THROMBOSIS RESEARCH 57; 31-38, 1990 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.


Francesco Rodeghiero, Giancarlo Castaman and Enrico Dini

Department of Hematology and Hemophilia and Thrombosis Center, San Bortolo Hospital, I-36100 Vicenza, Italy (Received 17.3.1989; accepted in revised form 10.10.1989 by Editor P.M. Mannucci) ABSTRACT L-Asparaginase, a widely used antileukemic agent, inhibits liver leading to hypofibrinogenemia and protein synthesis severe reduction of with a hypoprothrombinemia together antithrombin III and protein C. An increased risk of thrombosis has been reported in leukemic patients treated with this agent. We measured fibrinopeptide A (FPA) changes in 14 patients with acute lymphoblastic leukemia during induction remission treatment with a protocol including L-Asparaginase (10,000 U/m2/daily intravenous for 14 days). At diagnosis, 9114 patients had FPA level above upper limit of normal range (mean = 4.1 ng/ml). After two days of therapy, FPA rose to 5.2 ng/ml and thereafter showed a slight increase throughout. Antithrombin III, protein C and fibrinogen dropped to its nadir on day 6 and 9. However, the ratio FPA/fibrinogen on a molar basis showed a three-fold increase during this days, demonstrating that the thrombin-dependent consumption of fibrinogen was also increased. In conclusion, our data show that activation of blood coagulation occurs in concomitance with the hemostatic derangement caused by L-Asparaginase. Replacement therapy with the recently available antithrombin III concentrates may be worthy of a clinical trial to test its effectiveness in preventing the thrombotic phenomena reported in these patients. _______________________________-_---------------------------------Key words: Fibrinopeptide A - L-Asparaginase - Acute Leukemia Thrombosis 31



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INTRODUCTION L-Asparaginase (L-ASP) is an effective antileukemic agent that inhibits neoplastic cell growth by hydrolizing L-asparagine - essential to tumoral cell - to L-aspartic acid (1,2). This agent is an essential component of the principal protocols in use for the treatment of acute lymphoblastic leukemia (ALL) in adults and children (1). During the recent years, several reports have described the occurrence of thromboembolic events in patients treated with this drug (3-8). L-ASP has been demonstrated to inhibit liver synthesis of several components, especially procoagulant (fibrinogen, prothrombin) and inhibitory proteins of clotting system (3-5,9-ll), in particular antithrombin III (AT-III) (3-5) and protein C (12-14). The marked and prolonged reduction of these inhibitors has been deemed to be the possible cause of thrombosis even if it occurs in concomitance with a substantial reduction also of some procoagulants, including fibrinogen and prothrombin. However, the existence of an hypercoagulable state, to which also the leukemic disease itself could contribute, has not been extensively investigated (11,15). Hypercoagulability could be also responsible, at least partially, for increased risk of thrombosis in these patients. In this study, we analyze a marker of thrombin activity, namely fibrinopeptide A (FPA), selectively cleaved by thrombin from amino termini of A chain of fibrinogen, to explore the possibility that subclinical intravascular coagulation may occur during the course of this therapy.

Patients Fourteen patients (9 males, 5 females, age 7 - 48 years) were included in the study. ALL was diagnosed on the basis of bone-marrow and peripheral blood examination and immunological characterization with monoclonal antibodies. Patients were treated with a single infusion of mg/m2, on day 1 plus doxorubicin, 30 vincristine, 2 mg, and L-asparaginase (Crasnitin, Bayer), 10,000 U/m2/daily i.v. from day 1 to day 14, and dexamethasone, 8 mg i.v., on days 1 to 21 (16). The pediatric cases were considered at high risk and treated using the adult protocol. Blood samples for coagulation studies and FPA measurement were taken before starting of chemotherapy and on days 3, 6, 9, 12 and 48 hours after the end of L-ASP treatment.

