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Classical swine fever (CSF) is a multi-systemic disease that can be accompanied by severe haemorrhagic lesions. The underlying pathogenetic mechanisms are still far from being understood, though disseminated intravascular coagulation (DIC) was discussed as a major factor. In the presented study, the direct thrombin inhibitor hirudin was used in an attempt to elucidate the role of the coagulation system in the pathogenesis of CSF-induced haemorrhagic lesions. Two groups of piglets ( n = 5) were infected with highly virulent CSF virus (CSFV) strain CSF0634. One group underwent daily treatment with hirudin, the other served as untreated challenge infection control. Assessment of clinical signs using a clinical score system, coagulation tests, and blood counts were performed daily. Both groups developed acute-lethal CSF with haemorrhagic lesions. Although changes in the coagulation system were seen in the late stages of CSFV infection, our results strongly suggest that DIC does not present the crucial event in the pathogenesis of haemorrhagic lesions.

Based on two fields, namely the placement and the calcium concentration, a chemo-mechanically coupled three-dimensional model, describing the contractile behaviour of smooth muscles, is presented by means of a strain energy function. The strain energy function (Schmitz and Böl, 2011) is additively decomposed into a passive part, relating to elastin and collagen, and an active calcium-driven part related to the chemical contraction of the smooth muscle cells. For the description of the calcium phase the four state cross-bridge model of Hai and Murphy (Hai and Murphy, 1988) has been implemented into the finite element method. Beside three-dimensional illustrative boundary-value problems demonstrating the features of the presented modelling concept, simulations on an idealised artery document the applicability of the model to more realistic geometries.

Anticoagulant efficacy of PEG-Hirudin in patients on maintenance hemodialysis. Background Heparins are currently the anticoagulants of choice in long-term hemodialysis (HD). Because of their shortcomings, including the increasing incidence of heparin-induced thrombocytopenia (HIT II), alternative anticoagulation is necessary. The study objectives were to provide safe and effective HD by investigating an appropriate PEG (polyethylene glycol)-Hirudin dosage regimen in patients on HD, as well as to compare the safety, tolerability, and efficacy of PEG-Hirudin with that of unfractionated heparin (UFH). Methods Twenty patients (12 males, 8 females, mean age 57.8years) with end-stage renal disease (ESRD) took part in the study. Dialysis sessions lasting a mean of 4.3hours (QB 250 to 300mL/min, QD 500mL/min) were performed 3 times a week with a Gambro GFS plus 16 dialyzer. Ten patients (group I) received UFH at 3 regular dialysis sessions (HD 1-3 ) followed by 5 dialysis sessions using PEG-Hirudin (HD 4-8 ). Another 10 patients (group II) received UFH at 3 regular dialysis sessions (HD 1-3 ) followed by 10 sessions on PEG-Hirudin (HD 4-13 ). The starting dose of PEG-Hirudin was a single bolus injection of 80 μg/kg BW (HD 4 ), except for the first patient, who received 50 μg/kg BW followed by a 12 μg/kg bolus. Before each of the following sessions (HD 5-13 ), an individualized PEG-Hirudin dose of between 26 to 65 μg/kg body weight (BW) (mean dose 41 μg/kg BW) was injected. PEG-Hirudin plasma and blood concentrations derived from anti-Iia activity and ecarin clotting time (ECT), respectively, activated partial thromboplastin time (aPTT), bleeding time, and arteriovenous (AV) fistula compression time were investigated to calculate the pharmacokinetic parameters or to assess anticoagulant efficacy. Results Mean predialysis PEG-Hirudin plasma concentrations increased up to a maximum of 488ng/mL in group I (HD 8 ) and up to 536ng/mL in group II (HD 8 ). Mean plasma concentrations measured at 5 minutes after the 1st (HD 4 ), 5th (HD 8 ), and 10th (HD 13 ) PEG-Hirudin injection ranged from 1076 to 1298ng/mL. Mean post-dialysis plasma levels ranged from 818 to 995ng/mL. Mean predialysis aPTT was not affected by UFH, but was prolonged by 46 to 56 seconds by PEG-Hirudin. Five minutes after injecting PEG-Hirudin or UFH, mean aPTT was prolonged to a maximum of 85 and 188 seconds, respectively. Mean post-dialysis aPTT values ranged from 60 to 68 seconds after PEG-Hirudin and 34 to 46 seconds after UFH. PEG-Hirudin was well tolerated; no serious adverse events or bleeding complications were observed. Safety assessments yielded no significant difference between the two anticoagulants. Conclusion This pilot study confirmed the usefulness and tolerability of a PEG-Hirudin dose regimen consisting of a single, fixed bolus dose of 80 μg/kg BW injected before starting the first dialysis session (HD 4 ) and followed by a dose titration period over at least 4 sessions (HD 5-8 ), which again was followed by a fixed maintenance dose period (HD 9-13 ). On the basis of PEG-Hirudin data from patients with various degrees of renal insufficiency but not undergoing hemodialysis and prior recombinant-hirudin (r-hirudin) experience, patients were titrated into an EC-controlled dose range that proved to be efficacious enough to prevent clotting and safe enough to prevent bleeding. Due to the favorable pharmacokinetic properties of PEG-Hirudin, a residual anticoagulant effect is maintained in the intervals between dialysis sessions, and this permanent state of anticoagulation may prevent vascular access complications as well as other vascular events.

