Search

Background Factor XIII (FXIII) is the final enzyme of the coagulation cascade. While the other enzymatic coagulation factors are proteases, FXIII belongs to the transglutaminase family. FXIIIa covalently crosslinks the fibrin clot and represents a promising target for drug development to facilitate fibrinolysis. However, no FXIII‐inhibiting compound has entered clinical trials. Here, we introduce the features of a peptidomimetic inhibitor of FXIIIa (ZED3197) as a potential drug candidate. Methods The potency of ZED3197 against FXIIIa and the selectivity against other human transglutaminases were characterized using transamidation and isopeptidase assays. The inhibition of fibrin crosslinking was evaluated by biochemical methods and thromboelastometry. Further, the pharmacology of the compound was explored in a rabbit model of venous stasis and reperfusion. Results ZED3197 proved to be a potent and selective inhibitor of human FXIIIa. Further, the compound showed broad inhibitory activity against cellular FXIIIA from various animal species. Rotational thromboelastometry in whole human blood indicated that the inhibitor, in a dose‐dependent manner, prolonged clot formation, reduced clot firmness, and facilitated clot lysis without affecting the clotting time, indicating minimal impact on hemostasis. In vivo, the novel FXIIIa inhibitor effectively decreased the weight of clots and facilitated flow restoration without prolongation of the bleeding time. Conclusions ZED3197 is the first drug‐like potent compound targeting FXIIIa, a yet untapped target in anticoagulation. Due to the function of FXIII downstream of thrombin the approach provides minimal impact on hemostasis. In vivo data imply that the inhibitor dissociates an antithrombotic effect from increased bleeding tendency.

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.

A model of functional nephrectomy and hepatectomy in rats was used to compare the pharmacokinetic profile of the direct competitive thrombin inhibitor argatroban in rats with renal, hepatic, or hepatorenal failure and rats with normal hepatic and renal function. The pharmacokinetics of argatroban in rats after an intravenous bolus was described by an open two-compartment model with first-order elimination. This profile was not affected in nephrectomized rats, but hepatectomized rats showed a markedly increase in area under the curve values, distribution, and elimination half-life in comparison with untreated rats as expected for a drug eliminated primarily by the liver. Unexpectedly, blood levels of argatroban were lower in rats with hepatorenal failure than in rats with only hepatic failure. Similar results were observed after subcutaneous administration of argatroban. We propose that argatroban might be converted from 21-(R) to 21-(S) diastereoisomer in the kidneys. The 21-(S) diastereoisomer has a higher antithrombotic activity than the R isoform and this 21-(S) isomer might be preferentially hepatically eliminated. This could explain the higher anticoagulant activity in blood of hepatectomized rats with normal renal function in comparison with rats with hepatorenal failure.

The ecarin chromogenic assay (HaemoSys ECA; JenAffin GmbH, Jena, Germany) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle of measurement: the activation of prothrombin by ecarin, a snake venom from Echis carinatus. In the ECA, the prothrombin activation products meizothrombin and meizothrombin-des-F1 cleave a chromogenic substrate. The activity of meizothrombin/ meizothrombin-des-F1 is inhibited in a concentration-dependent manner by direct thrombin inhibitors. The ECA-T is an assay for quantitative determination of active site directed synthetic thrombin inhibitors. For argatroban ECA-T, there is a very precise and sensitive method of quantitative drug monitoring. Only very low interindividual variations were found compared with activated partial thromboplastin time and even ECT. ECA-T is independent of variations in prothrombin levels of the plasma samples. Heparin does not influence the measuring result. ECA-T is performed on manual coagulation analyzers with an option for optical measurement but can also be applied to automated laboratory systems. By the use of ECA-T as a drug-monitoring method, the drug safety of argatroban, most of all in critically ill patients, can be increased.

Hirudin – the long and stony way from an anticoagulant peptide in the saliva of medicinal leech to a recombinant drug and beyond - A historical piece

The story of success of aspirin has lasted for more than 100 years. Initially used as an analgetic with anti-inflammatory, antirheumatic as well as antipyretic effects, aspirin has been widely used

ZusammenfassungDas zentrale Gerinnungsenzym, die Serinproteinase Thrombin, kann über die Protease-aktivierten Rezeptoren 1 und 4 der Tumorzellen in deren Wachstum modulierend eingreifen. Das Thrombin ist im Tumormikroenvironment permanent verfügbar, Meizothrombin wird an einem tumorspezifischen Aktivierungskomplex aus Prothrombin generiert und kann ebenfalls das Tumorzellwachstum via PAR-1 und dem 7-Transdomänen-Protein-Rezeptor-Signalweg beeinflussen. PEG-gekoppelte direkte Thrombininhibitoren, die eine spezielle pharmakokinetische Charakteristik aufweisen und für eine langzeitige Wirksamkeit im extrazellulären Wasserraum designt wurden, kontrollieren die Serinproteinaseaktivität im Tumormikroenvironment und haben damit eine hohe potenzielle tumortherapeutische Wirksamkeit. In xenografischen Tumormodellen hat diese neue Substanzklasse einen signifikanten kanzerostatischen Effekt gezeigt.

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.

ZusammenfassungDer Platelet ADhesion Assay (PADA) ist eine POCT-fähige Methode zur quantitativen Bestimmung der Plättchenadhäsivität. Durch spezielle Polymerpartikel und Testbedingungen, die den physiologischen Verhältnissen im Organismus angepasst sind, wird aus einer Vollblutprobe direkt der aktuelle Funktionszustand der Blutplättchen bestimmt.Innerhalb kurzer Zeit, mit geringem Geräteaufwand und minimalem Probenvolumen ist auch ein therapeutisches Monitoring von Glykoprotein(GP)-IIb/IIIa- und ADP-Rezeptor-Antagonisten möglich. Während bei Gesunden die Dosis/Wirkungskurven der GP-IIb/IIIa-Antagonisten nur gering variieren, schwanken sie bei thrombophilen Patienten stark. Wirkungsunterschiede bis zur Arzneimittelresistenz treten auch beim ADP-Rezeptor-Antagonisten Clopidogrel auf. Ein therapeutisches Monitoring der GP-IIb/IIIa- und ADP-Rezeptorantagonisten ist essenziell und mittels PADA realisierbar – auch als Langzeit-Monitoring für ADP-Rezeptorantagonisten sowie zur Detektion einer Arzneimittelresistenz. Mit dem PADA ist eine individuelle Ex-vivo-Dosisrelation für GP-IIb/IIIa-Antagonisten möglich. Der PADA erlaubt die Diagnostik einer pathologisch veränderten Plättchenfunktion thrombophiler Patienten und durch die einfache Handhabung auch die Therapieüberwachung über längere Zeit.

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.

The ecarin clotting time (ECT) is a meizothrombin generation test that allows for precise quantification of direct thrombin inhibitors. The ECT has demonstrated its usefulness for more than 10 years in biochemical-pharmacological investigations as well as in clinical research and in the clinical routine. It has proved valuable especially as a drug-monitoring method in r-hirudin therapy. This test has been adjusted to clinical requirements by numerous modifications. Following the description of the biochemical background and the measuring principle of the ECT, this article gives a short survey of several modifications of the ECT for both preclinical and clinical use, e.g., for biochemical investigations, as a point-of-care method and for cardiac surgery. Advantages and disadvantages of these methods are discussed.

Hirudin is the anticoagulative product of the salivary glands of the medical leech Hirudo medicinalis. It is characterized by a direct, bifunctional inhibition mechanism and a high, exclusive specificity and a strong ability to bind to thrombin (tight binding). Further characteristics are the organic-chemical structure of hirudin (peptide), which allows only parenteral administration; the missing metabolism in the organism; and the exclusive glomerular filtration of hirudin in kidneys as the effective elimination mechanism. Recombinant hirudin (r-hirudin) is a product of genetic engineering that is identical to the hirudin found in nature and has the same biochemical and pharmacological characteristics as natural hirudin.

1. This study investigates, whether in addition to the protease-activated receptor-1 (PAR-1), PAR-4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR-1 and PAR-4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. 2. mRNAs for both, PAR-1 and PAR-4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT - PCR). 3. Treatment of the SMC with GYPGQV (200 microM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. 4. Stimulation of the SMC with 10 nM thrombin or 200 microM SFLLRN caused a time-dependent activation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 microM of GYPGQV induced a prolonged phosphorylation of ERK-1/2 with a maximum at 60 min. These data suggest that PAR-1 and PAR-4 are activated by thrombin at distinct concentrations and with distinct kinetics. 5. GYPGQV stimulated [(3)H]-thymidine incorporation in SMC. At 500 microM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 microM SFLLRN, respectively. 6. These data indicate that a functionally active PAR-4 is present in SMC and, in addition to PAR-1, might contribute to thrombin-induced mitogenesis.

