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Platelet-Independent defect in hemostasis associated with sirolimus use
Platelet-Independent Defect in Hemostasis Associated With Sirolimus Use T. Rampino, M. Marasa`, P.M. Malvezzi, G. Soccio, E. Roscini, G. Gamba, P. Noris, M. Alessiani, and A. Dal Canton ABSTRACT Sirolimus is currently used to prevent rejection of solid organ transplant, and sirolimuseluting stents have shown promise for the prevention of coronary artery restenosis. Thrombocytopenia is a well-known adverse effect of sirolimus limiting its use. Herein we report on a patient in whom sirolimus caused a platelet-independent hemostasis defect. The patient was a 52-year-old woman who underwent renal transplant with consequent normal kidney function. The immunosuppressive regimen included basiliximab, steroids, and cyclosporine induction later shifted to sirolimus and mycophenolate due to biopsy findings of tubular necrosis on day 6 posttransplantation. At discharge the serum creatinine was 0.7 mg/dL. Four months after transplantation the patient was admitted to our hospital because of fever (37.5°C to 38°C), anorexia, and asthenia. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 ⫻ 103/L, PLT 123 ⫻ 103/L, liver function tests normal, LDH 720 mU/mL, fibrinogen 628 mg/dL, D-dimer 0.42 ng/mL, FDP ⬎ 40 ng/mL, INR 1.10, PT 87%, aPTT 40 seconds. Cultures and tests for infection were negative. Serum sirolimus level was 25.9 ng/mL. The following day the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. Multiple bleeding times (Ivy test) performed before the renal biopsy were repeatedly over 30 minutes (normal 3 to 5 minutes), despite normal platelet count and platelet function studies. There was no spontaneous aggregation and in vitro aggregation was normal (collagen, ADP, adrenalin, and ristocetin induced). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis. Suspension of sirolimus treatment was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), and normalization of bleeding time.
S
IROLIMUS IS AN IMMUNOSUPPRESSIVE agent recently introduced for prophylaxis of renal graft rejection. Sirolimus (rapamycin) was isolated from a strain of Streptomyces hygroscopicus primarily as an antifungal agent.1 Further studies revealed its antiproliferative and immunosuppressive properties. Its molecular mechanism makes sirolimus a potent inhibitor of antigen-induced proliferation of T cells, B cells, and antibody production1: namely, the drug binds to an intracellular protein FKBP (FK-binding protein) forming a complex with mTOR (mammalian target of rapamycin), a protein kinase involved in growth and proliferation of various cell types. Inhibited mTOR function prevents protein synthesis and cell cycle progression through the G1 phase.2–5 Up-regulation of IL-2 transcription by the growth factor–mediated activation of mTOR is also inhibited.6 To date sirolimus has been used in three ways7: to spare 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.03.015 700
calcineurin inhibitors (cyclosporine or tacrolimus); as the principal immunosuppressant after calcineurin inhibitor nephrotoxicity; as the primary immunosuppressive agent following transplantation. In fact, sirolimus seems to allow the reduction or withdrawal of calcineurin inhibitors to reduce the number and severity of rejection episodes and the risk of chronic allograft nephropathy.1,8,9 Recent data show that sirolimus-eluting stents prevent From the Unit of Nephrology, Dialysis, and Transplantation (T.R., M.M., P.M.M., G.S., E.R., A.D.C.), Department of Internal Medicine (G.G., P.N.), and Institute of Hepato-pancreatic Surgery (M.A.), IRCCS Policlinico San Matteo and University, Pavia, Italy. Address reprint requests to Teresa Rampino, MD, Unit of Nephrology, Dialysis and Transplantation, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. E-mail: [email protected] © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 700 –702 (2004)
PLATELET-INDEPENDENT DEFECT
