European Heart Journal (1992) 13, 1641-1644
Lack of platelet-activating factor release during reversible myocardial ischaemia D. THOMAS*
From the Departments of Cardiology* and Hematology§, Hopital Pitie-Salpetriere, and the INSERM unit I5Q\, Hopital Lariboisiere, Paris, and Henri Beaufour Institute%, Le Plessis-Robinson, France KEY WORDS: Angina pectoris, PAF, eicosanoids, myocardial ischaemia. Platelet-activatingfactor (PAF) is involved in experimental models of myocardial ischaemia, and PAF infusion can cause thromboxane release. Thromboxane is produced during brief episodes of reversible myocardial ischaemia in patients with coronary heart disease. To learn whether PAF synthesis is associated with thromboxane production in mild myocardial ischaemia, we performed rapid atrial pacing in four patients with angina pectoris which caused chest pain, ST segment depression (AST= —1-8 + 0-2 mm) andlactate excretion in the coronary sinus (percent lactate extraction decreasedfrom 20 + 6% to —15±9%). Thromboxane B2 was produced causing a positive transmyocardial gradient (from 88 ± 154 pg . ml~' baseline to 1770 ± 1407 pg . ml~' at the peak) but there was no PA F release into coronary sinus blood. In four other patients we determined whether more pronounced ischaemia could be associated with PAF synthesis. Coronary sinus blood was sampled before and during balloon occlusion of a major coronary artery: PA F was not detected in coronary sinus, whereas percent lactate extraction decreasedfrom 24 + 6% to —63+_22% (n = 4). We conclude that PAF plays a minor role in short episodes of reversible ischaemia and does not participate in thromboxane production. Introduction Myocardial ischaemia is associated with a cascade of metabolic events including the release of thromboxane A2 (Tx A2) into the coronary circulation, especially in unstable angina'1"31, variant angina'451 and myocardial infarction'1 6J1. We and others have reported the coronary production of TxA2 during brief episodes of myocardial ischaemia in patients with stable angina'48"101. This platelet-derived mediator is formed from arachidonic acid which is produced, along with lyso-phospholipids, by the action of phospholipase A2 on membrane phospholipids. The lyso-phospholipid lyso-PAF is the precursor and the metabolite of PAF which could play a role in coronary heart disease. Different circulating or resident cells can release PAF and this mediator induces platelet and leukocyte activation, causes contraction of smooth muscle, decreases coronary blood flow and contractile function in animal models, and can induce thromboxane release1""'31. Moreover, PAF production has been reported in ischaemia myocardium'13"151 and has been detected in the coronary sinus blood of a few patients with coronary artery disease (CAD) undergoing pacinginduced ischaemia'161. We hypothesized that in patients with angina pectoris PAF may be associated with thromboxane release during reversible episodes of myocardial ischaemia. Mild ischaemia was induced by atrial pacing, whereas more pronounced ischaemia, although brief and reversible, was studied during coronary artery angioplasty. Submitted for publication on 17 December 1991 and in revised form 26 May 1992 Correspondence' G Montalescot, MD, PhD, Catherization Laboratory, Department of Cardiology, Hopital Pitie-Salpetriere, 47 Bl de 1'Hopital, 75651 Paris Cede* 13, France. 0195-668X/92/121641 + 04 $08.00/0
Methods PATIENT POPULATION
Eight patients without previous myocardial infarction, all of whom gave their informed consent for the study protocol, were included in this study. In the first group of patients who underwent atrial stimulation (group I, n = 4), all had typical effort angina pectoris and a positive exercise stress test. These patients presented a significant ischaemic ST-segment depression after relatively mild exercise (> 1 mm at 80% or less of the predicted maximal heart rate). The condition was stable without chest pain during the 48 h preceding catheterization. They received neither antianginal medication for 24 h nor drugs inhibiting cyclooxygenase (no aspirin) for 10 days before catheterization. In the second group of patients (group II, n = 4), we performed percutaneous coronary angioplasty (PTCA) for a tight stenosis (> 75% of the luminal diameter) in a major coronary artery. Aspirin (250 mg per day) was prescribed only in patients of group II undergoing PTCA. CARDIAC CATHETERIZATION
Atrial pacing After an overnight fast, a 7 Fr USCI pacing and sampling catheter was introduced via a peripheral vein into the coronary sinus (CS) and a 7 Fr polyurethane (pigtail) catheter was placed into the ascending aorta for arterial blood sampling. The lines were kept patent by intermittent flushing with saline without heparin. A 12lead ECG was continuously recorded. After a 10-min rest, we started CS pacing at the rate of 90 beats. min~', progressively increasing to 150 beats. min"1 over 3 min and then this rate was maintained. When atrioventricular © 1992 The European Society of Cardiology
Downloaded from https://academic.oup.com/eurheartj/article-abstract/13/12/1641/651306 by Western Sydney University Library user on 14 January 2019
