Biomedical subjects
Publications and source records attributed to F Markwardt.
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The acute release of tissue-type plasminogen activator t-PA from the vascular endothelium is of decisive importance for the prevention of intravascular fibrin deposits. A dose-dependent t-PA release from the isolated perfused vascular preparations may be induced by mediators (platelet-activating factor, bradykinin, histamine) adrenergic and cholinergic transmitters (isoprenaline, acetylcholine), thrombin, heparin and analogues, and 1-desamino-8-D-arginine-vasopression (DDAVP). Most of the compounds were shown to enhance the t-PA activity also in animal experiments (rats, rabbits, mini pigs). The pharmacologic stimulation of the t-PA release may be convenient for short-term thrombosis, prophylaxis and partial thrombolysis. Presently, this could only be achieved by unfractionated and low molecular weight heparins which have been shown to release t-PA.
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In contrast to thrombin the fibrinogen coagulant effect of the thrombin-like enzyme batroxobin in vitro and in vivo is not inhibited by the specific thrombin inhibitor hirudin. The haemostyptic effect of batroxobin has been studied in rats after bleeding had been induced by corresponding hirudin dosages. Dependent on batroxobin concentration bleeding time was shortened by local application of batroxobin containing solutions. Strong bleeding induced by i.v. injection of 5 mg r-hirudin/kg was stopped almost immediately when a batroxobin concentration of 40 BU/ml was used. Thrombin was less active to stop bleeding after r-hirudin administration than batroxobin.
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.
Using biochemically defined conditions on a fast kinetic centrifugal analyzer the effect of recombinant hirudin (rH) and unfractionated heparin (UH) on thrombin and factor Xa generation was investigated. Diluted fibrinogen deficient human plasma was incubated with increasing concentrations of the anticoagulants and protease generation was initiated either by extrinsic (EA; thromboplastin/calcium chloride) or intrinsic (IA; ellagic acid/cephaloplastin/calcium chloride) activation of the coagulation process. Generation of thrombin or factor Xa was measured continuously by amidolytic assays using the specific chromogenic substrates Spectrozyme TH and Spectrozyme FXa. By means of calibration curves for thrombin and factor Xa the IC50 values for the inhibition of the proteases were calculated. It was found that rH and UH were nearly equally effective in inhibiting both the thrombin and factor Xa formation after IA, whereas in EA system rH produced a stronger inhibition on thrombin generation than UH, which in general showed a more pronounced effect after intrinsic than after extrinsic activation. The results suggest that, with regards to thrombin and factor Xa generation, rH does not exhibit a much higher activity than UH. This may be an expression that thrombin-mediated positive feedback-reactions are not influenced by rH as strongly as expected when using a highly specific and selective thrombin inhibitor. Furthermore, it can be concluded that protease generation assays may be useful in the characterization of anticoagulants/antithrombotics.
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Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.
The generation of thrombin in citrated, r-hirudinized and heparinized whole blood (final concentrations 10, 25, 50 micrograms/ml) was studied in the presence or absence of various activators. Whole blood from healthy volunteers was activated either by glass or dextran sulfate (contact activation; CA) as well as by thromboplastin/calcium chloride (extrinsic activation; EA) and by ellagic acid/cephaloplastin/calcium chloride (intrinsic activation; IA) and incubated 10 (CA, EA, IA) and 30 (CA) min at 37 degrees C. After the incubation period plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT) were measured utilizing ELISA methodology. After EA or IA no blood clotting occurred in all r-hirudinized samples and in heparinized blood at the highest concentration used. Despite inhibition of clotting F1 + 2 levels were increased both in r-hirudinized and in heparinized blood. However, in blood anticoagulated with heparin F1 + 2 levels after EA, IA and especially after CA were markedly lower than in r-hirudinized blood. Furthermore, in r-hirudinized blood an increase of TAT levels was found. The enhancement of F1 + 2 and TAT levels by r-hirudin was concentration-dependent and was lower in the higher r-hirudin concentration. The results indicate that in r-hirudinized whole blood significant amounts of thrombin can be generated which may elicit thrombin-mediated feedback mechanisms.
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The naturally occurring anticoagulant from medicinal leeches, hirudin, which we isolated and biochemically analyzed 30 years ago as a miniprotein with specific antithrombin activity, has afterwards been employed for scientific and diagnostic purposes in hematology. Pure hirudin proved to be an antithrombotic agent of high quality that displays an antithrombotic action dependent upon its blood level. After intravenous injection, it is distributed in the extracellular space and is almost completely eliminated through the kidneys by glomerular filtration in a biologically active form. The efficacy of hirudin in preventing venous and arterial thrombosis and disseminated intravascular coagulation was demonstrated in various animal models. Clinical pharmacological studies corroborated the specific pharmacodynamic and pharmacokinetic properties of hirudin found in animal experiments. Genetic engineering led to the availability of sufficient quantities of recombinant hirudin (r-hirudin) for clinical purposes. Pharmacologic profiling of r-hirudin showed that both its pharmacokinetic and pharmacodynamic characteristics are very similar to those of native hirudin. Clinical pharmacological studies with r-hirudin revealed that, at single therapeutically relevant doses, r-hirudin is a well-tolerated and potent anticoagulant without any detectable side effects and allergic reactions. Further preclinical studies of r-hirudin should concentrate on identifying possible indications for use, on the development of r-hirudin preparations and derivatives, and on the development of antidotes for hirudin.
Pharmacological profiling of recombinant hirudin (r-hirudin) has shown that this selective tight-binding thrombin inhibitor is a potent, well-tolerated anticoagulant. Clinical pharmacological studies were performed in human volunteers after single and repeated doses of 0.1-0.5 mg/kg. Generally, administration of r-hirudin was tolerated without side effects. Thrombin time and partial thromboplastin time were prolonged dependent on the r-hirudin level in plasma. Platelet counts, fibrinogen level and fibrinolytic system remained unchanged. Bleeding time was not prolonged. On intravenous injection, r-hirudin was rapidly distributed into the extracellular space and eliminated, with a dose-dependent half-life of 1-2 h (first-order kinetics). After subcutaneous administration, the rH level in blood reached plateau values within 60-120 min. The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance.
Various reactions of disseminated intravascular coagulation (DIC) were experimentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concentrations of recombinant hirudin (r-hirudin; 20-200 ng/ml) were sufficient for the inhibition of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhibitor.