- Email: [email protected]
19.3 Cardiopulmonary bypass without systemic heparinization in a dog model
Wednesday, September 24. 1997
1400-l 700
Session 1Q-Cardiac Research/Endovascular Co-Chairmen:T. Itoh, Reginald Lord Mountbatton Room 19.1 ET-l Peptide Levels and Endothelin Converting Enzyme-l Gene Expression in Cardiac Surgical Patients F. HORKAY, I. SZOKODI, H. BOHNEMEIER, 0. KADECKI, M.LAZNE, J.LEPPALUOTO, 0. VUOLTEENAHO,,H. RUSKOAKHO, B. MERKELY, A.]UHASZ-NAGY, l? MARTIN and M. T6TH, Budapest, Hungary Endothelin-1 (ET-l) has been described as a potent vasoconstrictor also exhibiting proliferative effects on vascular tissues. ET-l is processed from the precursor big endothelin-1 being cleaved by the endothelin converting enzyme-l (ECE-1). ET-l levels were studied in the pericardial fluid (PF), plasma, atria1 and ventricular tissue of 16 patients (six males, 52 r 7 years, 10 females, 58 + 3 years) undergoing cardiac surgery. Extracted samples (Sep-Pak C18) were measured by RIA for ET-l-like immunoreactivity. Plasma ET-l was 3.33 + 0.51 pg/ ml (range: 0.72-7.15pg/ml). ET-l was higher in the PF (73.11 pg/ml, P c 0.05) than in the plasma, the highest in NYHA II (103.2Opg/ml) intermediate in NYHA II-III (73.14pg/ml) and the lowest in NYHA III (61.13pg/ml). The PF/plasma ratio was 36 + 12 (range 8-200). HPLC showed one peak corresponding to human ET-l. ECE-1 gene expression was investigated in heart samples of 34 patients. ECE-1 mRNA was extracted from atria1 tissues, reverse transcribed and the cDNA was determined by a quantitative PCR (qPCR) assay. The qPCR was performed with an internal standard using a deletion mutant of ECE-1 and correlated to a housekeeping gene by coquantitation of glyceraldehydephosphate-dehydrogenase. Standards of cloned ECE-1 cDNA with known concentrations were also quantified by qPCR. The ECE-1 expression levels in all tissues were inferred by comparison to this ECE-1 cDNA standard. ECE-1 expression did not differ in the whole collective (61.4 + 27.7fg/pl), with the exception of the angina pectoris (AP) group that showed lower expression level (35.5 + 22.5fg/p.l, n = 6, I’ < 0.05). According to the results ET-l levels are the highest in the PF of all human fluids tested so far. An inverse relationship exists to the NYHA class of patients. A lower expression of ECE-1 in AP could be due to mechanisms compensating vasoconstrictive effects of ET-l that would else worsen AP symptoms.
19.2 Differential Expression of Sodium Pump Isoforms in Hypertension H. WEBER, G. WOZNIAK, I.H. AKINTURK, F.W HEHRLEZN and G. SCHEINER-BOBIS, Giessen, Germany The sodium pump (Na+, K+-ATPase) from animal cell membranes is the enzyme responsible for the maintenance of membrane potential, for the function of secondary active transporters and for osmoregulation of the cell. Since inhibition of the enzyme by cardiac glycosides results in increased contractility of the heart muscle and increased blood pressure, we were interested in whether there is a correlation between hypertension and expression of the various isoforms of the sodium pump. To investigate this possibility, we examined the expression of mRNA coding for cwl, (~2 and a3 subunits of the sodium pump in 50 mg tissue samples from the right atrium of hypertensive or normotensive patients who were undergoing cardiac surgery. After reverse transcription and subsequent amplification of ol-, o2-, or a3-specific cDNA fragments in the presence of [a-32+P]dCTP, quantification of the amplified fragments was carried out by the Phosphor-ImagerTM technique. The results obtained show that the cul subunit mRNA is similarly expressed in normotensive or hypertensive patients. The o2 subunit mRNA, however, is increased 3-fold in hypertensive patients. In the same group the a3 isoform is also significantly increased, although not as dramatically as the u2 isoform. This investigation additionally shows a 2.5-fold increase in the expression of mRNA coding for the Na+/Ca’+ exchanger and a 2-fold increase of mRNA coding for the Ca” ATPase from plasma membranes of hypertensive patients. The expression of the sarcoplasmic reticulum Ca2+ATPase is the same in both groups. The results obtained so far appear to indicate a defective Na’ metabolism in the tissues of hypertensive patients. The localization of this defect would help in understanding the molecular mechanisms that are associated with the phenomenon of hypertension and would possibly lead to the development of a diagnostic test for the early recognition of the disease and preventive treatment.
19.3 Cardiopulmonary Bypass Without Systemic Heparinization in a Dog Model M.SAKAI, H. OHTEKZ, K. NAZTO and T. ITOH, Saga, Japan The aim of this study was to evaluate the effects of the combination of a heparin-coated extracorporeal circuit and argatroban as an anticoagulant
96
CARDIOVASCULAR SURGERY SEPTEMBER 1997
Materials and methods: Study ( 1) six mongrel dogs (average weight 22kg) were anethetized and cardiopulmonary bypass was established with cannulation through the right atria1 appendage and ascending aorta, The cardiopulmonary circuit was made of standard untreated vinyle chlorid tubing and a membrane lung blood oxygenator. Extracorporeal circulation was performed using non-pulsatle flow at a rate of 40mI/kg/ min for 2 h, followed by a 60min observation period. Argatroban (Novastan, Tokyo Tanabe Inc.) was administered, Celite activated clotting time (ACT) and arterial gas analyses were performed beforehand, 10 min thereafter. Each circuit was examined visually for evidence of clots or fibrin formation Study (2) The study included 15 mongrel dogs with a mean body weight of 22 kg. The dogs were randomly divided into three groups: in Group l(n = 5) no coating circuit was used and heparin used (2 mg/kg) as an anticoagulant; in Group 2(tz = 5) coating circuit was used and heparin used 2 mg/kg) as an anticoagulant; in Group 3(n = 5) coating circuit was used and argatroban used, (2.0mg + lOpg/kg/min,) as an anticoagulant. The effect of the three systems on the coagulation, fibrinolytic and complement systems were investigated Results: Study (1) Monitoring of argatroban during ACT resulted in a dose response. The ACT values fell immediately after CPB, reaching near-normal levels within 60min and no clots were noted in the extracorporeal circuit or in the surgical field during CPB. Study (2) In group 3, TAT complex, FDP showed constant decreases during CPB compaired with other groups and preserved platelet. Comment: We applied a new antithrombogenetic material, which is recently developed in Toyobo, Japan. Argatroban is a selective thrombin inhibitor synthesized in Japan. Argatroban exhibits no dependance on antithrombin3 and there is possibly no need to neutralize the anticoagulant activity as protamine to heparin due to the short half-life. This study clearly demonstrated that the combination use of new heparincoated circuits and argatroban as an anticoagulant results in reduced concentrations of TAT, FDP, which means this system is safe and reduces the activation of coagulation and fibrinolyric systems.
hemostasis (surgical tissue planes and vascular suture lines). The unique position of Factor IX/IXa in the coagulation cascade renders it an ideal target in this setting. We prepared active site-blocked Factor IXa (IXai) using dansyl glu gly arg chlormethylketone. Our previous observations in a baboon model of CPB revealed that the use of e&her H or FIXai resulted in similar hemodynamics throughout 1 h of CPB, as well as no significant clot formation or fibrin/platelet deposition in the bypass circuit (confirmed by scanning EM). Importantly, however, the use of IXai resulted in significantly decreased blood loss in the thoracic cavity through 3 h of observation postoperatively (P < 0.05). Similar concerns about the use of H and excess intraoperative bleeding exist in the setting of peripheral arterial repair, especially with the increased use of synthetic patch angioplasty (pdytetrafluoroethylene [PTFE] or dacron). We therefore tested IXai in a New Zealand rabbit aortotomy model with PTFE patch (2 x 6 mm) in 37 rabbits. Animals received either H (SOIU/kg; n = 16) or IXai (300 &kg; p1 = 21). Blood loss was measured [using preweighed gauze) and time to hemostasis recorded. Compared to H, the use of IXai resulted in significantly diminished blood loss (6.97 ~tr4.4g versus 2.72 r 2.5g; P <: 0.003) and time to hemostasis (176 2 46 set versus 120 t .i8 set; P c: 0.05). To assess long term patency and thrombosis, I2 rabbits (H; n = 6 and IXai; n = 6) have been observed up to 2 months postoperatively. No differences were observed between H or IXai; 100% of the grafts were patent with no evidence of intimal hyperpiasia by histological analysis. Serial measurement of Thrombin-Antithrombin complex (‘PAT), a sensitive/ specific marker of the action of Factor Xa on ptotbrombin revealed no evidence of thrombin generation in either group (postoperatively through 2 months). In a large animal model, six dogs (three H, three IXai) underwent PTFE patch repair oI a carotid aortotomy (2 x 8mm) with similar results; compared to H, IXai resulted in significantly reduced blood loss (39.1 -c S.Sg versus 20.7 + 12.9g, P < 0.1j.3) and time to hemostasis (230 * 17sec versus 162 % 34sec; I’ ‘: 0.05). At 1 month postoperatively, ultrasonographic studies revealed ali grafts to be patent and serial measurement of TAT revealed no evidence of thrombin generation in either group. These results suggest that IXai may be a safe and effective alternative to heparin to avoid the morbidity and occasional mortality of perioperative hemorrhage in cardiovascular surgery
19.4 Active-site Blocked Factor IXa: An Alternative to Heparin in Cardiovascular Surgery Which Results in Decreased Operative Blood Loss T.B. SPANIER, M.C. OZ, 1.0. MADIGAN, D.M. STERN, E.A. ROSE, A.M. SCHMIDT and R. NOWYGROD, New York, New York, USA.
