Tukostaipumus ja suora trombiinin estäjä lepirudiini : kliinisiä ja monitorointinäkökulmia

Thrombophilia (TF) predisposes both to venous and arterial thrombosis at a young age. TF may also impact the thrombosis or stenosis of hemodialysis (HD) vascular access in patients with end-stage renal disease (ESRD). When involved in severe thrombosis TF may associate with inappropriate response to anticoagulation. Lepirudin, a potent direct thrombin inhibitor (DTI), indicated for heparin-induced thrombocytopenia-related thrombosis, could offer a treatment alternative in TF. Monitoring of narrow-ranged lepirudin demands new insights also in laboratory. The above issues constitute the targets in this thesis. We evaluated the prevalence of TF in patients with ESRD and its impact upon thrombosis- or stenosis-free survival of the vascular access. Altogether 237 ESRD patients were prospectively screened for TF and thrombogenic risk factors prior to HD access surgery in 2002-2004 (mean follow-up of 3.6 years). TF was evident in 43 (18%) of the ESRD patients, more often in males (23 vs. 9%, p=0.009). Known gene mutations of FV Leiden and FII G20210A occurred in 4%. Vascular access sufficiently matured in 226 (95%). The 1-year thrombosis- and stenosis-free access survival was 72%. Female gender (hazards ratio, HR, 2.5; 95% CI 1.6-3.9) and TF (HR 1.9, 95% CI 1.1-3.3) were independent risk factors for the shortened thrombosis- and stenosis-free survival. Additionally, TF or thrombogenic background was found in relatively young patients having severe thrombosis either in hepatic veins (Budd-Chiari syndrome, BCS, one patient) or inoperable critical limb ischemia (CLI, six patients). Lepirudin was evaluated in an off-label setting in the severe thrombosis after inefficacious traditional anticoagulation without other treatment options except severe invasive procedures, such as lower extremity amputation. Lepirudin treatments were repeatedly monitored clinically and with laboratory assessments (e.g. activated partial thromboplastin time, APTT). Our preliminary studies with lepirudin in thrombotic calamities appeared safe, and no bleeds occurred. An effective DTI lepirudin calmed thrombosis as all patients gradually recovered. Only one limb amputation was performed 3 years later during the follow-up (mean 4 years). Furthermore, we aimed to overcome the limitations of APTT and confounding effects of warfarin (INR of 1.5-3.9) and lupus anticoagulant (LA). Lepirudin responses were assessed in vitro by five specific laboratory methods. Ecarin chromogenic assay (ECA) or anti-Factor IIa (anti-FIIa) correlated precisely (r=0.99) with each other and with spiked lepirudin in all plasma pools: normal, warfarin, and LA-containing plasma. In contrast, in the presence of warfarin and LA both APTT and prothrombinase-induced clotting time (PiCT®) were limited by non-linear and imprecise dose responses. As a global coagulation test APTT is useful in parallel to the precise chromogenic methods ECA or Anti-FIIa in challenging clinical situations. Lepirudin treatment requires multidisciplinary approach to ensure appropriate patient selection, interpretation of laboratory monitoring, and treatment safety. TF seemed to be associated with complicated thrombotic events, in venous (BCS), arterial (CLI), and vascular access systems. TF screening should be aimed to patients with repeated access complications or prior unprovoked thromboembolic events. Lepirudin inhibits free and clot-bound thrombin which heparin fails to inhibit. Lepirudin seems to offer a potent and safe option for treatment of severe thrombosis. Multi-centered randomized trials are necessary to assess the possible management of complicated thrombotic events with DTIs like lepirudin and seek prevention options against access complications.Veren poikkeava tukostaipumus altistaa nuorena