- Email: [email protected]
Effect of aspirin and ticlopidine on plasma tissue factor levels in stable and unstable angina pectoris
Effect of Aspirin and Ticlopidine on Plasma Tissue Factor Levels in Stable and Unstable Angina Pectoris Jean Marco, MD, Robert A.S. Arie¨ns, PhD, Jean Fajadet, MD, Irene M. Bossi, MD, Isabelle Marco, MD, Monique Bernies, MD, Salvatore M. Romano, MD, Francesco Donatelli, MD, Gabri M. Brambilla, MD, Francesco Somalvico, PhD, Daniela Mari, MD, and Luisa Gregorini, MD Patients with unstable angina have an increased activation of the coagulation system. Aspirin and ticlopidine given in combination may potentiate each other by the combination of different action mechanisms and may reduce the risk of coronary occlusion and clinical instability. Plasma tissue factor (TF) levels collected into the stenotic coronary artery may be an index of TF expression within the vasculature. In 160 patients undergoing angioplasty for a 81 ⴞ 5% coronary lesion, we measured TF in blood samples collected from a vein and from the coronary ostium. Immediately after and 10 minutes after the dilation procedures the samples were withdrawn also beyond the lesion. Heparin 150 U/kg was given as an anticoagulant. All patients were pretreated
with 250 mg/day of aspirin. One hundred twenty patients were randomly assigned to receive 24, 48, or 72 hours of ticlopidine treatment (250 mg/twice daily). TF levels did not increase during angioplasty but there was a significantly higher TF expression in unstable than in stable patients, irrespective of the invasiveness of debulking procedures. When ticlopidine was given for 72 hours, TF levels were similar to normal laboratory values both in stable and unstable patients. This combined antiplatelet pretreatment may be of benefit in unstable angina patients, with a favorable cost/benefit ratio. 䊚2000 by Excerpta Medica, Inc. (Am J Cardiol 2000;85:527–531)
nstable angina is a dynamic clinical situation in which recurrence of symptoms correlates with U plaque ulceration, coagulation activation, and throm-
let activation in patients undergoing angioplasty.18 In the present study we investigated whether aspirin and ticlopidine pretreatment, given in combination in patients with stable and unstable angina, may reduce plasma TF levels in the most unfavorable clinical condition of endothelium abrasion and plaque fissuring as during angioplasty.
bus formation.1–3 An angioscopic study of the plaques has shown that ulcerations are present in 55% and thrombus in 68% of the coronary arteries responsible for unstable angina.4 The lipid-rich atherosclerotic core exposed to circulating blood by plaque rupture triggers the coagulation activation.5,6 Coronary stenting7,8 and rotational atherectomy9 are revascularization techniques that extensively abrade endothelium and plaque,10,11 thus exposing the tissue factor (TF) present within the necrotic lipid-rich core12–15 to factor VII. TF may be responsible for activation of the coagulation cascade, and accordingly could be involved in the pathogenesis of acute events.15–17 Trace amounts of TF are also present in plasma,14 and these plasma levels may reflect TF expression within the vasculature. We have recently shown that aspirin, heparin, and 72 hours of ticlopidine pretreatment efficiently reduced both thrombin generation and plateFrom Clinique Pasteur, Toulouse, France; and Cardiovascular Science and Internal Medicine Department, Ospedale Maggiore-IRCCS, University of Milan, Milan, Italy. This study was supported in part by institutional funds from the University of Milan (MURST 60%), Milan, Italy, and by the research funds from Clinic Pasteur, Toulouse, France. Manuscript received April 26, 1999; revised manuscript received and accepted September 29, 1999. Address for reprints: Luisa V. Gregorini, MD, Cardiovascular Science Department, Ospedale Maggiore IRCCS, Universita` di Milano, Via Francesco Sforza 35, 20122 Milano, Italy. E-mail: [email protected]. ©2000 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 85 March 1, 2000
METHODS We enrolled in the study 160 consecutive patients who represented a typical sample of our angioplasty population. Among them 80 had stable angina and 80 had spontaneous durable recurrent episodes of chest pain and were classified as unstable.1,2 The mean age was 63.5 ⫾ 11.6 (18 women) and 64.4 ⫾ 11.3 (12 women) in patients with stable and unstable angina, respectively. The indication to angioplasty was given in the presence of a coronary lesion that occluded the vessel by 79 ⫾ 4% and 83 ⫾ 5% (mean ⫾ SD) in patients with stable and unstable angina, respectively. Among the unstable patients, 18 had non–insulindependent diabetes. All patients signed an informed written consent to allow blood sampling, and the Clinique Pasteur ethical committee had previously approved the study. Pharmacologic treatment: All patients were pretreated with 250 mg/day of aspirin. Twenty of 80 patients who had stable angina and an apparently regular, short, and smooth lesion were pretreated only with aspirin and the lesion was dilated by conventional percutaneous transluminal coronary angioplasty (PTCA). Twenty of 80 patients who had unstable 0002-9149/00/$–see front matter PII S0002-9149(99)00805-X
527
