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Thein vitro effects of the proteolytic enzyme brinase on ascites tumour cells were studied histochemically. Dissociation of protein from phospholipid attachment in surface and cytoplasmic membranes was seen, accompanied by activation of lysosomes in the cytoplasm. These changes are similar to those found by other authors in ascites cells exposed to specific antibody and indicate that changes in membrane lipid function should be envisaged in assessing the effects of proteolytic treatment of cells. Lysosomal activation also resulted from exposure of the ascites cells to tragacanth, a metaphase-blocking polysaccharide.Two patients with melanoma showed substantially increased lysosomal activity in their lymphocytes afterin vitro treatment with brinase.

Both coumarin (1,2-benzopyrone) and warfarin (4-hydroxycoumarin) have been shown to prevent the recurrence of malignant melanoma. Their action is macrophage-dependent and the dosage is critical. In 1984 a multicentre, prospective, randomised, double-blind trial of coumarin, given as a daily 50-mg dose for 2 years after surgery in patients with high-risk melanoma, was started. the patients had lesions greater than 1.70 mm thick and TNM stage IB or stage II disease. To date there are 4 recurrences in the coumarin-treated group of 13 patients, and 10 recurrences in the placebo-treated group of 14 patients (P0.01). There were no toxic effects.

A method has been developed for the direct determination of 7-hydroxycoumarin (7HC) and 7-hydroxycoumarin-glucuronide (7HCG) in urine without sample clean-up. Separation was carried out in 90% 100 mmol l-1 phosphate buffer, 11 mmol l-1 deoxycholic acid (sodium salt) and 10% acetonitrile, on a 47 cm uncoated silica capillary at 20 kV with detection of the analytes at 320 nm. The linear detection range for concentration versus peak area for the assay is from 0 to 100 micrograms ml-1 for both analytes, with a limit of quantitation of 2 micrograms ml-1 for 7HC and 5 micrograms ml-1 for 7HCG in urine. Inter- and intra-assay results showed sr values in peak areas of between 0.5 and 13%. The method was applied to the direct determination of 7HC and the glucuronide conjugate in urine from two volunteers administered with 250 mg of coumarin. The samples were also analysed by another CE method and by using HPLC. There was no statical difference between the results determined by each of the methods. Up to 83% of the coumarin administered was excreted as 7HC or 7HCG. The majority of the 7HC excreted was in the glucuronide form (98%) with 2% occurring as free 7HC.

Peripheral lymphocytes from normal subjects and from patients with impaired T cell function were compared with respect to sialic acid. Mean values for total and neuraminidase-susceptible sialic acid were abnormally high in metastatic cancer patients. Spontaneous release of sialic during incubation of lymphocytes in saline was measured for nine metastatic cancer patients and three non-cancers (two anergy, one sarcoidosis). Mean values for both groups were significantly elevated. Correlation between sialic acid release and clinical status was observed in 11 out of the 12 cases tested. Spontaneously released sialic acid was mainly in protein-bound form in normal and cancer cases but the non-cancer group showed high proportions in free and non-protein-bound form. This latter phenomenon was also observed in all groups, normal and diseased, on exposure of the cells to protease 1 from A. oryzae.

The T lymphocytes populations of 22 young healthy adults, 21 healthy middle aged and older blood donors, 35 non-pregnant women of child bearing age and 14 patients with advanced malignant disease were assessed and compared. It was found that the mean T cell counts in the middle aged and older controls were significantly lower than in the healthy young adults and were further reduced in the patients with malignant disease. The addition of the proteolytic agent brinase (protease 1 obtained from Aspergillus oryzae) to the rosetting test increased the T cell counts signficantly in all groups. This was mot marked in the older age groups and the patients with malignant disease. The proteolytic agent is shown to exert its effect on the lymphocytes in the test. Slow intravenous infusion of either brinase or streptokinase into patients with malignant disease is shown to result in increased T lymphocyte counts pari passu with a restoration of skin allergy. The significance of these findings and possible mode of action of the proteolytic agents in increasing T cell activity are discussed.