In addition to heparin, the standard medication for prophylaxis and therapy of thromboembolism, several other substances have been developed and tested for clinical use with the aim of decreasing or eliminating side effects. Most of all, hirudin, a direct antithrombin (AT), has proved to be effective. To define the therapeutic range and to avoid underdosage or overdosage, clinical monitoring is necessary. For monitoring of hirudin, thrombin time (TT) is not suited because of the missing linearity of the standard curve. Activated partial thromboplastin time (aPTT) can be used only in the lower hirudin level range, where the standard curve is quite linear. However, high and toxic hirudin levels cannot be determined using aPTT. Another drawback is a high variation in single measurements and in the normal value of patients. Methods using chromogenic substrates are suited for determination of hirudin in plasma but cannot be used at bedside. Especially for monitoring of hirudin, the ecarin clotting time (ECT) was developed. The standard curve is linear over the entire concentration range. There are no influences by other coagulation parameters or anticoagulants. For both acute clinical situations and long-term monitoring, this method capable of point-of-care therapy (POCT) will be the method of choice.

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.

1. This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinase-activated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar effect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. 3. The specific PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5. Trypsin (2 nM) as well as SLIGRL (100 microm) activated the nuclear factor KB (NFkappaB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkappaB by thrombin (10 nM) was detected after 4-5 h. 6. These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkappaB via either PAR-1 or PAR-2 does not predict mitogenesis.

Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2-terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.

Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca2+]i mobilization, Ins(1,4,5)P3 level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [3H]thymidine incorporation in MIA PaCa-2 cells. Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca2+]i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 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.

In this article, the "tethered ligand" thrombin receptor was identified on human pancreatic tumor cells, MIA PaCa-2, using immunofluorescence studies with a monoclonal anti-thrombin receptor antibody. Pharmacological characterization, using 3H-labeled thrombin receptor activating peptide-6 (TRAP-6) as radioligand, demonstrated a single class of high-affinity binding sites (KD = 9.1+/-1.8 x 10(-7) M) and a binding capacity of 13.9+/-0.7 fmol/mg protein. These binding sites represent functional thrombin receptors, as shown by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, protein kinase C translocation from cytosol to the cell membrane, and stimulation of DNA synthesis in MIA PaCa-2 cells. These results provide the first identification of tethered ligand thrombin receptor in human pancreatic cancer cells and suggest thrombin receptor involvement in mechanisms of human pancreatic tumor progression.

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg(51)-Thr(52)/Arg(54)-Asp(55) in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s(-1)), Arg(155)-Ser(156) in prothrombin (k = 2 x 10(4) M-1 s(-1)), and Arg(284)-Thr(285) in prethrombin 1 (k = 0.02 x 10(4) M-1 s(-1)). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg(51)-Thr(52)/Arg(54)-Asp(55)) was not detectable, whereas cleavage at Arg(155)-Ser(156) (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg(284)-Thr(285) (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t(1/2) = similar to 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 mu M meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s(-1) in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg(284)-Thr(285) in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des 1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s(-1)) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s(-1)). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s(-1)), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s(-1)) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s(-1), we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

Rat glioma C6 cells have been demonstrated to be a suitable model in the investigation of PAR-1-type thrombin receptors in brain. However, anti-PAR-1 antibodies, which should be very helpful tools in studying PAR-1 in rat cells, have not been available up until now. Therefore, we prepared a monoclonal anti-thrombin receptor antibody (Mab COR7-6H9) directed against the peptide sequence GRAVYLNKSRFPPMPPPPFISEDASG in the N-terminus below the thrombin cleavage site of the rat PAR-1-type thrombin receptor. Using this antibody, we demonstrated the presence of PAR-1 binding sites on the plasma membrane of rat glioma C6 cells both with confocal laser fluorescence and with scanning electron microscopy. In addition, Mab COR7-6H9 was shown to block PAR-1-mediated transmembranal signaling as demonstrated by measurement of free intracellular calcium and cyclic AMP. This novel anti-PAR-1 antibody is therefore likely to be a very helpful tool in studying PAR-1-type thrombin receptors in rat brain.

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.

BACKGROUND Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined. METHODS In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of α-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated. RESULTS Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 ± 2.2 × 10-7 M) and a binding capacity of 27 ± 3.4 fmol/mg protein. The function of these binding sites was demonstrated by α-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, α-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide. CONCLUSIONS To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism. Cancer 1997; 80:2068-74. © 1997 American Cancer Society.

