The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Andreas Greinacher - One of the best experts on this subject based on the ideXlab platform.
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Lubenow N. Recombinant Hirudin in clinical practice: focus on lepirudin. Circulation 2001;103:1479–84
2016Co-Authors: Andreas Greinacher, Md Norbert LubenowAbstract:Abstract—Clinical applications for recombinant Hirudins have been investigated for the past 10 years. The first indication for which a Hirudin—lepirudin—has been approved is treatment of heparin-induced thrombocytopenia (HIT). Also, the recently completed trials for use of lepirudin in unstable angina indicate a potentially new indication. This review describes pharmacology and clinical applications of lepirudin with an emphasis on HIT and unstable angina. An overview of usage of lepirudin in acute coronary syndromes is given, as well as a summary of rare indications for lepirudin, such as extracorporeal circulation, for which comprehensive data are lacking. (Circulation. 2001;103:1479-1484.) Key Words: recombinant Hirudin n platelets n thrombosis n cardiovascular diseases n heparin Hirudin was used for the first parenteral anticoagulation inhumans in 19091 and as the anticoagulant for the first hemodialysis in humans.2 Soon after, heparin became avail-able and has since become the most widely used drug for parenteral anticoagulation. Heparin, however, can induce a life-threatening adverse immune reaction, heparin-induced thrombocytopenia (HIT), which up to 3 % of patients receiv-ing unfractionated heparin (UFH) for.5 days will develop.
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Practical Transfusion Medicine - Heparin-induced thrombocytopenia
Journal of Thrombosis and Haemostasis, 2009Co-Authors: Andreas GreinacherAbstract:Recombinant Hirudins have a definite role in the treatment of patients with heparin-induced thrombocytopenia (HIT). The most important adverse effects are haemorrhages and the induction of antiHirudin antibodies. Major haemorrhages were not significantly increased in patients with HIT compared with a historical control group, but prospective data comparing Hirudin and heparinoids such as danaparoid are lacking. The definition of the optimal method for monitoring and the availability of an antidote for Hirudin would probably increase safety with this drug. To date, haemofiltration using high-flux filter systems is the only way to remove an overdosage of Hirudin from the circulation. In patients with renal impairment requiring Hirudin treatment, it therefore seems safer to start with a low dose that is subsequently adjusted according to the activated partial prothromboplastin time or ecarin clotting time.
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the direct thrombin inhibitor Hirudin
Thrombosis and Haemostasis, 2008Co-Authors: Andreas Greinacher, Theodore E WarkentinAbstract:This review discusses the pharmacology and clinical applications of Hirudin, a bivalent direct thrombin inhibitor (DTI). Besides the current major indication for Hirudin – anticoagulation of patients with heparin-induced thrombocytopenia (HIT) – the experience with Hirudin in other indications, especially acute coronary syndromes, are briefly presented. Hirudins have been formally studied prior to their regulatory approval; however, important information on their side effects and relevant preventative measures only became available later. Therefore, current recommendations and dosing schedules for Hirudin differ considerably from the information given in the package inserts. Drawbacks of Hirudin and important precautions for avoiding potential adverse effects are discussed in detail in the third part of this review.
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Recombinant Hirudin in clinical practice : Focus on lepirudin
Circulation, 2001Co-Authors: Andreas Greinacher, Norbert LubenowAbstract:Clinical applications for recombinant Hirudins have been investigated for the past 10 years. The first indication for which a Hirudin-lepirudin-has been approved is treatment of heparin-induced thrombocytopenia (HIT). Also, the recently completed trials for use of lepirudin in unstable angina indicate a potentially new indication. This review describes pharmacology and clinical applications of lepirudin with an emphasis on HIT and unstable angina. An overview of usage of lepirudin in acute coronary syndromes is given, as well as a summary of rare indications for lepirudin, such as extracorporeal circulation, for which comprehensive data are lacking.