1. Human cancer tissues are characterised by deposition of fibrin. 2. Aqueous extracts of cancer tissue contain labile factors which cause coagulation in buffered diluted human plasma. 3. The coagulative factors of cancer tissue have been fractionated by chromatography on DEAE cellulose into four main groups, A, B, C and D. 4. Comparable extracts of normal human tissues show either negligible coagulative activity or a small amount of activity corresponding roughly to group B. 5. Starch block electrophoresis of cancer tissue extract separates a fast-moving (prealbumin) thermolabile factor chromatographically identical with group C. 6. Extracts of foetal membrane (chorion) contain thermolabile factors corresponding chromatographically with groups B, C and D. 7. The three main coagulative factors of chorion extract have been isolated in active form by large scale chromatography on DEAB cellulose. 8. The nitrogen content of the factors has been found to be significantly lower than in most proteins. 9. The factors have been examined by starch gel electrophoresis. 10. The factors do not coagulate fibrinogen and, therefore, do not contain thrombin.

TWO methods for the production of venous thrombiin vivo are described. In the first instance radiopaque thrombi were formed. The disadvantages of this method are presented and the reasons for abandoning the technique are discussed. In the second method “physiological” thrombi were producedin vivo and a method of radiographically visualising dissolution of the thrombus is described. The advantages of this method and its potential in the evaluation and comparison of thrombolytic agents are demonstrated. The thrombolytic substance used was the Protease 1 of Aspergillus oryzae. Reports that this agent is thrombolytic are confirmed.

The effect of exercise on the dilute blood clot lysis time (B.L.T.), plasma fibrinogen, and serum anti plasmin has been investigated in two groups of healthy volunteers and in patients with multiple sclerosis.

A cytopathic effect on HeLa cells has been shown to be produced by fibrinolytic agents, protamine sulphate and chlorpropamide and by the alpha1 globulin and albumin electrophoretic fractions of saline placental extract.

The value of the oral anticoagulant warfarin sodium and fibrinolytic agents is discussed in relation to cancer surgery. A controlled trial of 128 patients showed that in a variety of recurrent cases the addition of warfarin to chemotherapy doubled the 2-year survival rate. The best results were obtained in postmenopausal patients with breast cancer. Warfarin depresses cellular immune responses which might militate against its use for cases undergoing "curative" surgery. Instead, induction of fibrinolysis by streptokinase or Brinase is suggested, because it increases the activity of the cellular immune mechanism. The results to date of an ongoing controlled randomized trial of streptokinase with surgery of tumors of the large bowel are presented, showing that the trends are in favor of streptokinase therapy; however, insufficient time has elapsed to make it, as yet, statistically significant. The action of streptokinase-induced plasmin and Brinase on lymphocytes is described.

EXPOSURE of Landschutz ascites tumour cells to the proteolytic enzymes trypsin or brinase was found to cause release of small but statistically significant amounts of cholesterol into the medium. Since treatment with these enzymes also increases the agglutinability of the cells towards specific antiserum, it is suggested that cholesterol loss may be among the membrane changes involved in this enhanced aggultinability

Brinase (an extract of Aspergillus Oryzae) was shown to rapidly digest human fibrinogen in vitro to aggregable degradation products with a molecular size range of 310,000 to 230,000 the latter fragments being more slowly digested to core fragments, Dbr and Ebr. The fibrinogen polypeptide chain susceptibility to Brinase attack was in the order Aα, γ, Bβ, Lysis of the Bβ chain seems to be the rate limiting step in the conversion of the high molecular weight fragments (MW 310,000–230,000) to the core fragments Dbr and Ebr. The conservation of NH2 terminal Tyrosine during fibrinogen digestion and the very transient existence of D dimer fragments during totally crosslinked fibrin lysis suggest that the carboxy end of the γ chain is prone to Brinase attack. The crosslinked α chains of fibrin, while resistant to plasmin, are vigorously digested by Brinase. The plasma of cancer patients being treated with Brinase contained degraded fibrinogen (lacking intact Aα chains) and their aggregates. These aggregates contained some crosslinked γ chains (γ-γ dimers) suggesting that Brinase in vivo exorcises both a lytic and coagulant effect. Thrombin mediated clots in all the plasmas examined contained no crosslinked α chains. Positive plasma ethanol gelation tests can be explained by the presence of the aggregable high molecular weight fragments observed during the in vitro lysis of fibrinogen by Brinase.

A method of testing for neutralisation of the cancer coagulative factor is described. Of a wide range of substances tested, protamine sulphate, placentalα 1 globulin and placental albumin were found to possess activity worthy of clinical investigation in the treatment of cancer. Plasmin is also highly active in counteracting the clotting effect of the cancer coagulative factor. Its probable mode of action is unspecific proteolysis, comparable with its effect on many of the recognised factors concerned in coagulation of the blood.