ilmaantuville laskimotukoksille ja harvemmin valtimotukoksille. Väitöskirjan tavoite oli kartoittaa tukostaipumuksen merkitystä vaikeiden tukosten yhteydessä ja dialyysiveritien tukos- ja ahtaumakomplikaatioissa vaikeaa kroonista munuaisten vajaatoimintaa sairastavilla potilailla. Selvitimme myös suoran trombiinin estäjän lepirudiinin tehoa vaikeiden perinteiselle hepariinihoidolle vastaamattomien tukosten hoidossa. Lepirudiinin virallinen käyttöaihe on nykyisin hepariinin aiheuttaman verihiutaleiden määrän laskuun liittyvän tukoksen hoito. Lepirudiinin kapea terapeuttinen alue edellyttää luotettavaa annosvasteen monitorointia. Nykyisin käytetyn menetelmän, aktivoitu osittainen hyytymisaika (APTT), puutteiden vuoksi selvitimme neljän muun spesifisen monitorointimenetelmän soveltumista lepirudiinin annosvasteiden arvioimiseen, myös kliinisesti tärkeiden sekoittavien tekijöiden, varfariinin (INR 1.5-3.9) ja lupus antikoagulantin (LA) yhteydessä. Kaikkiaan 237:lta potilaalta määritettiin verikokein tukostaipumusta ja tukoksille altistavia tekijöitä ennen dialyysiveritieleikkausta vuosina 2002-2004 (seuranta-aika ka. 3.6 v). Tukostaipumus todettiin 43:lla (18%) potilaista, useimmiten miehillä (23 vs. 9%, p=0.009). Tunnetut geenimutaatiot FV Leiden and FII G20210A esiintyivät 4%:lla, poikkeamatta normaaliväestöstä. Veriteiden tukos- ja ahtaumavapaa elinkaari 1-vuoden kohdalla oli 72%. Naissukupuoli (hasardisuhde, 2.5; 95% LV 1.6-3.9) ja poikkeava tukostaipumus (hasardisuhde 1.9, 95% LV 1.1-3.3) olivat itsenäisiä riskitekijöitä tukosten ja ahtaumien lyhentämälle veritien elinkaarelle. Poikkeava tukostaipumus todettiin myös suhteellisen nuorilla potilailla, joilla oli vaikea tukos (maksalaskimon tukos, Budd-Chiari oireyhtymä, BCS sekä 6 kriittinen verisuonikirurgiaan soveltumaton alaraajaiskemia). Lepirudiinin tavanomaisten käyttöaiheiden ulkopuolista tehoa ja turvallisuutta arvioitiin näiden erittäin vaikeiden tukosten hoidossa tilanteessa, jossa muita parantavia hoitomahdollisuuksia ei ollut ja hoitovaste perinteiselle antikoagulaatiolle oli heikko. Lepirudiinihoito osoittautui turvalliseksi ja tehokkaaksi. Vuotokomplikaatioita ei ilmaantunut ja kaikki potilaat toipuivat. Ainoastaan yksi alaraaja amputoitiin 3 vuotta myöhemmin 4 vuoden seuranta-aikana. Lepirudiinin annosvasteen arvio oli luotettavinta kromogeenisillä menetelmillä (ecarin chromogenic assay, ECA ja anti-Factor IIa), jotka korrelloivat erinomaisesti sekä keskenään (r=0.99) että lepirudiinipitoisuuksien kanssa kaikissa olosuhteissa: normaaliplasmassa, varfariinia ja LA-sisältävissä plasmoissa. APTT ja protrombiinin indusoima hyytymisaika (PiCT®) kuvastivat epälineaarisesti lepirudiinin annosvastetta. Kyseiset menetelmät eivät soveltuneet käytettäviksi LA:a sisältävässä plasmassa. Poikkeava tukostaipumus assosioitui hankaliin laskimo- (BCS) ja valtimotukoksiin (kriittinen alaraajaiskemia) sekä dialyysiveritien tukoksiin. Tukostaipumuksen kartoittaminen kannattaa suunnata potilaisiin, joilla on historiassa riskitekijöitä kuten aiemman veritien tukkeutuminen tai ilman altistetta ilmantunut laskimo- tai valtimotukos. Lepirudiini estää vapaata ja myös hyytymään sitoutunutta trombiinia toisin kuin hepariini, mikä osin selittänee sen tehoa hepariinihoidolle huonosti vastanneissa tukoksissa. Veren hyytymisjärjestelmän globaalisen mittarin APTT:n käyttö sitä tarkempien kromogeenisten monitorointimenetelmien rinnalla on avuksi haastavissa kliinisissä tilanteissa. Moniammatillinen yhteistyö laboratorion ja kliinikkojen välillä on ensiarvoisen tärkeää lepirudiinihoitojen toteutuksessa ja vasteiden arvioinnissa. Siten turvataan tehokas tukosten hoito, mutta estetään vuoto-ongelmia. Monikeskuksiset tutkimukset ovat tarpeen suorien trombiinin estäjien merkityksen arvioinnissa vaikeiden tukosten hoidossa sekä dialyysiveritien tukosten ja ahtaumien ehkäisyssä ja hoidossa