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Heparin-Induced Thrombocytopenia
BioDrugs, 2000Co-Authors: Norbert Lubenow, Andreas GreinacherAbstract:Recombinant Hirudins have a definite role in the treatment of patients with heparin-induced thrombocytopenia (HIT). The most important adverse effects are haemorrhages and the induction of antiHirudin antibodies. Major haemorrhages were not significantly increased in patients with HIT compared with a historical control group, but prospective data comparing Hirudin and heparinoids such as danaparoid are lacking. The definition of the optimal method for monitoring and the availability of an antidote for Hirudin would probably increase safety with this drug. To date, haemofiltration using high-flux filter systems is the only way to remove an overdosage of Hirudin from the circulation. In patients with renal impairment requiring Hirudin treatment, it therefore seems safer to start with a low dose that is subsequently adjusted according to the activated partial prothromboplastin time or ecarin clotting time. Even in special circumstances, such as cardiopulmonary bypass or dialysis, Hirudins can be applied successfully if care is taken to monitor their effects meticulously. There are many other indications in which Hirudins have shown feasibility (e.g. acute coronary syndromes) but available data preclude definite conclusions.
Jan-peter Hildebrandt - One of the best experts on this subject based on the ideXlab platform.
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Cell-free synthesis of the Hirudin variant 1 of the blood-sucking leech Hirudo medicinalis
Scientific Reports, 2020Co-Authors: Doreen A. Wüstenhagen, Christian Müller, Phil Lukas, Jan-peter Hildebrandt, Simone A. Aubele, Stefan KubickAbstract:Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor Hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant Hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active Hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature Hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized Hirudin showed a considerably higher anti-thrombin activity compared to recombinant Hirudin produced in bacterial cells.
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The Hirudin-like factors HLF3 and HLF4—hidden Hirudins of European medicinal leeches
Parasitology Research, 2020Co-Authors: Christian Müller, Phil Lukas, Dana Sponholz, Jan-peter HildebrandtAbstract:The Hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis , Hirudo verbana , and Hirudo orientalis , respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of Hirudin and HLF molecules for thrombin inhibition.
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Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different
Parasitology Research, 2019Co-Authors: Phil Lukas, Jan-peter Hildebrandt, Robert Wolf, Bernhard H. Rauch, Christian MüllerAbstract:Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and Hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo , but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of Hirudin, a new class of putative leech saliva components, the Hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria . Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional Hirudins (HM3 and HM4) of Hirudinaria manillensis . While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the Hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis . Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of Hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.
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Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches
The Journal of parasitology, 2019Co-Authors: Christian Müller, Phil Lukas, Sarah Lemke, Jan-peter HildebrandtAbstract:Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is Hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of Hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of Hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the Hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both Hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of Hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the Hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.
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Hirudins and Hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships
Parasitology Research, 2017Co-Authors: Christian Müller, Sarah Lemke, Martin Haase, Jan-peter HildebrandtAbstract:Haematophagous leeches express a broad variety of bioactive factors that are released from the salivary gland cells into the wound of a host during feeding. Among these, Hirudin is probably the best studied factor and, moreover, the only one that has successfully made the transition from nature to clinical use. Many components of the leech saliva still remain either poorly characterized or yet completely unknown. Only recently, a new class of leech-derived factors has been discovered in Hirudo medicinalis , the Hirudin-like factors (HLFs). HLFs comprise typical structural features of Hirudin but lack others. We were able to verify the expression of HLFs not only in two additional species of the genus Hirudo , but also in Hirudinaria manillensis . Various phylogenetic analyses based on gene and protein sequences support a sister group relationship between Hirudins and HLFs. Although potential molecular targets of HLFs remain unknown, the presence of multiple isoforms in individual leeches of different genera points to key functions in the regulation of several processes associated with the blood-sucking life style of leeches.
Karlgeorg Fischer - One of the best experts on this subject based on the ideXlab platform.