angina were pretreated only with aspirin, but to prevent possible complications they were given low molecular weight heparin in a weight–adjusted dose (enoxaparin 120 IU/kg) before PTCA. After coronary stenting they were treated with ticlopidine and aspirin for 1 month. All other patients received ticlopidine 250 mg twice daily in addition to aspirin.18 Ticlopidine pretreatment was randomly given to patients after diagnostic coronary angiography (1 of 4 patients in the stable or unstable angina group) for 24, 48, or 72 hours to find the time course of antiplatelet treatment. Antianginal treatment such as nitrates and calcium antagonists (diltiazem 180 to 360 mg/day) or -adrenergic blockers (atenolol 50 mg/day) were given in doses able to reduce recurrent ischemic episodes. Angioplasty procedure: To dilate the lesion, conventional PTCA was performed in 40 patients with stable angina, coronary stenting in 110 cases, and rotational atherectomy in 10 patients (Rotablator, Heart Technology, Bellevue, Washington). The choice of technique to perform for dilating the stenosis was left to the clinician after the anatomy of the plaque to treat and the presence of calcium was established.7–9 Ninety patients had left anterior descending, 20 left circumflex, and 44 right coronary artery lesions, and 6 of them had a left main trunk lesion. Either the radial (90 patients) or the femoral (70 patients) artery approach was used to perform angioplasty. Heparin (150 U/kg) was given intravenously as anticoagulant after obtaining the basal venous and coronary ostium blood samples. Isosorbide dinitrate (3 mg) was administered intracoronarily as vasodilator. One or multiple stents were implanted in vessels with a reference diameter ⱖ3.0 mm. High-pressure inflations were performed to overextend the struts of the stents. No acute or subacute closure was observed after coronary stenting. After coronary stenting ticlopidine, 250 mg once daily was given for 1 month. Aspirin was given for life. Blood sampling: To collect blood samples from the distal dilated coronary artery and from the coronary ostium, a probing catheter with an internal lumen of 0.018 inch was inserted immediately after the last balloon inflation through the monorail side. As soon as collected blood samples were added to ice-chilled plastic tubes containing sodium citrate (9:1 vol.:vol.). The blood samples were again withdrawn 10 minutes after the dilation procedures. The blood samples were centrifuged at 2,000 g for 15 minutes at 4°C. The supernatant platelet-poor plasma was stored at ⫺80°C until analysis. TF was measured in duplicate by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (ImmuBind tissue factor ELISA Kit, American Diagnostica Inc., Greenwich, Connecticut). Thrombin-antithrombin (TAT) was measured by enzyme immunoassay using commercially available kits (Behring Enzygnost Diagnostic Inc.: ELISA-TAT Micro, Marburg, Germany). All plasma assessments were performed in a blinded manner and in duplicate. Statistical analysis: Results are expressed as mean ⫾ SD. One- or 2-way analysis of variance was 528 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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performed with the SPSS 6.1 package, (SPSS Inc., Chicago, Illinois). To assess statistical significance between groups and times of treatment, the Tukey test for multiple comparisons was applied, with a p value ⬍0.05 considered significant. The plot of the differences between the 2 plasma sample assays against the mean differences was evaluated by estimating the consistent bias between measurements (mean ⫾ 2 SDs), as described by Bland and Altman.19 The significance of the mean differences was evaluated using a paired 2-tailed Student’s t test.
RESULTS No difference was found between TF levels obtained in blood samples collected from the coronary ostium before the procedure and those obtained from the coronary ostium or from the dilated coronary segment beyond the lesion immediately after or 10 minutes after angioplasty (Figure 1 and Table I). In addition, the samples obtained from a peripheral vein were not different from those obtained from the guiding catheter. No differences were observed in plasma TF antigen levels among patients undergoing the different dilation or debulking procedures. Accordingly, data reported in Figure 1 and in Table I were pooled. A significant difference in TF levels was found only when patients were divided by angina status, stable, or unstable. This difference was no longer significant when ticlopidine pretreatment was administered for ⱖ72 hours. In our patients, the revascularization procedure did not, per se, increase TF levels. The plot of the differences between the values from 2 TF samples versus the mean of the 2 measurements showed a mean difference of 0.565 ⫾ 10.755 pg/ml, with good agreement because all points but 2 were within ⫾ 2 SDs (Figure 2) (r ⫽ 0.994; p ⫽ 0.60). Thrombin-antithrombin complexes: The TAT levels were not different in venous and in arterial baseline samples (Figure 3 and Table II), indicating that the catheter did not activate the coagulation in the presence of the combined anticoagulant and antiplatelet treatment given. During the revascularization procedures, the degree of coagulation activation did not change significantly, with no differences found between basal venous and arterial levels of TAT compared with immediately after and 10 minutes after PTCA levels. Significantly higher TAT levels were found in patients not treated or pretreated with ticlopidine for ⱕ24 hours than in patients given ⱖ72 hours of ticlopidine pretreatment. No difference was observed between the samples obtained in duplicate (p ⫽ 0.477). The variability between the 2 measurements estimated in 100 cases showed a mean difference of 0.60 ⫾ 0.834 g/ml (Figure 2), with all values but 3 within ⫾ 2 SDs.
DISCUSSION Our findings show that combined antiplatelet pretreatment with aspirin and ticlopidine and intravenous heparin reduced plasma TF levels and thrombin generation in patients with unstable angina, irrespective of plaque abrasion or dissections or of metal struts. MARCH 1, 2000
FIGURE 1. Mean plasma TF levels (pg/ml ⴞ SD) collected before PTCA and soon after dilation procedures. TF normal laboratory values: 141 ⴞ 45 pg/ml. h ⴝ hours of ticlopidine pretreatment; S ⴝ stable; T ⴝ ticlopidine; U ⴝ patients with unstable angina.
TABLE I Plasma Tissue Factor Levels in Patients With Unstable and Stable Angina Pectoris Before PTCA Angina Pectoris Unstable
T No T
No.
