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Hemorrhagic potential of combined diltiazem and recombinant tissue-type plasminogen activator administration
Hemorrhagic potential of combined diltiazem and recombinant tissue-type plasminogen activator administration In the Thrombolysis In Myocardial Infarction (TIMI) phase II study, use of calcium channel antagonists at study entry was associated with an increased risl< of intracerebral hemorrhage. Whether the observed association was due solely to chance, underlying cerebrovascular disease, or an effect of calcium channel antagonists themselves was not determined. Accordingly, blood loss from standardized ear incisions was measured in six groups of anesthetized New Zealand white rabbits: (1) saline control, (2) intravenous diltiazem (20 /Lig/kg/min X 60 minutes), (3) intravenous recombinant tissue-type plasminogen activator (rTPA) (1.0 mg/kg over 60 minutes, 10% bolus), (4) diltiazem plus rTPA, (5) diltiazem daily for 3 consecutive days, and (6) diltiazem (3 days) plus rTPA given on day 3. The combination of rTPA plus diltiazem (3 days) resulted in significantly more blood loss than rTPA alone, diltiazem (60-minute infusion), or rTPA plus diltiazem (60-minute infusion) (p = 0.003). Similarly, diltiazem (3 days) resulted in more blood loss than either agent alone or rTPA plus diltiazem (60-minute infusion) (p < 0.05). Thus, in this animal model, prolonged exposure to diltiazem with or without rTPA was associated with increased bleeding. The potential for chronic use of oral calcium channel antagonist to increase hemorrhagic risk after rTPA administration requires further investigation. (AM HEART J 1993;126:11-14.)
Richard C. Becker, MD, Ronald Caputo, MD, Steven Ball, RN, Jeanne M. Corrao, RN, MS, Stephen Baker, MS, and Joel M. Gore, MD Worcester, Mass.
Thrombolytic therapy is used widely in the treatment of acute myocardial infarction. The evidence that timely thrombolysis significantly decreases short-^'^ and long-term^'® mortality is compelling. The use of thrombolytic agents, however, is associated with a small but identifiable risk of bleeding.'' Intracerebral hemorrhage, the most feared and serious complication of thrombolysis, although it occurs in only a small number of patients, can have devastating consequences.® In the Thrombolysis in Myocardial Infarction phase II (TIMI II) trial, intracerebral hemorrhage was documented in 0.4 % of patients treated with recombinant tissue-type plasminogen activator (rTPA) (100 mg) given in combination with intravenous heparin and aspirin. Interestingly, use of
From the Thrombosis Research Center, Division of Cardiovascular Medicine, University of Massachusetts Medical School. Received for publication Nov. 25, 1992; accepted Jan. 8, 1993. Reprint requests: Richard C. Becker, MD, Thrombosis Research Center, University of Massachusetts Medical School, Worcester, MA 01655. Copyright ® 1993 by Mosby-Year Book, Inc. 0002-8703/93/$1.00 + .10 4 / 1 / 4 6 3 9 6
calcium channel antagonists at study entry was associated with an increased frequency of intracerebral hemorrhage. Whether this association was due to chance, accompanying cerebrovascular disease, or to a direct effect of calcium channel antagonists could not be determined. The purpose of this study was to distinguish these possibilities further by investigating the effect of calcium channel antagonists on the hemostatic mechanism when administered with rTPA. METHODS
A rabbit model was chosen for our series of experiments. In an earlier study^ we were able to show that blood loss from standardized ear incisions was not influenced by the presence of a jugular vein thrombus. Thus, although the initial design called for a venous thrombosis model, it was excluded from the present study. The method for assessing blood loss was derived from the work of Carter et aL^" and Pietraszek et al.^^ In brief, New Zealand white rabbits were anesthetized with Ketaset (ketamine hydrochloride) and Rompun (xylazine hydrochloride). The ears were shaved. One ear was then immersed for 10 minutes in a 37° C water bath. Under trans-
11
July 1993 American Heart Journal
12 Becker et al. Table I. Total blood loss from standardized ear incisions in six experimental groups Total blood loss Treatment 1 2 3 4 5 6
(N = 6) (N = 11) (N = 10) (N = 5) (N = 6) (N = 6)
group
M
SD
75.4 76.4 78.7 76.7 380.8* 278.5*
53.8 77.2 115.8 80.0 199.5 203.4
*p < 0.05.