G. MONTALESCOT*, J. MACLOUFI, G. DROBFNSKI*, J. M. MENCIA-HUERTAJ, A. ANKRI§, Y. GROSGOGEAT* AND
1642 G. Montalescot et al.
Percutaneous transluminal coronary angioplasty We placed a 7 Fr USCI sampling catheter into the coronary sinus. An 8 Fr guiding catheter was placed into the coronary orifice and baseline samples were drawn from the coronary sinus and the sidearm of the arterial introducer. The first balloon inflation was short (15 s) and at low pressure (3 bars). We performed a second inflation of the balloon at a higher pressure over 1-2 min according to the clinical and electrical signs of ischaemia; the balloon was then deflated and samples were drawn simultaneously from the artery and the CS. Blood samples for prostaglandins measurements were transferred immediately to ice until completion of the procedure. EXTRACTION OF BLOOD SAMPLES AND BIOASSAY OF PAF
One millilitre of whole arterial blood was collected in glass test tubes containing 4 ml of absolute ethanol. The samples were stored overnight at 4°C then centrifuged at 400 g for 15 min. The supernatants were collected, transferred into glass test tubes and dried by centrifugation under reduced pressure (Savant, Hicksville, New York, U.S.A.). The dried residue was resuspended in 200 u.1 of 0 1 5 M NaCl in distilled water (pH 7-4) containing 0-25% fatty acid-free bovine albumin (BSA, Sigma, Chem. Co, St Louis, MO, U.S.A.). The samples were stored at — 20°C until assayed for PAF activity. Following thawing, the samples were allowed to stand at room temperature for at least 1 h with intermittent vortexing. PAF activity was measured by a platelet aggregation assay*17181. Rabbit platelets were prepared by differential centrifugations'1718', were washed once, then incubated at room temperature in the presence of 01 mM aspirin lysin-salt (Aspegic, Lab Egic, France) and subsequently washed twice more. Washed platelets (2-5 xlO8) stirred at 1100 rpm in a final volume of 400 \i\ of Tyrode's buffer containing 0-25% fatty acid-free bovine saline albumin (BSA) and the adenosine diphosphate (ADP) scavenger complexes, creatine phosphate (1 mM, Sigma) and creatine phosphokinase (10U .ml"', Sigma) were aggregated in a Chronolog aggregometer (Coultronics, Margency, France). Variations of light transmission were recorded'181. PAF activity was calculated over the linear portion of a calibration curve performed with known amounts (5 to 25 pg) of synthetic PAF (1 -0-alkyl-2-acetyl«j-g]yceryl-3-phosphorylcholine, Bachem, Buddendorf, Switzerland). The threshold sensitivity of this bioassay was equivalent to 5 pg of synthetic PAF, i.e. 20 pg in the blood extracts. In some experiments, the possible presence of substance(s) in the blood extracts interfering
with the assay was investigated by comparison with the aggregation induced by 15 pg of synthetic PAF. Aliquots (100 ul) of the blood extracts were resuspended in 0-25% fatty acid-free BSA in 015 M NaCl in distilled water (pH 7-4) and were added to 400 ul of absolute methanol and stored at — 20cC for 24 h. The samples were centrifuged at 1500 £ for 30 min and supernatants were collected and resolved by high performance liquid chromatography, as previously described'18'. The fractions eluting at the retention time of authentic PAF (i.e. 16-18 min) were collected and dried under reduced pressure. To these tubes we added 200 ul of 0-25% fatty acidfree BSA in 015 M NaCl in distilled water (pH 7-4) prior to bioassay for PAF activity as described above. EICOSANOID MEASUREMENTS
