A regimen for antithrombin III substitution in patients with acute lymphoblastic leukemia under treatment with L-asparaginase.

BACKGROUND AND METHODS: Seventeen adult patients with acute lymphoblastic leukemia (ALL) treated with L-asparaginase (20,000 IU/m2 on six alternate days) were infused with antithrombin III (AT III) concentrates (Kybernin P, Behring). Substitution therapy was aimed at increasing the reduced AT III concentration usually found in these patients, since AT III deficiency is thought to be associated with an increased risk of thrombosis. Two schedules of AT III administration, different in dosage, timing and duration were evaluated. The first 7 patients (group A) received a fixed dose of 2,000 U every day for 6 times, starting with the second L-asparaginase (L-ase) infusion, independently of their plasma AT III levels. In the following 10 patients (group B), 20-25 U/Kg b.w. were administered daily for 7 times only when the plasma AT III level was lower than 60% with plasma fibrinogen higher than 100 mg/dl and platelet count higher than 50 x 10(9)/l, or when AT III was below 40%. Thirteen patients who received L-ase without AT III substitution served as controls. RESULTS AND CONCLUSIONS: Both substitution regimens resulted in mean plasma AT III nadir values significantly (p less than 00.1) higher than in the controls. Our data suggest that, in ALL patients receiving L-ase according to the L20 protocol, satisfactory plasma AT III levels may be assured with infusions of 20-25 U/Kg b.w./day for 7-10 days, starting by day 2 of L-ase treatment.

Platelet function and interferon alpha-2a treatment in essential thrombocythaemia.

The effects of interferon (IFN) alpha-2a treatment on platelet function were evaluated in 20 patients affected by essential thrombocythaemia (ET). Baseline data documented the well-known abnormalities of in vitro platelet aggregation and the constant presence of a delta-storage pool deficiency. The therapy in all patients reduced the platelet count, and in the majority of them caused a partial improvement of in vitro platelet aggregation. Although the mean intraplatelet ADP level improved during treatment, it always remained below the normal range documenting persistence of the delta-storage pool deficiency. The plasma beta-TG levels, which initially were high, significantly decreased during treatment, but the beta-TG ratio and the platelet beta-TG values always remained within the normal range--this suggests an absence of platelet activation either before or during therapy. Our results demonstrate that, despite significantly reducing the platelet count, IFN alpha-2a treatment only partially corrects the qualitative platelet abnormalities in ET.

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes.

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.

Prompt plasma-exchange treatment and coma reversibility in two patients with thrombotic thrombocytopenic purpura.

We report on the cases of two women with acute thrombotic thrombocytopenic purpura (TTP) whose clinical courses were characterized by the onset of a coma state. Prompt commencement of plasma-exchange (PE) treatment led to complete hematological and neurological remission, which can still be observed without any maintenance therapy. No CNS abnormalities were observed in either patient using brain CT and NMR scans.

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation.

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m2 over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50-60% of pretreatment values after 2-5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however due to C4b-binding protein levels higher than total PS levels (p less than 0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 less than p less than 0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.

Ticlopidine in the treatment of thrombotic thrombocytopenic purpura: report of two cases.

The observation of two clinical cases make possible an evaluation of the potential therapeutic activity of platelet function inhibitors in thrombotic thrombocytopenic purpura (TTP). In particular, the clinical and hematological effects of ticlopidine (TC), employed alone in two TTP patients, are reported. The mechanism of action of this peculiar antiplatelet drug is mainly represented by the inhibition of fibrinogen binding on the platelet surface. In the first patient, a 45-year-old female in whom plasma-exchange (PE) and corticosteroids (C) led to a partial remission (platelets 80 x 10(9)/l), treatment with TC at a dose of 750 mg/day was carried out, and after 6 weeks a normal platelet count was observed. A complete remission was maintained for 31+ months, even after reduction of the TC dose to 250 mg/day. In the second patient, an 18-year-old female affected by relapsing TTP, a complete remission obtained with PE and C was maintained for 19 months in concomitance with TC treatment, started at a dose of 750 mg/day and lowered to 250 mg/day. After 11 months of treatment at this low dosage there was a relapse (platelets 20 x 10(9)/l), but the increase of the TC dose to 750 mg/day in a few weeks induced a complete remission again. These data, in accord with a few other recent preliminary reports, suggest that TC, even alone, may play an interesting role in the management of TTP patients.

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

Bioluminescent flow sensors: L-lactate dehydrogenase activity determination in serum.

The catalytic activity of serum L-lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L-lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37 degrees C and from 3 to 2000 U/l at 25 degrees C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.