illumination, five full-thickness incisions were made with a Bard-Parker No. 11 scalpel blade (Becton Dickinson Infusion Systems, Lincoln Park, N.J.). Care was taken to avoid the major vessels. Blood from the incisions was collected in a beaker containing 25 ml of 0.15 mol/L sodium chloride and hemolyzed by the addition of 2.5 ml of Triton X-100 solution (Sigma Diagnostics, St. Louis, Mo.). The optical density of the solution was measured photometrically at 546 with a Karl-Zeiss spectrophotometer (Spectronic 710, Bausch & Lomb, Rochester, N.Y.). Total blood loss was quantitated by means of a standard curve constructed with known volumes of rabbit blood. Six groups of animals were studied: group 1 received a 60-minute infusion of 0.9 % sodium chloride (total volume 15 cc); group 2 received intravenous diltiazem (Marion Merrell Dow, Kansas City, Mo.) at a dose of 20 /ig/kg/min for a total of 60 minutes; group 3 received intravenous rTPA (Genentech, Inc., South San Francisco, Calif.) at a dose of 1 mg/kg given over 60 minutes (10 % of the total dose was given as a bolus); group 4 received intravenous diltiazem (20 ^g/kg/min X 60 minutes) plus rTPA (1 mg/kg over 60 minutes; 10% bolus); group 5 received intravenous diltiazem (20 jug/kg/min X 60 minutes daily for 3 consecutive days); and group 6 received intravenous diltiazem (20 /xg/kg/min X 60 minutes daily for 3 consecutive days followed by rTPA (1 mg/kg over 60 minutes; 10 % bolus) given on day 3. In each experiment the ear incisions were made within 30 minutes of completing the study drug infusions. Arterial blood pressure was monitored continuously in a series of pilot experiments designed to assess the animals' response to intravenous diltiazem. The doses selected for the present study were chosen to produce steady-state concentrations spanning the range observed during oral diltiazem and intravenous rTPA therapy in humans.^^"'* Statistical analysis. The distributional characteristics of blood loss were evaluated with frequency histograms and the Kolmogorov-Smirnov goodness-of-flt test for normality. Differences in blood loss between groups of rabbits were evaluated with one-way analysis of variance. Pairwise comparisons between treatment groups were performed with the Student-Newman-Keuls multiple-comparison procedure. Statistical significance was defined as p < 0.05. RESULTS
Total blood loss quantitated among the six treatment groups is presented in Table I. Significant dif-
ferences were found across all groups by analysis of variance (p = 0.003). rTPA alone was associated with minimal bleeding, and total blood loss was not significantly different from that observed in either the saline control animals or those receiving a 1-hour infusion of diltiazem. Similarly, 1-hour infusions of diltiazem plus rTPA did not result in significantly more blood loss than either rTPA alone or diltiazem alone. In contrast, diltiazem infused daily for 3 consecutive days followed by a 1-hour infusion of rTPA caused significantly more blood loss than rTPA alone, diltiazem alone, or rTPA combined with a single 1-hour infusion of diltiazem (p = 0.003). Diltiazem infused daily for 3 consecutive days resulted in significantly more blood loss than either a 1-hour infusion of diltiazem, rTPA alone, or rTPA combined with diltiazem (1-hour infusion) (p < 0.05). DISCUSSION
Calcium channel antagonists are used commonly in the treatment of patients with coronary heart disease including stable angina pectoris, unstable angina, and acute myocardial infarction. Although their role in acute myocardial infarction is limited,^^ they may offer some benefit after thrombolytic therapy by reducing reperfusion injury, which is theorized to be a calcium-mediated phenomenon.^^' ^' The findings of our study suggest, however, that combined administration of calcium channel antagonists and thrombolytic agents may increase the risk of hemorrhagic complications. Furthermore, they suggest that longterm use of calcium channel antagonists in particular may be associated with increased risk. In circulating blood, platelets are bathed in a medium containing calcium ions at high concentrations. Platelets themselves contain calcium, which is stored primarily in mitochondria, a-granules, and dense granules, in the dense tubular system, and in the inner surface of the plasma membrane. Platelet activation is associated with a prompt increase in free calcium within the platelet cytosol. In turn, calcium acts as a secondary messenger for activation of receptortriggered phospholipase C. Increased cytosolic calcium also is involved in the activation of protein kinase C and phospholipase A2.^^' ^^ Calcium channel antagonists prevent the influx of calcium in response to several platelet activators.^" They also may increase intracellular cyclic adenosine monophosphate levels (21), antagonize calmodulin activation,^^ reduce thromboxane A2 synthesis,^^ inhibit serotonin uptake,^^"^® inhibit platelet-activating factor, and neutralize glycoprotein Ilb/IIIa, which in addition to being a vital surface receptor for platelet aggregation can function as a calcium channel as well.27
Volume 126, Number 1
Becker et al.