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Hemofiltration of Recombinant Hirudin by Different Hemodialyzer Membranes: Implications for Clinical Use
2015Co-Authors: Kerstin Benz, Matthias A Nauck, Karlgeorg FischerAbstract:Recombinant Hirudin (lepirudin) is a potent direct thrombin inhibitor that is used particularly for treatment of immune-mediated heparin-induced thrombocytopenia. Because Hirudin is almost exclusively eliminated by the kidneys, its half-life is markedly prolonged in patients with severe renal insufficiency. Therefore, these patients are at risk for bleeding, particularly because no antidote is available. To use Hirudin safely in patients who are on renal replacement therapy, knowledge of Hirudin-sieving characteristics of different hemodialyzers is required. Data on this issue are sparse and in part contradictory. Eight different conventional low-flux and high-flux hemodialyzers were tested in an in vitro circuit with ultrafiltrate reinfusion. In each experiment, lepirudin concentration was repetitively measured during 3 h in the prefilter, the postfilter, and the filtration line using a chromogenic assay. On the basis of these data, sieving coefficients were calculated. All high-flux hemodialyzers tested allowed filtration of Hirudin yet with marked differences in steady-state sieving (sieving coefficients in whole blood: polysulfone [PS] 0.97 0.03; polymethylmethacrylate [PMMA] 0.75 0.02; polyarylethersulfone 0.73 0.02; polyamide 0.49 0.02). None of the low-flux hemodialyzer membranes tested (cuprophane, hemophane, PS, and PMMA) showed significant Hirudin filtration. Owing to marked differences in Hirudin-sieving characteristics, choice of the appro-priate hemodialyzer membrane is an important determinant of bleeding risk in dialysis-dependent patients who are treated with Hirudin. In case of overdosage or bleeding complications, hemofiltration via PS membranes is recommended to reduce plasma Hirudin concentration. Hirudin dosage should be adapted not only to the clinical situation but also to the Hirudin
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hemofiltration of recombinant Hirudin by different hemodialyzer membranes implications for clinical use
Clinical Journal of The American Society of Nephrology, 2007Co-Authors: Kerstin Benz, J Bohler, Matthias A Nauck, Karlgeorg FischerAbstract:Recombinant Hirudin (lepirudin) is a potent direct thrombin inhibitor that is used particularly for treatment of immune-mediated heparin-induced thrombocytopenia. Because Hirudin is almost exclusively eliminated by the kidneys, its half-life is markedly prolonged in patients with severe renal insufficiency. Therefore, these patients are at risk for bleeding, particularly because no antidote is available. To use Hirudin safely in patients who are on renal replacement therapy, knowledge of Hirudin-sieving characteristics of different hemodialyzers is required. Data on this issue are sparse and in part contradictory. Eight different conventional low-flux and high-flux hemodialyzers were tested in an in vitro circuit with ultrafiltrate reinfusion. In each experiment, lepirudin concentration was repetitively measured during 3 h in the prefilter, the postfilter, and the filtration line using a chromogenic assay. On the basis of these data, sieving coefficients were calculated. All high-flux hemodialyzers tested allowed filtration of Hirudin yet with marked differences in steady-state sieving (sieving coefficients in whole blood: polysulfone [PS] 0.97 ± 0.03; polymethylmethacrylate [PMMA] 0.75 ± 0.02; polyarylethersulfone 0.73 ± 0.02; polyamide 0.49 ± 0.02). None of the low-flux hemodialyzer membranes tested (cuprophane, hemophane, PS, and PMMA) showed significant Hirudin filtration. Owing to marked differences in Hirudin-sieving characteristics, choice of the appropriate hemodialyzer membrane is an important determinant of bleeding risk in dialysis-dependent patients who are treated with Hirudin. In case of overdosage or bleeding complications, hemofiltration via PS membranes is recommended to reduce plasma Hirudin concentration. Hirudin dosage should be adapted not only to the clinical situation but also to the Hirudin-sieving characteristics of the assigned dialyzer.
