Kallikrein Inhibitor Units Research Articles

Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass

The use of aprotinin to reduce blood loss after cardiopulmonary bypass is under debate. Concern has been raised about the renal effects of aprotinin. We administered a mean aprotinin dose of 4.2 x 10(6) kallikrein-inhibiting units to 13 patients with coronary disease undergoing cardiopulmonary bypass for 74 +/- 5 minutes (mean +/- standard error of the mean); 13 comparable patients having cardiopulmonary bypass served as control subjects, and all were studied postoperatively for 24 hours. Aprotinin reduced postoperative blood loss by 50% (p = 0.0082). Two of the 13 patients who received aprotinin needed one red cell unit each versus a total of 18 units in eight of 13 control patients (p = 0.0096). Blood pressure, hemoglobin value and serum protein concentration were higher after operation in the aprotinin group (p less than 0.05 to p less than 0.01). Platelet counts did not differ, but plasma thromboxane was lower in aprotinin recipients (p less than 0.001). In control patients fibrinogen degradation products (D dimer) doubled, and alpha 2-antiplasmin activity was halved during and after cardiopulmonary bypass (p less than 0.01 to p less than 0.001), whereas aprotinin patients showed no changes. The complement breakdown products C4a, C3a, and C3dg as well as C9 neoantigen increased from prebypass baseline in both groups (p less than 0.001); the increment of C3a and C3dg was greater in the aprotinin than in the control patients (p less than 0.001). Serum electrolytes, osmolality, and creatinine remained normal in both groups of patients. Creatinine clearance was normal or above normal and virtually identical in both groups. Osmolar clearance and fractional sodium excretion were higher in the aprotinin group than in the control group shortly after cardiopulmonary bypass (p less than 0.05 to p less than 0.01); renal function was unremarkable the next morning. No adverse clinical effects attributable to aprotinin were seen. In summary, aprotinin offers advantages for cardiopulmonary bypass.

Effects of sample handling on adrenocorticotropin concentration measured in canine plasma, using a commercially available radioimmunoassay kit

SUMMARY A commercially available radioimmunoassay (ria) kit for measurement of human adrenocorticotropin (hacth) was validated for use in dogs. Assay sensitivity was 3 pg/ ml. Intra-assay coefficient of variation (× 100; cv) for 3 canine plasma pools was 3.0 (mean ± sd, 33 ± 0.99 pg/ ml), 4.2 (71 ± 2.4 pg/ml) and 3.7 (145 ± 3.7 pg/ml) %. Interassay cv for 2 plasma pools measured in 6 assays was 9.8 (37 ± 3.6 pg/ml) and 4.4 (76 ± 3.4 pg/ml) %, respectively. Dilutional parallelism was documented by assaying 2 pools of canine plasma at 3 dilutions and correcting the measured result for dilution. Corrected mean concentrations for the first pool were 33 (± 0.99), 36 (± 4.3), and 33 (± 6.8) pg/ml; corrected mean concentrations for the second pool were 145 (± 5.4), 141 (± 10.8) and 125 (± 3.4) pg/ml. Recovery of 1-39hacth added to canine plasma (6.25, 12.5, 25.0, 50.0, and 100.0 pg/ml) was linear and quantitative (slope = 0.890, R2 = 0.961). To test whether anticoagulant or the protease inhibitor, aprotinin, influences acth concentration in canine plasma, acth was measured in canine blood collected in 4 tubes containing anticoagulant: heparin (h), heparin + 500 kallikrein inhibitor units (KIU) of aprotinin/ml (ha), edta (e), and edta + aprotinin (ea). Plasma acth concentration was the same when samples containing h and ha, or ha and e were compared, and was significantly (P < 0.01) lower in samples containing ea. Plasma storage at - 20 C for 1 week or 1 month was not associated with significant change in acth concentration in canine plasma, using any of the 4 anticoagulant treatments. Plasma acth concentration measured after 6 months’ storage at —20 C was significantly (P < 0.01) lower for all anticoagulants used. Synthetic 1-39hacth added to canine blood was accurately recovered (88 to 109%, n = 3) from plasma containing edta, with or without aprotinin, whereas percentage recovery was overestimated by 18 to 91% in heparinized plasma. Plasma acth concentrations in edta- treated canine blood kept at 4 or 22 to 25 C for 15 to 90 minutes prior to centrifugation at 8 C were not significantly different. Plasma acth concentration in canine plasma was affected by storage tube material. Concentration of acth in canine plasma stored in borosilicate glass tubes for 1 week or 1 month at — 70 C was significantly higher than initial acth values (P ≤ 0.01), but was unchanged over time in plasma stored in polypro-pylene or polystyrene tubes. Sample handling procedures affect canine plasma acth concentration measured by use of the ria kit. Optimal sample handling conditions for plasma acth measurement in dogs include use of edta anticoagulant, blood collected at 20 to 25 C (room temperature) followed by centrifugation within 15 to 90 minutes, and plasma storage in plastic (not glass) tubes for not longer than 1 month at −20 C.