Thromboxane B2 (TxB2) concentrations were measured in patients undergoing atrial stimulation but not in patients who underwent PTCA; they were under aspirin regimen which has been proved to be effective in preventing acute reocclusion. To measure plasma TxB2 concentrations, 4 ml of blood were collected in test tubes containing ethylene-diethine tetra acetic-acid (EDTA) and indomethacin (5mM and 10 UM final concentration respectively). Samples were immediately transferred to ice, centrifuged at 3000 g for 10 min and the plasma stored at - 70°C until further analyses. TxB2 and 6-keto-PGFla analyses were performed by enzyme immunoassay1191. OTHER MEASUREMENTS
Arterial and CS blood was drawn (2 ml samples) into glass test tubes containing 80 UM of fluoride/EDTA reagent. After centrifugation at 3000 # for 10 min, lactate levels were measured on supernatants by enzymatic assay (Boehringer Mannheim, Germany). We calculated the percent lactate extraction (%L) by subtracting the CS from the arterial concentration and dividing by the arterial concentration. Total white blood cells (WBC), and erythrocyte concentration, haematocrit, haemoglobin, and platelet concentrations were measured by an automated counter (ELT 800, Ortho Diagnostics, U.S.A.). STATISTICS
All data are expressed as mean±SEM. Changes of variables within groups were evaluated by a paired t-test. Significance was declared at an alpha level of 005. Results EPISODES OF MYOCARDIAL ISCHAEMIA
Atrial stimulation (group I) caused a significant decrease of % lactate extraction (> 5%) and ST-segment depression ( > l m m ) in all effort angina patients. Maximum changes of ST-segment from baseline were measured in the same lead at the peak of pacing-induced ischaemia. Mean ST-segment depression induced by pacing was - 1 -8 ± 0-2 mm. The percent lactate extraction in these angina patients decreased from +20 ± 6 % at baseline to — 15 ± 9% at the maximal pacing rate, then to
Downloaded from https://academic.oup.com/eurheartj/article-abstract/13/12/1641/651306 by Western Sydney University Library user on 14 January 2019
block was induced, atropine (1/2 mg i.v.) was administered. Our criteria to cease atrial stimulation were either chest pain, significant ST-segment depression ( > 1 mm), or a 10 min period of pacing at the maximum rate (150 beats . min~'). Three sets of blood samples were collected: at rest before pacing, during the last minute of pacing and immediately after pacing was discontinued. At each measurement interval, blood samples were taken simultaneously from the aorta and CS then placed onto ice.
PA F and myocardial ischaemia 1643
not measure significant changes of plasma 6-keto-PGFla levels in patients during atrial stimulation; all plasma 6-keto-PGF 1 a levels remained below 300 pg. ml"'.
20 0 i
-20
\i
-60 -80
*
i
Baseline
M
i Peak of ischaemia
1 Post-pacing
Figure I Changes of % lactate extraction after pacing (group I, O; n = 4) and PTCA (group II, • ; n = 4) in patients with angina pectoris.
