Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer.

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.

Inhibition of human peptidyl dipeptidase (angiotensin I converting enzyme: kininase II) by human serum albumin and its fragments.

Purified peptidyl dipeptidase (angiotensin I converting enzyme or kininase II) from human lung or hog kidney is inhibited by commercially prepared plasma protein preparations, by human serum albumin and by the additive albumin stabilizer, acetyltryptophan. After the initial steps of purification, albumin was detected by immunodiffusion as a component in human lung peptidyl dipeptidase preparation. Fragment C of albumin (sequence 124-298) is a more potent inhibitor than the parent molecule (Ki = 1.7 X 10(-5)M). Reduction and carboxymethylation of five of the six S-S bridges in Fragment C yield the most potent noncompetitive inhibitor (Ki = 3 X 10(-6)M). Reduction of the sixth bridge raises the K1. This indicates that maintenance of the tertiary structure in Fragment C is of importance for the inhibition. Neither albumin nor Fragment C are substrates of the enzyme. Fragment C and its derivative also inhibit the inactivation of bradykinin by the purified human enzyme and by the peptidyl dipeptidase on the surface of intact cultured human endothelial cells.

Induction of fibrinolysis by polyanions in human plasma.

The fibrinolytic potency of several polyanions was comparatively investigated. Fibrinolytic activity was measured in a whole plasma assay using H-D-Val-Leu-Lys-pNA (S-2251) as chromogenic substrate and by a fibrin plate assay using plasminogen rich fibrin plates. In the chromogenic substrate assay potent fibrinolytic polyanions comprised dextran sulfate, GAGPS, pentosan polysulfate, polyanethol sulfate, l-carrageenan and i-carrageenan. Chondroitin sulfates A, B, C, keratan sulfate, ribonucleic acid, k-carrageenan and heparin were weakly fibrinolytic. Hyaluronic acid and lipopolysaccharide from E. coli were inactive. Similar results were obtained when fibrinolytic activity was measured by a fibrin plate assay. All polyanions except lipopolysaccharide produced lysis zones. Induction of fibrinolytic activity in human plasma was shown to be at least partially dependent on Hageman factor. In factor XII deficient plasma no fibrinolysis was induced by any of the polyanions when measured in the fibrin plate assay. In the chromogenic substrate assay corn Hageman factor inhibitor (CHFI) inhibited the activation of S-2251 cleaving enzyme by GAGPS, pentosan polysulfate, polyanethol sulfate, heparin, and ribonucleic acid near completely. The activation by dextran sulfate was inhibited by 45%. Heparin, pentosan polysulfate and GAGPS, three polyanions of therapeutic interest were separately compared. In both assays GAGPS proved the most potent activator, while pentosan polysulfate exhibited 83% and 44% and heparin 32% and 14% of GAGPS fibrinolytic activity in the chromogenic substrate test and the fibrin plate assay, respectively.

Interaction of the sulfated lactobionic acid amide LW 10082 with thrombin and its endogenous inhibitors.

The synthetic polyanion LW 10082 prolonged thrombin clotting times when human or bovine thrombin were used to induce clotting. In the clotting time prolongation LW 10082 was 15 times more potent in the presence of human thrombin than of bovine thrombin. In an amidolytic thrombin inhibition assay, LW 10082 inhibited bovine and human thrombin when plasma was present. Here, too, thrombin inhibition was much more pronounced when human thrombin was used. When purified antithrombin III was used, neither human nor bovine thrombin were inhibited by LW 10082. LW 10082 proved to be a potent inhibitor of human thrombin in the presence of heparin cofactor II in an amidolytic assay. The time course of the inhibition was slow and at least 10 minutes of incubation were required to obtain complete inhibition of thrombin by an excess of HC II and LW 10082. The anticoagulant activity of LW 10082 in the APTT was unchanged in the presence of anti-antithrombin III antibodies while the potency of heparin was clearly reduced. This indicates that the overall anticoagulant effect of LW 10082 was as well independent of antithrombin III.

Anticoagulant and antithrombotic properties of synthetic sulfated bis-lactobionic acid amides.

Sulfated bis-lactobionic acid amides, a new class of polyanions with heparin-like properties, were synthesized and their antithrombotic and anticoagulant activities were determined. Compared to heparin and Fragmin, a low molecular weight heparin, the substances exhibited moderate to low anticoagulant activities in aPTT, thrombin clotting time and Heptest assays. In amidolytic assays no anti-IIa activity and only exceedingly low anti-Xa activity was observed. The antithrombotic activity of the bis-lactobionic acid amides was determined using two thrombosis models. In rabbit and rat models thrombi were induced by a combination of endothelial damage and reduction of blood flow or only by endothelial damage. At least one of the bis-lactobionic acid amides (LW 10082) exhibited a considerable antithrombotic activity which was similar to low molecular weight heparin.