Seminal plasma proteins of fertile and infertile men analyzed by two dimensional electrophoresis

Sperm-free seminal plasma from seminal fluid ejaculate of fertile and infertile men obtained in the presence and absence of aprotinin (500 kallikrein inhibitor units per milliliter) was analyzed by two-dimensional electrophoresis followed by silver staining. To evaluate postliquefaction proteolytic breakdown of seminal plasma proteins, protease inhibitors were added to the semen at 15, 30, and 60 minutes after ejaculation. Most seminal plasma proteins in normospermic men (n = 4) had molecular weights of 30,000 to 70,000 and were similar to those present in serum. The major non-serum protein in seminal plasma of all men was a basic product with an approximate molecular weight of 40,000. A group of proteins (molecular weights = 20,000 to 23,000) in seminal plasma analyzed immediately after liquefaction was detected in oligospermic men (n = 4) but not in normospermic men (n = 4) or azoospermic men (n = 4). When protease inhibitor was added to normospermic semen at greater than or equal to 15 minutes after liquefaction, these proteins (molecular weight = 20,000 to 23,000) and another group of proteins (molecular weights = 40,000 to 43,000) were readily identifiable but were further enhanced in the absence of protease inhibitors. These findings suggest that oligospermic men may have accelerated proteolysis of sperm or seminal plasma proteins that may contribute to subfertility.

The Effects of Aprotinin, a Kallikrein Inhibitor, on Renin Release and Urinary Sodium Excretion in Mild Essential Hypertensives

The effects of aprotinin on renin release and renal function were evaluated in 24 male essential hypertensive patients, on unrestricted (n = 17) and on chronic low as well as on high sodium intake. Aprotinin (1 X 10(6) kallikrein inhibitor units) or saline (200 ml) were infused in all patients for 6 h. Blood samples were taken for plasma renin activity (PRA) and 6-h urine collections were obtained for active and inactive kallikrein, sodium and potassium excretion measurement. In patients on unrestricted sodium diet, aprotinin had no effect on blood pressure (BP), glomerular filtration rate, renal plasma flow, urinary sodium and potassium excretion. However, an inverse relationship was found between pretreatment urinary sodium excretion and the per cent reduction of the latter after aprotinin. A significant reduction in urinary sodium excretion was induced by aprotinin in patients on high sodium intake, whereas no change was observed in the same patients when on a low sodium diet. Aprotinin reduced the urinary excretion of active kallikrein by 81% and the active to total kallikrein ratio from 24 to 6%. Infusion of aprotinin induced a significant decline in active renin but did not modify inactive renin levels in patients on unrestricted sodium diet as well as in patients on low or high sodium intake. Our data suggest that the inhibition of kallikrein and/or other serine proteases by aprotinin can interfere with renal release of active renin and also support the hypothesis that the renal kallikrein system exerts a regulatory control on sodium excretion in salt replete hypertensives.