The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca2+ and at low ionic strength (K-m = 0.085 mu M and k(cat) = 0.68 s(-1) at I congruent to 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the K-m (K-m = 0.8 mu M and k(cat) = 1.03 s(-1) at I congruent to 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired. (C) 1998 Elsevier Science Ltd.

Effects of thrombin on brain cells, including change of neurite outgrowth and astrocyte shape, are described, but the molecular mechanisms are unclear. We investigated the effects of human alpha-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca2+]i, phosphoinositide hydrolysis, and protein kinase C in rat glioma C6 cells. Stimulation of C6 cells with both alpha-thrombin and TRAP-6 resulted in [Ca2+]i mobilization, [3H]Inositol phosphate response, and enhanced immunoreactivity of the protein kinase C (PKC) isoenzymes alpha, beta, gamma, delta, and epsilon. Results suggest that alpha-thrombin and TRAP-6 activate at least partially the same intracellular signaling pathways in rat glioma C6 cells, which is evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma C6 cells.

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl-P3-Pro-4-amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L-configuration. Among this series, several derivatives inhibit thrombin with Ki values of less than 1 nM. Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin-like serine proteases. X-ray analysis of the most potent analogue in complex with thrombin demonstrated that the N-terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.

Increased platelet reactivity may contribute to the development of coronary artery disease and myocardial infarction. The aim of the present study was to assess platelet adhesiveness in different stages of coronary artery disease using the platelet adhesion assay (PADA). In addition, the acute effect of coronary angiography and stent implantation on platelet adhesiveness was examined.85 patients with stable coronary artery disease (SAP - stable angina pectoris) and 19 patients with acute myocardial infarction (AMI) were enrolled in the study. 35 volunteers served as controls. Blood sampling in SAP and AMI patients was done before coronary angiography to measure the adhesion index (AI) using the PADA. In 33 patients additional blood was drawn at the end of coronary angiography. In 6 patients blood samples were drawn also after stent implantation.In blood samples from patients with an AMI the AI was significantly higher than in blood samples from the SAP group (p0.01) and the control group (p0.01). Coronary angiography and stent implantation did not result in significant acute changes in mean AI however results show an interindividual variability in initial values as well as in AI changes during procedures.This is the first study to show that PADA, a quick and simple laboratory method for the quantitative determination of platelet adhesiveness, is able to detect platelet hyper-adhesiveness in patients with AMI.

SummaryAutoimmune thrombocytopenia (ITP) is an acquired autoimmune disease characterised by isolated persistent thrombocytopenia and normal megakaryopoiesis. This definition also applies to heparin-induced thrombocytopenia (HIT II), a frequent side effect of heparin treatment. In HIT II, the immunogen is a coagulation active complex of heparin and platelet factor 4 (PF4). By now, diagnostics of HIT II is often material and time consuming. Three groups of patients were investigated for HIT II antibodies (HIT II-AB): 54 hospitalised stroke patients, 87 hospitalised cardiac patients, and 71 patients on chronic haemodialysis, all treated with heparin. Furthermore, 100 healthy volunteers were investigated. For detection of HIT II-AB the innovative whole blood test PADA-HIT (PADA: platelet adhesion assay) was used. PADA-HIT quantifies the interaction of IgG antibodies with FcγIIA receptors by comparing the activation state of platelets in citrated and heparinised whole blood. The occurrence of HIT II-AB in blood was very high with 44 % of stroke patients, 69% of cardiac patients and 38% of haemodialysis patients compared to only 15% of healthy volunteers. This demonstrates a high incidence and a rapid onset of HIT II-AB in patients being acutely treated with heparin. HIT II is one of the most frequent and severe autoimmune diseases bearing a great thrombosis risk. PADA-HIT represents an innovative diagnostic method for detection of autoimmune antibodies of IgG type that are directed against platelet factor 4 (PF4)-heparin-complex. By early and fast diagnostics and appropriate treatment severe complications of HIT II can be prevented.