coronary artery restenosis.10 Due to its ability to inhibit cytokine and growth factor–mediated proliferation of lymphocytes and smooth muscle cells, sirolimus reduces neointimal proliferation when slowly released by coronary stents,11,12 avoiding the interventions needed for the high percentage of restenosis after percutaneous coronary revascularization. Laboratory abnormalities more often related to sirolimus treatment are thrombocytopenia, leukopenia, hypertriglyceridemia, hypercholesterolemia, and hepatic disorders.13,14 There is evidence of a correlation between the occurrence of these effects and whole-blood concentrations of sirolimus, especially with trough concentrations above 15 ng/ mL.15,16 Thrombocytopenia can be explained by the sirolimus-induced enhancement of agonist (ADP/thrombin equivalent)-mediated platelet aggregation in a time- and dose-dependent manner.17 In the literature there is no evidence of any correlation between sirolimus and hemostasis disorders, except thrombocytopenia. Herein we describe the case of a 52-year-old woman in whom sirolimus caused a primary platelet-independent hemostasis disorder, as indicated by an elongation of bleeding time. CASE REPORT A 52-year-old woman with polycystic kidney disease underwent cadaver kidney transplantation. The procedure rapidly normalized renal function. Before the procedure the platelet count and routine coagulation values (INR, aPTT, PT, and fibrinogen) were normal. The immunosuppressive regimen was based on basiliximab, steroids, and cyclosporine. On day 6 following transplantation a sudden fall in diuresis occurred, associated with hypertension (240/120 mm Hg), fever (38.3°C), and an increased serum creatinine. Because a kidney biopsy showed evidence of acute tubular necrosis, cyclosporine treatment was suspended and substituted with sirolimus and mycophenolate. The bleeding time performed before the biopsy was 5 minutes (Ivy test). Renal function started to improve on day 20; by discharge the serum creatinine was 0.7 mg/dL. About 3 months following transplantation the patient was admitted to our hospital because of fever (37.5°C to 38°C), anorexia, and asthenia. Physical examination was normal; blood pressure 145/90 mm Hg, pulse 64 bpm. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 ⫻ 103/L (neutrophil 44%, lymphocyte 44%, eosinophil 8.3%), platelets 123 ⫻ 103/L, total protein 5.8 g/dL, and albumin 3.4 g/dL, normal liver tests function, fibrinogen 628 mg/dL, INR 1.10, PT 87%. Negative results were obtained for CMV antigen (and viral load), EBV, Legionella, and Brucella serologies. The urinary sediment and culture tests as well as (urine, blood, and stool) were negative. Abdomen ultrasound and thorax radiography did not evidence any alteration. Thereafter the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. The bleeding time (Ivy test) performed before a programmed renal biopsy was repeatedly elongated beyond 30 minutes. The blood smear showed some helmet cells. Further blood analysis showed normal haptoglobin and bilirubin levels with an increased LDH (720 mU/mL), D-dimer (0.42 ng/mL), and FDP (⬎40 ng/mL). Platelet function studies (on PRP with 115 ⫻ 109 plt/L) showed
701 the absence of spontaneous aggregation but normal in vitro– induced aggregation. In vitro platelet aggregation performed with the Born method was induced with collagen (20 and 4 g/mL), ADP (20 and 5 mol/L), adrenalin (10 mol/L), and ristocetin (1.5 mg/mL). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis, with presence in serum of some substances interfering with thrombin and fibrin formation. In particular these studies showed (limits in brackets): aPTT 40 seconds (21 to 33), PT 87.5% (70 to 100), INR 1.09, thrombin time 26.6 seconds (⬍21), reptilase time 28.3 seconds (⬍20), Russell viper venom time (RVVT) 85 seconds (54), KCT 90.2 seconds (43.9), RVVT ratio miscele 1:1 (Pt:N/N)/Pt ⫽ 1.17, KCT ratio miscele 1:1 (Pt:N)/Pt ⫽ 1.2; FVIII C 105% (60 to 200), vWF ristocetin cofactor 112% (60 to 200), fibrinogen 634 mg/dL. The patient was taking atorvastatin, which was discontinued as well as atenolol, doxazosin, ranitidine, levothyroxin, erythropoietin alpha, mycophenolate, and sirolimus. Blood sirolimus level was 25.9 ng/mL. Suspension of sirolimus was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), normalization of coagulation tests in 7 days and of bleeding time in 2 weeks.
