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.

Non-invasive monitoring of coronary artery reconstruction by use of Doppler shift ultrasound.

Percutaneous recording of flow signals from coronary by-passes was carried out by use of a Doppler flow meter. The signals were recorded on a tape recorder and audiofrequency analysis was performed by use of a sonograph instrument. Twenty-six patients were examined one week to two years after coronary by-pass surgery, signals from 22 patients could be classified as either being "normal" or "abnormal". Abnormal signals were recorded from 8 patients. Comparison of patients with abnormal signals to those with normal Doppler findings showed that the patient group with abnormal signals had also had a less pronounced increase in blood flow subsequent to peroperative injection of papaverin into the bypass, than the normal patient group. The reported findings merit an extended, longitudinal investigation of patients undergoing coronary by-pass surgery.

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.

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.

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.

Thrombolytic treatment with i.v. brinase of advanced arterial obliterative disease of the limbs.

The material includes 17 patients suffering from different degrees of chronic peripheral arterial disease (11 chronic patients stage III and IV, two patients with acute arterial occlusion, and four patients stage II). Presence and extent of arterial occlusion was ascertained by initial arteriography. In twelve of the patients amputation had been considered. The patients were treated by a series of i.v. infusions of brinase, a proteolytic enzyme from Aspergillus oryzae. The brinase inhibitor capacity in plasma was determined by the azocollagen technique. Dosage of brinase was calculated to retain a rest-inhibitor capacity in order to avoid free proteolytic activity. In five patients the enzyme was also given preoperatively by intra-arterial instillation prior to a series of i.v. brinase infusions. Thirteen patients showed clinical improvement after brinase treatment. The condition of two patients remained unchanged, and in two patients amputation could not be avoided. In fourteen patients the treatment results were followed by measurement of peripheral systolic blood pressure. In ten patients obvious increase of the peripheral systolic blood pressure was observed. Cutaneous microcirculation was studied in seven patients by i.v. sequential fluorescein angiography and signs of improved microcirculation (appearance time, intensity and/or extent of fluorescence) were found in all examined patients. One patient with acute arterial occlusion of the right leg with obstruction of blood flow from the external iliac artery showed complete disobliteration after a series of i.v. brinase infusions. Bleeding complications associated with brinase treatment were not observed in the material. In three patients brinase treatment was discontinued because of complications (2 brinase, 1 heparin).

Dosage of i.v. brinase in man based on brinase inhibitor capacity and coagulation studies.

In 17 patients a total of 148 brinase infusions were given. All patients but two had anticoagulant treatment (dicoumarol or heparin) during the brinase series. Dosage of brinase was based on determination of brinase inhibitor capacity in plasma (azocollagen technique) prior to infusion of the enzyme. Iv infusion of brinase lowered inhibitors in plasma. Good correlation was found between expected lowering and measured inhibitor values after brinase infusion. The stipulated safety margin for brinase inhibitor capacity could be maintained. alpha 1-antitrypsin showed irregular changes and alpha 2-macroglobulin values were decreased by iv infusions of brinase. Fibrinogen values were somewhat lowered after iv brinase infusion. Values for fibrinogen degradation products increased and the ethanol gelation test became positive. Thrombotest values were lowered and the activated partial thromboplastin time was prolonged in patients receiving dicoumarol and iv heparin respectively. Changes in other coagulation parameters were insignificant. In two patients transient renal failure was recorded, laboratory data indicated intravascular coagulation as a possible cause. None of these two patients were on anticoagulant treatment. One patient developed a haematoma of the forefoot during massive iv heparin treatment during a series of iv infusions of brinase. Bleeding complications due to brinase treatment were not observed.