19.5 Endovascular Surgery in Cardiovascular Diseases M. OKADA, M. YOSHZDA, y. TSUJI, M. NAKAMURA and Y MATOBA, K&r, t-fyogo, Japan
Heparin (H) has been the mainstay of antithrombotic therapy in cardiopulmonary bypass (CPB) and arterial repair procedures. Because of its multiple sites of action in the coagulation cascade, both intravascular (desired effect) and extravascular (undesired side effect) hemostasis are impaired, often resulting in significant intraoperative blood loss. We tested the hypothesis that selective inhibition of intravascular coagulation without significant impairment of extravascular hemostasis would prevent clotting intraluminally (extracorporeal circuit and at the site of vascular repair) while preserving extrinsic
CARDIOVASCULAR SURGERY
SEPTEMBER 1997
Recently much attention has been paid to endovascular surgery. Since I980 we have studied laser angioplasty for patients with peripheral and coronary arterial disease. The aim of this technique is to keep a wide-opening of the occlusive arterial lumen. Based on the our excellent experimental studies, laser has been clinically applied for 224 patients with intermittent claudication renal failure and angina1 attack. Among them, there was one transmyocardial revascularization. Laser angioplasty was performed for 112 patients with stenotic or occlusive lesions more than 75% of diameter of the peripheral and the coronary arteries angiographically. They consisted of
97
1400-l 700
Session 1Q-Cardiac Research/Endovascular Co-Chairmen:T. Itoh, Reginald Lord Mountbatton Room 19.1 ET-l Peptide Levels and Endothelin Converting Enzyme-l Gene Expression in Cardiac Surgical Patients F. HORKAY, I. SZOKODI, H. BOHNEMEIER, 0. KADECKI, M.LAZNE, J.LEPPALUOTO, 0. VUOLTEENAHO,,H. RUSKOAKHO, B. MERKELY, A.]UHASZ-NAGY, l? MARTIN and M. T6TH, Budapest, Hungary Endothelin-1 (ET-l) has been described as a potent vasoconstrictor also exhibiting proliferative effects on vascular tissues. ET-l is processed from the precursor big endothelin-1 being cleaved by the endothelin converting enzyme-l (ECE-1). ET-l levels were studied in the pericardial fluid (PF), plasma, atria1 and ventricular tissue of 16 patients (six males, 52 r 7 years, 10 females, 58 + 3 years) undergoing cardiac surgery. Extracted samples (Sep-Pak C18) were measured by RIA for ET-l-like immunoreactivity. Plasma ET-l was 3.33 + 0.51 pg/ ml (range: 0.72-7.15pg/ml). ET-l was higher in the PF (73.11 pg/ml, P c 0.05) than in the plasma, the highest in NYHA II (103.2Opg/ml) intermediate in NYHA II-III (73.14pg/ml) and the lowest in NYHA III (61.13pg/ml). The PF/plasma ratio was 36 + 12 (range 8-200). HPLC showed one peak corresponding to human ET-l. ECE-1 gene expression was investigated in heart samples of 34 patients. ECE-1 mRNA was extracted from atria1 tissues, reverse transcribed and the cDNA was determined by a quantitative PCR (qPCR) assay. The qPCR was performed with an internal standard using a deletion mutant of ECE-1 and correlated to a housekeeping gene by coquantitation of glyceraldehydephosphate-dehydrogenase. Standards of cloned ECE-1 cDNA with known concentrations were also quantified by qPCR. The ECE-1 expression levels in all tissues were inferred by comparison to this ECE-1 cDNA standard. ECE-1 expression did not differ in the whole collective (61.4 + 27.7fg/pl), with the exception of the angina pectoris (AP) group that showed lower expression level (35.5 + 22.5fg/p.l, n = 6, I’ < 0.05). According to the results ET-l levels are the highest in the PF of all human fluids tested so far. An inverse relationship exists to the NYHA class of patients. A lower expression of ECE-1 in AP could be due to mechanisms compensating vasoconstrictive effects of ET-l that would else worsen AP symptoms.
