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To test the efficacy of a single intraocular injection of various doses of recombinant desulphatohirudin variant 1 (Revasc) in a rabbit model after lensectomy-vitrectomy surgery.Standard fragmatome lensectomies and core vitrectomies were performed on Dutch Belted rabbits. In a masked fashion, 17 control eyes received single intraocular injections of lactated Ringer's solution, and the treated eyes, with eight eyes per treatment group, received single intraocular injections of hirudin at doses of 0.02 microgram, 2 micrograms, 10 micrograms, 50 micrograms, or 200 micrograms.Intraocular injections of hirudin were effective in preventing postoperative fibrin formation at doses of 2 micrograms. There was no notable intraoperative bleeding. With an injection of 2.0 micrograms hirudin there was no hemorrhage at any time after surgery; with an injection of 200 micrograms hirudin, however, there was notable bleeding.Hirudin is effective in the prevention of postoperative fibrin formation with a single intraocular injection of 2.0 micrograms after lensectomy-vitrectomy surgery in the rabbit model. There was no bleeding at the effective dose of 2.0 micrograms; at 100 times the effective dose, however, there was significant postoperative bleeding.

Recombinant hirudin is currently being developed as a potential prophylactic and therapeutic antithrombotic drug in various clinical indications such as angina and deep venous thrombosis. In this report, we have discussed the production of specific polyclonal antibodies to recombinant hirudin (rH) and the development of two ELISA methods to measure rH concentrations in biological fluids: a sandwich and a competitive ELISA method. Intra- and inter-assay variations in the two methods are extremely low (3-7%). The competitive ELISA method is rapid, simple and highly reproducible. Saturation binding curves, selection of appropriate incubation times, recovery of different hirudin variants and reactivity in the presence of thrombin are discussed. The methods can be easily adapted to monitor hirudin concentrations in the clinical laboratory for diagnostic purposes as well as for performing pharmacokinetic studies.

Polyion complex (PIC) micelles based on methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-chitosan) and Arg-Gly-Asp conjugated poly(ethylene glycol)-grafted-chitosan (RGD-PEG-g-chitosan) were designed as carriers for platelet-targeted delivery of recombinant hirudin variant-2 (rHV2). The rHV2-loaded plain PIC micelles (mPIC micelles) and RGD conjugated PIC micelles (RGD-PIC micelles) were successfully prepared with mean size of 30.9 +/- 0.5 nm and 41.9 +/- 1.8 nm, and their encapsulation efficiencies were 76.90 +/- 0.84% and 81.08 +/- 0.85%, respectively. The pharmacokinetics experiments showed that the mean retention time (MRT) of rHV2 encapsulated in both kinds of micelles was significantly prolonged, especially for mPIC micelles. The confocal laser scanning microscopy intuitively proved the specific binding of RGD-PIC micelles to platelets. The efficacies of rHV2-loaded RGD-PIC micelles were greatly better than those of rHV2-loaded mPIC micelles and rHV2 solution in aspect of anticoagulation and inhibition of platelet aggregation. These results suggested platelet-targeting specificity of RGD-PIC micelles and that RGD-PIC micelles could potentially be used as a carrier for platelet-targeted delivery and long circulation of rHV2.

Leeching is an art dating back at least to ancient Egypt. It reached its zenith in the late 18th and early 19th centuries. The antithrombotic quality of leech saliva was first noted by Haycraft in 1884 and the active anticoagulant ingredient isolated in 1904 by Jacoby. He gave this agent the name ‘hirudin’. Hirudin was isolated in pure crystalline form by Markwardt in 1957 and first produced in quantity by genetic engineering in 1986. The salivary glands of several species of leech also contain other biologically active substances which are currently undergoing investigation.