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Methods Prothrombin time, partial thromboplastin time, fibrinogen, serum fibrinogen-fibrin degradation products (FDP) and ethanol gelation test were determined as previously described (17). AT-III activity was measured as heparin cofactor activity using chromogenic substrate (S-2238, Kabi). Protein C antigen was measured using an ELISA method (Stago, Paris). For FPA assay, blood was collected without stasis using a 19 gauge needle: after discarding the first 10 ml of blood, two subsequent samples of 2.5 ml were collected into polypropylene syringes containing 0.25 ml of an anticoagulant mixture composed of aprotinin (Trasylol, Bayer) and heparin (Liquemin, Roche) at 1,000 U/ml final concentration. After gentle mixing, the samples were immediately transferred to new polypropylene tubes and platelet poor plasma (PPP) obtained by centrifugation at 1,500 g at 4" C for 20 minutes. In order to remove fibrinogen and its degradation products, PPP was adsorbed by a double volume of bentonite (Sigma, St. Louis) 90 mg/ml in Tris 0.05 M, NaCl 0.1 M, ovalbumin (Sigma, St. Louis) 1 g/l, pH 8.9 and supernatants removed and stored at -40" C. FPA content was measured by radioimmunoassay with a commercial kit (Mallinkrodt, St. Louis, USA). The lowest FPA value obtained in the two consecutive samples was recorded and the results differing more than 30 % were excluded. FPA level determined in 30 healthy subjects aged 18 to 62 was 3.0 ng/ml or less (median value 1.4 ng/ml, range 0.4 - 3.0 ng/ml). The median FPA/fibrinogen calculated on a molar basis, assuming 340,000 D molecular weight for fibrinogen and 1,535 D for FPA, was 0.12 x 10s3 (range 0.05 0.3 x 10-3).


At presentation, disseminated intravascular coagulation (DIC), diagnosed according to previously reported criteria (17), was present in a single case with hyperleukocytic (158,OOO/pl) T-cell ALL (PT 1.8 INR, fibrinogen 80 mg/dL; FDP > 20 < 40 ng/ml, AT-III 103 %, FPA 26.2 ng/ml). In the whole group of patients, mean fibrinogen was 265.7 f 72.6 mg/dl and mean AT-III was 94.5 + 9.5 %. Apart from the above reported patient, none of the remaining cases had fibrinogen < 200 mg/dl and AT-III < 80 %. FPA higher than 3.0 ng/ml was present in 9/14 (64%) of patients. Figure 1 reports the mean fibrinogen, AT-III and FPA levels during the period of treatment with L-ASP.


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. and showed a slight increase at the end of therapy (80.2 + 24 X). Mean FPA showed a slight increase on day 3 of chemotherapy (5.2 ng/ml vs 4.1 ng/ml) and thereafter progressively declined towards near-normal values at the end of therapy (3.8 ng/ml on day 6, 3.9 ng/ml on day 9, 3.1 ng/ml on day 12 and 2.5 ng/ml 48 hour after the end of therapy). However, the ratio FPAfFibrinogen, on a molar basis, showed the maximum increase between days 6 and 9 indicating that during this period the maximum thrombin-dependent turnover of fibrinogen occurs (table 1). Table 1. Mean FPA and fibrinogen molar concentration and its ratio during L-Asparaginase treatment ____________________~~_~~~~~~~~~~~~~~_~~~_~~~__~~~~~~~~~~~~~~~~~~~~~~~~~~ DAYS OF L-ASPARAGINASE ADMINISTRATION (day 1 to 14) ____________________~~~~~~~~~~~~~~~~~~~~~_~~~_~~~~~~~~~~~~~~~~~-~~~~-~~-0 3 6 9 12 16 ______________________~~~_~~~~~~~~~~~~~~~~~ ____________________~~~~~~~~~~ FPA (nM)







Fibrinogen (nM) x 103







Ratio FPA/Fibrinogen 0.33 0.72 0.96 1.09 0.61 0.29 (x 10-3) _________________________________________________~________~__~~~_~~~~~~~~

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Protein C antigen was measured in 6 patients throughout the course of chemotherapy and showed a pattern similar to that observed for AT-III, with the maximum decrease on days 6 (mean= 67 + 24 %) and 9 (mean= 72 + 29 X). Two patients showed clinical evidence of thrombosis. A 15-year-old boy (the one with DIC and hyperleucocytosis at diagnosis) developed signs of thrombophlebitis in both thighs on day 7 after starting chemotherapy and 1251-fibrinogen test showed a marked uptake in these regions. AT-III level ranged from 77 % to 108 %, protein C from 68 % to 100% and fibrinogen from 20 to 60 mg/dl in the immediately preceding days. A 14-year-old girl developed painful swelling of her right leg on 10th day after starting chemotherapy. A diagnosis of thrombophlebitis was made and calcium-heparin (12,500 U twice daily by subcutaneous route) was started. severe However, 4 days later the patient developed cianosis and respiratory distress and died. Autopsy revealed pulmonary hemorrhagic infarction. AT-III level on the day preceding the thrombosis was 48 %, protein C 45 % and fibrinogen 100 mg/dl.