Antithrombotic and anticoagulant properties of synthetic polyanions: sulfated bis-aldonic acid amides.

Fifteen polyanionic bis-aldonic acid amides were synthesized by condensation of the corresponding aldonic acids and alpha,omega-diamines and by subsequent sulfation of the intermediates. The compounds were completely sulfated and exhibited molecular weights between 1450 and 2600 daltons. The antithrombotic activity of these synthetic heparin analogs was evaluated in a rat jugular vein clamping induced thrombosis model. Several compounds pounds were potent antithrombotic substances comparable in potency to the low molecular weight heparin Fraxiparin. Some structure activity relationships on their pharmacologic action could be deduced. The antithrombotic activity of a representative agent was confirmed using the Wessler stasis model in rabbits. In the in vitro tests the bis-aldonic acid amides exhibited moderate to low anticoagulant effects depending on the assay used. In the activated partial thromboplastin time assay, the anticoagulant potency of these compounds was between 2- and 30-fold lower than that of heparin. The anti-factor Xa activity of the experimental substances was at least 50 times lower than that of heparin, whereas the compounds were devoid of anti-factor IIa activity when measured in an amidolytic assay. The anticoagulant effects of these agents were dependent on the length of the spacing and on the type of aldonic acid moieties. These bisaldonic acid amides represent a novel class of potent antithrombotic substances without any anti-factor IIa activities and only very low anti-factor Xa activities, suggesting that these antiprotease actions are not a prerequisite for the antithrombotic action of polyanionic substances.

Antithrombotic action of recombinant hirudin in a venous thrombosis model.

The dose- and time-dependent antithrombotic activity of recombinant hirudin (r-hirudin) was examined in a new animal model of venous thrombosis. For the evaluation of the antithrombotic activity of r-hirudin, an occluding thrombus was produced by repeated clamping of the rat jugular vein. The number of clampings necessary to induce thrombosis served as a measure of antithrombotic activity. The gradual reduction of blood flow was registered by Doppler sonography. In order to estimate the relative antithrombotic activity of r-hirudin, its potency in this model was compared with other antithrombotic substances. r-Hirudin proved to be less potent than heparin after intravenous administration. Its half-life after subcutaneous administration was relatively short; however, the antithrombotic potency after subcutaneous administration was comparable to that of heparin.

Influence of various irrigation fluids on articular cartilage.

When bovine articular cartilage was incubated in Ringer's solution, considerable amounts of proteoglycan were washed out of the cartilage. Thus, 4% of the proteoglycan was lost into the medium during an incubation of 4 hours. Subsequently, it was found that ionic aqueous media like saline or Ringer's solution extracted much more proteoglycan than ion-free media like distilled water or a variety of carbohydrate containing solutions. In separate experiments, it could be shown that solutions of 20% sorbitol and 2% mannitol exhibited particularly low proteoglycan extracting properties. It was found that the extraction of proteoglycan was dependent on the ion concentration in the aqueous media. The extraction of proteoglycan by sodium chloride was negligible at NaCl concentrations of 0.1% and lower. Proteoglycan loss from cartilage was only induced at 0.9% NaCl. Using intact rat femoral heads it could be shown that the elution of proteoglycan from cartilage occurred as well when the cartilage was intact. Here, the elution occurred at a slower rate but the differences between ionic and ion-free solutions were greater. By electronmicroscopic examination of bovine cartilage incubated in different media, it was observed that Ringer's solution induced a more uneven and rougher appearance of the cartilage surface than did 10% mannitol solution, indicating that probably a denudation of collagen fibers occurs on the loss of proteoglycan from the cartilage. Because the observed proteoglycan washout occurred within rather short periods of contact of the cartilage with the medium, it is concluded that this may be of relevance for the clinical situation during arthroscopic procedures. The use of preferably isotonic carbohydrate solutions like 5% mannitol is suggested to prevent unnecessary loss of proteoglycan from hyaline cartilage.

Biochemical studies on sulfated lactobionic acid amides.