The effect of recombinant hirudin (r-hirudin, LU 52369) and unfractionated heparin on recombinant tissue-type plasminogen activator (rt-PA; LU 50232) thrombolysis and reocclusion rates after successful thrombolysis was studied in a copper-coil-induced thrombus model in the iliac artery of anesthetized rabbits. Simultaneous administration of rt-PA and recombinant hirudin (r-hirudin) at a dose not affecting the partial thromboplastin time (PTT) increased the number of recanalized arteries significantly. The incidence of reocclusion was drastically reduced from 100% to less than 25%. At a dose increasing PTT twofold, unfractionated heparin had no effect on the incidence of reperfusion and reocclusion. These experimental data indicate that the combination of rt-PA with low-dose r-hirudin may be useful for the prevention of early reocclusion in thrombolytic therapy.

Recombinant hirudin (r-hirudin) inhibited the thrombin-induced aggregation and 14C-serotonin secretion of human platelets in the same concentration range as native hirudin. In r-hirudinized blood, lower spontaneous platelet aggregation was found than in citrated or heparinized blood. Except for a demonstrable aggregation-potentiating effect, adrenaline did not induce aggregation in r-hirudin- or in citrate-anticoagulated blood. The lowest platelet adhesion to glass surface was found in r-hirudinized plasma. The ADP-induced aggregation was nearly the same in the three differently anticoagulated plasma samples: however, desaggregation predominated in r-hirudinized plasma. Adrenaline caused only a slight aggregation in r-hirudinized plasma. ADP and adrenaline caused 14C-serotonin secretion in citrated plasma only. The collagen- as well as the PAF- and arachidonic-acid-induced aggregation did not differ significantly in the three plasma samples. Hence, r-hirudin is suitable for studying platelet functions at physiological calcium concentrations.

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.

Several laboratory methods are available to measure the anticoagulant activity of recombinant hirudin (r-hirudin), a potent thrombin inhibitor. These assays include clot-based, amidolytic, immunologic and physicochemical techniques. Although r-hirudin, like heparin, is an effective anticoagulant, the mechanism of action of the two agents is different. Thus it is not surprising that the global tests, such as the prothrombin time (PT), partial thromboplastin time (APTT) and the Heptest (Haemachem, Inc., St. Louis, Mo., USA), do not show adequate responses to r-hirudin. In the range of 0.5-10.0 microgram/ml, where full anticoagulation is achieved, as determined by animal models of thrombosis, these assays show little to no prolongation of the time to clot. In order to find a more suitable assay system, modifications of the above assays were evaluated. The diluted APTT and diluted Heptest showed linear concentration-dependent responses to lower levels of r-hirudin with an enhanced sensitivity than that of the classical assays. On the other hand, the diluted thrombin time was too sensitive. Whole-blood clotting assays, ACT and thrombelastograph, effectively measured r-hirudin levels up to 25 micrograms/ml. The amidolytic anti-factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective particularly when modified to decrease the sample:thrombin ratio. This assay may be useful in quality control since it is biochemically defined, and reagents are easily standardized. The relevance of the results of the anti-IIa assay to clinical conditions, however, remains to be determined. Thrombin generation assays have limited value in monitoring the anticoagulant effect of r-hirudin since the effect of thrombin inhibition by r-hirudin on coagulation feedback mechanisms, and thus the effect on thrombin generation, appears to be minimal. Immunologic methods such as ELISA and RIA are under development, but they may only be useful for the direct quantitation of absolute levels of r-hirudin and not for monitoring the clinical anticoagulant action. Furthermore, these assays are only sensitive to sub-microgram/ml levels. Therefore, thrombin-based clotting and amidolytic assays may at present be the best choice for evaluating the functional, clinical antithrombotic effects of r-hirudin.

The purification and identification of recombinant hirudin (r‐hirudin) (rHV2‐Lys47) and its several C‐terminal proteolytic degradation derivatives, produced by Pichia pastoris, were described. The high‐purity rHV2‐Lys47 of above 99% and its three degradation products were obtained by a straightforward two‐step chromatography procedure, a combination of cation exchange and reverse phase chromatography, with a recovery yield of 74% for hirudin. The purified rHV2 had the predicted N‐terminal amino acid sequence and the derivatives were the degradation products of hirudin, short of one to three amino acid residues at C‐terminal.

We report a case in which intravenous heparin, given for the treatment of acute deep vein thrombosis, precipitated an acute allergic reaction, and an alternative anticoagulant, recombinant hirudin, provided rapid and successful therapeutic intervention.