CONCLUSION
The observed elongation of bleeding time was not dependent on platelet or van Willebrand factor deficiency or dysfunction. A possible explanation is an alteration of fibrin and thrombin formation caused by a ill-defined anticoagulant, as demonstrated by the hemostasis studies. Suspension of sirolimus resulted in regression of all symptoms, thus confirming that the drug may have an anticoagulant effect. Furthermore, we suspect a possible effect of sirolimus on endothelium, as suggested by the rise in LDH, FDP, and D-dimer as well as the presence of helmet cells in the blood smear. The fact that sirolimus is able to reduce neointimal proliferation supports this hypothesis. REFERENCES 1. Sehgal SN: Sirolimus: its discovery, biological properties, and mechanism of Action. Transplant Proc 35(Suppl 3A):7S, 2003 2. Brown EJ, Alberts MW, Shin TB, et al: A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369: 756, 1994 3. Sabers CJ, Martin MM, Brunn GJ, et al: Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270:815, 1995 4. Sabatini DM, Erdjument-Bromage H, Lui M, et al: RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin dependent fashion and is homologous to yeast TORs. Cell 78:35, 1994 5. Jefferies HBJ, Reinhard C, Kozma SC, et al: Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family. Proc Natl Acad Sci USA 91:4441, 1994 6. Lai JH, Tan TH: CD28 signaling causes a sustained downregulation of I kappa B alpha which can be prevented by the immunosuppressant rapamycin. J Biol Chem 269:30077, 1994 7. Kreis H, et al: New strategies to reduce nephrotoxicity. Transplantation 72(Suppl):99, 2001 8. Johnson RWG, Kreis H, Oberbauer R, et al: Sirolimus allows early cyclosporine withdrawal in renal transplantation resulting in improved renal function and lower blood pressure. Transplantation 72:777, 2001
702 9. Podbielski J, Schoenberg L: Use of sirolimus in kidney transplantation. Prog Transplant 11:29, 2001 10. Morice MC, Serruys P, Sousa J, et al: A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 346:1773, 2002 11. Gallo R, Padurean A, Jayaraman T, et al: Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation 99: 2164, 1999 12. Sousa JE, Costa MA, Abizaid A, et al: Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries. Circulation 103:192, 2001 13. Charpentier B, Groth CG, Morales JM, et al: Bicetre hospital experience with sirolimus-based therapy in human renal
RAMPINO, MARASA`, MALVEZZI ET AL transplantation: the Sirolimus European Renal Transplant Study. Transplantation Proc 35(Suppl 3A):58S, 2003 14. Hong JC, Kahan BD: Sirolimus-induced thrombocytopenia and leukopenia in renal transplant recipients: risk factors, incidence, progression, and management. Transplantation 69:2085, 2000 15. Meier-Kriesche HU, Kaplan B: Toxicity and efficacy of sirolimus: relationship to whole-blood concentration. Clin Ther 22(Suppl B):B93, 2000 16. Kahan BD, Napoli KL, Kelly PA, et al: Therapeutic drug monitoring of sirolimus: correlation with efficacy and toxicity. Clin Transplant 14:97, 2000 17. Babinska A, Markell MS, Salifu MO, et al: Enhancement of human platelet aggregation and secretion induced by rapamycin. Nephrol Dial Transplant 13:3153, 1998
S
IROLIMUS IS AN IMMUNOSUPPRESSIVE agent recently introduced for prophylaxis of renal graft rejection. Sirolimus (rapamycin) was isolated from a strain of Streptomyces hygroscopicus primarily as an antifungal agent.1 Further studies revealed its antiproliferative and immunosuppressive properties. Its molecular mechanism makes sirolimus a potent inhibitor of antigen-induced proliferation of T cells, B cells, and antibody production1: namely, the drug binds to an intracellular protein FKBP (FK-binding protein) forming a complex with mTOR (mammalian target of rapamycin), a protein kinase involved in growth and proliferation of various cell types. Inhibited mTOR function prevents protein synthesis and cell cycle progression through the G1 phase.2–5 Up-regulation of IL-2 transcription by the growth factor–mediated activation of mTOR is also inhibited.6 To date sirolimus has been used in three ways7: to spare 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.03.015 700
calcineurin inhibitors (cyclosporine or tacrolimus); as the principal immunosuppressant after calcineurin inhibitor nephrotoxicity; as the primary immunosuppressive agent following transplantation. In fact, sirolimus seems to allow the reduction or withdrawal of calcineurin inhibitors to reduce the number and severity of rejection episodes and the risk of chronic allograft nephropathy.1,8,9 Recent data show that sirolimus-eluting stents prevent From the Unit of Nephrology, Dialysis, and Transplantation (T.R., M.M., P.M.M., G.S., E.R., A.D.C.), Department of Internal Medicine (G.G., P.N.), and Institute of Hepato-pancreatic Surgery (M.A.), IRCCS Policlinico San Matteo and University, Pavia, Italy. Address reprint requests to Teresa Rampino, MD, Unit of Nephrology, Dialysis and Transplantation, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. E-mail: [email protected] © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 700 –702 (2004)