Dissociation of urinary kallikrein activity and salt and water excretion in the rat

Experiments were designed to examine the relationship, if any, between urinary kallikrein activity (amidolytic assay) and sodium and water excretion in 12-wk-old Munich-Wistar rats. Five groups of animals were studied: euvolemic, saline-expanded and water-loaded anesthetized rats, and euvolemic and saline-expanded conscious restrained rats. Following surgery, animals were allowed to stabilize (60-180 min) and reach a steady-state urine flow. By design, basal sodium and/or water excretion varied markedly among groups as a function of hydration state. Group means for sodium excretion and urine flow ranged from 0.8 to 12.4 mu eq/min and 6 to 112 microliter/min, respectively. In contrast, neither active nor total urinary kallikrein activity differed significantly among the five groups. In anesthetized euvolemic rats, intravenous administration of aprotinin produced a dose-dependent decrease in urinary kallikrein activity. The greatest inhibition of 93 +/- 3% (active) and 72 +/- 10% (total) was observed with a dose of 5,000 kallikrein inhibiting units (KIU)/kg and 1,000 KIU X kg-1 X min-1. This dose produced a significant decrease in active and total kallikrein activity in each group (P less than 0.001). However, sodium and water excretion were unchanged in aprotinin-treated rats and similar to values in vehicle-treated time-control groups. Linear regression analysis revealed no significant correlations between urinary kallikrein activity and sodium excretion or urine flow either among or within groups. These results indicate that urinary kallikrein activity is not related to acute sodium and water homeostasis in anesthetized or conscious rats.

The effects of the protease inhibitor, aprotinin, on the course of shock induced by endotoxin in cats

The administration of endotoxin derived from Escherichia coli to anaesthetized cats resulted, within the first 5 min, in an initial increase in right atrial pressure and a reduction in systemic arterial blood pressure. Over the next 2 h shock was characterized by a reduced cardiac output, tachycardia, reduced arterial pH and an increased level of lactate. The survival rate at the end of the 8 h experimental period was only 10%. The protease inhibitor aprotinin (Trasylol), given as a continuous intravenous infusion 1000 kallikrein inhibitor units (k.i.u.) kg-1h-1 together with a bolus of 40,000 k.i.u. kg-1, significantly inhibited the severity and incidence of the initial endotoxin response (increase in right atrial pressure and systemic hypotension), perhaps suggesting the direct or indirect involvement of kinins. Aprotinin did not reduce the delayed effects of endotoxin (sustained reduction in cardiac output, lacticacidosis), nor did it improve survival at 8 h. Indeed, there was some evidence that aprotinin exaggerated the delayed effects of endotoxin in this model.

Guanidinobenzoatase on the surface of tumour cells

Guanidinobenzoatase is a recently described serine enzyme (Steven & Al-Ahmad, 1983) that hydrolyses two active-site titrants for trypsin, namely 4-methylumbelliferyl p-guanidi nobenzoate (Coleman et al.. 1976) and 4-nitrophenyl pguanidinobenzoate (Chase & shaw, 1967). Unlike an active-site titration, the hydrolysis of these two substrates by guanidinobenzoatase is continuous over a 2h period. Each of these compounds acts as a competitive substrate for guanidinobenzoatase when the other compound is employed as the prime substrate in the enzyme assay. Guanidinobenzoatase is inhibited by a-N-benzoyl-DL-arginine ethyl ester and is unable to cleave this substrate for trypsin-like esterases and can therefore be distinguished from the esterase described by Walsh & Nadler (1980) that is assayed with tr-N-benzoyl-DL-arginine ethyl ester and also cleaves 4meth ylumbelliferyl-p-guanidinobenzoate. Guanidinobenzoatase is present in the extracellular fluid surrounding Ehrlich-ascites-tumour cells grown in mice (Steven & Al-Ahmad, 1982) and in the tumour fluid obtained from V,-carcinoma implanted in the leg muscles of rabbits (McCroskery et al., 1975). Normal rabbit muscle and rabbit serum lack guanidinobenzoatase, but an enzyme similar to guanidinobenzoatase is present in normal rabbit intestine and extracts of intestinal tissue. The enzyme can be easily purified from tumour fluids employing a two-step affinity Sepharose procedure. The tumour fluid ( 5 ml) is first mixed with loop1 of aprotinin (lo00 kallikrein-inhibitory units; Trasylol) in order to inhibit proteinases that may be present. The tumour fluid is then equilibrated with 2ml of damp Sepharose-2B in order to adsorb an enzyme which attaches to Sepharose and which rapidly degrades starch. Guanidinobenzoatase is not bound to Sepharose-2B but is bound to a second support made of agmatine-Sepharose-4B. The unbound protzins are eluted from the agmatine-Sepharose-4B with iso-osmotic NaCl and the guanidinobenzoatase is finally eluted with 0.2 M-Na,SO,, pH 6.0. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate demonstrated the presence of three polypeptide chains derived from guanidinobenzoatase. The guanidinobenzoatase eluted from agmatine-Sepharose-4B was successfully employed to raise antisera in rabbits against the mouse enzyme; these sera caused inhibition of guanidinobenzoatase assayed with 4meth ylumbelliferyl p-guanidinobenzoate. Agar gels (190) were prepared in 0.1 M-phosphate buffer,