After genetically engineered recombinant DNA desulfatohirudin (r-hirudin) had been investigated as to its pharmacokinetic behavior and blood level course in nephrectomized dogs, the compound was studied for its capability to prevent thrombus formation in the extracorporeal circulation. Beagle dogs underwent bilateral functional nephrectomy followed by experimental hemodialysis. r-Hirudin content in blood, fibrinogen level as well as platelet count were determined before and during the dialysis. Furthermore, the blood loss during the experiment was measured as well as the mean arterial pressure and the pressure in the blood line system. Intravenous administration of the thrombin inhibitor resulted in initial distribution in the extracellular space (distribution phase 90 min) followed by retarded decrease of the r-hirudin blood level (t½β ∼ 6–8 h) which is due to the missing renal excretion of the inhibitor. This caused a long-lasting, dose-dependent anticoagulant effect, which is not only characterized by the prevention of increasing pressure before the capillary dialyzer but also by the reduced drop in fibrinogen and platelets during hemodialysis. The required dose of r-hirudin (0.5 mg/kg) is within a range where bleeding complications will not occur yet.

Diagnosis of a hyper- or hypocoagulable state has been very difficult. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample.

At this time no practical laboratory method for the measurement of the current functional state of blood platelets is available. A new innovative platelet adhesion assay (PADA) is described here. With only a short time requirement and minimal equipment, the PADA provides quantitative measurements of platelet adhesiveness. Only 0.5 mL of freshly drawn citrated whole blood is needed, to which special polymer particles are added. A defined shear grade is induced by a short period of shaking the sample. Proteins of the blood sample, especially fibrinogen, and thereafter also activated platelets, bind to the specific polymer surface. Following platelet counts both in the sample and in a control (blood without particles), the adhesion index (AI) is calculated as a quantitative measure of platelet adhesiveness. In healthy volunteers, a mean AI of 52+/-12 was measured. AI was shown to be nearly independent of the number of platelets in the sample (100 to 350 k/ microL), the hematocrit (44 to 25%), and the fibrinogen content (1.5 to 5 g/L). Age of the volunteers had only a minor influence on the AI. PADA was shown to be a simple reliable laboratory method for the detection of disturbed platelet function. The test requires low analytical efforts compared with other diagnostic platelet function tests.

The platelet adhesion assay (PADA) is an innovative method for the detection of both normal, pathologically increased or decreased platelet adhesiveness. Adhesion is the first important phase of platelet activation, followed by shape change and aggregation. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to a lesser extent also by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, and since the PADA uses polymer particles that become coated with fibrinogen, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdosing, potentially leading to bleeding complications. Ex vivo, citrated whole blood from healthy volunteers and patients was spiked with increasing GPIIb/IIIa inhibitor concentrations and PADA was measured. Comparing these results with GPIIb/IIIa receptor occupancy, determined by FACS, a basic consistency of the data was shown. Via intracellular signaling, the adenosine diphosphate (ADP) receptor mechanism is closely involved in the activation of GPIIb/IIIa receptors so that also ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can be quantitatively determined by the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were realized. Furthermore, patients having partial or full clopidogrel resistance were identified. Overdoses can be detected as well.

Dipetalogastin is a potent thrombin inhibitor from Dipetalogaster maximus. The cDNA of dipetalogastin codes for a large protein which consists of six Kazal-type domains. There are three tandem, homologous regions each including two domains. Three biologically active recombinant proteins rDI, rDII and rDIII each corresponding to one region of the dipetalogastin cDNA were expressed, purified and investigated with regard to their biological activities. rDI and rDII with molecular masses of 12,660 and 12,911 Da, respectively, proved to be potent thrombin inhibitors. The investigation of their influences on amidolytic activities of different serine proteases showed no inhibition of factor Xa (FXa) and alpha-chymotrypsin. At a large molar excess of rDI and rDII over the enzymes only low effects on the activities of trypsin and plasmin were observed. rDIII differs much from the both others. An inhibition of thrombin was found only at a molar excess of rDIII over the enzyme. Furthermore, an inhibition of trypsin and low effects on plasmin were detected at a molar excess of inhibitor over these enzymes. These results indicate that rDIII is active against thrombin, trypsin and plasmin, and finally possesses no specificity for only one serine proteinase.

The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus , and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass ( M r =7560) and a thrombin inhibitory activity ( K i =446 f M ) comparable to r-hirudin.

