neutrophil elastase may be proposed to be one of the reasons why TFPI levels decreased in the present study. The TFPI levels are also to a great extent dependent on low-density lipoproteins [19]. However, it is difficult to consider that plasma low-density lipoprotein varies during cardiac arrest
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and CPR. The last possibilities are that hypoxia and ischaemia may reduce the synthesis of TFPI from endothelial cells or enhance the clearance of TFPI. We used heparin for the purpose of anticoagulation of the arterial catheter inserted into the patients. The heparin being used in the flushing device may induce a significant increase of the TFPI levels after resuscitation in patients with ROSC [19,20,22]. Massive fibrin formation and consecutive impairment of fibrinolysis contribute to reperfusion disorder after cardiac arrest [2]. Tissue factorinduced activation of the extrinsic coagulation pathway as well as the low levels of TFPI observed in the present study may accelerate the derangement of vital organ dysfunction after cardiac arrest. Thomas and colleagues [6] demonstrated that tissue factor-mediated fibrin formation contribute to no-reflow in noncapillary microvessels and also proved that the antitissue factor monoclonal antibody TF9-6B4 increases the microvascular reflow after focal cerebral ischemia and reperfusion. Taylor and his group [23] has energetically clarified that antitissue factor antibody, recombinant TFPI, and active site-inhibited factor VIIa protect animals with sepsis-induced disseminated intravascular coagulation from organ failure and death. Recombinant TFPI also prevents venous thrombosis, rethrombosis after successful thrombolysis, and fibrin deposition on procoagulant subendothelial matrix [19]. The common predominator for all these conditions is the exposure of tissue factor, either by monocytes or by endothelial damage, to circulatory blood [19]. In conclusion, we demonstrated persistent elevation of tissue factor levels associated with low levels of TFPI during and after CPR in patients with outof-hospital cardiac arrest. These results indicate that activation of the extrinsic coagulation pathway without adequate TFPI generation may contribute to thrombin activation and fibrin formation after whole-body ischemia and reperfusion.
2.
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7.
8.
9.
10.
References 1. Bo¨ttiger BW, Motsch J, Bo¨hrer H, Bo¨ker T, Aulmann M, Nawroth PP, Martin E. Activation of blood coagulation after cardiac arrest is not balanced adequately by activation of en-
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dogenous fibrinolysis. Circulation 1995;92: 2572–8. Gando S, Kameue T, Nanzaki S, Nakanishi Y. Massive fibrin formation with consecutive impairment of fibrinolysis in patients with outof-hospital cardiac arrest. Thromb Haemost 1997;77:278–82. Østerud B, Bajaj MS, Bajaj SP. Sites of tissue factor pathway inhibitor (TFPI) and tissue factor expression under physiologic and pathologic conditions. Thromb Haemost 1995;73: 873–5. Gertler JP, Abbott WM. Current research review. Prothrombotic and fibrinolytic function of normal and perturbed endothelium. J Surg Res 1992;52:89–95. Jaffe EA. Biochemistry, immunology, and cell biology of endothelium. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, editors. Hemostasis and thrombosis. Basic principles and clinical practice, 3d ed. Philadelphia: JB Lippincott Company; 1994. p 718–44. Thomas WS, Mori E, Copeland BR, Yu JQ, Morrissey JH, del Zoppo G. Tissue factor contribute to microvascular defects after focal cerebral ischaemia. Stroke 1993;24:847–54. Petersen LC, Valentin S, Hedner U. Regulation of the extrinsic pathway system in health and disease: The role of factor VIIa and tissue factor pathway inhibitor. Thromb Res 1995; 79:1–47. American Heart Association. Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiac Care. JAMA 1992;268:2171–302. Cummins RO, Chamberlain DA, Abramson NS, Allen M, Baskett P, Becker L, Bossaert L, Delooz H, Dick W, Eisenberg M, Evans T, Holmberg S, Kerber R, Mullie A, Ornato JP, Sandoe E, Skulberg A, Tunstall-Pedoe H, Swanson R, Theis WH. Recommended guidelines for uniform reporting of data from outof-hospital cardiac arrest: The Utstein style. Ann Emerg Med 1991;20:861–74. Rapaport SI, Rao LVM. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscler Thromb 1992;12:1111–21. Francis JL, Carvalho M, Francis DA. The clinical value of tissue factor assays. Blood Coagl Fibrinolysis 1995;6:S37–S44. Gando S, Nanzaki S, Sasaki S, Kemmotsu O.