We measured no significant changes from baseline of WBC and platelet concentrations in either CS or arterial blood after atrial stimulation. Therefore pacing-induced myocardial ischaemia was not associated with any significant CS-arterial cell gradient. Similarly, there were no changes of WBC and platelet counts following balloon inflation during PTCA. No changes of haematocrit and erythrocyte count were measured in any group. Discussion
Acute and brief episodes of myocardial ischaemia induced by pacing were associated with thromboxane release into coronary sinus plasma. The acute myocardial
Lack of platelet-activating factor release during reversible myocardial ischaemia D. THOMAS*
From the Departments of Cardiology* and Hematology§, Hopital Pitie-Salpetriere, and the INSERM unit I5Q\, Hopital Lariboisiere, Paris, and Henri Beaufour Institute%, Le Plessis-Robinson, France KEY WORDS: Angina pectoris, PAF, eicosanoids, myocardial ischaemia. Platelet-activatingfactor (PAF) is involved in experimental models of myocardial ischaemia, and PAF infusion can cause thromboxane release. Thromboxane is produced during brief episodes of reversible myocardial ischaemia in patients with coronary heart disease. To learn whether PAF synthesis is associated with thromboxane production in mild myocardial ischaemia, we performed rapid atrial pacing in four patients with angina pectoris which caused chest pain, ST segment depression (AST= —1-8 + 0-2 mm) andlactate excretion in the coronary sinus (percent lactate extraction decreasedfrom 20 + 6% to —15±9%). Thromboxane B2 was produced causing a positive transmyocardial gradient (from 88 ± 154 pg . ml~' baseline to 1770 ± 1407 pg . ml~' at the peak) but there was no PA F release into coronary sinus blood. In four other patients we determined whether more pronounced ischaemia could be associated with PAF synthesis. Coronary sinus blood was sampled before and during balloon occlusion of a major coronary artery: PA F was not detected in coronary sinus, whereas percent lactate extraction decreasedfrom 24 + 6% to —63+_22% (n = 4). We conclude that PAF plays a minor role in short episodes of reversible ischaemia and does not participate in thromboxane production. Introduction Myocardial ischaemia is associated with a cascade of metabolic events including the release of thromboxane A2 (Tx A2) into the coronary circulation, especially in unstable angina'1"31, variant angina'451 and myocardial infarction'1 6J1. We and others have reported the coronary production of TxA2 during brief episodes of myocardial ischaemia in patients with stable angina'48"101. This platelet-derived mediator is formed from arachidonic acid which is produced, along with lyso-phospholipids, by the action of phospholipase A2 on membrane phospholipids. The lyso-phospholipid lyso-PAF is the precursor and the metabolite of PAF which could play a role in coronary heart disease. Different circulating or resident cells can release PAF and this mediator induces platelet and leukocyte activation, causes contraction of smooth muscle, decreases coronary blood flow and contractile function in animal models, and can induce thromboxane release1""'31. Moreover, PAF production has been reported in ischaemia myocardium'13"151 and has been detected in the coronary sinus blood of a few patients with coronary artery disease (CAD) undergoing pacinginduced ischaemia'161. We hypothesized that in patients with angina pectoris PAF may be associated with thromboxane release during reversible episodes of myocardial ischaemia. Mild ischaemia was induced by atrial pacing, whereas more pronounced ischaemia, although brief and reversible, was studied during coronary artery angioplasty. Submitted for publication on 17 December 1991 and in revised form 26 May 1992 Correspondence' G Montalescot, MD, PhD, Catherization Laboratory, Department of Cardiology, Hopital Pitie-Salpetriere, 47 Bl de 1'Hopital, 75651 Paris Cede* 13, France. 0195-668X/92/121641 + 04 $08.00/0
Methods PATIENT POPULATION
Eight patients without previous myocardial infarction, all of whom gave their informed consent for the study protocol, were included in this study. In the first group of patients who underwent atrial stimulation (group I, n = 4), all had typical effort angina pectoris and a positive exercise stress test. These patients presented a significant ischaemic ST-segment depression after relatively mild exercise (> 1 mm at 80% or less of the predicted maximal heart rate). The condition was stable without chest pain during the 48 h preceding catheterization. They received neither antianginal medication for 24 h nor drugs inhibiting cyclooxygenase (no aspirin) for 10 days before catheterization. In the second group of patients (group II, n = 4), we performed percutaneous coronary angioplasty (PTCA) for a tight stenosis (> 75% of the luminal diameter) in a major coronary artery. Aspirin (250 mg per day) was prescribed only in patients of group II undergoing PTCA. CARDIAC CATHETERIZATION
Atrial pacing After an overnight fast, a 7 Fr USCI pacing and sampling catheter was introduced via a peripheral vein into the coronary sinus (CS) and a 7 Fr polyurethane (pigtail) catheter was placed into the ascending aorta for arterial blood sampling. The lines were kept patent by intermittent flushing with saline without heparin. A 12lead ECG was continuously recorded. After a 10-min rest, we started CS pacing at the rate of 90 beats. min~', progressively increasing to 150 beats. min"1 over 3 min and then this rate was maintained. When atrioventricular © 1992 The European Society of Cardiology
Downloaded from https://academic.oup.com/eurheartj/article-abstract/13/12/1641/651306 by Western Sydney University Library user on 14 January 2019