Heparin-induced thrombocytopenia (HIT) is a severe complication following the application of heparin; antibodies against complexes of heparin and PDF4 initiate activation of platelets. This may lead to massive thrombembolism, which is associated with a slight and transient drop of platelets in HIT I or a drop below 50% after approximately 5 days in HIT II. Further administration of heparin has to be strictly avoided in these patients. Immunologic evidence for HIT can easily be obtained by the heparin-induced platelet aggregation assay. If anticoagulation is necessary, different, alternative drugs are available. Recombinant hirudin (r-hirudin) is a well-established drug for safe anticoagulation. Monitoring is possible by estimating the plasma level of r-hirudin from the ecarin-clotting time. We report a case of a patient with prosthetic aortic valve endocarditis and HIT II who suffered from massive postoperative bleeding requiring massive substitution of blood components and coagulants caused by free circulating r-hirudin due to the use of a hemofilter.

We present a patient with coronary heart disease and a heparin- induced thrombocytopenia , who was successfully treated by coronary ar- tery bypass grafting (CABG) using recombinant hirudin as an anticoagu- lant for cardiopulmonary bypass (CPB) instead of heparin. (Eur J Car- dio-thorac Surg (1996) 10: 386-388)

Recombinant hirudin and a shortened synthetic analogue, with the amino acid sequence of D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, are specific thrombin inhibitors which in a concentration-dependent manner inhibit thrombus formation as well as clot propagation both in vitro and in vivo. In comparison to the analogue, lower molar concentrations of rhirudin affected doubling of aPTT and TT as well as inhibition of thrombin amidolytic activity or thrombin-induced platelet aggregation in vitro. In the rat wire coil-induced thrombosis model, a 50% thrombo-protective effect may be brought about with doses of 0.043 μmol/kg of rhirudin and 1.43 μmol/kg of the synthetic peptide. However, doubling of bleeding times is caused, on average, by dosages of between 0.143 and 0.43 μmol/kg rhirudin or approximately 0.143 μmol/kg of the analogue. Treatment groups included animals revealing significant prolongation of bleeding times as well as nonresponders. Despite the 10-fold longer impact on aPTT after application of rhirudin, the extent of mean bleeding time prolongation is identical to that of the analogue.

Leeches have been in medical use for many years. Hirudin, the anticoagulant obtained from the medicinal leech has been purified, characterized and can now be produced by recombinant (r) technology. R-hirudin is a potent inhibitor of thrombin and is therefore a potentially valuable anticoagulant and antithrombotic drug. This article reviews the current status of r-hirudin in this role and compares the pharmacokinetics, mechanism of action and clinical efficacy of this agent with heparin. The methods available for laboratory assessment and clinical monitoring of r-hirudin and the possible ways of antagonizing its effects are also discussed. Finally, the potential clinical applications of r-hirudin are outlined, although further laboratory and clinical studies, together with a fall in the cost of this compound are required before r-hirudin can be more widely accepted as an anticoagulant and antithrombotic agent.

The naturally occurring anticoagulant from medicinal leeches, hirudin, which we isolated and biochemically analyzed 30 years ago as a miniprotein with specific anti-thrombin 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.

The effect of two recombinant hirudins, HBW 023 (Hoechst AG, Frankfurt, FRG) and CGP 39393 [recy(tyr 63)] (Ciba Geigy, Basel, Switzerland) on various platelet functions has been investigated in blood samples of healthy volunteers anticoagulated with hirudin at concentrations of 10, 30, 50 μg/ml blood. Spontaneous aggregation was rarely observed in hirudinized platelet-rich plasma (PRP) compared to citrate-PRP. In comparison with citrate-PRP, both hirudins reduced the maximal response to epinephrine, but had no influence on the maximal response to ADP. Both hirudins reduced platelet adhesion to siliconized glass and inhibited platelet spreading compared to citrate-PRP. If hirudin was added to citrate-PRP, platelet count, epinephrine-induced aggregation, platelet spreading and adhesion remained unchanged while the maximal response to ADP increased. If citrate was added to hirudin-PRP, the maximal response to epinephrine as well as platelet spreading and adhesion increased to the extent observed in citrate-PRP. The effects of hirudin on platelet function are more pronounced than those of unfractionated heparin.