Peripheral Vein
20
553.3 ⫾ 91
Stable
No T
20
193.3 ⫾ 95
Unstable
24 h T
20
425.1 ⫾ 172
Stable
24 h T
20
Unstable
48 h T
20
162.1 ⫾ 75 †
263.3 ⫾ 87
Soon After Dilation
Coronary Ostium
Coronary Ostium
544.3 ⫾ 81
§
562.7 ⫾ 61
§
194.6 ⫾ 87
‡ ‡ *
184.9 ⫾ 88 276.8 ⫾ 123
Beyond the Lesion 563.3 ⫾ 57
§
182.7 ⫾ 64
413.0 ⫾ 167 §
‡ 155.1 ‡
*
⫾ 97
227 ⫾ 125
‡157.3 ‡
*
⫾ 107
264.2 ⫾ 120
48 h T
20
158.8 ⫾ 58
146.0 ⫾ 41
135.5 ⫾ 67
136.6 ⫾ 65
Unstable
72 h T
20
148.7 ⫾ 103
171.0 ⫾ 144
155.3 ⫾ 105
151.4 ⫾ 127
Stable
72 h T
20
88.2 ⫾ 60
100.8 ⫾ 58
NS
77.7 ⫾ 46
555.5 ⫾ 93
§
NS
80.7 ⫾ 55
§
252.5 ⫾ 103
412.2 ⫾ 179 §
Stable
NS
Beyond the Lesion
225.3 ⫾ 51
415.1 ⫾ 159 §
10 Min Later
410.3 ⫾ 111 §
‡170.7 ‡
*
⫾ 186
282 ⫾ 132
§ ‡ ‡ *
130.1 ⫾ 59 152.7 ⫾ 42 NS
79.5 ⫾ 40
NS
*p ⬍0.05 unstable versus stable; †p ⬍0.05, 48- versus 24-hour treatment; ‡p ⬍0.001, 72- versus 24-hour treatment; §p ⬍0.001 unstable versus stable. Values are expressed as mean ⫾ SD (pg/ml). T ⫽ ticlopidine.
Higher plasma TF levels and TAT complexes were found in patients with unstable angina receiving 24 hours of ticlopidine pretreatment than in patients with stable angina. Also, after 72 hours of ticlopidine pretreatment, TF levels tended to be higher in patients with unstable than stable angina, but the difference was no longer significant and was similar to normal laboratory values in both groups. Our findings are in agreement with previous studies that showed that monocytes express higher TF amounts in patients with unstable coronary syndromes.13,14 TF antigen and activity were also greater in plaque specimens obtained by directional atherectomy when patients had unstable angina and myocardial infarction than in patients with
stable angina.17 Plasma TF levels may be directly related to TF expression within the vasculature and the atherosclerotic lesion. Therefore, plasma TF levels may be an index of thrombotic risk and acute coronary syndromes in a condition of plaque rupture as in patients with unstable angina.4 In fact, TF plasma levels paralleled the plasma TAT complexes, indicating that increased activation of the TF coagulation pathway is involved in acute coronary syndromes. Arterial specimens showed that a simple balloon friction abrades the endothelium, and that angioplasty ruptures the fibrous tissue.10 Accordingly, one can hypothesize that TF present in atherosclerotic lesions14,15 may be released into the circulation during
CORONARY ARTERY DISEASE/PLASMA TISSUE FACTOR IN UNSTABLE ANGINA
529
FIGURE 2. The plot of the mean differences of TF and TAT plasma samples’ (y-axis) corresponding mean values (x-axis) is shown. Mean differences (solid line) and ⴞ 2 SDs from the difference (dashed line) (Bland Altman test) are represented.
FIGURE 3. Mean plasma TAT complexes ⴞ SD collected before PTCA and immediately after dilation procedures. TAT normal laboratory values 141 ⴞ 45 pg/ml. Abbreviations as in Figure 1.
the dilation procedures. However, we did not find significantly increased plasma TF levels after dilation procedures in samples collected from either the proximal coronary artery or just beyond the dilated lesion. Based on these results, we hypothesize that dilation procedures, per se, do not release significant quantities of TF into the circulation when patients are treated with a combination of antiplatelet drugs and heparin. Whether or not the dilation procedure releases TF into circulation, abrasion of the plaque very likely leads to exposure of functionally active TF14,15 of which high concentrations are present in lesions of patients with myocardial infarction and unstable angina.15 Lower TF plasma levels were found in patients receiving 72 hours of ticlopidine treatment than in patients receiv530 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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ing no ticlopidine or receiving ticlopidine for ⬍24 hours. Apparently, the effect was independent of the type of atherosclerotic lesion and instability or severity of the clinical situation. These results indicate that ticlopidine treatment for at least 72 hours reduces TF plasma levels both in stable and in unstable patients. Hence, ticlopidine not only inhibits platelet aggregation, but it also significantly lowers plasma TF levels and therefore may inhibit intravascular TF expression and subsequent coagulation activation. How thienopyridines reduce TF plasma levels is not elucidated at present. One possibility might be that TF plasma levels are directly proportional to the amount of TF expressed on monocytes and endothelial cells. The reduction in plasma TF we found could therefore be MARCH 1, 2000
TABLE II Thrombin-Antithrombin Complexes Angina Pectoris Unstable
Before PTCA T No T
No.