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Significant correlations between tissue factor and thrombin markers in trauma and septic patients with disseminated intravascular coagulation. Thromb Haemost 1998;79:1111–5. Brozna JP. Cellular regulation of tissue factor. Blood Coagl Fibrinolysis 1990;1:415–26. Goto O, Asano T, Koide T, Takakura K. Ischemic brain edema following occlusion of the middle cerebral artery in the rat. I: The time courses of the brain water, sodium and potassium content and blood-brain barrier permeability to I125-albumin. Stroke 1985;16:101–9. Mackman N. Regulation of the tissue factor gene. Thromb Haemost 1997;78:747–54. Østerud B. Tissue factor: Complex biological role. Thromb Haemost 1997;78:755–8. Semeraro N, Colucci M. Tissue factor in health and disease. Thromb Haemost 1997;78:759–64. Compeau CG, Ma J, DeCampos KN, Waddell
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TK, Brisseau GF, Slutsky AS, Rotstein OD. In situ ischaemia and hypoxia enhance alveolar macrophage tissue factor expression. Am J Respir Cell Mol Biol 1994;11:446–55. Sandset PM. Tissue factor pathway inhibitor (TFPI)—An update. Haemostasis 1996;26 (Suppl):154–65. Broze Jr GJ. Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coagl Fibrinolysis 1995;6:S7–S13. Gando S, Tedo I. Increased neutrophil elastase release in patients with cardiopulmonary arrest: Role of elastase inhibitor. Intensive Care Med 1995;21:636–40. Lindahl AK, Sandset PM, Abildgaard U. The present status of tissue factor pathway inhibitor. Blood Coagl Fibrinolysis 1992;3:439–49. Taylor Jr FB. Tissue factor and thrombin in posttraumatic systemic inflammatory response syndrome. Crit Care Med 1997;25:1774–5.
REGULAR ARTICLE
Tissue Factor and Tissue Factor Pathway Inhibitor Levels during and after Cardiopulmonary Resuscitation Satoshi Gando, Satoshi Nanzaki, Yuji Morimoto, Shigeaki Kobayashi and Osamu Kemmotsu Department of Anesthesiology and Intensive Care, Hokkaido University School of Medicine, Kita-ku, N15 W7, Japan. (Received 20 January 1999 by Editor A. Takada; revised/accepted 7 April 1999)
Abstract Disseminated intravascular coagulation frequently occurs after global ischemia and reperfusion due to cardiac arrest. The present study was performed to demonstrate the role of tissue factor for coagulation pathway activation, as well as to investigate the precise time course of tissue factor pathway inhibitor (TFPI) during and after cardiopulmonary resuscitation (CPR). Thirty-two of out-of-hospital cardiac arrest patients were classified into two groups, those who achieved return of spontaneous circulation (ROSC) (n513) and those without ROSC (n519). Ten normal healthy volunteers served as control subjects. Serial levels of tissue factor and TFPI were measured during and after cardiac arrest and CPR. In patients with ROSC, cardiac arrest and CPR led to persistent increases in the levels of tissue factor that peaked 6 hours after arrival at the Emergency Department. Tissue factor levels in patients without ROSC also showed marked elevations compared to those of the control subjects. In both groups, the levels of TFPI were significantly lower than those in the control subjects. However, we could not find differences in the levels of the two markers between the patients Abbreviations: TFPI, tissue factor pathway inhibitor; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation. Corresponding author: Satoshi Gando, MD, Department of Anesthesiology and Intensive Care, Hokkaido University School of Medicine, N15 W7, Kita-ku, Sapporo, 060 Japan. Tel: 181 (11) 716 1161; Fax: 181 (11) 716 9666; E-mail: ,[email protected] dai.ac.jp..