FPA A is a small peptide of 1535 D selectively cleaved from Aachain of fibrinogen by thrombin. Its increase has been assumed to be a marker of intravascular thrombin activity in several diseases. In particular, the peptide has been demonstrated to be a sensitive index of subclinical blood activation in acute leukemia (18-20). In a previous paper (20), we have demonstrated that the increase of this fragment in patients still in clinical complete remission could anticipate the recurrence of the disease in the next l-2 months. Moreover, it was showed that, albeit to a lesser extent, thrombin generation occurs also in ALL (20). L-ASP has been demonstrated to interfer with both coagulation and fibrinolysis (9,lO). In particular, it is well known that it is able to decrease the level of the natural anticoagulant system, in particular of AT-III and protein C (3-5, 12-14). Despite this occurs in concomitance with a marked decrease of procoagulant components (e.g. fibrinogen and prothrombin), this hemostatic derangement has been thought to be at least partially responsible for the increased risk of thrombosis, reported in leukemic patients treated with this agent. However, the documentation that clotting system is activated in this situation is lacking. In one study (ll), it has been shown that FPA increases after the starting of chemotherapy in ALL using L-ASP. In our study FPA was thoroughly




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monitored during the course of therapy with L-ASP. As previously reported by others (3-5,9, 12-14), we found a marked decrease of fibrinogen and moderate of AT-III and protein C during this treatment, particularly evident on day 6 and 9 after starting of chemotherapy. FPA slightly increased after 48 hours from the starting of chemotherapy (5.2 ng/ml vs 4.1 ng/ml). In the study of Legnani et al (ll), a greater increase of FPA was reported but the peptide was measured immediately after L-ASP, which was administered by the intramuscular route. Although the level of FPA did not significantly increase throughout the course of the treatment with L-ASP, the ratio FPA/fibrinogen did increase and reached its peak (three-fold basal) in concomitance with the maximum decrease of AT-III and protein C (table 1). This would mean that, in concomitance with the inhibition of liver synthesis of natural inhibitors, a real activation of blood coagulation occurs, probably favoured also by the ongoing blast cell lysis. Our data are in partial contrast with those of Bauer et al (15). These authors using sensitive markers of thrombin generation, namely the F1+2 prothrombin activation fragment and thrombin-antithrombin III complexes measured by specific radioimmunoassays, demonstrated that the level of these markers did not change during the treatment with L-ASP and suggested that other factors would be responsible for the thrombotic phenomena, for example old age or previous history of thrombosis. Our study differs from the study of Bauer et al, since their patients were treated in remission for consolidation and with a different schedule. Moreover, our patients who developed thrombosis were young and other risk factors were not present. The results of this study offer a rationale to the replacement treatment with the now available AT-III (and possibly protein C) concentrates in leukemic patients during L-ASP treatment. Our results support its use since a low grade intravascular clotting has been demonstrated in these patients, as measured with sensitive marker like FPA, and the concomitant low level of natural inhibitors makes thrombosis likely to occur. It would be interesting to undertake a study to assess the effectiveness of substitutive therapy with AT-III concentrates in controlling thrombin generation during L-ASP treatment, before its use is suggested in clinical practice.


1. Capizzi, R.L., Bertino, J.R.. Skeel, R.T., Creasey, W.A., Zanes, R., Olayon, C., Peterson, R.G., Handschumacher, R.E. L-asparaginase: clinical, biochemical, pharmacological, and immunological studies. Ann.

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Intern. Med. 74: 893-901, 1971. 2. Haskell, C.M., Canellos, G.P., Leventhal, B.G., Carbone, P.P., Block, J.B., Serpick, A.A., Selawry, O.S. L-asparaginase: therapeutic and toxic effects in patients with neoplastic disease. N. Eng. J. Med. 281: 1028-1034, 1969. 3. Pitney, W.R., Phadke, K.P., Dean, S. Antithrombin III deficiency during asparaginase therapy. Lancet i: 493-494, 1980. 4. Vellenga, E., Mulder, N.H., Nieweg, H.O. Antithrombin III deficiency during asparaginase therapy. Lancet i: 649-650, 1980. 5. Conard, J., Cazenave, B., Maury, J., Horellou, M.H., Samama, M. L-asparaginase, antithrombin III and thrombosis. Lancet i: 1091, 1980.