Peripheral Vein 38.63 ⫾ 10
20
Stable
No T
20
Unstable
24 h T
20
Stable
24 h T
20
Unstable
48 h T
20
‡
Coronary Ostium
6.65 ⫾ 2.1
‡
§
†
7.0 ⫾ 3.9 5.87 ⫾ 2.7
Stable
48 h T
20
3.91 ⫾ 0.7
3.61 ⫾ 1.7
Unstable
72 h T
20
4.40 ⫾ 1.5
4.45 ⫾ 2.3
Stable
72 h T
20
Beyond Lesion
‡
15.53 ⫾ 5.3
15.11 ⫾ 7.1
‡
11.9 ⫾ 7.1
‡ §
Coronary Ostium
§
10.9 ⫾ 6.2
‡
11.91 ⫾ 3.2
†
34.3 ⫾ 9.5
§
7.99 ⫾ 2.7
6.92 ⫾ 1.7
Soon After Dilation
*
3.03 ⫾ 0.7
3.00 ⫾ 0.7
§
5.73 ⫾ 3.1 7.90 ⫾ 3.1
§
10.22 ⫾ 3.5
‡ ‡
§ ‡
*
5.71 ⫾ 3.1
‡ 11.52 §
§
3.51 ⫾ 1.2 3.35 ⫾ 1.1 2.36 ⫾ 0.5
§
6.56 ⫾ 3.2
‡
⫾ 3.5
8.29 ⫾ 2.3 4.68 ⫾ 2.1
‡ §
‡ ‡
*
3.92 ⫾ 1.7 NS
3.25 ⫾ 1.5
NS
2.53 ⫾ 0.3
*p ⬍0.05 unstable versus stable; unstable versus stable; †p ⬍0.05; ‡p ⬍0.001; §p ⬍0.001. T ⫽ ticlopidine.
caused by inhibition of TF expression on these cells by ticlopidine. This effect could be similar to, but independent of, the reduction of glycoprotein IIb-IIIa expression induced on platelets by ticlopidine. Alternatively, plasma levels of TF may reflect concentration of the enzyme responsible for TF release into the circulation. The inhibitory effect of ticlopidine on TF could be through reduction of this releasing mechanism. Either effect may be independent of the effect on platelet biochemistry. Ticlopidine reduces platelet aggregation by inhibiting adenosine diphosphate–induced platelet activation, serotonin release from platelets, and expression of the fibrinogen receptor on the platelet membrane.20,21 TF has a structure similar to interferon receptors, and is placed in the cytokine receptor superfamily.22,23 Therefore, TF functions as a cellular receptor of factor VII,24 and because ticlopidine inhibits expression of platelet fibrinogen receptors, it may inhibit expression of TF in a similar fashion. 1. Braunwald E. Unstable Angina: a classification. Circulation 1989;80:410 – 414. 2. Buja LM, Willerson JT. Clinicopathologic correlates of acute ischemic heart syndromes. Am J Cardiol 1984;54:1349 –1354. 3. Merlini PA, Bauer KA, Oltrona L, Ardissino D, Cattaneo M, Belli C, Mannucci PM, Rosenberg RD. Persistent activation of coagulation mechanisms in unstable angina and myocardial infarction. Circulation 1994;90:61– 68. 4. de Feyter PJ, Ozaki Y, Baptista J, Escaned J, Di Mario C, de Jaegere PP, Serruys PW, Roelandt JR. Ischemia related lesion characteristics in patients with stable or unstable angina. A study with intracoronary angioscopy and ultrasound. Circulation 1995;92:1408 –1413. 5. Fuster V, Steele PM, Chesebro JH. Role of platelets and thrombosis in coronary atherosclerotic disease and sudden death. J Am Coll Cardiol 1985;5: 175B-184B. 6. Bleifeld W, Hamm CW, Braunwald E, eds. Unstable Angina. New York: Springer-Verlag, 1990. 7. Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P, et al, for the Benestent Study Group. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med 1994;331: 489 – 495. 8. Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K,
Veltri L, Ricci D, Nobuyoshi M, Cleman M, Heuser R, Almond D, Teirstein RS, Fish D, and the STRESS investigators. A randomized comparison of coronarystent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994;331:496 –501. 9. Warth DC, Leon MB, O’Neill W, Zacca N, Polissar NL, Buchbinder M. Rotational atherectomy multicenter registry: acute results, complications and 6-month angiographic follow-up in 709 patients. J Am Coll Cardiol 1994;24: 641– 648. 10. Waller BF. “Crackers, breakers, stretchers, drillers, scrapers, shavers, burners, welders and melters”: the future treatment of the atherosclerotic coronary artery disease, a clinical morphologic assessment. J Am Coll Cardiol 1989;13: 969 –987. 11. Mintz GS, Potkin BN, Karen G, Satler LF, Pichard AD, Kent KM, Popma JJ, Leon MB. Intravascular ultrasound evaluation of the effect of rotational atherectomy in obstructive atherosclerotic coronary artery disease. Circulation 1992;86: 1383–1393. 12. Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989;86:2839 –2843. 13. Fuster V, Fallon JT, Nemerson Y. Coronary thrombosis. Lancet 1996; 348(suppl):S7-S10. 14. Nemerson Y. Tissue factor and hemostasis. Blood 1988;71:1– 8. 15. Neri Serneri GG, Abbate R, Gori AM, Attansio M, Martini F, Giusti B, Dabazzi P, Poggesi L, Modesti PA, Trotta F, Rostagno C, Boddi M, Gensini GF. Transient intermittent lymphocyte activation is responsible for the instability of angina. Circulation 1992;86:790 –797. 16. Marmur JD, Thiruvikraman SV, Fyfe BS, Arabinda G, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation 1996;94:1226 –1232. 17. Ardissino D, Merlini PA, Arie¨ns RAS, Coppola R, Bramucci E, Mannucci PM. Tissue-factor antigen and activity in human atherosclerotic plaques. Lancet 1997;349:769 –771. 18. Gregorini L, Marco J, Fajadet J, Bernies M, Cassagneau B, Brunel P, Bossi IM, Mannucci PM. Ticlopidine and aspirin pretreatment reduces coagulation and platelet activation during coronary dilation procedures. J Am Coll Cardiol 1997; 29:13–20. 19. Bland MJ, Altman DG. Statistical method for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–309. 20. McTavish D, Faulds D, Goa KL. Ticlopidine an up dated review of its pharmacology and therapeutic use in platelet dependent disorders. Drugs 1990; 40:238 –259. 21. Harker LA, Bruno JJ. Ticlopidine’ s mechanism of action on human platelets. In: Hass WK, Easton JD, eds. Ticlopidine, Platelets and Vascular Disease. New York: Springer Verlag, 1991:41–51. 22. Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad SCI USA 1990;87:6934 – 6938. 23. Edgington TS, Mackman N, Brand K, Ruff W. The structural biology of expression and function of tissue factor. Thromb Haemost 1991;66:67–79. 24. Pendurthi UR, Alok D, Rao LVM. Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: up-regulation of poly (A) polymerase. Proc Natl Acad Sci USA 1997;94:12598 –12603.