Following a course of therapy with protease 1. four cases of acute leukaemia were shown to have complement dependent cytotoxic autoantibodies against their own leucocytes and leukaemic cells. Their presence was suspected clinically in a further case. Whole blood transfusion resulted in severe depression of total white cell counts and platelets in three cases. Another case went into remission after a whole blood transfusion. An adult case of acute myeloblastic leukaemia responded well to protease 1 and developed autoantibodies in the fourth week of therapy, but was not affected by packed cell transfusion. It is suggested that these, cases were deficient in complement.

The coagulative activity of cancers is in general higher, often much higher, than that of the tissues from which they spring.

Landschutz ascites tumour cells treated in vitro with Protease I showed no changes in intracellular K and Na levels when the cells were analysed directly at the end of the 60-min incubation period. If, however, the cells were washed twice in the saline medium before analysis significant losses of K, apparently in exchange for Na, were found to take place. Controls incubated in the absence of Protease I and similarly treated showed no such K loss. Viability tests, using lissamine green, indicated that the observed changes were not due to dead cells. These findings are interpreted as being due to an alteration in the plasma membrane associated with increased sensitivity to mechanical trauma, which in turn leads to secondary loss of K. Increases in UV absorbance in the 260 nm region were noted in the supernatant medium of treated cells. Normal mice weighing 30 g tolerated intraperitoneal injections of 0.3 mg Protease I and under, in repeated daily doses, but a single injection of 0.4 (i.e. 13 mg/kg) was lethal within 2 hours. The presence of tumour cells (7.5 mg dry wt) intraperitoneally was found to protect mice against repeated daily injections of this normally lethal dose, indicating that the enzyme is quickly adsorbed onto the tumour cells. No effect on tumour growth in vivo was found when single injections of Protease I were administered intraperitoneally at the same time as the tumour, but when additional doses of the enzyme were given on the two subsequent days, the 7-day yield of tumour cells was significantly increased. Les Effets de la Protease I D'aspergzllus Oryzae (Brinase) Sur la Permeabilite de la Membrane et la Croissance des Cellules de la Tumeur Ascitique de Landschutz Des cellules de tumeur ascitique de Landschutz traitees in vitro avec de la protease I n'ont subi aucune modification quant aux niveaux intracellulaires de K et de Na lorsqu'elles ont ete directement analysees a la fin d'une periode d'incubation de 60 minutes. Toutefois, si les cellules etaient rincees deux fois en milieu salin avant l'analyse, on pouvait observer des pertes importantes de K, apparemment compensees par du Na. Des cellules temoins incubees en l'absence de protease I et traitees de la měme facon n'ont subi aucune perte de K. Les tests de viabilite utilisant le vert de lissamine ont montre que les modifications observees ne sont pas dues aux cellules mortes. Ces constations indiquent qu'il se produit une alteration de la membrane plasmatique associee a une sensibilite accrue a un trauma mecanique, qui a son tour entraǐne une perte secondaire de K. Une augmentation de l'absorption d'ultraviolets dans la zone des 260 nm a ete constatee dans le milieu surnageant des cellules traitees. Des souris normales d'un poids de 30 g ont tolere des injections intraperitoneales de 0.3 mg de protease I au maximum s'il s'agissait de doses quotidiennes, mais une seule injection de 0.4 mg (soit 13 mg/kg) a entraǐne la mort en l'espace de deux heures. La presence de cellules tumorales (7.5 mg, poids sec) dans le peritoine protege les souris contre les injections quotidiennes de cette dose qui normalement est mortelle, ce qui indique que l'enzyme est rapidement adsorbe sur les cellules tumorales. On n'a remarque aucun effet sur la croissance tumorale in vivo lorsque des injections intraperitoneales uniques de protease I ont ete effectuees en měme temps que des injections de cellules tumorales, mais lorsque des doses supplementaires de l'enzyme ont ete administriees les deux jours suivants, le rendement des cellules tumorales a considerablement augmente apres sept jours.

1 Two thousand, one hundred and seventy-three patients with cancer or chronic infections were treated with coumarin in a clinical trial. 2 0.37% of these patients developed elevated liver enzyme levels during therapy with coumarin. 3 This hepatitis was probably a form of idiosyncratic hepatotoxicity and may have been immune in origin.