PLATELET-INDEPENDENT DEFECT
coronary artery restenosis.10 Due to its ability to inhibit cytokine and growth factor–mediated proliferation of lymphocytes and smooth muscle cells, sirolimus reduces neointimal proliferation when slowly released by coronary stents,11,12 avoiding the interventions needed for the high percentage of restenosis after percutaneous coronary revascularization. Laboratory abnormalities more often related to sirolimus treatment are thrombocytopenia, leukopenia, hypertriglyceridemia, hypercholesterolemia, and hepatic disorders.13,14 There is evidence of a correlation between the occurrence of these effects and whole-blood concentrations of sirolimus, especially with trough concentrations above 15 ng/ mL.15,16 Thrombocytopenia can be explained by the sirolimus-induced enhancement of agonist (ADP/thrombin equivalent)-mediated platelet aggregation in a time- and dose-dependent manner.17 In the literature there is no evidence of any correlation between sirolimus and hemostasis disorders, except thrombocytopenia. Herein we describe the case of a 52-year-old woman in whom sirolimus caused a primary platelet-independent hemostasis disorder, as indicated by an elongation of bleeding time. CASE REPORT A 52-year-old woman with polycystic kidney disease underwent cadaver kidney transplantation. The procedure rapidly normalized renal function. Before the procedure the platelet count and routine coagulation values (INR, aPTT, PT, and fibrinogen) were normal. The immunosuppressive regimen was based on basiliximab, steroids, and cyclosporine. On day 6 following transplantation a sudden fall in diuresis occurred, associated with hypertension (240/120 mm Hg), fever (38.3°C), and an increased serum creatinine. Because a kidney biopsy showed evidence of acute tubular necrosis, cyclosporine treatment was suspended and substituted with sirolimus and mycophenolate. The bleeding time performed before the biopsy was 5 minutes (Ivy test). Renal function started to improve on day 20; by discharge the serum creatinine was 0.7 mg/dL. About 3 months following transplantation the patient was admitted to our hospital because of fever (37.5°C to 38°C), anorexia, and asthenia. Physical examination was normal; blood pressure 145/90 mm Hg, pulse 64 bpm. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 ⫻ 103/L (neutrophil 44%, lymphocyte 44%, eosinophil 8.3%), platelets 123 ⫻ 103/L, total protein 5.8 g/dL, and albumin 3.4 g/dL, normal liver tests function, fibrinogen 628 mg/dL, INR 1.10, PT 87%. Negative results were obtained for CMV antigen (and viral load), EBV, Legionella, and Brucella serologies. The urinary sediment and culture tests as well as (urine, blood, and stool) were negative. Abdomen ultrasound and thorax radiography did not evidence any alteration. Thereafter the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. The bleeding time (Ivy test) performed before a programmed renal biopsy was repeatedly elongated beyond 30 minutes. The blood smear showed some helmet cells. Further blood analysis showed normal haptoglobin and bilirubin levels with an increased LDH (720 mU/mL), D-dimer (0.42 ng/mL), and FDP (⬎40 ng/mL). Platelet function studies (on PRP with 115 ⫻ 109 plt/L) showed
701 the absence of spontaneous aggregation but normal in vitro– induced aggregation. In vitro platelet aggregation performed with the Born method was induced with collagen (20 and 4 g/mL), ADP (20 and 5 mol/L), adrenalin (10 mol/L), and ristocetin (1.5 mg/mL). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis, with presence in serum of some substances interfering with thrombin and fibrin formation. In particular these studies showed (limits in brackets): aPTT 40 seconds (21 to 33), PT 87.5% (70 to 100), INR 1.09, thrombin time 26.6 seconds (⬍21), reptilase time 28.3 seconds (⬍20), Russell viper venom time (RVVT) 85 seconds (54), KCT 90.2 seconds (43.9), RVVT ratio miscele 1:1 (Pt:N/N)/Pt ⫽ 1.17, KCT ratio miscele 1:1 (Pt:N)/Pt ⫽ 1.2; FVIII C 105% (60 to 200), vWF ristocetin cofactor 112% (60 to 200), fibrinogen 634 mg/dL. The patient was taking atorvastatin, which was discontinued as well as atenolol, doxazosin, ranitidine, levothyroxin, erythropoietin alpha, mycophenolate, and sirolimus. Blood sirolimus level was 25.9 ng/mL. Suspension of sirolimus was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), normalization of coagulation tests in 7 days and of bleeding time in 2 weeks.