6. Priest, J.R., Ramsey, M.R., Latchan, R.E., Krivit, W. Thrombotic and hemorrhagic strokes complicating early therapy for childhood acute lymphoblastic leukemia. Cancer 46: 1548-1554, 1980. 7. Steinherz, P.G., Miller, P.L., Ghavimi, F., Allen, J.C., Miller, D.R. Dural sinus thrombosis in children with acute lymphoblastic leukemia.- J Am. Med. Ass. 246: 2837-2839, 1981. 8. Barbui, T., Rodeghiero, F., Meli, S., Dini, E. Fatal pulmonary embolism and antithrombin III deficiency in adult lymphoblastic leukaemia during L-asparaginase therapy. Acta Haemat. 69: 188-191, 1983. 9. Gralnick, H.R., Henderson, E. Hypofibrinogenemia and coagulation factor deficiences with L-asparaginase treatment. Cancer 27: 1313-1320, 1971. 10. Vallenga, E., Kluft, C., Mulder, NH, Wijngaards, G., Niewey H.O. The influence of L-asparaginase on the fibrinolytic system. Br. J. Haematol. 57: 247-254, 1984. 11. Legnani, C., Palareti, G., Pession, A., Poggi, M., Vecchi, V., Bianchini, B., Coccheri, S. Intravascular coagulation phenomena associated with prevalent fall in fibrinogen and plasminogen during L-asparaginase treatment in leukemic children. Haemostasis 18: 179-186, 1988. 12. Barbui, T., Finazzi, G., Vigano, S., Mannucci, P.M. L-asparaginase




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lowers protein C antigen. Thromb. Haemostas. 52, 216, 1984. 13. Conard, J., Horellou, M.H., Van Dreden, P., Potevin, F., Zittoun, R., Samama, M. Decrease in protein C in L-asparaginase-treated patients. Br. J. Haematol. 59: 725-727, 1985. 14. Vellenga, E., Broekmans, A.W., Kluft, C. Thrombotic complications during L-asparaginase related to protein C deficiency ? Thromb. Haemostas. 53: 443, 1985. 15. Bauer, K.A., Teitel, J.M., Rosenberg, R.D. L-Asparaginase induced antithrombin III deficiency: evidence against the production of a hypercoagulable state. Thromb. Res. 29: 437-442, 1983. 16. Lister, T.A., Whitehouse, J.M.A., Brearley, R.L., Beard, M.E.J., Wrigley, P.F.M., Paxton, A.M., Freeman, J.E., Woodruff, R.K., Malpas, J.S., Crowther, D. Combination chemotherapy for acute lymphoblastic leukaemia in adults. Br. Med. J. i: 199-203, 1978. 17. Rodeghiero, F., Mannucci, P.M., Viganb, S., Barbui, T., Gugliotta. L., Cortellaro, M., Dini, E. Liver dysfunction rather than intravascular coagulation as the main cause of low protein C and antithrombin III in acute leukemia. Blood 63: 965-969, 1984. 18. Myers, T.J., Rickles, F.R., Barb, C.E., Cronlund, M. Fibrinopeptide A in acute leukemia: relationship of activation of blood coagulation to disease activity. Blood 57: 518-525, 1981. 19. Gugliotta, L., Viganb, S., D'Angelo, A., Guarini, A., Tura, S., Mannucci, P.M. High fibrinopeptide A (FPA) levels in acute non-lymphocytic leukemia are reduced by heparin administration. Thromb. Haemostas. 52: 301-304, 1984.

20. Rodeghiero, F., Castaman, G., Soffiati, G., Quaglio, R., Castronovo S., Cortesi, S., Dini, E. Clinical significance of fibrinopeptide A in acute lymphocytic and non-lymphocytic leukemia. Blut 59, 177-183, 1989.

Fibrinopeptide A changes during remission induction treatment with L-asparaginase in acute lymphoblastic leukemia: evidence for activation of blood coagulation.

L-Asparaginase, a widely used antileukemic agent, inhibits liver protein synthesis leading to hypofibrinogenemia and hypoprothrombinemia together with...
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