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A reproducible standardized method for measuring capillary bleeding time (CBT) in dogs is presented. The skin was punctured at the toe from a forelimb of non-anaesthetized dogs parallel to the edge of the horny skin of the pad. Before the procedure, a cuff of a sphygmometer was placed above the antibrachium and a hyperaemie agent was applied to the shaved area. The normal CBT in non-anaesthetized dogs was 2.25 ± 0.76 min (x ± SD). After injection of acetylsalicylic acid (ASA) (20 mg/kg body weight), CBT was prolonged up to 25 min. Platelet aggregation in response to 10 μg collagen/ml was decreased in parallel after treatment with ASA. [ABSTRACT FROM AUTHOR]

The aim of the present study was to test the haemostyptic properties of a phospholipid complex with platelet factor-3 (PF-3) activity in platelet-dependent haemostatic disorders. The substance was investigated in an animal model comprising six investigational groups (five dogs each). A thrombocytopathy was created in the dogs belonging to groups 1-5 by intravenous administration of 20 mg/kg body weight (BW) acetylsalicylic acid (ASA). Two hours later a human albumin solution (5%) (control group) or a phospholipid complex with PF-3 activity was injected or infused intravenously in different dosages. In dogs pre-treated with ASA, injection of 2 ml/kg BW of the phospholipid complex shortened capillary bleeding time, which was prolonged as a consequence of ASA-treatment. This effect lasted for 4 h at least. The capability of the platelets to aggregate increased 5 min after intravenous injection of the phospholipid without differences in the respective groups. At the same time platelet counts dropped to approximately 50%, but increased again distinctly after 30 min. When the phospholipid complex was administered to clinically healthy dogs who had not been treated with ASA, this resulted in prolongation of the capillary bleeding time as well as a significant platelet drop in the peripheral blood. Although these were only short-term effects after administration of the phospholipid complex, a disadvantageous effect cannot be excluded in patients suffering from haemorrhagic diathesis. For this reason, a phospholipid complex with PF-3 activity cannot be recommended as a therapeutic agent for platelet-dependent coagulation disorders in the dog. [ABSTRACT FROM AUTHOR]

Patients with hypercholesterolemia and with coronary atherosclerosis have increased plasma levels of plasminogen activator inhibitor (PAI)-1. PAI-1 and low-density lipoproteins (LDL) are also present in the walls of atherosclerotic vessels, where they participate in the development and remodeling of the atherosclerotic plaques. We investigated the influence of LDL on the apical (luminal) and basolateral (subendothelial) secretion of PAI-1 by human umbilical vein endothelial cells in a two-compartment cell-culture model. Confluent cells were incubated with LDL either in the apical compartment or in the basal compartment. Cells incubated with culture medium served as controls. A significantly higher concentration of PAI-1 was found in both the apical (P = 0.025) and the basal compartment (P = 0.025) if cells were incubated with LDL on the basolateral side. In contrast, incubation of the cells with LDL apically resulted in an increased PAI-1 concentration only in the apical compartment (P = 0.028) and not in the basal compartment. Our findings indicate that the LDL particles that reach the subendothelial space can induce an increased release of PAI-1 by endothelial cells into the vessel lumen and also contribute to the release of PAI-1 into the subendothelial space and thus to the process of atherosclerotic plaque remodeling., (Copyright 2003 Wiley-Liss, Inc.)

Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N- and O-linked glycans of the endothelial cell thrombin receptor. While N-linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate-like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin-mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly-cationic basic proteins, whereas the CSGAG-deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O-linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post-translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell-related anticoagulant potential in different vascular beds.