G. MONTALESCOT*, J. MACLOUFI, G. DROBFNSKI*, J. M. MENCIA-HUERTAJ, A. ANKRI§, Y. GROSGOGEAT* AND
1642 G. Montalescot et al.
Percutaneous transluminal coronary angioplasty We placed a 7 Fr USCI sampling catheter into the coronary sinus. An 8 Fr guiding catheter was placed into the coronary orifice and baseline samples were drawn from the coronary sinus and the sidearm of the arterial introducer. The first balloon inflation was short (15 s) and at low pressure (3 bars). We performed a second inflation of the balloon at a higher pressure over 1-2 min according to the clinical and electrical signs of ischaemia; the balloon was then deflated and samples were drawn simultaneously from the artery and the CS. Blood samples for prostaglandins measurements were transferred immediately to ice until completion of the procedure. EXTRACTION OF BLOOD SAMPLES AND BIOASSAY OF PAF
One millilitre of whole arterial blood was collected in glass test tubes containing 4 ml of absolute ethanol. The samples were stored overnight at 4°C then centrifuged at 400 g for 15 min. The supernatants were collected, transferred into glass test tubes and dried by centrifugation under reduced pressure (Savant, Hicksville, New York, U.S.A.). The dried residue was resuspended in 200 u.1 of 0 1 5 M NaCl in distilled water (pH 7-4) containing 0-25% fatty acid-free bovine albumin (BSA, Sigma, Chem. Co, St Louis, MO, U.S.A.). The samples were stored at — 20°C until assayed for PAF activity. Following thawing, the samples were allowed to stand at room temperature for at least 1 h with intermittent vortexing. PAF activity was measured by a platelet aggregation assay*17181. Rabbit platelets were prepared by differential centrifugations'1718', were washed once, then incubated at room temperature in the presence of 01 mM aspirin lysin-salt (Aspegic, Lab Egic, France) and subsequently washed twice more. Washed platelets (2-5 xlO8) stirred at 1100 rpm in a final volume of 400 \i\ of Tyrode's buffer containing 0-25% fatty acid-free bovine saline albumin (BSA) and the adenosine diphosphate (ADP) scavenger complexes, creatine phosphate (1 mM, Sigma) and creatine phosphokinase (10U .ml"', Sigma) were aggregated in a Chronolog aggregometer (Coultronics, Margency, France). Variations of light transmission were recorded'181. PAF activity was calculated over the linear portion of a calibration curve performed with known amounts (5 to 25 pg) of synthetic PAF (1 -0-alkyl-2-acetyl«j-g]yceryl-3-phosphorylcholine, Bachem, Buddendorf, Switzerland). The threshold sensitivity of this bioassay was equivalent to 5 pg of synthetic PAF, i.e. 20 pg in the blood extracts. In some experiments, the possible presence of substance(s) in the blood extracts interfering
with the assay was investigated by comparison with the aggregation induced by 15 pg of synthetic PAF. Aliquots (100 ul) of the blood extracts were resuspended in 0-25% fatty acid-free BSA in 015 M NaCl in distilled water (pH 7-4) and were added to 400 ul of absolute methanol and stored at — 20cC for 24 h. The samples were centrifuged at 1500 £ for 30 min and supernatants were collected and resolved by high performance liquid chromatography, as previously described'18'. The fractions eluting at the retention time of authentic PAF (i.e. 16-18 min) were collected and dried under reduced pressure. To these tubes we added 200 ul of 0-25% fatty acidfree BSA in 015 M NaCl in distilled water (pH 7-4) prior to bioassay for PAF activity as described above. EICOSANOID MEASUREMENTS