with ROSC and those without ROSC. In conclusion, we demonstrated persistent elevation of the tissue factor levels associated with low TFPI during and after CPR in patients with out-of-hospital cardiac arrest. These results indicate the activation of the extrinsic coagulation pathway without adequate TFPI generation, which may contribute to thrombin activation and fibrin formation after whole-body ischemia and reperfusion. 1999 Elsevier Science Ltd. All rights reserved. Key Words: Tissue factor; Tissue factor pathway inhibitor; Cardiac arrest; Cardiopulmonary resuscitation
F
or global ischemia due to cardiac arrest there is frequent evidence of disseminated intravascular coagulation [1]. We previously demonstrated that during and after out-of-hospital cardiac arrest, fibrinopeptide A—a marker of thrombin activation—markedly elevated, followed by an increase in plasminogen activator inhibitor, which provides evidence of impaired fibrinolysis [2]. These data suggest that hypoxia, ischemia, and reperfusion accelerate coagulation, then suppress fibrinolysis. The physiologic trigger for coagulation appears to be the formation of complex between tissue factor and factor VII/VIIa. The VIIa tissue factor complex initiates the extrinsic coagulation pathway by activating both factors IX and X, leading to thrombin generation and fibrin formation [3]. An important inhibitor of tissue factor-initiated coagulation is the tissue factor pathway inhibitor (TFPI) [3]. Tissue hypoxia and ischemia cause endothelial
0049-3848/99 $–see front matter 1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00073-0
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S. Gando et al./Thrombosis Research 96 (1999) 107–113
damage, increase in tissue factor activity, and production of the factor Xa activator [4,5]. Using a baboon model of focal cerebral ischemia, Thomas and colleagues [6] showed that tissue factor-mediated events contribute to the no-reflow phenomenon in noncapillary microvessels after focal cerebral ischemia and reperfusion. Recently plasma TFPI levels in various diseases such as cardiovascular disease, blood disease, thromboembolic disease, and disseminated intravascular coagulation have been reported [7]. Endothelium under inflammatory conditions may synthesize increased amounts of TFPI [3]; however, few data are available for the TFPI levels during ischemia and reperfusion. To test the hypothesis that the exposure of tissue factor to the circulation contributes to intravascular coagulation, and to investigate the precise time course of the tissue factor and TFPI levels during and after human cardiac arrest, plasma levels of tissue factor and TFPI were prospectively measured.
1. Materials and Methods 1.1. Patients Approval for this study was obtained from the Ethics Committee of our institution. Informed consent was not obtained, because of the emergency situation with rare opportunity for family consultation. Cardiopulmonary arrest was defined as the absence of spontaneous respiration or palpable pulse and unresponsiveness. Patients over 16 years of age with cardiopulmonary arrest outside the hospital were included in this study. Patients were excluded if they were over 90 years of age, had terminal illness, hypothermia, trauma, or if they had clear signs of irreversible arrest (such as rigor mortis or dependent lividity). Ten normal volunteers (seven males/three females, age 3162 years) served as control subjects.
suscitation (CPR) procedure followed the 1992 Standards and Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiac Care [8]. ECG was monitored and blood pressure was continuously measured using a femoral arterial catheter. Patients showing a recovery of blood pressure and pulse for more than 1 hour were defined as return of spontaneous circulation (ROSC), whether or not catecholamines were used. The patients who died during CPR without ROSC were defined as death. All patients were administered about 700– 2000 IU heparin/day for the anticoagulation of the arterial catheter. Characteristics of the Emergency Medical Service system and Emergency Department were described according to the Utstein Style [9]. Patient outcome at the time of hospital discharge was evaluated based on cerebral and overall performance category [9].