American Heart Journal
Calcium channel antagonists have been shown to inhibit platelet aggregation in vitro.^*'^^ In humans, ex vivo studies have shown that most subjects taking therapeutic doses of these compounds exhibit a reduced response to platelet agonists.^^ In animal models, calcium channel antagonists reduce thrombus formation in femoral artery grafts^^ and prolong the template bleeding time, a marker of platelet activity and hemostatic potential.^^ The calcium channel antagonist diltiazem was chosen for our study because of its widespread use in clinical practice and because of evidence from previous investigations that its antiplatelet effects may be more potent than those of other compounds in response to common agonists, including adenosine diphosphate, collagen, and epinephrine.^^' ^^ We were able to show that a brief exposure to diltiazem did not increase blood loss; however, daily exposure for 3 consecutive days (included to simulate chronic ingestion) was associated with a significant increase in blood loss either with or without rTPA administration. Our observations are consistent with previous findings from animal experiments showing that prolonged incubation of platelets with calcium channel antagonists results in a greater inhibitory effect than does a brief incubation.^^ Patients participating in the TIMI II study received intravenous heparin, aspirin, and rTPA. Many patients were given |8-blockers as well. Although we are not aware of an additive or potentiating effect of calcium channel antagonists on heparin which increases bleeding risk, this possibility has not been actively investigated. In contrast, diltiazem has been shown to potentiate the inhibitory effect of aspirin on platelet aggregation.^^' ^® Furthermore, diltiazem administered in combination with (^-adrenergic receptor blockers results in an additive inhibitory effect on platelet function.^'' Thus a treatment regimen including an anticoagulant, a fibrinolytic agent, and several platelet inhibitors would be expected to have profound effects on the hemostatic mechanism and as a result hemorrhagic risk. Limitations. Our study was designed to investigate an association between calcium channel antagonists and hemorrhagic risk after rTPA administration. An established animal model was employed; however, extrapolation of the findings to humans, particularly to the occurrence of spontaneous hemorrhagic events such as intracerebral bleeding, should be made with caution. Furthermore, extrapolation to other calcium channel antagonists or thrombolytic agents may not be appropriate. Last, the model chosen for our study may be more sensitive to abnormalities in primary hemostasis than to fibrinolytic-mediated bleeding.^^ Therefore we may have underestimated the true
13
hemorrhagic risk existing in clinical situations where calcium channel antagonists and thrombolytic agents are administered concomitantly. Clinical implications. The major adverse effect of thrombolytic therapy is bleeding, which occurs in approximately 5 % and 30 % of patients treated with noninvasive and invasive strategies, respectively. The most serious hemorrhagic event is intracerebral hemorrhage, with a frequency reported to be between 0.2 % and 1.6 % .^ The identification of risk factors for intracerebral hemorrhage has been difficult, and the mechanisms involved have been poorly defined. Mounting evidence suggests, however, that spontaneous bleeding in the central nervous system may be unique, differing from spontaneous events in other organs. It is conceivable that calcium channel antagonists increase the risk of intracerebral hemorrhage through their antiplatelet effects, by preventing vasoconstriction of intracerebral vessels (altering autoregulation) , or by one or more undefined mechanisms specific to the central nervous system. Further investigation of these areas must be undertaken. We thank Jack Ansell, MD, for scientific guidance. REFERENCES 1. Gruppo Italiano per lo Studio Delia Streptochinasi Nell'inf arte Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986; 1:397-401. 2. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomized trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet 1988;8:34960. 3. The ASSET Study Group. Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction. Lancet 1988;3:525-30. 4. AIMS Trial Study Group. Effect of intravenous APSAC on mortality after acute myocardial infarction: preliminary report of aplacebo-controUed clinical trial. Lancet 1988;12:545-9. 5. Gruppo Italiano per lo Studio Delia Streptochinasi Nell'inf arto Miocardico (GISSI). Long-term effects of intravenous thrombolysis in acute myocardial infarction: final report of the GISSI study. Lancet 1987;17:871-4. 6. Williams DO, Braunwald E, Knatterud G, Babb J, Bresnahan J, Greenberg MA, Raizner A, Wasserman A, Robertson T, Ross R, and the TIMI Investigators. One-year results of the Thrombolysis in Myocardial Infarction investigation (TIMI) phase II trial. Circulation 1992;85:533-42. 7. Bovill EG, Terrin ML, Stump DC, Berke AD, Frederick M, CoUen D, Feit F, Gore JM, Hillis LD, Lambrew CT, Leiboff R, Mann KG, Markis JE, Pratt CM, Sharkey SW, Sopko G, Tracy RP, Chesebro JH, for the TIMI Investigators. Hemorrhagic events during therapy with recombinant tissue-type plasminogen activator, heparin, and aspirin for acute myocardial infarction. Ann Intern Med 1991;115:256-65. 8. Gore JM, Sloan M, Price TR, Randall AMY, Bovill E, CoUen D, Forman S, Knatterud G, Sopko G, Terrin ML, and the TIMI Investigators. Intracerebral hemorrhage, cerebral infarction, and subdural hematoma after acute myocardial infarction and thrombolytic therapy in the Thrombolysis in Myocardial Infarction study. Circulation 1991;83:448-59. 9. Becker RC, Corrao JM, Ball SP, Klassen V, Baker SP, Ansell J. The impact of intravascular thrombus formation on hemor-
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17. 18. 19. 20.
21. 22.
23.
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rhagic complications following recombinant tissue-type plasminogen activator administration. J Appl Cardiol 1991 ;6:221-7. Carter CJ, Kelton JG, Hirsch J, Cerskus A, Santos AV, Gent M. The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. Blood 1982;59:1239-45. Pietraszek MH, Chabielska E, Buczko W. Effects of verapamil, diltiazem and nifedipine on some parameters of hemostasis in the rat. Thromb Res 1988;50:325-8. Yeh SJ, Kou HC, Lin FC, Hung JS, Wu D. Effects of oral diltiazem in paroxysmal supraventricular tachycardia. Am J Cardiol 1983;52:271-8. Shenasa M, Fromer M, Faugere G, Nadeau R, Leblanc RA, Lambert C, Sadr-Ameli MA. Efficacy and safety of intravenous and oral diltiazem for Wolff-Parkinson-White syndrome. Am J Cardiol 1987;59:301-6. Tiefenbrunn AJ, Robinson AK, Kurneck PB, Ludbrook PA, Sobel BE. Clinical pharmacology in patients with evolving myocardial infarction of tissue-type plasminogen activator produced by recombinant DNA technology. Circulation 1985;71:110-16. Lamas GA, Pfeffer MA, Hamm P, Wertheimer J, Rouleau JL, Braunwald E, for the SAVE Investigators. Do the results of randomized clinical trials of cardiovascular drugs influence medical practice? N Engl J Med 1992;327:241-54. Garcia-Dorado D, Theroux P, Fernandez-Aviles F, Blizaga J, Solares J, Galinanes M. Diltiazem and progression of myocardial ischemic damage during coronary artery occlusion and reperfusion in porcine hearts. J Am Coll Cardiol 1987;10:90611. Becker RC, Gore JM. Adjunctive use of beta-adrenergic blockers, calcium antagonists and other therapies in coronary thrombolysis. Am J Cardiol 1991;67:25A-31A. Ashby B, Daniel JL, Smith JB. Mechanisms of platelet activation and inhibition. Hematol Oncol Clin North Am 1990;4:126. Murer EH. The role of platelet calcium. Semin Hematol 1985;22:313-23. Avdonin PV, Menshikov MY, Svitina-Ulitina IV, Tkachuk VA. Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine. Thromb Res 1988;52:587-97. Moore JB, Fuller BL, Falotico R, Tolman EL. Inhibition of rabbit platelet phosphodiesterase activity and aggregation by calcium channel blockers. Thromb Res 1985;40:401-11. Epstein PM, Fiss K, Hachisu R, Andrenyak DM. Interaction of calcium antagonists with cyclic AMP phosphodiesterases and calmodulin. Biochem Biophys Res Commun 1982;105: 1142-9. Davi G, Noso S, Fiore M. Effects by nifedipine on thrombox-
July 1993 American Heart Journal
ane synthesis in vitro and in vivo. Thromb Res 1982;28:837-42. 24. Lee SL, Long K, Veda S, Fanburg BL. Verapamil inhibits serotonin uptake of endothelial cells in culture by a mechanism unrelated to Ca^* channel blockade. J Cell Physiol 1987; 132:178-82. 25. Affulter H, Burkard WP, Pletscher A. Verapamil an antagonist at 5-hydroxytryptamine receptors of human platelets. Eur J Pharmacol 1985;108:157-62. 26. Baudouin-Legros M, Dard B, Guichency P, Meyer P. Double action of nicardipine on serotonin release from rat platelets. J Cardiovasc Pharmacol 1987;10:287-92. 27. Rybak MEM, RenzuUi LA. Effect of calcium channel blockers on platelet GPIIb-IIIa as a calcium channel in liposomes: comparison with effects on the intact platelet. Thromb Res 1991;65:131-6. 28. Takahara K, Kuroiwa A, Matsushima T. Effects of nifedipine on platelet function. AM HEART J 1985;109:4-7. 29. Greer lA, Walker JJ, McLaren M. Inhibition of whole blood platelet aggregation by nicardipine, and synergism with prostacyclin in vitro. Thromb Res 1986;41:509-18. 30. Jones CR, Pasanisi G, Elliott HL, Reid JL. Effect of verapamil and nisoldipine on human platelets: in vivo and in vitro studies. Br J Clin Pharmacol 1985;20:191-6. 31. Schacter M. Do calcium antagonists affect platelets? Eur Heart J 1987;8(suppl K):75-82. 32. Pumphrey CW, Fuster V, Dewanjee MK, Chesebro JH, VKetstra RE, Kaye MP. Comparison of the antithrombotic action of calcium antagonist drugs with dipyridamole in dogs. Am J Cardiol 1983;51:591-5. 33. Kiyomoto A, Sasaki Y, Odawara A, Morita T. Inhibition of platelet aggregation by diltiazem. Circ Res 1983;52(suppl I): 115-9. 34. Mehta P, Mehta J, Ostrowski N, Brigmon L. Inhibitory effects of diltiazem on platelet activation caused by ionophore A23187 plus ADP or epinephrine in subthreshold concentrations. J Lab Clin Med 1983;102:332-9. 35. Altman R, Scazziota A, Dujovne C. Diltiazem potentiates the inhibitory effect of aspirin on platelet aggregation. Clin Pharmacol Ther 1988;44:320-5. 36. Ring ME, Corrigan Jr J J , Fenster PE. Effects of oral diltiazem on platelet function: alone and in combination with "low dose" aspirin. Thromb Res 1986;44:391-400. 37. Ring ME, Corrigan Jr JJ, Fenster PE. Antiplatelet effects of oral diltiazem, propranolol, and their combination. J Clin Pharmacol 1987;24:615-20. 38. Racanelli AL, Diemer MJ, Dobies AC, Dubin JR, Reilly TM. Comparison of recombinant plasminogen activator inhibitor-2 and epsilon amino caproic acid in a hemorrhagic rabbit model. Thromb Hemost 1992;67:692-6.
Richard C. Becker, MD, Ronald Caputo, MD, Steven Ball, RN, Jeanne M. Corrao, RN, MS, Stephen Baker, MS, and Joel M. Gore, MD Worcester, Mass.