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with 250 mg/day of aspirin. One hundred twenty patients were randomly assigned to receive 24, 48, or 72 hours of ticlopidine treatment (250 mg/twice daily). TF levels did not increase during angioplasty but there was a significantly higher TF expression in unstable than in stable patients, irrespective of the invasiveness of debulking procedures. When ticlopidine was given for 72 hours, TF levels were similar to normal laboratory values both in stable and unstable patients. This combined antiplatelet pretreatment may be of benefit in unstable angina patients, with a favorable cost/benefit ratio. 䊚2000 by Excerpta Medica, Inc. (Am J Cardiol 2000;85:527–531)
nstable angina is a dynamic clinical situation in which recurrence of symptoms correlates with U plaque ulceration, coagulation activation, and throm-
let activation in patients undergoing angioplasty.18 In the present study we investigated whether aspirin and ticlopidine pretreatment, given in combination in patients with stable and unstable angina, may reduce plasma TF levels in the most unfavorable clinical condition of endothelium abrasion and plaque fissuring as during angioplasty.
bus formation.1–3 An angioscopic study of the plaques has shown that ulcerations are present in 55% and thrombus in 68% of the coronary arteries responsible for unstable angina.4 The lipid-rich atherosclerotic core exposed to circulating blood by plaque rupture triggers the coagulation activation.5,6 Coronary stenting7,8 and rotational atherectomy9 are revascularization techniques that extensively abrade endothelium and plaque,10,11 thus exposing the tissue factor (TF) present within the necrotic lipid-rich core12–15 to factor VII. TF may be responsible for activation of the coagulation cascade, and accordingly could be involved in the pathogenesis of acute events.15–17 Trace amounts of TF are also present in plasma,14 and these plasma levels may reflect TF expression within the vasculature. We have recently shown that aspirin, heparin, and 72 hours of ticlopidine pretreatment efficiently reduced both thrombin generation and plateFrom Clinique Pasteur, Toulouse, France; and Cardiovascular Science and Internal Medicine Department, Ospedale Maggiore-IRCCS, University of Milan, Milan, Italy. This study was supported in part by institutional funds from the University of Milan (MURST 60%), Milan, Italy, and by the research funds from Clinic Pasteur, Toulouse, France. Manuscript received April 26, 1999; revised manuscript received and accepted September 29, 1999. Address for reprints: Luisa V. Gregorini, MD, Cardiovascular Science Department, Ospedale Maggiore IRCCS, Universita` di Milano, Via Francesco Sforza 35, 20122 Milano, Italy. E-mail: [email protected]. ©2000 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 85 March 1, 2000
METHODS We enrolled in the study 160 consecutive patients who represented a typical sample of our angioplasty population. Among them 80 had stable angina and 80 had spontaneous durable recurrent episodes of chest pain and were classified as unstable.1,2 The mean age was 63.5 ⫾ 11.6 (18 women) and 64.4 ⫾ 11.3 (12 women) in patients with stable and unstable angina, respectively. The indication to angioplasty was given in the presence of a coronary lesion that occluded the vessel by 79 ⫾ 4% and 83 ⫾ 5% (mean ⫾ SD) in patients with stable and unstable angina, respectively. Among the unstable patients, 18 had non–insulindependent diabetes. All patients signed an informed written consent to allow blood sampling, and the Clinique Pasteur ethical committee had previously approved the study. Pharmacologic treatment: All patients were pretreated with 250 mg/day of aspirin. Twenty of 80 patients who had stable angina and an apparently regular, short, and smooth lesion were pretreated only with aspirin and the lesion was dilated by conventional percutaneous transluminal coronary angioplasty (PTCA). Twenty of 80 patients who had unstable 0002-9149/00/$–see front matter PII S0002-9149(99)00805-X
527
angina were pretreated only with aspirin, but to prevent possible complications they were given low molecular weight heparin in a weight–adjusted dose (enoxaparin 120 IU/kg) before PTCA. After coronary stenting they were treated with ticlopidine and aspirin for 1 month. All other patients received ticlopidine 250 mg twice daily in addition to aspirin.18 Ticlopidine pretreatment was randomly given to patients after diagnostic coronary angiography (1 of 4 patients in the stable or unstable angina group) for 24, 48, or 72 hours to find the time course of antiplatelet treatment. Antianginal treatment such as nitrates and calcium antagonists (diltiazem 180 to 360 mg/day) or -adrenergic blockers (atenolol 50 mg/day) were given in doses able to reduce recurrent ischemic episodes. Angioplasty procedure: To dilate the lesion, conventional PTCA was performed in 40 patients with stable angina, coronary stenting in 110 cases, and rotational atherectomy in 10 patients (Rotablator, Heart Technology, Bellevue, Washington). The choice of technique to perform for dilating the stenosis was left to the clinician after the anatomy of the plaque to treat and the presence of calcium was established.7–9 Ninety patients had left anterior descending, 20 left circumflex, and 44 right coronary artery lesions, and 6 of them had a left main trunk lesion. Either the radial (90 patients) or the femoral (70 patients) artery approach was used to perform angioplasty. Heparin (150 U/kg) was given intravenously as anticoagulant after obtaining the basal venous and coronary ostium blood samples. Isosorbide dinitrate (3 mg) was administered intracoronarily as vasodilator. One or multiple stents were implanted in vessels with a reference