Encapsulation of the thrombolytic enzyme, brinase, in photosensitized erythrocytes: a novel thrombolytic system based on photodynamic activation.

In order to circumvent many of the problems associated with the systemic administration of agents used in thrombolytic therapy, it was decided to investigate the possibility of using erythrocytes as carriers and delivery vehicles for these agents. The enzyme brinase, a fibrinolytic enzyme produced by Aspergillus oryzae, was loaded into rabbit erythrocytes using electroporation. The loading index for this enzyme was found to be 60% and incorporation appeared to be relatively stable over a period of 4 h. In order to facilitate the predetermined release of the loaded component from the erythrocytes, they were photosensitized using haematoporphyrin derivative (HPD) and release was demonstrated within 5 min of photoactivation. Inclusion of the loaded, photosensitized system into clotting blood and subsequent exposure to light demonstrated almost complete lysis of the clot. We believe that this system exhibits potential for use in thrombolytic therapy.

Properties of the complex between alpha 2-macroglobulin and brinase, a proteinase from Aspergillus oryzae with thrombolytic effect.

The proteinase, brinase (Mr approximately 35000), from Aspergillus oryzae, which has been used in therapeutic attempts as a thrombolytic agent in arterial thrombosis, binds to purified human alpha 2-macroglobulin (alpha 2M) with a stoichiometry of 1.7-1.9 mol of enzyme/mol inhibitor. This binding leads to quantitative cleavage of the bait region of the inhibitor and to release of 3.6 thiol groups per molecule of alpha 2M, reflecting cleavage of the thioester bonds. The reaction with brinase is accompanied by a similar conformational change of alpha 2M as the reaction with trypsin, as shown by gradient gel electrophoresis and spectroscopic analyses. Brinase thus binds to alpha 2M in a similar manner as most small proteinases. However, in the complex formed at saturation of alpha 2M with brinase, the enzyme retains considerable proteolytic activity against macromolecular substrates, corresponding to about 25% of that of the free enzyme with fibrin as substrate. This finding indicates that the trapping of brinase by alpha 2M is less efficient than that of smaller proteinases. The complex formed at equimolar concentrations of the reactants has appreciably lower, although still significant, activity, amounting to 5-10% of that of free brinase against fibrin. This proteolytic activity of alpha 2M-brinase complexes against high-molecular-weight substrates most likely accounts for the thrombolytic effect of brinase in vivo. The observations also indicate that this thrombolytic activity increases more than proportionally to the brinase concentration as the latter is increased to approach saturation of alpha 2M in plasma.

Studies of protease and protease inhibitors in familial amyloidotic polyneuropathy.

Serum levels of 6 protease inhibitors, alpha 1-antitrypsin, Cl inactivator, alpha 2-macroglobulin, antithrombin-3, alpha 1-antichymotrypsin and inter-alpha-trypsin inhibitor were measured in patients with familial amyloidotic polyneuropathy (FAP) and a control group without neurologic disease. No significant differences were observed between the 2 groups. The proteolytic effect of brinase, an enzyme from Aspergillus oryzae, on amyloid tissue sections from patients with FAP was also evaluated. Amyloid fibrils were degraded by brinase, while the tissue structure remained fairly intact.

Degradation of amyloid proteins with protease I from Aspergillus oryzae. In vivo increase in SAA clearance rate after enzyme infusion.

The effect of the thrombolytic enzyme protease I from Aspergillus oryzae (brinase) on the amyloid protein AA was investigated. The effect of the enzyme on purified, low molecular weight protein AA was very high. Protein AA in intact amyloid fibrils suspended in neutral buffer was also degraded by the enzyme, although at a much lower rate. Amyloid fibrils in tissue sections could also be attacked and removed, leaving the tissue structure fairly intact. The in vivo effect of brinase on protein SAA was demonstrated in rabbits by an increase in the clearance rate of SAA after enzyme infusion.

Total and surface-located sialic acid levels in normal and leukaemic lymphocytes: relationship to T and B cell nature and to location in vivo.

Unfractionated normal blood lymphocytes (predominantly T lymphocytes) showed significantly elevated total and neuraminidase-susceptible sialic acid compared with CLL lymphocytes (predominantly B lymphocytes). T-enriched fractions from normal blood contained about three-fold the sialic acid content of the corresponding T-depleted fractions, and similarly low values were also obtained for T-depleted fractions from tonsils. Thus the reduced sialic acid levels in CLL lymphocytes appear to be due to their B cell nature. In contrast to the situation in normal peripheral venous blood, tonsillar T and B cells did not differ markedly in sialic acid content, suggesting that for T cells maturity and/or location in vivo are important determinants of this parameters. Results with calf thymocytes were in agreement with this. Sialic acid was estimated in malignant B and T lymphoblasts. Shedding of sialic acid-containing moieties from lymphocytes was also examined.