19.2 Differential Expression of Sodium Pump Isoforms in Hypertension H. WEBER, G. WOZNIAK, I.H. AKINTURK, F.W HEHRLEZN and G. SCHEINER-BOBIS, Giessen, Germany The sodium pump (Na+, K+-ATPase) from animal cell membranes is the enzyme responsible for the maintenance of membrane potential, for the function of secondary active transporters and for osmoregulation of the cell. Since inhibition of the enzyme by cardiac glycosides results in increased contractility of the heart muscle and increased blood pressure, we were interested in whether there is a correlation between hypertension and expression of the various isoforms of the sodium pump. To investigate this possibility, we examined the expression of mRNA coding for cwl, (~2 and a3 subunits of the sodium pump in 50 mg tissue samples from the right atrium of hypertensive or normotensive patients who were undergoing cardiac surgery. After reverse transcription and subsequent amplification of ol-, o2-, or a3-specific cDNA fragments in the presence of [a-32+P]dCTP, quantification of the amplified fragments was carried out by the Phosphor-ImagerTM technique. The results obtained show that the cul subunit mRNA is similarly expressed in normotensive or hypertensive patients. The o2 subunit mRNA, however, is increased 3-fold in hypertensive patients. In the same group the a3 isoform is also significantly increased, although not as dramatically as the u2 isoform. This investigation additionally shows a 2.5-fold increase in the expression of mRNA coding for the Na+/Ca’+ exchanger and a 2-fold increase of mRNA coding for the Ca” ATPase from plasma membranes of hypertensive patients. The expression of the sarcoplasmic reticulum Ca2+ATPase is the same in both groups. The results obtained so far appear to indicate a defective Na’ metabolism in the tissues of hypertensive patients. The localization of this defect would help in understanding the molecular mechanisms that are associated with the phenomenon of hypertension and would possibly lead to the development of a diagnostic test for the early recognition of the disease and preventive treatment.
19.3 Cardiopulmonary Bypass Without Systemic Heparinization in a Dog Model M.SAKAI, H. OHTEKZ, K. NAZTO and T. ITOH, Saga, Japan The aim of this study was to evaluate the effects of the combination of a heparin-coated extracorporeal circuit and argatroban as an anticoagulant
96
CARDIOVASCULAR SURGERY SEPTEMBER 1997
Materials and methods: Study ( 1) six mongrel dogs (average weight 22kg) were anethetized and cardiopulmonary bypass was established with cannulation through the right atria1 appendage and ascending aorta, The cardiopulmonary circuit was made of standard untreated vinyle chlorid tubing and a membrane lung blood oxygenator. Extracorporeal circulation was performed using non-pulsatle flow at a rate of 40mI/kg/ min for 2 h, followed by a 60min observation period. Argatroban (Novastan, Tokyo Tanabe Inc.) was administered, Celite activated clotting time (ACT) and arterial gas analyses were performed beforehand, 10 min thereafter. Each circuit was examined visually for evidence of clots or fibrin formation Study (2) The study included 15 mongrel dogs with a mean body weight of 22 kg. The dogs were randomly divided into three groups: in Group l(n = 5) no coating circuit was used and heparin used (2 mg/kg) as an anticoagulant; in Group 2(tz = 5) coating circuit was used and heparin used 2 mg/kg) as an anticoagulant; in Group 3(n = 5) coating circuit was used and argatroban used, (2.0mg + lOpg/kg/min,) as an anticoagulant. The effect of the three systems on the coagulation, fibrinolytic and complement systems were investigated Results: Study (1) Monitoring of argatroban during ACT resulted in a dose response. The ACT values fell immediately after CPB, reaching near-normal levels within 60min and no clots were noted in the extracorporeal circuit or in the surgical field during CPB. Study (2) In group 3, TAT complex, FDP showed constant decreases during CPB compaired with other groups and preserved platelet. Comment: We applied a new antithrombogenetic material, which is recently developed in Toyobo, Japan. Argatroban is a selective thrombin inhibitor synthesized in Japan. Argatroban exhibits no dependance on antithrombin3 and there is possibly no need to neutralize the anticoagulant activity as protamine to heparin due to the short half-life. This study clearly demonstrated that the combination use of new heparincoated circuits and argatroban as an anticoagulant results in reduced concentrations of TAT, FDP, which means this system is safe and reduces the activation of coagulation and fibrinolyric systems.