diameter ⱖ3.0 mm. High-pressure inflations were performed to overextend the struts of the stents. No acute or subacute closure was observed after coronary stenting. After coronary stenting ticlopidine, 250 mg once daily was given for 1 month. Aspirin was given for life. Blood sampling: To collect blood samples from the distal dilated coronary artery and from the coronary ostium, a probing catheter with an internal lumen of 0.018 inch was inserted immediately after the last balloon inflation through the monorail side. As soon as collected blood samples were added to ice-chilled plastic tubes containing sodium citrate (9:1 vol.:vol.). The blood samples were again withdrawn 10 minutes after the dilation procedures. The blood samples were centrifuged at 2,000 g for 15 minutes at 4°C. The supernatant platelet-poor plasma was stored at ⫺80°C until analysis. TF was measured in duplicate by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (ImmuBind tissue factor ELISA Kit, American Diagnostica Inc., Greenwich, Connecticut). Thrombin-antithrombin (TAT) was measured by enzyme immunoassay using commercially available kits (Behring Enzygnost Diagnostic Inc.: ELISA-TAT Micro, Marburg, Germany). All plasma assessments were performed in a blinded manner and in duplicate. Statistical analysis: Results are expressed as mean ⫾ SD. One- or 2-way analysis of variance was 528 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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performed with the SPSS 6.1 package, (SPSS Inc., Chicago, Illinois). To assess statistical significance between groups and times of treatment, the Tukey test for multiple comparisons was applied, with a p value ⬍0.05 considered significant. The plot of the differences between the 2 plasma sample assays against the mean differences was evaluated by estimating the consistent bias between measurements (mean ⫾ 2 SDs), as described by Bland and Altman.19 The significance of the mean differences was evaluated using a paired 2-tailed Student’s t test.
RESULTS No difference was found between TF levels obtained in blood samples collected from the coronary ostium before the procedure and those obtained from the coronary ostium or from the dilated coronary segment beyond the lesion immediately after or 10 minutes after angioplasty (Figure 1 and Table I). In addition, the samples obtained from a peripheral vein were not different from those obtained from the guiding catheter. No differences were observed in plasma TF antigen levels among patients undergoing the different dilation or debulking procedures. Accordingly, data reported in Figure 1 and in Table I were pooled. A significant difference in TF levels was found only when patients were divided by angina status, stable, or unstable. This difference was no longer significant when ticlopidine pretreatment was administered for ⱖ72 hours. In our patients, the revascularization procedure did not, per se, increase TF levels. The plot of the differences between the values from 2 TF samples versus the mean of the 2 measurements showed a mean difference of 0.565 ⫾ 10.755 pg/ml, with good agreement because all points but 2 were within ⫾ 2 SDs (Figure 2) (r ⫽ 0.994; p ⫽ 0.60). Thrombin-antithrombin complexes: The TAT levels were not different in venous and in arterial baseline samples (Figure 3 and Table II), indicating that the catheter did not activate the coagulation in the presence of the combined anticoagulant and antiplatelet treatment given. During the revascularization procedures, the degree of coagulation activation did not change significantly, with no differences found between basal venous and arterial levels of TAT compared with immediately after and 10 minutes after PTCA levels. Significantly higher TAT levels were found in patients not treated or pretreated with ticlopidine for ⱕ24 hours than in patients given ⱖ72 hours of ticlopidine pretreatment. No difference was observed between the samples obtained in duplicate (p ⫽ 0.477). The variability between the 2 measurements estimated in 100 cases showed a mean difference of 0.60 ⫾ 0.834 g/ml (Figure 2), with all values but 3 within ⫾ 2 SDs.
DISCUSSION Our findings show that combined antiplatelet pretreatment with aspirin and ticlopidine and intravenous heparin reduced plasma TF levels and thrombin generation in patients with unstable angina, irrespective of plaque abrasion or dissections or of metal struts. MARCH 1, 2000
FIGURE 1. Mean plasma TF levels (pg/ml ⴞ SD) collected before PTCA and soon after dilation procedures. TF normal laboratory values: 141 ⴞ 45 pg/ml. h ⴝ hours of ticlopidine pretreatment; S ⴝ stable; T ⴝ ticlopidine; U ⴝ patients with unstable angina.
TABLE I Plasma Tissue Factor Levels in Patients With Unstable and Stable Angina Pectoris Before PTCA Angina Pectoris Unstable
T No T
No.
Peripheral Vein
20
553.3 ⫾ 91
Stable
No T
20
193.3 ⫾ 95
Unstable
24 h T
20
425.1 ⫾ 172
Stable
24 h T
20
Unstable
48 h T
20
162.1 ⫾ 75 †
263.3 ⫾ 87
Soon After Dilation
Coronary Ostium
Coronary Ostium
544.3 ⫾ 81
§
562.7 ⫾ 61
§
194.6 ⫾ 87
‡ ‡ *
184.9 ⫾ 88 276.8 ⫾ 123
Beyond the Lesion 563.3 ⫾ 57
§
182.7 ⫾ 64
413.0 ⫾ 167 §
‡ 155.1 ‡
*
⫾ 97
227 ⫾ 125
‡157.3 ‡
*
⫾ 107
264.2 ⫾ 120
48 h T
20
158.8 ⫾ 58
146.0 ⫾ 41
135.5 ⫾ 67
136.6 ⫾ 65
Unstable
72 h T
20
148.7 ⫾ 103
171.0 ⫾ 144
155.3 ⫾ 105
151.4 ⫾ 127
Stable
72 h T
20
88.2 ⫾ 60
100.8 ⫾ 58
NS
77.7 ⫾ 46
555.5 ⫾ 93
§
NS
80.7 ⫾ 55
§
252.5 ⫾ 103
412.2 ⫾ 179 §
Stable
NS
Beyond the Lesion
225.3 ⫾ 51
415.1 ⫾ 159 §
10 Min Later
410.3 ⫾ 111 §
‡170.7 ‡
*
⫾ 186
282 ⫾ 132
§ ‡ ‡ *
130.1 ⫾ 59 152.7 ⫾ 42 NS
79.5 ⫾ 40
NS
*p ⬍0.05 unstable versus stable; †p ⬍0.05, 48- versus 24-hour treatment; ‡p ⬍0.001, 72- versus 24-hour treatment; §p ⬍0.001 unstable versus stable. Values are expressed as mean ⫾ SD (pg/ml). T ⫽ ticlopidine.