Collagenase inhibition in the healing colon.

A randomized controlled trial was performed to assess the effect of intravenous aprotinin (Trasylol) on the healing of experimental colonic anastomoses in the rabbit following a standard left colonic resection anastomosis. Assessment of tensile strength was by means of both bursting pressure and breaking strength. Those animals subjected to bursting pressure assessment received intravenous aprotinin 80 000 KIU (kallikrein inhibitory units) at the time of anaesthesia, and postoperatively 160 000 KIU per day given in divided doses for three days. Control animals received saline placebo. A further group of animals received a lower loading and maintenance aprotinin dose (40 000 KIU and 60 000 KIU per day respectively) with control animals receiving saline. Breaking strength was employed as the means of assessment. The mean bursting pressures were 47.7 +/- 2.9 mmHg and 37.5 +/- 3.4 mmHg for aprotinin and controls respectively (P less than 0.05). The mean difference in collagen content of the anastomosis compared to the resected specimen was +1.25 +/- 0.50 microgram/mg and -1.02 +/- 0.47 microgram/mg for aprotinin and placebo groups (P less than 0.005). The mean breaking strength in the aprotinin group was 169.6 +/- 74.5 g and 110.0 +/- 65.9 g for the saline group (P less than 0.02). The mean difference in collagen content of the anastomosis compared to the resected specimen was +0.95 +/- 0.69 microgram/mg and -1.5 +/- 0.78 microgram/mg for the aprotinin and saline groups respectively (P less than 0.05). The significant elevation of both bursting pressure and breaking strength assessments, with a significant improvement in the collagen content of the anastomoses, may be the result of collagenase inhibition following the use of intravenous aprotinin in the experimental model.

Effect of the Kallikrein Inhibitor Aprotinin on Myocardial Ischemia and Necrosis in Man.

The effect of administration of aprotinin, a serine esterase inhibitor capable of inactivating kallikrein, on the extent and severity of acute myocardial ischemic injury and subsequent necrosis, was studied in 25 patients. Another group of 25 patients who did not receive aprotinin served as a control group.We administered 100,000 kallikrein inhibitor units (KIU) of aprotinin as a bolus dose, followed by a continuous infusion (4 ml/min) that contained 10,000 KIU/kg in 240 ml of dextrose/water solution, to all 25 patients admitted to the hospital within 30 to 60 minutes after the onset of acute myocardial ischemia.To measure the effect of aprotinin, three parameters were studied; the sum of S-T segment elevations (ΣST), the development of Q waves, and the prediction of infarct size by measuring the disappearance rate of creatine phosphokinase(MB CPK isoenzyme).The average ΣST in the treated group decreased from 40.5 ± 7.00 mv to 12.95 ±4.60 mv (P < 0.01); in contrast the control group's ΣST did not change significantly, from 54.25 ± 8.02 to 51.7 ± 6.8. Deeper Q waves evolved in the control group compared to the treated group: ΔQ (6 hours) = 1.0 ST (15 min) +1.19 (25 patients, r = 0.78); and in the treated group ΔQ (6 hours) = 0.66 ST (15min) + 0.91 (25 patients, r = 0.65) (P < 0.025).In the control group the estimated infarct size was 57.4 ± 4 CPK-gram equivalents(CPK-g-Eq). There was significantly less damage in the treated group: 19 ± 2 CPK-g-Eq (P < 0.01). Thus we conclude that aprotinin diminishes myocardial damage.

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor. The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor. Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.

Effects of aprotinin on organ cultures of the rat's kidney.