Thrombolytic therapy is used widely in the treatment of acute myocardial infarction. The evidence that timely thrombolysis significantly decreases short-^'^ and long-term^'® mortality is compelling. The use of thrombolytic agents, however, is associated with a small but identifiable risk of bleeding.'' Intracerebral hemorrhage, the most feared and serious complication of thrombolysis, although it occurs in only a small number of patients, can have devastating consequences.® In the Thrombolysis in Myocardial Infarction phase II (TIMI II) trial, intracerebral hemorrhage was documented in 0.4 % of patients treated with recombinant tissue-type plasminogen activator (rTPA) (100 mg) given in combination with intravenous heparin and aspirin. Interestingly, use of
From the Thrombosis Research Center, Division of Cardiovascular Medicine, University of Massachusetts Medical School. Received for publication Nov. 25, 1992; accepted Jan. 8, 1993. Reprint requests: Richard C. Becker, MD, Thrombosis Research Center, University of Massachusetts Medical School, Worcester, MA 01655. Copyright ® 1993 by Mosby-Year Book, Inc. 0002-8703/93/$1.00 + .10 4 / 1 / 4 6 3 9 6
calcium channel antagonists at study entry was associated with an increased frequency of intracerebral hemorrhage. Whether this association was due to chance, accompanying cerebrovascular disease, or to a direct effect of calcium channel antagonists could not be determined. The purpose of this study was to distinguish these possibilities further by investigating the effect of calcium channel antagonists on the hemostatic mechanism when administered with rTPA. METHODS
A rabbit model was chosen for our series of experiments. In an earlier study^ we were able to show that blood loss from standardized ear incisions was not influenced by the presence of a jugular vein thrombus. Thus, although the initial design called for a venous thrombosis model, it was excluded from the present study. The method for assessing blood loss was derived from the work of Carter et aL^" and Pietraszek et al.^^ In brief, New Zealand white rabbits were anesthetized with Ketaset (ketamine hydrochloride) and Rompun (xylazine hydrochloride). The ears were shaved. One ear was then immersed for 10 minutes in a 37° C water bath. Under trans-
11
July 1993 American Heart Journal
12 Becker et al. Table I. Total blood loss from standardized ear incisions in six experimental groups Total blood loss Treatment 1 2 3 4 5 6
(N = 6) (N = 11) (N = 10) (N = 5) (N = 6) (N = 6)
group
M
SD
75.4 76.4 78.7 76.7 380.8* 278.5*
53.8 77.2 115.8 80.0 199.5 203.4
*p < 0.05.
illumination, five full-thickness incisions were made with a Bard-Parker No. 11 scalpel blade (Becton Dickinson Infusion Systems, Lincoln Park, N.J.). Care was taken to avoid the major vessels. Blood from the incisions was collected in a beaker containing 25 ml of 0.15 mol/L sodium chloride and hemolyzed by the addition of 2.5 ml of Triton X-100 solution (Sigma Diagnostics, St. Louis, Mo.). The optical density of the solution was measured photometrically at 546 with a Karl-Zeiss spectrophotometer (Spectronic 710, Bausch & Lomb, Rochester, N.Y.). Total blood loss was quantitated by means of a standard curve constructed with known volumes of rabbit blood. Six groups of animals were studied: group 1 received a 60-minute infusion of 0.9 % sodium chloride (total volume 15 cc); group 2 received intravenous diltiazem (Marion Merrell Dow, Kansas City, Mo.) at a dose of 20 /ig/kg/min for a total of 60 minutes; group 3 received intravenous rTPA (Genentech, Inc., South San Francisco, Calif.) at a dose of 1 mg/kg given over 60 minutes (10 % of the total dose was given as a bolus); group 4 received intravenous diltiazem (20 ^g/kg/min X 60 minutes) plus rTPA (1 mg/kg over 60 minutes; 10% bolus); group 5 received intravenous diltiazem (20 jug/kg/min X 60 minutes daily for 3 consecutive