Thromboxane B2 (TxB2) concentrations were measured in patients undergoing atrial stimulation but not in patients who underwent PTCA; they were under aspirin regimen which has been proved to be effective in preventing acute reocclusion. To measure plasma TxB2 concentrations, 4 ml of blood were collected in test tubes containing ethylene-diethine tetra acetic-acid (EDTA) and indomethacin (5mM and 10 UM final concentration respectively). Samples were immediately transferred to ice, centrifuged at 3000 g for 10 min and the plasma stored at - 70°C until further analyses. TxB2 and 6-keto-PGFla analyses were performed by enzyme immunoassay1191. OTHER MEASUREMENTS
Arterial and CS blood was drawn (2 ml samples) into glass test tubes containing 80 UM of fluoride/EDTA reagent. After centrifugation at 3000 # for 10 min, lactate levels were measured on supernatants by enzymatic assay (Boehringer Mannheim, Germany). We calculated the percent lactate extraction (%L) by subtracting the CS from the arterial concentration and dividing by the arterial concentration. Total white blood cells (WBC), and erythrocyte concentration, haematocrit, haemoglobin, and platelet concentrations were measured by an automated counter (ELT 800, Ortho Diagnostics, U.S.A.). STATISTICS
All data are expressed as mean±SEM. Changes of variables within groups were evaluated by a paired t-test. Significance was declared at an alpha level of 005. Results EPISODES OF MYOCARDIAL ISCHAEMIA
Atrial stimulation (group I) caused a significant decrease of % lactate extraction (> 5%) and ST-segment depression ( > l m m ) in all effort angina patients. Maximum changes of ST-segment from baseline were measured in the same lead at the peak of pacing-induced ischaemia. Mean ST-segment depression induced by pacing was - 1 -8 ± 0-2 mm. The percent lactate extraction in these angina patients decreased from +20 ± 6 % at baseline to — 15 ± 9% at the maximal pacing rate, then to
Downloaded from https://academic.oup.com/eurheartj/article-abstract/13/12/1641/651306 by Western Sydney University Library user on 14 January 2019
block was induced, atropine (1/2 mg i.v.) was administered. Our criteria to cease atrial stimulation were either chest pain, significant ST-segment depression ( > 1 mm), or a 10 min period of pacing at the maximum rate (150 beats . min~'). Three sets of blood samples were collected: at rest before pacing, during the last minute of pacing and immediately after pacing was discontinued. At each measurement interval, blood samples were taken simultaneously from the aorta and CS then placed onto ice.
PA F and myocardial ischaemia 1643
not measure significant changes of plasma 6-keto-PGFla levels in patients during atrial stimulation; all plasma 6-keto-PGF 1 a levels remained below 300 pg. ml"'.
20 0 i
-20
\i
-60 -80
*
i
Baseline
M
i Peak of ischaemia
1 Post-pacing
Figure I Changes of % lactate extraction after pacing (group I, O; n = 4) and PTCA (group II, • ; n = 4) in patients with angina pectoris.
We measured no significant changes from baseline of WBC and platelet concentrations in either CS or arterial blood after atrial stimulation. Therefore pacing-induced myocardial ischaemia was not associated with any significant CS-arterial cell gradient. Similarly, there were no changes of WBC and platelet counts following balloon inflation during PTCA. No changes of haematocrit and erythrocyte count were measured in any group. Discussion
Acute and brief episodes of myocardial ischaemia induced by pacing were associated with thromboxane release into coronary sinus plasma. The acute myocardial