The importance of granulocytes and/or platelets in endotoxin-induced generalized intravascular coagulation was studied as well as thrombocytopenic rabbits. Neutropenia and thrombocytopenia were induced by oral administration of busulphan. Generalized intravascular coagulation, as indicated by renal glomerular microclot formation, was initiated by two intravenous injections of endotoxin. Granulocyte counts before the second injection of endotoxin were most significantly related to activation of intravascular coagulation whereas platelet counts either before the first or second injection of endotoxin were not definitely related to the activation process. Renal glomerular microclots occurred in rabbits after two injections of endotoxin even when the platelet counts were between 500 and 5000/mul. These experiments indicated that granulocytes but not platelets are essential to the activation of endotoxin-induced intravascular coagulation.

The in vivo behaviour of urea-soluble fibrin monomer (FM) was compared with that of fibrinogen in rabbits. Purified rabbit 125I-fibrinogen was injected into 36 unanaesthetized rabbits. Three days later the rabbits received either purified rabbit 131I-FM I mg/kg body weight, which corresponds to cI% of the circulating plasma fibrinogen pool 131I-fibrinogen, or buffered urea only. The distribution volume of FM was 43.4 +/- 6.9 ml/kg and of fibrinogen 43.5 +/- 7.7 ml/kg (mean +/- SD). The elimination curve of urea-soluble FM as represented by the clottable 131I-radioactivity. plotted on a semi-logarithmic paper, consisted of an initial steep decay within the first 6 h and a slow flattening of the slope 6 to 24 h after injection of FM. Although a terminal single-exponential slope, as observed in fibrinogen elimination, could not be computed for the 24 h following FM injection the mean half-life time of the last segment of the clottable-radioactivity curve, between 12 h and 24 h, was 12 h. Within 24 h a mean of 83-5% of the injected FM were removed from the circulating blood. The elimination characteristics of fibrinogen were not influenced by the injected FM. Control experiments showed that buffered 3.0 M urea, the solvent of FM, does not influence distribution volume and elimination of 125I-fibrinogen. The distribution of 131I as well as 125I-radioactivities in organs representing FM and fibrinogen respectively did not differ from each other. Elevated levels of 131I-radioactivity, however, were found in the urine after FM injection suggesting an accelerated elimination of FM in comparison to fibrinogen. Fibrinolysis inhibition with high doses of aprotinin did not significantly reduce the urinary excretion of 131I-radioactivity representing breakdown of FM. Furthermore, inhibition of the fibrinolytic system had no influence on the elimination characteristics of either fibrinogen or FM. In order to explain the results obtained in this study the following theory is proposed. Intravenously injected FM forms complexes with fibrinogen and fibrinolytic degradation products. The complexes continuously increase in size until they dissociate into fibrin oligomers and carrier proteins. The oligomers are eliminated whereas the carrier proteins recycle.

The behaviour of labelled des-AB fibrin in plasma was studied by gel filtration after it had been injected into rabbits. Purified rabbit [125I]des-AB fibrin was prepared by clotting of [125I]fibrinogen by thrombin and solubilizing the formed clot in buffered 3 M urea. Gel filtration of this material on urea-equilibrated columns showed a single peak identical to the elution profile of fibrinogen. This indicated the existence of monomeric des-AB fibrin. When plasma from rabbits injected with monomeric [125I]des-AB fibrin and [131I]fibrinogen was gel-filtered through plasma-equibrated columns, two separate peaks of radioactivities were obtained. The first peak eluted mainly with the void volume and contained [125I]des-AB fibrin whereas the second peak eluting within the fractionation range contained [131I]fibrinogen. Identical elution profiles were obtained in in vitro studies when monomeric [125I]des-AB fibrin was mixed with plasma containing [131I]fibrinogen and gel-filtered through plasma-equilibrated columns. We conclude from these studies that monomeric des-AB fibrin formed high-molecular weight aggregates or changed its conformation posing as a larger molecule than fibrinogen when injected into rabbits. No complex formation between des-AB fibrin and fibrinogen was observed as [131I]fibrinogen was not incorporated into des-AB fibrin aggregates. Thus, soluble des-AB fibrin can circulate in the blood without forming fibrin-fibrinogen complexes.