Recent advances in microvascular techniques have significantly enhanced the success rate of these procedures. Microvascular free tissue transfers at present have a 85–90% success rate. This, however, still leaves a 10–15% failure rate due primarily to thrombosis of the blood vessels at the anastomotic site. Previous work in the field has shown that anticoagulants such as heparin can reduce the rate of thrombosis. However, heparin can also cause on-site bleeding. Hirudin, a potent thrombin inhibitor of 65 amino acids, is available through recombinant technology. Our current experimental work utilized 75 male Sprague-Dawley rats for assessing the efficacy of hirudinized saline versus saline alone and heparinized saline as an irrigant in a microsurgery model of anastomosis. After exposing the right femoral artery in a standard fashion, the artery was clamped and a standard transverse division of the vessel was made. The vessel ends were irrigated with one of the three solutions then anastomosed. Vessels were observed for patency over a 3-day period. Initial results showed comparable performance of hirudinized saline and heparized saline, both of which were better than saline, for preventing thrombosis. From these initial data, and the reduced bleeding risk associated with hirudin, this agent appears to be a useful anticoagulant in microsurgery.

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.

Recombinant hirudin (r-hirudin) is currently under development as an anticoagulant for use in surgery, therapeutic anticoagulation, disseminated intravascular coagulation and other pathologic states involving the generation of thrombin. Circulating levels of r-hirudin as an antithrombotic agent range from 2 to 20 μg/ml (0.1–1.0 mg/kg) as determined in an animal model of stasis thrombosis. In order to establish a relationship between the r-hirudin circulating level and bleeding, we utilized a rabbit ear blood loss model. r-Hirudin did not produce any loss of blood at dosages up to 20 μg/ml i.v. (1.0 mg/kg). When the circulating levels were maintained at 20 μg/ml for periods of up to 3 h, no increase in blood loss was observed. At 50 and 100 μg/ml initial circulating levels (2.5 and 5.0 mg/kg) a dose-dependent increase in the blood loss was observed which was equivalent to that observed with 1.25 and 2.5 mg/kg i.v. heparin. Such levels of r-hirudin are not expected in clinical usage. In contrast to heparin, the anticoagulant actions of r-hirudin were not neutralized by protamine sulfate, platelet factor 4, other polycationic agents and heparinase. In our studies, the blood loss induced by > 2.0 mg/kg i.v. dosages of r-hirudin in an animal model was neutralized by the administration of an activated pro thrombin complex concentrate at 25 U/ kg. In a similar experimental setting, r-factor Vila was also partially effective. These studies suggest that r-hirudin anticoagulation may not require neutralization, since bleeding effects are not observed at effective antithrombotic dosages in individuals with normal hemostatic status. However, in individuals with a hemostatic deficit, r-hirudin may augment bleeding. In circumstances where excessive bleeding might occur, however, r-hirudin can be partially neutralized by activated prothrombin complex concentrates.

SummaryHirudin prolongs the APTT when added to normal plasma and the extent of prolongation depends on the type of reagent used. The aim of this study was to compare the dose-response curves of 10 widely used APTT reagents for linearity and parallelism. On each of 10 working days a normal pooled plasma was mixed with increasing amounts of recombinant hirudin (HBW023) ranging from 0 to 5 μg/ml and tested for APTT by photo optical coagulometer. Within each working day, clotting times were measured in duplicate and the order of testing with each reagent was changed every day. Results were expressed as ratios of clotting times with hirudin to clotting times without hirudin, and the values plotted against the hirudin concentration on a log-log scale. The dose-response curves for all reagents were linear over 0.3–1.2 μg/ml. The reagent-related slopes ranged from 0.225 ± 0.003 to 0.303 ± 0.003 (mean ± SE) and were significantly different. Precision studies indicated that the least sensitive reagent was also the least precise. These findings indicate that the clotting time values obtained for patients treated with hirudin will vary depending on the APTT reagent used.