Thrombopoietin (TPO) plays a crucial role in megakaryocyte development. TPO signalling, which is mediated by its receptor Mpl, includes Janus kinase, (JAK) signal transducer and activator of transcription (STAT) and Shc/Ras/mitogen-activated protein kinase (MAPK) pathways. The precise nature of these signalling routes has not been clarified in detail up until now. We investigated the effect of TPO on activation of cAMP-dependent protein kinase (PKA) and its involvement in MAPK signalling in human megakaryoblastic leukaemia CMK cells. For estimation of PKA activity, phosphorylation of a PKA-specific peptide substrate was assayed in CMK cell lysates. Since activation of PKA is associated with translocation of its catalytic subunit alpha (C-PKA) into the cell nucleus, Western blot analysis of nuclear fractions with an anti-C-PKA antibody was additionally performed. The activation of TPO-induced MAPK activation and the effect of the PKA inhibitor H-89 was measured using immunoblotting with a monoclonal anti-pERK antibody. TPO enhanced cAMP and induced activation of PKA in CMK cells. In addition, H-89 partly blocked TPO-induced MAPK activation in CMK cells. Our results indicate a novel TPO-triggered signalling event, activation of the cAMP/PKA pathway in human megakaryoblastic CMK cells. This signal transduction route seems to be involved in TPO-induced MAPK signaling.

Cellular effects of thrombin are mediated by members of a new subfamily of G protein-coupled receptors designated proteinase-activated receptors (PARs) with the prototype PAR-1. Investigation of PAR-1-induced signaling has been shown to be very important in clarifying thrombin's role in cell metabolism, differentiation, and growth. We evaluated connection of PAR-1 with the cAMP/PKA pathway in SNB-19 glioblastoma cells. Alpha-thrombin and the synthetic PAR-1 agonist SFLLRN stimulated PKA as shown by increased PKA activity and translocation of the catalytic PKA alpha subunits (PKA(cat)alpha) into the nucleus. However, no effect on cAMP could be observed. PKA(cat)alpha was found to be associated with nuclear factor-kappa B (NF-kappaB) p65 and its inhibitor protein IkappaB in SNB-19 cells. After PAR-1 stimulation, this association was markedly diminished. We conclude that PAR-1 mediates PKA activation without altering cAMP levels but includes NF-kappaB-associated PKA(cat)alpha in SNB-19 glioblastoma cells. This is the first evidence for a cAMP-independent PKA signaling by a G protein-coupled receptor.

Thrombin induces well-characterized effects on normal and neoplastic brain cells by interaction with protease-activated receptor (PAR)-type thrombin receptors. However, nothing is known about the function of intermediate enzymes of prothrombin activation recently shown to evoke PAR-1-mediated signaling in smooth muscle cells. Therefore, we investigated the effect of recombinant human meizothrombin (rMT), one of thrombin's catalytically active precursor enzymes in the prothrombin cleavage cascade, on calcium mobilization in human SNB-19 glioblastoma cells. By using reverse-transcription polymerase chain reaction, immunofluorescence studies with a monoclonal anti-PAR-1 antibody and calcium measurements, SNB-19 cells were shown to express functional PAR-1-type thrombin receptors. PAR-1 is not only a receptor for thrombin in SNB-19 cells but was also activated by rMT very effectively. Under the conditions used in our experiments, SNB-19 cells stimulated with thrombin after rMT challenge were unable to elicit a new calcium response and vice versa. In addition, both rMT and thrombin induced no further calcium signal after that observed with the PAR-1-activating peptide SFLLRN. Therefore, rMT and thrombin seem to activate calcium signaling by similar mechanisms including PAR-1. Our results demonstrate rMT as a potent activator of PAR-1-type thrombin receptors in SNB-19 glioblastoma cells, suggesting a function of catalytically active thrombin precursor enzymes in cells of glial origin.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.

A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood-sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a protein of 344 amino-acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains. Each domain shows a double-headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood-sucking insect Rhodnius prolixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and II to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal-type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood-sucking insect which belongs to the Kazal-type family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of 12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The Ki value of the recombinant dipetalogastin was determined to be 49.3 +/- 22.28 fM.

SummaryWhile effects of alpha-thrombin have been well characterized in different cell types, the biological function of intermediates of prothrombin activation is still undefined. Meizothrombin could be shown to be a potent agonist for vascular contraction which seems to be mediated by an interaction with the vascular smooth muscle. To explore this effect at intracellular signaling level, we used rat aortic smooth muscle cells and investigated the effect of the intermediates formed by cleavage of human prothrombin with ecarin, the prothrombin activator from Echis carinatus venom, on mobilization of free intracellular calcium. The ecarin-activated prothrombin induced very rapidly transient calcium mobilization in SMC´s comparable to that observed with alpha-thrombin. We conclude that meizothrombin/ meizothrombin desF1 (MT/MT desF1) are the most likely candidates for this effect. Furthermore, our results suggest the involvement of PAR-1-type thrombin receptors in MT/MT desF1-induced calcium signaling in rat aortic smooth muscle cells.