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The Role of Recombinant Hirudins in the Management of Thrombotic Disorders
BioDrugs, 2004Co-Authors: Karlgeorg FischerAbstract:Native Hirudin is the most potent natural direct thrombin inhibitor currently known; it is capable of inhibiting not only fluid phase, but also clot-bound thrombin. Recombinant technology now allows production of recombinant Hirudins (r-Hirudins), which are available in sufficient purity and quantity with essentially unaltered thrombin-inhibitory potency. As thrombin is known to play a key role in a number of thrombotic disorders, numerous studies focused on the impact of r-Hirudins on the clinical course in these diseases. R-Hirudins provided significantly more stable anticoagulation than standard heparin, but demonstrated a relatively narrow therapeutic range with relevant bleeding risk even at clinically effective doses. In doses that are not associated with an increased bleeding risk, r-Hirudins often failed to demonstrate significant superiority to heparin. To date, r-Hirudins have a definite role in the treatment of heparin-induced thrombocytopenia, where they markedly reduce the high risk of thrombosis. For prophylaxis of deep vein thrombosis, r-Hirudins have been shown to be superior to both unfractionated and low molecular weight heparin, but are not extensively used in this indication. In acute coronary syndromes, a definite role of r-Hirudins has not yet been firmly established. When applied in an appropriate dose as adjunct to thrombolysis in patients with acute myocardial infarction, randomized, controlled trials did not show a consistent benefit of r-Hirudins, especially in the long-term. In patients undergoing coronary balloon angioplasty for acute coronary syndromes, promising effects in the early postprocedural phase did not translate to an improved outcome after 6 months. In patients with unstable angina pectoris, efficacy and safety of r-Hirudins as primary antithrombotic therapy are still under debate. In the future, r-Hirudins are to be compared with alternative or additional potent antithrombotic agents or treatment strategies. This comparison will ultimately lead to their final placement in the management of thrombotic disorders.
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Anti-Hirudin antibodies alter pharmacokinetics and pharmacodynamics of recombinant Hirudin.
Thrombosis and haemostasis, 2003Co-Authors: Karlgeorg Fischer, Volker Liebe, Renata Hudek, Lukas Piazolo, Karl K. Haase, Martin Borggrefe, G. HuhleAbstract:Recombinant Hirudin (r-Hirudin) is a potent direct thrombin inhibitor with immunogenic properties. Anti-Hirudin antibodies (aHAb) are detected in up to 74% of patients treated with r-Hirudin for more than 5 days. aHAb may alter the pharmacokinetics and pharmacodynamics of r-Hirudin. The effects of aHAb on the pharmacokinetics of r-Hirudin were investigated in rats receiving r-Hirudin intravenously either without aHAb (controls), 15 min after intravenous administration of non-specific antibodies or aHAb, and after pre-incubation with aHAb. When both were compared to controls and pre-treatment with non-specific antibodies, aHAb significantly altered the pharmacokinetics of r-Hirudin with similar effects in both approaches: In the presence of aHAb, the volume of distribution in a steady state and total plasma clearance were diminished, while the half-life of elimination was prolonged. Both the maximum r-Hirudin plasma concentration and the area under the curve were increased. In addition, r-Hirudin filtration by high-flux hemodialyzer membranes (polysulfone,AN69) was investigated 1) in the absence of aHAb, 2) in the presence of non-specific mouse antibodies, and 3) in the presence of three monoclonal aHAb. In the absence of aHAb, both hemodialyzers allowed for significant r-Hirudin filtration. Non-specific mouse antibodies did not markedly affect r-Hirudin filtration. By contrast, all three aHAb almost completely hindered r-Hirudin filtration. aHAb varied in their capacity to neutralize r-Hirudin. In conclusion, aHAb markedly alter the pharmacokinetics of r-Hirudin leading to r-Hirudin accumulation. In the presence of aHAb, hemofiltration does not allow for rapid reduction of r-Hirudin concentration. aHAb are capable of modifying pharmacodynamics of r-Hirudin. Close monitoring of aHAb-positive patients treated with r-Hirudin is considered mandatory.
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Hirudin in renal insufficiency.