Introduction : The anticoagulant r-hirudin lepirudin is eliminated exclusively via the kidneys. We examined the C-terminal amino acid degradation of lepirudin by the proximal kidney tubulus cells in humans as well as the antithrombotic efficacy of the metabolites and quantified the metabolite portions. Materials and methods : In vitro metabolites of lepirudin were produced by adding 250 μg lepirudin to urine of three healthy volunteers and a concentration of 100 ml fresh urine to 1.5 ml and subsequent separation by high performance liquid chromatography. Anticoagulant activities of the mass spectrometrically identified metabolites were measured by ecarin clotting time and protein determination with bicinchoninic acid. In 10 healthy volunteers 1 mg lepirudin was administered intravenously, urine was collected during the following 2 h. The urine amount containing 50 μg lepirudin measured by ecarin clotting time was enzyme-inactivated and measured analogously to the in vitro samples. Results : The in vitro generated metabolites were shortened amino acid by amino acid at the C-terminal end, up to five amino acids. Their anticoagulant activity was reduced to 92.6% (M 64 ), 80.1% (M 63 ) and 74.4% (M 60,61,62 ) in comparison to lepirudin. Lepirudin (57.9±8.6%) was eliminated unchanged via the kidneys. Identical to the in vitro situation metabolite fragments were built in the distribution M 64 =8.1±5.7%, M 63 =21.1±6.5%, and M 60,61,62 =12.9±4.5%. Conclusions : Lepirudin is metabolized spontaneously in more than 10-fold concentrated urine. Metabolization of lepirudin takes place in the proximal tubulus cells as well. In vitro, the degradation takes place amino acid by amino acid, but in vivo even dipeptides and perhaps tripeptides are degraded.

This study focused on proteinase-activated receptor-2 (PAR-2) in primary cultured human meningioma cells. Stimulation of these cells with the serine proteinase trypsin resulted in a dose-dependent transient calcium response. Since the specific PAR-2 agonist peptide SLIGRL also induced [Ca2+]i mobilization in human meningioma cells and successive application of SLIGRL and trypsin elicited no new calcium signal we conclude that trypsin-induced calcium signaling is mediated by PAR-2 in human meningioma cells. To our knowledge, this is the first report describing functional PAR-2-type receptors in human brain tumor cells.

Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C 6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C 6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma C 6 cells with CCK-8S resulted in activation of protein kinase C isozymes βI, βII, γ and ζ. The participation of protein kinase C in the CCK-8S-induced effect on C 6 cell growth was demonstrated by measurement of [ 3 H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C 6 cells by a protein kinase C-dependent mechanism.

Cholecystokinin (CCK) is known to stimulate pancreatic cancer cell growth, but no detailed CCK receptor subtype characterization and investigation of CCK receptor-mediated cellular responses in human pancreatic cancer cells have been reported thus far. In this study, CCK binding sites were identified in human pancreatic cancer cells (MIA-PaCa-2) using radioligand binding studies. Pharmacological characterization demonstrated a single class of high-affinity CCK sites on MIA-PaCa-2 cells (326 +/- 18 pM, receptor density 16.9 +/- 2.3 fmol/mg protein). These CCK binding sites displayed a typical CCKB binding profile as shown in competition studies by using different CCK-related compounds and non-peptide CCK antagonists discriminating between CCKA and CCKB sites. CCKB receptor-connected effector systems have been characterized in MIA-PaCA-2 cells, and their involvement in CCK-8S-induced proliferative effects on MIA-PaCa-2 cells has been demonstrated.

The antithrombin effect of heparin, hirudin and 4-amidinophenylpyruvic acid (APPA) was studied in rat plasma in vitro and in vivo. Equieffective doses of these inhibitors were used to prevent thrombin-induced disseminated intravascular coagulation (DIC) in rats. Changes in blood indicative of consumption coagulopathy and the decrease in oxygen consumption may be prevented by previous administration of the inhibitors. The several inhibitors exert different effects on microthrombosis in various organs. Hirudin possesses a stronger effect than heparin. The synthetic inhibitor APPA inhibits microthrombosis in all organs investigated (lungs, kidneys, suprarenals, spleen, liver) as long as a sufficient inhibitor level is present.

The role of platelets in thrombin-induced disseminated intravascular coagulation (DIC) was studied in rats. Thrombin was infused in an amount causing a pronounced decrease in fibrinogen concentration, increase in plasma hemoglobin concentration, reduction of total oxygen consumption of the animals, and microthrombus formation. During thrombin infusion, platelet counts decreased and the platelets remaining in the circulating blood showed a reduced aggregability and a disturbed serotonin uptake. Busulfan-induced thrombocytopenia or administration of acetylsalicylic acid (ASA) did not prevent microthrombosis but reduced the increase in plasma hemoglobin. The oxygen consumption did neither drop in thrombocytopenic nor in ASA-treated animals. Thus, platelets do not seem to be involved in thrombin-induced microthrombosis, but they are considered to be mediators of secondary reactions in the course of DIC.