hemostasis (surgical tissue planes and vascular suture lines). The unique position of Factor IX/IXa in the coagulation cascade renders it an ideal target in this setting. We prepared active site-blocked Factor IXa (IXai) using dansyl glu gly arg chlormethylketone. Our previous observations in a baboon model of CPB revealed that the use of e&her H or FIXai resulted in similar hemodynamics throughout 1 h of CPB, as well as no significant clot formation or fibrin/platelet deposition in the bypass circuit (confirmed by scanning EM). Importantly, however, the use of IXai resulted in significantly decreased blood loss in the thoracic cavity through 3 h of observation postoperatively (P < 0.05). Similar concerns about the use of H and excess intraoperative bleeding exist in the setting of peripheral arterial repair, especially with the increased use of synthetic patch angioplasty (pdytetrafluoroethylene [PTFE] or dacron). We therefore tested IXai in a New Zealand rabbit aortotomy model with PTFE patch (2 x 6 mm) in 37 rabbits. Animals received either H (SOIU/kg; n = 16) or IXai (300 &kg; p1 = 21). Blood loss was measured [using preweighed gauze) and time to hemostasis recorded. Compared to H, the use of IXai resulted in significantly diminished blood loss (6.97 ~tr4.4g versus 2.72 r 2.5g; P <: 0.003) and time to hemostasis (176 2 46 set versus 120 t .i8 set; P c: 0.05). To assess long term patency and thrombosis, I2 rabbits (H; n = 6 and IXai; n = 6) have been observed up to 2 months postoperatively. No differences were observed between H or IXai; 100% of the grafts were patent with no evidence of intimal hyperpiasia by histological analysis. Serial measurement of Thrombin-Antithrombin complex (‘PAT), a sensitive/ specific marker of the action of Factor Xa on ptotbrombin revealed no evidence of thrombin generation in either group (postoperatively through 2 months). In a large animal model, six dogs (three H, three IXai) underwent PTFE patch repair oI a carotid aortotomy (2 x 8mm) with similar results; compared to H, IXai resulted in significantly reduced blood loss (39.1 -c S.Sg versus 20.7 + 12.9g, P < 0.1j.3) and time to hemostasis (230 * 17sec versus 162 % 34sec; I’ ‘: 0.05). At 1 month postoperatively, ultrasonographic studies revealed ali grafts to be patent and serial measurement of TAT revealed no evidence of thrombin generation in either group. These results suggest that IXai may be a safe and effective alternative to heparin to avoid the morbidity and occasional mortality of perioperative hemorrhage in cardiovascular surgery
19.4 Active-site Blocked Factor IXa: An Alternative to Heparin in Cardiovascular Surgery Which Results in Decreased Operative Blood Loss T.B. SPANIER, M.C. OZ, 1.0. MADIGAN, D.M. STERN, E.A. ROSE, A.M. SCHMIDT and R. NOWYGROD, New York, New York, USA.
19.5 Endovascular Surgery in Cardiovascular Diseases M. OKADA, M. YOSHZDA, y. TSUJI, M. NAKAMURA and Y MATOBA, K&r, t-fyogo, Japan
Heparin (H) has been the mainstay of antithrombotic therapy in cardiopulmonary bypass (CPB) and arterial repair procedures. Because of its multiple sites of action in the coagulation cascade, both intravascular (desired effect) and extravascular (undesired side effect) hemostasis are impaired, often resulting in significant intraoperative blood loss. We tested the hypothesis that selective inhibition of intravascular coagulation without significant impairment of extravascular hemostasis would prevent clotting intraluminally (extracorporeal circuit and at the site of vascular repair) while preserving extrinsic
CARDIOVASCULAR SURGERY
SEPTEMBER 1997
Recently much attention has been paid to endovascular surgery. Since I980 we have studied laser angioplasty for patients with peripheral and coronary arterial disease. The aim of this technique is to keep a wide-opening of the occlusive arterial lumen. Based on the our excellent experimental studies, laser has been clinically applied for 224 patients with intermittent claudication renal failure and angina1 attack. Among them, there was one transmyocardial revascularization. Laser angioplasty was performed for 112 patients with stenotic or occlusive lesions more than 75% of diameter of the peripheral and the coronary arteries angiographically. They consisted of
97