days); and group 6 received intravenous diltiazem (20 /xg/kg/min X 60 minutes daily for 3 consecutive days followed by rTPA (1 mg/kg over 60 minutes; 10 % bolus) given on day 3. In each experiment the ear incisions were made within 30 minutes of completing the study drug infusions. Arterial blood pressure was monitored continuously in a series of pilot experiments designed to assess the animals' response to intravenous diltiazem. The doses selected for the present study were chosen to produce steady-state concentrations spanning the range observed during oral diltiazem and intravenous rTPA therapy in humans.^^"'* Statistical analysis. The distributional characteristics of blood loss were evaluated with frequency histograms and the Kolmogorov-Smirnov goodness-of-flt test for normality. Differences in blood loss between groups of rabbits were evaluated with one-way analysis of variance. Pairwise comparisons between treatment groups were performed with the Student-Newman-Keuls multiple-comparison procedure. Statistical significance was defined as p < 0.05. RESULTS
Total blood loss quantitated among the six treatment groups is presented in Table I. Significant dif-
ferences were found across all groups by analysis of variance (p = 0.003). rTPA alone was associated with minimal bleeding, and total blood loss was not significantly different from that observed in either the saline control animals or those receiving a 1-hour infusion of diltiazem. Similarly, 1-hour infusions of diltiazem plus rTPA did not result in significantly more blood loss than either rTPA alone or diltiazem alone. In contrast, diltiazem infused daily for 3 consecutive days followed by a 1-hour infusion of rTPA caused significantly more blood loss than rTPA alone, diltiazem alone, or rTPA combined with a single 1-hour infusion of diltiazem (p = 0.003). Diltiazem infused daily for 3 consecutive days resulted in significantly more blood loss than either a 1-hour infusion of diltiazem, rTPA alone, or rTPA combined with diltiazem (1-hour infusion) (p < 0.05). DISCUSSION
Calcium channel antagonists are used commonly in the treatment of patients with coronary heart disease including stable angina pectoris, unstable angina, and acute myocardial infarction. Although their role in acute myocardial infarction is limited,^^ they may offer some benefit after thrombolytic therapy by reducing reperfusion injury, which is theorized to be a calcium-mediated phenomenon.^^' ^' The findings of our study suggest, however, that combined administration of calcium channel antagonists and thrombolytic agents may increase the risk of hemorrhagic complications. Furthermore, they suggest that longterm use of calcium channel antagonists in particular may be associated with increased risk. In circulating blood, platelets are bathed in a medium containing calcium ions at high concentrations. Platelets themselves contain calcium, which is stored primarily in mitochondria, a-granules, and dense granules, in the dense tubular system, and in the inner surface of the plasma membrane. Platelet activation is associated with a prompt increase in free calcium within the platelet cytosol. In turn, calcium acts as a secondary messenger for activation of receptortriggered phospholipase C. Increased cytosolic calcium also is involved in the activation of protein kinase C and phospholipase A2.^^' ^^ Calcium channel antagonists prevent the influx of calcium in response to several platelet activators.^" They also may increase intracellular cyclic adenosine monophosphate levels (21), antagonize calmodulin activation,^^ reduce thromboxane A2 synthesis,^^ inhibit serotonin uptake,^^"^® inhibit platelet-activating factor, and neutralize glycoprotein Ilb/IIIa, which in addition to being a vital surface receptor for platelet aggregation can function as a calcium channel as well.27
Volume 126, Number 1
Becker et al.