1.3. Study Protocol Blood samples were collected using an arterial catheter immediately after arrival at the Emergency Department (time point 1). The initial 5 mL of every blood were discarded. The second blood samples were drawn when CPR was discontinued due to death (in cases of death) or at 30 minutes after arrival (in cases of ROSC) (time point 2). In the cases of ROSC, additional blood samples were taken 60 minutes after (time point 3), and 6 hours (time point 4) and 24 hours after (time point 5) the ROSC had been achieved. The measured parameters are as follows. Tissue factor antigen concentration (tissue factor) was measured by ELISA (Tissue Factor ELISA kit, American Diagnostica, Greenwich, CT, USA). Reference values for normal healthy control subjects were 135.468.1 pg/mL (n510). Total TFPI antigen concentration were measured by ELISA (TFPI ELISA kit, American Diagnostica, Greenwich, CT, USA). Reference values for normal healthy control subjects were 118.0612.3 ng/mL (n510).
1.2. Treatment Protocol 1.4. Statistical Analysis All patients were given artificial ventilation using a bag-valve-mask unit or esophageal tracheal double lumen airway (Combitube, Sheridan, NY, USA) with 100% oxygen and cardiac massage by the Emergency Medical Service. Cardiopulmonary re-
All data were expressed as mean6SEM. A statistical software package (Stat View 4.5, Abacus Concepts, Berkeley, CA, USA) was used for all statistical calculation analysis. Differences between more
109
S. Gando et al./Thrombosis Research 96 (1999) 107–113
Table 1. Clinical characteristics of the patients and Emergency Medical Service system
Age (years) Male/female Witnessed arrest (yes/no) Bystander CPR (yes/no) Initial rhythm at ED (vf/asystole/other) Outcome (survived/died) Time interval* 1 2 3 4 5
ROSC (n513)
Death (n519)
p value
6863 9/4 8/5 2/11
6063 16/3 16/3 1/18
0.0949 0.5677 0.2988 0.7284
5/3/5 3/10
3/11/5 0/19
0.3370 –
6.360.5 26.162.3 14.163.1 – 36.164.8
6.260.6 27.661.8 – 30.462.9 58.263.5
0.5387 0.4887 – – 0.001
ROSC indicates return of spontaneous circulation; CPR, cardiopulmonary resuscitation; ED, Emergency Department. Values are mean6SEM. *1, interval between the call receipt and the vehicle stops; 2, interval between the vehicle stops and the arrival at the ED; 3, interval between the arrival at the ED and the ROSC; 4, interval between the arrival at the ED and the cessation of CPR; 5, total CPR time.
than two matched samples were tested by Friedman’s test, followed by Wilcoxon signed-rank test. When two groups were being compared, the MannWhitney U test or chi-square test were used. A p,0.05 was considered statistically significant.
mance category54.0) and the third patient had good overall and cerebral performance categories (overall performance category5cerebral performance category51.0).
2.2. Changes in Tissue Factor and TFPI
2. Results 2.1. Baseline Characteristics Characteristics of the patients and Emergency Medical Service system are shown in Table 1. We studied 32 patients, 13 of whom successfully achieved ROSC while the remaining 19 patients died without ROSC. Except total CPR time, baseline characteristics between the two groups were the same. None of the patients in either group were taking drugs that affect blood coagulation. Table 2 shows the etiology of the cardiac arrest. There were statistical differences in the dose of epinephrine (p50.0004) and the use of other catecholamines (p50.002) between the two groups (Table 3). With regards to the outcome of the patients with ROSC, 10 died in a period of 2.3860.9 days and three were discharged from the hospital. At the time of discharge, two of the three patients were in a vegetative state (overall performance category5cerebral perfor-
Figure 1 shows the results of the measurement of tissue factor. In patients with ROSC, tissue factor levels at time point 1 (at the arrival to the Emergency Department) through time point 5 (24 hours after arrival) were significantly higher than those in the control subjects. The levels reached the highest values at time point 4 (6 hours after arrival), which showed a significant statistical difference from time
Table 2. Etiology of the cardiac arrest ROSC
Death
6
13 (12)
3 3 0 1 0
2 0 3 0 1
Cardiac (presumed) Noncardiac Central nervous system Respiratory Vascular Other Unknown ROSC indicates return of spontaneous circulation.