CONCLUSION
The observed elongation of bleeding time was not dependent on platelet or van Willebrand factor deficiency or dysfunction. A possible explanation is an alteration of fibrin and thrombin formation caused by a ill-defined anticoagulant, as demonstrated by the hemostasis studies. Suspension of sirolimus resulted in regression of all symptoms, thus confirming that the drug may have an anticoagulant effect. Furthermore, we suspect a possible effect of sirolimus on endothelium, as suggested by the rise in LDH, FDP, and D-dimer as well as the presence of helmet cells in the blood smear. The fact that sirolimus is able to reduce neointimal proliferation supports this hypothesis. REFERENCES 1. Sehgal SN: Sirolimus: its discovery, biological properties, and mechanism of Action. Transplant Proc 35(Suppl 3A):7S, 2003 2. Brown EJ, Alberts MW, Shin TB, et al: A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369: 756, 1994 3. Sabers CJ, Martin MM, Brunn GJ, et al: Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270:815, 1995 4. Sabatini DM, Erdjument-Bromage H, Lui M, et al: RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin dependent fashion and is homologous to yeast TORs. Cell 78:35, 1994 5. Jefferies HBJ, Reinhard C, Kozma SC, et al: Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family. Proc Natl Acad Sci USA 91:4441, 1994 6. Lai JH, Tan TH: CD28 signaling causes a sustained downregulation of I kappa B alpha which can be prevented by the immunosuppressant rapamycin. J Biol Chem 269:30077, 1994 7. Kreis H, et al: New strategies to reduce nephrotoxicity. Transplantation 72(Suppl):99, 2001 8. Johnson RWG, Kreis H, Oberbauer R, et al: Sirolimus allows early cyclosporine withdrawal in renal transplantation resulting in improved renal function and lower blood pressure. Transplantation 72:777, 2001
702 9. Podbielski J, Schoenberg L: Use of sirolimus in kidney transplantation. Prog Transplant 11:29, 2001 10. Morice MC, Serruys P, Sousa J, et al: A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 346:1773, 2002 11. Gallo R, Padurean A, Jayaraman T, et al: Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation 99: 2164, 1999 12. Sousa JE, Costa MA, Abizaid A, et al: Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries. Circulation 103:192, 2001 13. Charpentier B, Groth CG, Morales JM, et al: Bicetre hospital experience with sirolimus-based therapy in human renal
RAMPINO, MARASA`, MALVEZZI ET AL transplantation: the Sirolimus European Renal Transplant Study. Transplantation Proc 35(Suppl 3A):58S, 2003 14. Hong JC, Kahan BD: Sirolimus-induced thrombocytopenia and leukopenia in renal transplant recipients: risk factors, incidence, progression, and management. Transplantation 69:2085, 2000 15. Meier-Kriesche HU, Kaplan B: Toxicity and efficacy of sirolimus: relationship to whole-blood concentration. Clin Ther 22(Suppl B):B93, 2000 16. Kahan BD, Napoli KL, Kelly PA, et al: Therapeutic drug monitoring of sirolimus: correlation with efficacy and toxicity. Clin Transplant 14:97, 2000 17. Babinska A, Markell MS, Salifu MO, et al: Enhancement of human platelet aggregation and secretion induced by rapamycin. Nephrol Dial Transplant 13:3153, 1998