American Heart Journal
Calcium channel antagonists have been shown to inhibit platelet aggregation in vitro.^*'^^ In humans, ex vivo studies have shown that most subjects taking therapeutic doses of these compounds exhibit a reduced response to platelet agonists.^^ In animal models, calcium channel antagonists reduce thrombus formation in femoral artery grafts^^ and prolong the template bleeding time, a marker of platelet activity and hemostatic potential.^^ The calcium channel antagonist diltiazem was chosen for our study because of its widespread use in clinical practice and because of evidence from previous investigations that its antiplatelet effects may be more potent than those of other compounds in response to common agonists, including adenosine diphosphate, collagen, and epinephrine.^^' ^^ We were able to show that a brief exposure to diltiazem did not increase blood loss; however, daily exposure for 3 consecutive days (included to simulate chronic ingestion) was associated with a significant increase in blood loss either with or without rTPA administration. Our observations are consistent with previous findings from animal experiments showing that prolonged incubation of platelets with calcium channel antagonists results in a greater inhibitory effect than does a brief incubation.^^ Patients participating in the TIMI II study received intravenous heparin, aspirin, and rTPA. Many patients were given |8-blockers as well. Although we are not aware of an additive or potentiating effect of calcium channel antagonists on heparin which increases bleeding risk, this possibility has not been actively investigated. In contrast, diltiazem has been shown to potentiate the inhibitory effect of aspirin on platelet aggregation.^^' ^® Furthermore, diltiazem administered in combination with (^-adrenergic receptor blockers results in an additive inhibitory effect on platelet function.^'' Thus a treatment regimen including an anticoagulant, a fibrinolytic agent, and several platelet inhibitors would be expected to have profound effects on the hemostatic mechanism and as a result hemorrhagic risk. Limitations. Our study was designed to investigate an association between calcium channel antagonists and hemorrhagic risk after rTPA administration. An established animal model was employed; however, extrapolation of the findings to humans, particularly to the occurrence of spontaneous hemorrhagic events such as intracerebral bleeding, should be made with caution. Furthermore, extrapolation to other calcium channel antagonists or thrombolytic agents may not be appropriate. Last, the model chosen for our study may be more sensitive to abnormalities in primary hemostasis than to fibrinolytic-mediated bleeding.^^ Therefore we may have underestimated the true
13
hemorrhagic risk existing in clinical situations where calcium channel antagonists and thrombolytic agents are administered concomitantly. Clinical implications. The major adverse effect of thrombolytic therapy is bleeding, which occurs in approximately 5 % and 30 % of patients treated with noninvasive and invasive strategies, respectively. The most serious hemorrhagic event is intracerebral hemorrhage, with a frequency reported to be between 0.2 % and 1.6 % .^ The identification of risk factors for intracerebral hemorrhage has been difficult, and the mechanisms involved have been poorly defined. Mounting evidence suggests, however, that spontaneous bleeding in the central nervous system may be unique, differing from spontaneous events in other organs. It is conceivable that calcium channel antagonists increase the risk of intracerebral hemorrhage through their antiplatelet effects, by preventing vasoconstriction of intracerebral vessels (altering autoregulation) , or by one or more undefined mechanisms specific to the central nervous system. Further investigation of these areas must be undertaken. We thank Jack Ansell, MD, for scientific guidance. REFERENCES 1. Gruppo Italiano per lo Studio Delia Streptochinasi Nell'inf arte Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986; 1:397-401. 2. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomized trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet 1988;8:34960. 3. The ASSET Study Group. Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction. Lancet 1988;3:525-30. 4. AIMS Trial Study Group. Effect of intravenous APSAC on mortality after acute myocardial infarction: preliminary report of aplacebo-controUed clinical trial. Lancet 1988;12:545-9. 5. Gruppo Italiano per lo Studio Delia Streptochinasi Nell'inf arto Miocardico (GISSI). Long-term effects of intravenous thrombolysis in acute myocardial infarction: final report of the GISSI study. Lancet 1987;17:871-4. 6. Williams DO, Braunwald E, Knatterud G, Babb J, Bresnahan J, Greenberg MA, Raizner A, Wasserman A, Robertson T, Ross R, and the TIMI Investigators. One-year results of the Thrombolysis in Myocardial Infarction investigation (TIMI) phase II trial. Circulation 1992;85:533-42. 7. Bovill EG, Terrin ML, Stump DC, Berke AD, Frederick M, CoUen D, Feit F, Gore JM, Hillis LD, Lambrew CT, Leiboff R, Mann KG, Markis JE, Pratt CM, Sharkey SW, Sopko G, Tracy RP, Chesebro JH, for the TIMI Investigators. Hemorrhagic events during therapy with recombinant tissue-type plasminogen activator, heparin, and aspirin for acute myocardial infarction. Ann Intern Med 1991;115:256-65. 8. Gore JM, Sloan M, Price TR, Randall AMY, Bovill E, CoUen D, Forman S, Knatterud G, Sopko G, Terrin ML, and the TIMI Investigators. Intracerebral hemorrhage, cerebral infarction, and subdural hematoma after acute myocardial infarction and thrombolytic therapy in the Thrombolysis in Myocardial Infarction study. Circulation 1991;83:448-59. 9. Becker RC, Corrao JM, Ball SP, Klassen V, Baker SP, Ansell J. The impact of intravascular thrombus formation on hemor-
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