Fragments of adult renal tissue were maintained as organ cultures for 7 days in HEPES-buffered medium 199, supplemented with 10% calf serum and antibiotics. Addition of aprotinin (10,000 kallikrein inhibitor units per ml) to the medium resulted in improved survival of the cells of the glomeruli and tubules and preserved the integrity of the glomerular, capsular and tubular basement membranes. Optimal results were obtained when aprotinin was present throughout the period of culturing. Inclusion of aprotinin in the medium for only the first 3 days in vitro slightly increased the numbers of surviving glomerular and tubular cells, but also promoted the growth of connective tissue in the explants and was detrimental to the basement membranes of the tubules. It is suggested that both the antiproteolytic and the carbohydrate-binding properties of aprotinin are involved in the mediation of the observed effects.

Effect of trasylol on packed cell volume and plasma kallikrein activation in acute Babesia argentina infection of splenectomised calves.

Three groups of four splenectomised calves, three months old, were infected with Babesia argentina parasites. One group was inoculated intravenously with 2000 kallikrein inhibitor units (kiu)/kg body weight of the broad-spectrum proteinase inhibitor, Trasylol, three times a day for 5 days commencing on the 3rd day after infection (early treatment). The same dose of Trasylol was given to a second group for 3 days commencing 8 days after infection (late treatment). The third group was untreated. Parasite multiplication rates were similar in the three groups. In the early treated group levels of activated kallikrein in plasma were significantly lower than those in the other two groups. The early treatment group also showed significantly higher levels of plasma kallikrein inhibitor. No significant differences in total plasma kallikrein levels were seen among the three groups. Packed cell volumes in the treated groups remained significantly higher than those in the controls. The implications of these findings are discussed.

Effects of aprotinin on organ cultures of the cerebellum of the adult rat

Cerebellar cortical explants taken from adult rats were maintained in organ culture for 5 days in the presence of aprotinin, a polypeptide inhibitor of proteolytic enzymes, in concentrations from 0.1 to 50,000 kallikrein inhibitor units (KIU) per ml. In explants cultured in media containing 1000–50,000 KIU/ml, Purkinje cells survived in greater numbers, and these cells and also those of the molecular layer resembled their counterparts in the uncultured cerebellum more closely than those in cultures from media without or with lower levels of aprotinin. The histological appearance of axons in the cultures was unaffected by the presence of aprotinin in the media. It is suggested that aprotinin might enhance the survival of neurons injured by the process of explantation as a consequence of the inhibition of lysosomal cathepsin D, though the carbohydrate-binding properties of the drug could also be involved.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

Mediators of inflammation in leukocyte lysosomes. II. Mechanism of action of lysosomal cationic protein upon vascular permeability in the rat.

The vascular permeability-increasing action of rabbit PMNL lysosomes has been studied in skin and cremaster muscle of the rat. Both an extract of frozen-thawed granules and a cathepsin-free cationic protein fraction of the granules (which had previously been demonstrated to cause leukocyte adhesion and emigration in vivo) induce increased vascular permeability in skin and muscle which resembles that produced by histamine or histamine-liberators with respect to the timing of the response and the predominant type of microvessel affected. Extracts of frozen-thawed lysosomes and the inflammatory lysosomal cationic protein both cause disruption of rat mesenteric mast cells in vitro, whereas a granule-free cytoplasmic fraction of PMN leukocytes and a non-inflammatory cationic protein fraction of the granules do not do so under identical test conditions. The mastocytolytic action of lysosomal materials in vitro is not inhibited in the presence of 10 kallikrein-inhibiting units of trasylol per ml. The mast cell rupturing fraction of PMNL granules (cationic protein) possesses no detectable peroxidase activity or acid-mucopolysaccharase activity. When compared with compound 48/80 on the basis of estimated molecular weight, the lysosomal cationic protein appears to be at least as active as the latter compound with respect to in vitro mastocytolytic potency. Chronic pretreatment of rats with an agent known to reduce tissue mast cell numbers causes marked suppression of the vascular permeability change normally induced in skin and muscle by lysosomal extracts and cationic protein. Similar results are obtained if lysosomal materials are tested in rats pretreated with an antihistaminic. These observations are discussed with respect to the mode of action of PMNL lysosomes in the early and late phases of local tissue-injury reactions.