Seminars in thrombosis and hemostasis, 2002Co-Authors: Karlgeorg FischerAbstract:Recombinant Hirudins (r-Hirudins) are potent direct thrombin inhibitors increasingly used for alternative anticoagulation, especially in heparin-induced thrombocytopenia. R-Hirudins are almost exclusively eliminated by the kidneys, and a close correlation between r-Hirudin clearance and endogenous creatinine clearance has been observed. Accordingly, the pharmacokinetics of r-Hirudin are altered in patients with renal insufficiency. A decline of renal r-Hirudin clearance is associated with an increase of r-Hirudin half-life and the area under the curve (AUC). Therefore, renal impairment necessitates reduction of r-Hirudin dose to avoid overdose or inadequate accumulation of the thrombin inhibitor. To this end, close monitoring of r-Hirudin anticoagulation is required, which at best is performed by measuring r-Hirudin blood levels by ecarin clotting times (ECT) or chromogenic assays, in addition to activated partial thromboplastin time (aPTT). Recent studies showed that r-Hirudin anticoagulation is feasible in acute or chronic renal failure treated with continuous or intermittent renal replacement therapy, if appropriate r-Hirudin dosing and adequate monitoring are warranted. High-volume hemofiltration with r-Hirudin-permeable hemodialyzers constitutes a valuable means to markedly reduce r-Hirudin blood concentration and total r-Hirudin body content in case of r-Hirudin overdose or r-Hirudin-associated bleeding. In the future, the hepatically eliminated direct thrombin inhibitor argatroban may facilitate alternative anticoagulation in patients with renal insufficiency.
Christian Müller - One of the best experts on this subject based on the ideXlab platform.
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Cell-free synthesis of the Hirudin variant 1 of the blood-sucking leech Hirudo medicinalis
Scientific Reports, 2020Co-Authors: Doreen A. Wüstenhagen, Christian Müller, Phil Lukas, Jan-peter Hildebrandt, Simone A. Aubele, Stefan KubickAbstract:Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor Hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant Hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active Hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature Hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized Hirudin showed a considerably higher anti-thrombin activity compared to recombinant Hirudin produced in bacterial cells.
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The Hirudin-like factors HLF3 and HLF4—hidden Hirudins of European medicinal leeches
Parasitology Research, 2020Co-Authors: Christian Müller, Phil Lukas, Dana Sponholz, Jan-peter HildebrandtAbstract:The Hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis , Hirudo verbana , and Hirudo orientalis , respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of Hirudin and HLF molecules for thrombin inhibition.
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Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different
Parasitology Research, 2019Co-Authors: Phil Lukas, Jan-peter Hildebrandt, Robert Wolf, Bernhard H. Rauch, Christian MüllerAbstract:Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and Hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo , but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of Hirudin, a new class of putative leech saliva components, the Hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria . Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional Hirudins (HM3 and HM4) of Hirudinaria manillensis . While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the Hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis . Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of Hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.
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Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches
The Journal of parasitology, 2019Co-Authors: Christian Müller, Phil Lukas, Sarah Lemke, Jan-peter HildebrandtAbstract:Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is Hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of Hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of Hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the Hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both Hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of Hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the Hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.
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Hirudins and Hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships
Parasitology Research, 2017Co-Authors: Christian Müller, Sarah Lemke, Martin Haase, Jan-peter HildebrandtAbstract:Haematophagous leeches express a broad variety of bioactive factors that are released from the salivary gland cells into the wound of a host during feeding. Among these, Hirudin is probably the best studied factor and, moreover, the only one that has successfully made the transition from nature to clinical use. Many components of the leech saliva still remain either poorly characterized or yet completely unknown. Only recently, a new class of leech-derived factors has been discovered in Hirudo medicinalis , the Hirudin-like factors (HLFs). HLFs comprise typical structural features of Hirudin but lack others. We were able to verify the expression of HLFs not only in two additional species of the genus Hirudo , but also in Hirudinaria manillensis . Various phylogenetic analyses based on gene and protein sequences support a sister group relationship between Hirudins and HLFs. Although potential molecular targets of HLFs remain unknown, the presence of multiple isoforms in individual leeches of different genera points to key functions in the regulation of several processes associated with the blood-sucking life style of leeches.