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S. Gando et al./Thrombosis Research 96 (1999) 107–113
Table 3. Clinical data and therapy during cardiopulmonary resuscitation
Epinephrine (mg) Other catecholamine (yes/no) Countershock (yes/no) Mean arterial pressure (mm Hg) Heart rate (beats/min)
ROSC (n513)
Death (n519)
p value
3.360.6 10/3 4/9 8868 11268
7.260.8 3/16 9/10 – –
0.0004 0.002 0.5669 – –
ROSC indicates return of spontaneous circulation.
point 1 (p,0.05). Tissue factor levels in the dead patients also showed marked differences compared to those in the control subjects. Figure 2 shows the changes in TFPI. At time points 1 and 2, the levels of the TFPI in both groups were significantly lower than those in the control subjects. After time point 2, TFPI values in patients with ROSC increased significantly compared to those of time point 1. p values for the Friedman test in patients with ROSC were 50.011 for tissue factor and 50.0036 for TFPI. We could not find any differences in the levels of the two markers between the patients with ROSC and those without ROSC.
3. Discussion
Fig. 1. Bar graphs showing changes in tissue factor antigen concentration (tissue factor) during and after cardiopulmonary resuscitation at time points 1, 2, 3, 4, and 5. See “Methods” for definitions of the time points. Open bar, control subjects; hatched bar, patients with return of spontaneous circulation (ROSC); dark stippled bar, patients without ROSC (death). *p,0.05, **p,0.01, 1p,0.001, 11p,0.0001 vs. control. #p,0.05 vs. time point 1.
Fig. 2. Bar graphs showing changes in tissue factor pathway inhibitor (TFPI) during and after cardiopulmonary resuscitation at time points 1, 2, 3, 4, and 5. See “Methods” for definitions of the time points. Open bar, control subjects; hatched bar, patients with return of spontaneous circulation (ROSC); dark stippled bar, patients without ROSC (death). *p,0.05 vs. control, #p,0.05 and p,0.01 vs. time point 1.
In the former study [2], we demonstrated that plasma fibrinopeptide A, a marker of thrombin activation, and a cross-linked fibrin degradation product (D-dimer), a marker of fibrin formation (and subsequent fibrinolysis), had persistently elevated from the arrival at the Emergency Department to 24 hours after the arrival in patients with out-of-hospital cardiac arrest. Bo¨ttiger et al. [1] also showed the activation of blood coagulation after cardiac arrest. The exposure of blood to tissue factor and the formation of factor VIIa tissue factor
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complexes appear to be the primary mechanism for initiating blood coagulation during hemostasis [3,10]. Activation of factor XII initiates the contact activation reactions that result in activation of factor XI; however, these reactions are only required for blood to clot normally in a glass test tube. Therefore, the contact activation reactions are not required for hemostasis [10]. The interpretation of plasma tissue factor antigen assay used in this study may be complicated by the fact that it is not clear whether tissue factor detectable by our assay is in a functional form. However, since tissue factor requires phospholipid for its activity, free soluble tissue factor in plasma does not reflect the potential for clotting activity [11]. Further, we proved significant correlations between plasma tissue factor and various thrombin markers such as prothrombin fragment F112, thrombin antithrombin III complex, fibrinopeptide A, and D-dimer in patients with sepsis and trauma [12]. In the present study, we did not measure thrombin markers. However, this evidence suggests that the elevated tissue factor levels are closely linked to the thrombin activation and fibrin formation during and after CPR observed in the former studies [1,2]. Several mechanisms by which cardiac arrest and resuscitation cause coagulation abnormalities are proposed. The impact of interventions during CPR, the amount of endogenously released and exogenously administered catecholamines, presence of indwelling catheter, and underlying etiology such as coronary artery disease may have influences on blood coagulation; however, we believe that the profound effect of cardiac arrest and resuscitation overshadow these effects. Tissue hypoxia and ischemia cause endothelial damage with a consequent increase in tissue factor activity and production of factor Xa activator [4,5]. We recently confirmed endothelial injury manifested by liberation of soluble thrombomodulin, a novel marker of endothelial injury, into the circulation in patients with cardiac arrest (unpublished data, 1998). The endothelial injury disrupts a barrier that normally separates the circulating blood from tissue factor, with the resultant binding of plasma factor VII to tissue factor [7,10,13]. Increased vascular permeability after ischemia and reperfusion observed by Goto et al. [14] may expose perivascular tissue factor to the plasma compartment with subsequent activation of the extrin-