Influence of brinase on fibrinogen: fibrin transition in vitro and in vivo studies. II. Induction of fibrin deposits by brinase in the kidneys of rats with inhibited fibrinolysis.

The influence of brinase on fibrinogen levels, ethanol gelation test (EGT), fibrin content of lungs and kidneys (125I-fibrinogen) and brinase inhibitor capacity was investigated in the rat. The animals were either treated with i.v. infusions of brinase (4 mg/kg), tranexamic acid (AMCA) + brinase or heparin + AMCA + brinase. A control group was given i.v. saline, a reference group AMCA + thrombin. Brinase caused a decrease in fibrinogen levels and an increase in the incidence of positive EGT, deposition of fibrin was not observed. Infusion of brinase into rats with inhibition fibrinolytic system (AMCA), caused a more pronounced decrease in fibrinogen levels, a further increase in the incidence of positive EGT and also fibrin deposition in the kidneys. In rats treated with AMCA + thrombin a similar decrease in fibrinogen levels was recorded, the EGT was positive in all animals, and extensive fibrin deposits were observed in the kidneys and lungs of the animals. Heparinization before infusion of AMCA + brinase antagonized the lowering in fibrinogen levels and the increase in incidence of positive EGT and, moreover, fibrin deposits were no longer observed.

Studies on the degradation of fibrinogen by proteolytic enzymes from the larvae of Lanomia achelous (Cramer).

Fibrinogen degradation products formed by the action of purified haemolymph and saliva of a Saturnidae caterpillar of the Lonomia genus were studies by immunoelectrophoresis and polyacrylamide/SDS gel electrophoresis. The pattern of degradation differ form the one described for plasmin, trypsin, brinase, and ochrase. The most striking difference being the rapid loss of the alpha chain in spite of the presence of the protease inhibitor aprotinin, and/or denaturalizing agents such as 8 M Urea and 2% SDS.

Influence of brinase on fibrinogen: fibrin transition in in vitro and in vivo studies. I. Influence of in vitro addition of brinase to plasma on the ethanol gelation test.

The incidence of positive ethanol gelation test (EGT) after addition of brinase (a proteolytic enzyme preparation from Aspergillus oryzae) to anticoagulated and non-anticoagulated human plasma was studied. In vitro addition of brinase to plasma causes positive EGT, and the incidence is dose-dependent. In plasma from warfarin-treated patients and/or after addition of heparin to plasma, prior to the addition of brinase, a significantly reduced incidence of positive EGT is observed. The incidence is lowest after heparin. Gels formed in the presence of heparin are easier susceptible to enzymatic degradation than those formed in the absence of heparin.

Clinical trial of brinase and anticoagulants as a method of treatment for advanced limb ischemia.

A randomized, single-blind trial of repeated intravenous infusion of Brinase was carried out in 70 petients with severe chronic limb ischemia, who were candidates for lumbar sympathectomy or amputation. The enzyme caused s significant increase in calf and ankle pressure index. At six months follow-up, the clinical results were statistically significant in favour of Brinase when all patients were considered, but not if 10 patients with Buerger's disease were omitted from the analysis. Patients treated with a combination of Brinase and coumarins had a better clinical outcome than patients receiving either treatment on its own.

Action of brinase on human fibrinogen and plasminogen.

Brinase added to human plasma in vitro caused a decrease in fibrinogen concentration, positive paracoagulation tests and formation of a friable clot in sequence. Agarose gel filtration of these samples revealed the presence of fibrinogen derivatives both larger and smaller than the parent molecule. Infusion of the enzyme in vivo resulted in a decreased fibrinogen level, a prolonged thrombin time and an increase in fibrinogen related antigen (FRA) in serum. The elution pattern of FRA in the plasma samples obtained after infusion of Brinase was similar to that of the in vitro samples. The plasma pool of fibrinogen was partially consumed by infusion of Brinase, but the turnover of plasminogen remained unaffected. Purified plasminogen was partially degraded by addition of the enzyme but this was accompanied by a generation of proteolytic activity. These findings confirm that Brinase induces a proteolytic degradation of fibrinogen in plasma without activation of the plasminogen-plasmin system. Exposure of polymerization site(s) in the fibrinogen molecule is probably responsible for the reported clot promoting effect of the enzyme.

Relief of chronic arterial obstruction using intravenous brinase. A control study.

A therapeutic trial using placebo or the thrombolytic enzyme brinase was carried out in a group of patients with chronic arterial obstruction. The patients were observed for 3 months before receiving six intravenous infusions of either saline or brinase over a period of 2 weeks. Ankle blood pressure, Doppler ultrasound scanning, and arteriography were used to establish diagnosis in the patients. No changes were observed during the 3-month pre-observation period. After six brinase infusions, recanalization of 17 out of 27 obstructed arterial segments was recorded and the number of patent segments increased from 11 to 27. No improvement was observed in the placebo-treated patients. The differences between brinase and placebo treatment was statistically significant.