Higher plasma TF levels and TAT complexes were found in patients with unstable angina receiving 24 hours of ticlopidine pretreatment than in patients with stable angina. Also, after 72 hours of ticlopidine pretreatment, TF levels tended to be higher in patients with unstable than stable angina, but the difference was no longer significant and was similar to normal laboratory values in both groups. Our findings are in agreement with previous studies that showed that monocytes express higher TF amounts in patients with unstable coronary syndromes.13,14 TF antigen and activity were also greater in plaque specimens obtained by directional atherectomy when patients had unstable angina and myocardial infarction than in patients with
stable angina.17 Plasma TF levels may be directly related to TF expression within the vasculature and the atherosclerotic lesion. Therefore, plasma TF levels may be an index of thrombotic risk and acute coronary syndromes in a condition of plaque rupture as in patients with unstable angina.4 In fact, TF plasma levels paralleled the plasma TAT complexes, indicating that increased activation of the TF coagulation pathway is involved in acute coronary syndromes. Arterial specimens showed that a simple balloon friction abrades the endothelium, and that angioplasty ruptures the fibrous tissue.10 Accordingly, one can hypothesize that TF present in atherosclerotic lesions14,15 may be released into the circulation during
CORONARY ARTERY DISEASE/PLASMA TISSUE FACTOR IN UNSTABLE ANGINA
529
FIGURE 2. The plot of the mean differences of TF and TAT plasma samples’ (y-axis) corresponding mean values (x-axis) is shown. Mean differences (solid line) and ⴞ 2 SDs from the difference (dashed line) (Bland Altman test) are represented.
FIGURE 3. Mean plasma TAT complexes ⴞ SD collected before PTCA and immediately after dilation procedures. TAT normal laboratory values 141 ⴞ 45 pg/ml. Abbreviations as in Figure 1.
the dilation procedures. However, we did not find significantly increased plasma TF levels after dilation procedures in samples collected from either the proximal coronary artery or just beyond the dilated lesion. Based on these results, we hypothesize that dilation procedures, per se, do not release significant quantities of TF into the circulation when patients are treated with a combination of antiplatelet drugs and heparin. Whether or not the dilation procedure releases TF into circulation, abrasion of the plaque very likely leads to exposure of functionally active TF14,15 of which high concentrations are present in lesions of patients with myocardial infarction and unstable angina.15 Lower TF plasma levels were found in patients receiving 72 hours of ticlopidine treatment than in patients receiv530 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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ing no ticlopidine or receiving ticlopidine for ⬍24 hours. Apparently, the effect was independent of the type of atherosclerotic lesion and instability or severity of the clinical situation. These results indicate that ticlopidine treatment for at least 72 hours reduces TF plasma levels both in stable and in unstable patients. Hence, ticlopidine not only inhibits platelet aggregation, but it also significantly lowers plasma TF levels and therefore may inhibit intravascular TF expression and subsequent coagulation activation. How thienopyridines reduce TF plasma levels is not elucidated at present. One possibility might be that TF plasma levels are directly proportional to the amount of TF expressed on monocytes and endothelial cells. The reduction in plasma TF we found could therefore be MARCH 1, 2000
TABLE II Thrombin-Antithrombin Complexes Angina Pectoris Unstable
Before PTCA T No T
No.
Peripheral Vein 38.63 ⫾ 10
20
Stable
No T
20
Unstable
24 h T
20
Stable
24 h T
20
Unstable
48 h T
20
‡
Coronary Ostium
6.65 ⫾ 2.1
‡
§
†
7.0 ⫾ 3.9 5.87 ⫾ 2.7
Stable
48 h T
20
3.91 ⫾ 0.7
3.61 ⫾ 1.7
Unstable
72 h T
20
4.40 ⫾ 1.5
4.45 ⫾ 2.3
Stable
72 h T
20
Beyond Lesion
‡
15.53 ⫾ 5.3
15.11 ⫾ 7.1
‡
11.9 ⫾ 7.1
‡ §
Coronary Ostium
§
10.9 ⫾ 6.2
‡
11.91 ⫾ 3.2
†
34.3 ⫾ 9.5
§
7.99 ⫾ 2.7
6.92 ⫾ 1.7
Soon After Dilation
*
3.03 ⫾ 0.7
3.00 ⫾ 0.7
§
5.73 ⫾ 3.1 7.90 ⫾ 3.1
§
10.22 ⫾ 3.5
‡ ‡
§ ‡
*
5.71 ⫾ 3.1
‡ 11.52 §
§
3.51 ⫾ 1.2 3.35 ⫾ 1.1 2.36 ⫾ 0.5
§
6.56 ⫾ 3.2
‡
⫾ 3.5
8.29 ⫾ 2.3 4.68 ⫾ 2.1
‡ §
‡ ‡
*
3.92 ⫾ 1.7 NS
3.25 ⫾ 1.5
NS
2.53 ⫾ 0.3
*p ⬍0.05 unstable versus stable; unstable versus stable; †p ⬍0.05; ‡p ⬍0.001; §p ⬍0.001. T ⫽ ticlopidine.