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sic coagulation pathway. In addition to constitutive expression, the tissue factor gene is inducibly expressed in several vascular cell types, including monocytes, endothelial cells, and smooth muscle cells [15]. P-selectin [16], oxygen-free radicals [17,18], and thrombin [2] generated during ischemia and reperfusion may induce tissue factor in monocytes and endothelial cells. Cytokines such as tumor necrosis factor-a and interleukin 1-b also induce the tissue factor gene [15]. However, we could not find any increase in these cytokines during and after CPR (unpublished data, 1998). Thus the above-mentioned factors (except cytokines) may contribute to the elevation of tissue factor observed in our present study. The TFPI is found in at least three intravascular pools [7,19]. A major pool is bound to the endothelial surface and the second pool is plasma TFPI. A smaller pool is circulating and is to a great extent in complex form with lipoproteins. A third pool of TFPI is located in platelet. Our assay used in the present study measures all parts of the TFPI. The endothelium is the principle site of synthesis responsible for maintaining the plasma levels of TFPI and the TFPI is removed from the plasma by the liver, kidney, and the low-density lipoprotein receptor-related protein-dependent mechanism [7,19]. The mechanisms by which cardiac arrest and CPR induced TFPI decrease are not precisely known. No patients were administered intravenous drip infusion before their arrival to the Emergency Department, so the effect of the hemodilution can be denied. Low levels of TFPI are occasionally seen in tissue factor-induced disseminated intravascular coagulation, but no unanimity has been achieved with regard to TFPI plasma level in patients diagnosed as having disseminated intravascular coagulation [7,19,20]. Neutrophil elastase cleaves TFPI between Thr87 and Thr88, which is within the polypeptide that links the first and second Kunitz domain [10]. This impairs the ability of TFPI to neutralize both factor VIIa tissue factor and factor Xa [10]. We already reported increased neutrophil elastase release in patients with cardiac arrest [21]. Thus, the neutrophil elastase may be proposed to be one of the reasons why TFPI levels decreased in the present study. The TFPI levels are also to a great extent dependent on low-density lipoproteins [19]. However, it is difficult to consider that plasma low-density lipoprotein varies during cardiac arrest
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and CPR. The last possibilities are that hypoxia and ischaemia may reduce the synthesis of TFPI from endothelial cells or enhance the clearance of TFPI. We used heparin for the purpose of anticoagulation of the arterial catheter inserted into the patients. The heparin being used in the flushing device may induce a significant increase of the TFPI levels after resuscitation in patients with ROSC [19,20,22]. Massive fibrin formation and consecutive impairment of fibrinolysis contribute to reperfusion disorder after cardiac arrest [2]. Tissue factorinduced activation of the extrinsic coagulation pathway as well as the low levels of TFPI observed in the present study may accelerate the derangement of vital organ dysfunction after cardiac arrest. Thomas and colleagues [6] demonstrated that tissue factor-mediated fibrin formation contribute to no-reflow in noncapillary microvessels and also proved that the antitissue factor monoclonal antibody TF9-6B4 increases the microvascular reflow after focal cerebral ischemia and reperfusion. Taylor and his group [23] has energetically clarified that antitissue factor antibody, recombinant TFPI, and active site-inhibited factor VIIa protect animals with sepsis-induced disseminated intravascular coagulation from organ failure and death. Recombinant TFPI also prevents venous thrombosis, rethrombosis after successful thrombolysis, and fibrin deposition on procoagulant subendothelial matrix [19]. The common predominator for all these conditions is the exposure of tissue factor, either by monocytes or by endothelial damage, to circulatory blood [19]. In conclusion, we demonstrated persistent elevation of tissue factor levels associated with low levels of TFPI during and after CPR in patients with outof-hospital cardiac arrest. These results indicate that activation of the extrinsic coagulation pathway without adequate TFPI generation may contribute to thrombin activation and fibrin formation after whole-body ischemia and reperfusion.
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