A series of sulfated bis-lactobionic acid amides was prepared. The compounds comprise highly charged poly-anions of very low molecular weight. Usually, the compounds contain 16 sulfate groups per molecule and are homogeneous, monodisperse substances. The molecular weights range from 2388 to 2514. The compounds were evaluated for anticoagulant activity using a number of standard tests. The compounds exhibited moderate to good APTT activity. Interestingly, the anti-Xa and anti-IIa activities were very low, particularly in amidolytic assays. Using prothrombin and Factor X activation assays, it was demonstrated that these compounds were more potent in these experimental settings than in standard anti-Xa or anti-IIa assays. Compared with heparin and LMW heparins, the bis-lactobionic acid amides were particularly active as inhibitors of the intrinsic system activation of Factor X. The bis-lactobionic acid amides exhibited potent anticomplement activity, being clearly superior to heparin and LMW heparins. For the most interesting substance, LW 10082, experimental results suggest that its anticoagulant activity is independent of antithrombin II. The anticoagulant activity of LW 10082 was readily neutralized by protamine sulfate. Platelet factor 4, however, did not reduce its anticoagulant activity. Because of these favorable properties, it is hoped that LW 10082 might prove to be an interesting alternative to heparin or LMW heparin in clinical use.

Derivatives of bis-aldonic acid amides and their anticoagulant and antithrombotic properties.

Aprosulate sodium was the first representative of a new class of synthetic polyanions showing antithrombotic efficacy in different animal models. In several clinical trails (Phase I) in human volunteers aprosulate demonstrated anticoagulant properties, too. Searching for other active substances of similar structure, a series of bis-aldonic acid amides were synthesized. These compounds exhibited interesting antithrombotic and anticoagulant activities. The pharmacodynamic activities of the compounds LW 10121, LW 10125, LW 10114, 10244, and LW 10168 are summarized in this article. These substances prolonged the APTT to 150% of the blank values at concentrations of 1.5 to 13.5 micrograms/mL. The thrombin time and anti-Xa test were only slightly influenced by concentrations up to 100 micrograms/mL. All compounds were investigated in a jugular vein hemostasis model in rats to examine their antithrombotic potential. They all had an antithrombotic activity lower than aprosulate, except compound LW 10121, which seemed to be a little more active. The subcutaneous injection of 10 mg/kg LW 10121 resulted in a longer duration of action than aprosulate and heparin. On the basis of the chemical structure and the profile of action, it is assumed that the new compounds may possess the same mode of action as aprosulate, but the mechanism of action may be different from heparin and low molecular weight heparins.

Effects of different irrigation liquids and times on articular cartilage: an experimental, biomechanical study.

To characterize the suitability of different solutions [6% Dextran, 5% sorbitol (I); 5% fructose (II); 5% mannitol (III); Ringer's solution (IV)] for arthroscopy, bovine knee articular cartilage specimens (n = 52) were immersed for 0, 2, 4, or 20 h before indentation creep testing, known as a sensitive probe for tissue degeneration. Immersion in liquid I for up to 20 h produced significant softening of articular cartilage [p < 0.05, Friedman two-way analysis of variance (ANOVA)]. Liquids II-III produced no statistically significant changes in the deformational characteristics of articular cartilage. After 2 h of immersion in liquid IV deformation increased and remained elevated over the observation period (p < 0.05, Friedman two-way ANOVA). Based on these results, the first and most remarkable softening of cartilage took place with Ringer's solution as compared with nonionic solutions. Therefore, the nonionic solutions, such as 5% fructose or mannitol, may have potential for use as an irrigation liquid during arthroscopic procedures.

[Pyridinoline-containing collagen degradation products in urine in coxarthrosis]. / Pyridinolinhaltige Kollagenbruchstücke im Urin bei Coxarthrose.

High-performance liquid chromatography of collagen degradation products in human urine for diagnosis of osteoarthrosis was introduced by Macek and Adam (6) in 1987. The aim of the present study was to demonstrate the clinical usefulness of this method in a group of 20 patients with osteoarthrosis of the hip and 10 healthy volunteers. There were no significant differences in number or intensity of the detected signals between the two groups. The results of this study show that the determination of collagen degradation products is not useful for diagnosis or for the control of therapy in patients with osteoarthrosis.

Pharmacologic profile of the antithrombotic and bleeding actions of sulfated lactobionic acid amides.

1. The introduced bis-lactobionic acid amides represent a new group of synthetic polyanions with a molecular weight of 2387 to 2514d. The compounds are homogeneous and chemically defined. 2. The in vitro blood coagulation parameters (APTT, Heptest, anti-Xa, thrombin time, and anti-IIa) are only marginally altered by these compounds. 3. In a rat model of venous thrombosis using endothelial damage as the thrombogenic stimulus, the antithrombotic activity of the bis-lactobionic acid amides was shown in a dose- and time-dependent manner. 4. LW 10082 and LW 10121 prolong the bleeding time at dosages about 10 times less than that of heparin. The safety/efficacy index appears to be wider for these compounds than for heparin. 5. In subchronic toxicologic studies in rats, LW 10082 was well tolerated up to a dosage of 100 mg/kg body weight. 6. The anticoagulant and antithrombotic activities of the bis-lactobionic acid amides decrease gradually with the increasing number of methylene groups in the alkylene chain.