caused by inhibition of TF expression on these cells by ticlopidine. This effect could be similar to, but independent of, the reduction of glycoprotein IIb-IIIa expression induced on platelets by ticlopidine. Alternatively, plasma levels of TF may reflect concentration of the enzyme responsible for TF release into the circulation. The inhibitory effect of ticlopidine on TF could be through reduction of this releasing mechanism. Either effect may be independent of the effect on platelet biochemistry. Ticlopidine reduces platelet aggregation by inhibiting adenosine diphosphate–induced platelet activation, serotonin release from platelets, and expression of the fibrinogen receptor on the platelet membrane.20,21 TF has a structure similar to interferon receptors, and is placed in the cytokine receptor superfamily.22,23 Therefore, TF functions as a cellular receptor of factor VII,24 and because ticlopidine inhibits expression of platelet fibrinogen receptors, it may inhibit expression of TF in a similar fashion. 1. Braunwald E. Unstable Angina: a classification. Circulation 1989;80:410 – 414. 2. Buja LM, Willerson JT. Clinicopathologic correlates of acute ischemic heart syndromes. Am J Cardiol 1984;54:1349 –1354. 3. Merlini PA, Bauer KA, Oltrona L, Ardissino D, Cattaneo M, Belli C, Mannucci PM, Rosenberg RD. Persistent activation of coagulation mechanisms in unstable angina and myocardial infarction. Circulation 1994;90:61– 68. 4. de Feyter PJ, Ozaki Y, Baptista J, Escaned J, Di Mario C, de Jaegere PP, Serruys PW, Roelandt JR. Ischemia related lesion characteristics in patients with stable or unstable angina. A study with intracoronary angioscopy and ultrasound. Circulation 1995;92:1408 –1413. 5. Fuster V, Steele PM, Chesebro JH. Role of platelets and thrombosis in coronary atherosclerotic disease and sudden death. J Am Coll Cardiol 1985;5: 175B-184B. 6. Bleifeld W, Hamm CW, Braunwald E, eds. Unstable Angina. New York: Springer-Verlag, 1990. 7. Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P, et al, for the Benestent Study Group. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med 1994;331: 489 – 495. 8. Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K,
Veltri L, Ricci D, Nobuyoshi M, Cleman M, Heuser R, Almond D, Teirstein RS, Fish D, and the STRESS investigators. A randomized comparison of coronarystent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994;331:496 –501. 9. Warth DC, Leon MB, O’Neill W, Zacca N, Polissar NL, Buchbinder M. Rotational atherectomy multicenter registry: acute results, complications and 6-month angiographic follow-up in 709 patients. J Am Coll Cardiol 1994;24: 641– 648. 10. Waller BF. “Crackers, breakers, stretchers, drillers, scrapers, shavers, burners, welders and melters”: the future treatment of the atherosclerotic coronary artery disease, a clinical morphologic assessment. J Am Coll Cardiol 1989;13: 969 –987. 11. Mintz GS, Potkin BN, Karen G, Satler LF, Pichard AD, Kent KM, Popma JJ, Leon MB. Intravascular ultrasound evaluation of the effect of rotational atherectomy in obstructive atherosclerotic coronary artery disease. Circulation 1992;86: 1383–1393. 12. Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989;86:2839 –2843. 13. Fuster V, Fallon JT, Nemerson Y. Coronary thrombosis. Lancet 1996; 348(suppl):S7-S10. 14. Nemerson Y. Tissue factor and hemostasis. Blood 1988;71:1– 8. 15. Neri Serneri GG, Abbate R, Gori AM, Attansio M, Martini F, Giusti B, Dabazzi P, Poggesi L, Modesti PA, Trotta F, Rostagno C, Boddi M, Gensini GF. Transient intermittent lymphocyte activation is responsible for the instability of angina. Circulation 1992;86:790 –797. 16. Marmur JD, Thiruvikraman SV, Fyfe BS, Arabinda G, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation 1996;94:1226 –1232. 17. Ardissino D, Merlini PA, Arie¨ns RAS, Coppola R, Bramucci E, Mannucci PM. Tissue-factor antigen and activity in human atherosclerotic plaques. Lancet 1997;349:769 –771. 18. Gregorini L, Marco J, Fajadet J, Bernies M, Cassagneau B, Brunel P, Bossi IM, Mannucci PM. Ticlopidine and aspirin pretreatment reduces coagulation and platelet activation during coronary dilation procedures. J Am Coll Cardiol 1997; 29:13–20. 19. Bland MJ, Altman DG. Statistical method for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–309. 20. McTavish D, Faulds D, Goa KL. Ticlopidine an up dated review of its pharmacology and therapeutic use in platelet dependent disorders. Drugs 1990; 40:238 –259. 21. Harker LA, Bruno JJ. Ticlopidine’ s mechanism of action on human platelets. In: Hass WK, Easton JD, eds. Ticlopidine, Platelets and Vascular Disease. New York: Springer Verlag, 1991:41–51. 22. Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad SCI USA 1990;87:6934 – 6938. 23. Edgington TS, Mackman N, Brand K, Ruff W. The structural biology of expression and function of tissue factor. Thromb Haemost 1991;66:67–79. 24. Pendurthi UR, Alok D, Rao LVM. Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: up-regulation of poly (A) polymerase. Proc Natl Acad Sci USA 1997;94:12598 –12603.
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