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.

Meizothrombin, the stable intermediate product of ecarin-induced prothrombin conversion, was investigated for its ability to bind hirudin in blood. After in vitro pre-incubation of rat plasma with ecarin, the prolongation of the thrombin time caused by hirudin was reduced. The extent of hirudin neutralization was found to be dependent on the duration of incubation with ecarin. In vivo, after bilateral nephrectomy in Wistar rats and following administration of hirudin at a dose of 1 or 5 mg/kg, the blood level of hirudin remained constant after 2 h. After infusion of ecarin following hirudin administration, the hirudin blood level dropped sharply, reaching significantly reduced values, and bleeding stopped. Platelet count and fibrinogen level in plasma remained unchanged in the experiments using ecarin-induced prothrombin conversion intermediate generation. It is concluded that meizothrombin, a naturally occurring prothrombin conversion intermediate, provides an effective agent to neutralize toxic blood levels of hirudin.

The pharmacokinetic properties of r-hirudin were studied in nine patients suffering from different degrees of renal insufficiency. To this end, r-hirudin was administered intravenously at dosages of 0.1 mg/kg. The elimination half-life t1/2 beta was determined in blood plasma and the cumulative r-hirudin excretion in urine was measured over 48 h. In healthy volunteers t1/2 beta was 0.9 +/- 0.2 h; the cumulative r-hirudin excretion in urine after 48 h amounted to 38 +/- 10% of the dose administered, most of this quantity was excreted during the first hours. In seven patients with chronic renal failure, t1/2 beta was 15 to 41 h; in three of these patients cumulative urinary r-hirudin excretion was increased to 70-80%, in four patients cumulative r-hirudin excretion in urine within 48 h amounted to 39 +/- 8%, but was delayed in time. In 2 bilaterally nephrectomized patients, t1/2 beta was 168 and 316 h, resp. The renal clearance of hirudin was significantly and linearly correlated with the creatinine clearance (r = 0.872). In all patients aPTT and bleeding time were only moderately prolonged. Because of the modified pharmacokinetic behaviour the administration of hirudin in patients with impaired renal function requires individually adjusted dosages or prolonged administration intervals.

Anti-IIa-Inhibitoren (Thrombininhibitoren) sind im Gegensatz zu Heparinen und oralen Antikoagulanzien in der Lage, die Proteaseaktivitat des Thrombins direkt zu hemmen und damit prazise in das Gerinnungsgeschehen einzugreifen. Direkte Thrombininhibitoren sind entweder biosimilar (r-Hirudine) oder synthetisch hergestellt (Bivalirudin, Argatroban, Dabigatran). Bereits seit 1997 ist r-Hirudin zugelassen, hat aber aufgrund seines engen therapeutischen Fensters und der Notwendigkeit eines Monitorings noch nicht die breite klinische Anwendung erfahren. Seit 2004 bzw. 2005 sind die synthetischen Thrombininhibitoren Bivalirudin und Argatroban verfugbar, mit Dabigatran folgte 2008 der erste orale synthetische Thrombininhibitor. Alle vier Substanzen haben eine geringe Plasmaproteinbindung und sind in der Lage, auch clotgebundenes Thrombin zu hemmen. Sie unterscheiden sich hinsichtlich des Applikationsweges, der Anwendungsdauer und der Elimination und bieten damit eine auf den Anwendungsfall abgestimmte prazise Gerinnungshemmung ohne die Gefahr einer HITII. Diese Vorteile sollten in Form der Entwicklung weiterer direkter Thrombininhibitoren genutzt werden.

The usability of recombinant hirudin as anticoagulant agent in haemodialysis was studied in nephrectomized dogs. To this end, we examined the capability of recombinant hirudin to penetrate the membranes of different capillary dialyzers used. Furthermore we investigated the pharmacokinetic behaviour of recombinant hirudin in nephrectomized dogs as well as its capability to prevent the activation of the clotting system and fibrin deposition during haemodialysis. The present results evidence the efficiency of recombinant hirudin in preventing thrombus formation in experimental haemodialysis and hence its suitability as anticoagulant in such extracorporeal circulation.