In vitro clot lysis as a potential indicator of thrombus resistance to fibrinolysis--study in healthy subjects and correlation with blood fibrinolytic parameters.

Using an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. 125I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37 degrees C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, alpha 2-antiplasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p < 0.001) and age (p < 0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-induced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.

Yield and collection efficiency.

Yield and collection efficiency are two important and strictly related aspects of an apheresis system. We have examined the various formulas, dwelling upon a few particular aspects of the collection of platelets, granulocytes and stem cells.

[Plasmapheresis in neuromuscular diseases. Personal cases]. / La plasmaferesi nelle malattie neuromuscolari. Casistica personale.

The authors used plasmapheresis to treat 8 patients with myasthenia gravis and 26 with polyradiculoneuropathy. In myasthenia the treatment was effective in 85% of the cases, as reported in other studies. Good results occurred in some 80% of the cases with acute and relapsing polyradiculoneuropathy. In all cases plasma-exchange was started in the early stage. In chronic polyradiculoneuropathy the treatment was less effective. The authors discuss the advantages, disadvantages and prospects of this kind of therapy.

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone).

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.

Plasma exchange in systemic lupus erythematosus.

Plasma exchange (PE) was performed in 8 of 14 patients with systemic lupus erythematosus (SLE) PE was instituted as an additional therapy during active disease in patients who did not respond to conventional therapy. The best results were obtained in plasmapherized patients; in fact, immune complex serum levels disappeared rapidly, whereas anti-DNA antibodies decreased slowly. The changes observed indicate that PE may be beneficial, especially when it is associated with drugs that can block antibody rebound. A variety of "technical factors" may influence PE therapy which therefore must be monitored appropriately.

Plasma-exchange in the treatment of cryoglobulinemia.

Plasma-exchange was used in 10 patients with mixed cryoglobulinemia. The procedure was used as a primary therapeutic tool to reduce cryoglobulin levels, and in combination with prednisone and cytotoxic drugs. The results show that PE alone is detrimental, although it may be an important adjunct to conventional therapy in MCG with progressive deterioration in the clinical condition, and in those patients who do not respond to drug therapy alone. The weak response to PE in MCG may be due to many technical variables, but mainly to the low percentage of circulating cryoglobulins removed.

In vitro effect of Leptospira icterohaemorrhagiae on human mononuclear leukocytes: comparison of virulent with non virulent strain.

The in vitro effect of a virulent and a non virulent strain of Leptospira interrogans serotype icterohaemorrhagiae on human peripheral mononuclear cells was investigated. After addition of bacteria to citrated whole blood the production of mononuclear cell procoagulant activity (tissue factor) was observed. Indeed mononuclear cells isolated from whole blood-bacteria mixtures after prolonged incubation shortened the recalcification time of normal plasma. The virulent strain induced a significantly higher procoagulant activity than non virulent and this effect was dependent on the number of bacteria. The production of tissue factor, a potent trigger of blood clotting, by Leptospira icterohaemorrhagiae could help to understand the mechanism(s) responsible for the activation of intravascular coagulation sometimes associated with leptospirosis.

[HBsAg and anti-HBs in thalassemia non-transfused patients (and in their relatives)]. / L'HBsAg e l'anti-HBs in soggetti con emopatia mediterranea non trasfusi (e nei loro familiari).

The sera obtained from 105 non transfused adult heterozygous thalassaemic subjects and from 119 members of 25 thalassaemic families have been tested with radioimmunoassay methods for the presence of HBsAg and anti-HBs. 11 out of 105 thalassaemic patients (10.4%) showed HBsAg in their serum and 41 (40%) anti-HBs antibodies. Family studies revealed that the incidence of HBsAg was 8.9% in 89 thalassaemic members and 10% in 30 non thalassaemic relatives. The incidence of anti-HBs was 34.8% and 13.3% respectively. The frequency of HBsAg in thalassaemic subjects, independently of blood transfusion as shown by our data, suggests some difficulty in eliminating the antigen. So these subjects become rather easily carriers of the antigen and source of the infection particularly in the family.