Job Harenberg - One of the best experts on this subject based on the ideXlab platform.
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Quantitative determination of PEG-Hirudin in human plasma using a competitive enzyme-linked immunosorbent assay.
Thrombosis research, 2000Co-Authors: X. H. Song, G. Huhle, Lianchun Wang, Job HarenbergAbstract:Polyethylene glycol (PEG) coupled r-Hirudin mutein is determined by biological methods-the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-Hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-Hirudin in human plasma. A PEG-Hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-Hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-Hirudin were between 12 and 22%. The correlation of the concentration of PEG-Hirudin determined with the ELISA and the ecarin clotting time was r = 0.902. No interactions between unfractionated heparin, low molecular-weight heparin, or phenprocoumon and PEG-Hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-Hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.
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Generation of Anti-Hirudin Antibodies in Heparin-Induced Thrombocytopenic Patients Treated With R-Hirudin
Circulation, 1999Co-Authors: X. H. Song, G. Huhle, Lianchun Wang, Ursula Hoffmann, Job HarenbergAbstract:Background—Hirudin is a small protein with strong thrombin inhibition that may be antigenic. The generation and disappearance of anti-Hirudin antibodies were investigated in patients with heparin-induced thrombocytopenia who were treated with recombinant Hirudin (r-Hirudin) for ≥5 days. Methods and Results—The IgA, IgE, IgG, and IgM isotypes of anti-Hirudin antibodies were determined by ELISA before and after the start of r-Hirudin therapy. A total of 56% of patients (13 of 23) developed ≥1 antibody isotype during therapy. No IgE antibodies were generated. IgA, IgG, and IgM antibodies were detected in 30% (7 of 23), 52% (12 of 23), and 17% (4 of 23) of patients, respectively. Four patients generated only IgG, 2 patients developed either IgM or IgG and IgM, 5 patients IgG and IgA, and 2 patients IgG, IgM, and IgA antibodies. IgM antibodies disappeared within 8 days of the cessation of r-Hirudin. IgA and IgG antibodies disappeared within 1 year in all but 1 patient. Binding of purified IgG to r-Hirudin in I...
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Capillary electrophoresis of r-Hirudin and a polyethylene glycol derivative of r-Hirudin (PEG-Hirudin).
Seminars in thrombosis and hemostasis, 1997Co-Authors: Reinhard Malsch, Anja Timmermann, Job HarenbergAbstract:UNLABELLED Recombinant (r-) Hirudins and PEG-Hirudin are currently tested for anticoagulant therapy. For their concentration measurement, radioimmunoassay and HPLC methods are available. The separation of r- and PEG-Hirudin is currently performed by HPLC. However, the sensitivity of the method is low. Capillary electrophoresis is a rapid, selective technique that requires low sample amounts. Our aim was the development of a capillary electrophoresis method to measure r- and PEG-Hirudin. The results are as follows: In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-Hirudin was separated from PEG-Hirudin in a purified system using a fused silica capillary (50 cm long and 75-micron i.d. and reversed polarity). A neutral capillary with a 20 mM tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r- from PEG-Hirudin. A linear correlation was found between the peak area and the concentration between 20 micrograms/mL and 10 mg/mL for Hirudin (r2 = 0.99) and between 1.25 and 10 mg/mL for PEG-Hirudin (r2 = 0.99). In human plasma mixtures, r- and PEG-Hirudin were completely separated. The linear correlation between the peak area and the concentration was r2 = 0.99. CONCLUSION In a fused silica capillary, r- and PEG-Hirudin are separated in a purified system. Capillary electrophoresis which is performed in a neutral capillary, resolves r- from PEG-Hirudin in a purified system, in plasma and in urine. The sensitivities of the methods are comparable. Capillary electrophoresis separates r- from PEG-Hirudin and may be applied to biologic systems to measure the concentration and purity of r- and PEG-Hirudin.