Biochemische und pharmakokinetische Charakterisierung zweier Varianten eines neuen, auf Dipetalogastin II basierenden, chimären Thrombininhibitors

Hirudin und Dipetalogastin II sind hochpotente, natürliche Thrombininhibitoren, die in der Therapie kardiovaskulärer Erkrankungen als auch in der Tumortherapie potentielle Einsatzgebiete aufweisen. In dieser Arbeit wurden zwei Varianten eines neuen, chimären Thrombininhibitors auf der Basis dieser beiden Inhibitoren charakterisiert. Beide Inhibitorvarianten wiesen eine starke Thrombinhemmung auf, wobei Variante 1 eine slow, tight-binding und Variante 2 eine klassisch kompetitive Inhibitionskinetik präsentierte

Beeinflussen die Antikoagulantien Heparin und Hirudin die Aktivierung des plasmatischen Gerinnungssystems unter kontinuierlicher Nierenersatztherapie

Die plasmatische Gerinnungshemmung ist entscheidend bei akutem Nierenversagen mit Notwendigkeit zur kontinuierlicher Nierenersatztherapie. Die Antikoagulation mit Heparin stellt eine Alternative zur Citrat- Antikoagulation dar. Bei der Unverträglichkeit beider Substanzen ist die Kenntnis über eine dritte Alternativsubstanz essentiell. In der vorliegenden Studie wurde kontinuierlich verabreichtes Heparin (UFH) mit diskon-tinuierlich verabreichtem Hirudin im Vergleich bei CVVH hinsichtlich der plasmatischen Gerinnungsaktivierung untersucht. Die intermittierende Hirudin Gabe kann zur Antikoagulation bei kontinuierlicher extrakorporaler Nierenersatztherapie sicher einge-setzt werden. Die diskontinuierliche Hirudin Anwendung war gegenüber der kontinu-ierlichen Heparin Applikation in Bezug auf Blutungskomplikationen nicht unterlegen. Die vorliegende Studie zeigte, dass unfraktioniertes Heparin bei der Hemmung der Gerinnungsaktivierung Vorteile besitzt. Unter kontinuierlicher Heparin Applikation ist eine engmaschige Anpassung der Heparin Dosis möglich. Dies ermöglicht im Vergleich der hier dargelegten Applikationsform des Hirudin weniger Gerinnungsaktivierung und damit längere Filterlaufzeiten. Die vorgelegte Studie wies nach, dass Hirudin hinsichtlich der dargelegten Applikationsform zu mehr plasmatischer Gerinnungsaktivität bezogen auf den Thrombin-Antithrombin-Komplex (TAT) führt.The plasmatic anticoagulation is crucial for the efficacy of renal replacement therapy due to acute renal failure. Heparin- Anticoagulation is an alternative to Citrate- Antico-agulation. An intolerance of both substances results in an important need to know a third alternative substance. The present study is a comparison between continuous application of unfractionated heparin versus discontinuous application of recombinant Hirudin due to continuous renal replacement therapy and the activation of plasmatic coagulation. The study showed Benefits in using continuous application of Heparin due to the adjustment. Results were a lower activation of plasmatic coagulation and therefore a longer filter runtime. Discontinuous applicated Hirudin is a safe treatment and noninferior to Heparin treatment related to bleeding complications. Hirudin showed a significant activation of plasmatic coagulation referred to Thrombin-Antithrombin-Complex (TAT)

An examination of the haemostatic changes induced in humans bitten by the Papuan Taipan (Oxyuranus scutellatus canni) and applications of the venom for use in the haemostasis diagnostic laboratory

An examination of the haemostatic changes induced in 84 patients bitten by the Papuan taipan (Oxyuranus scutellatus canni), 13 patients bitten by the death adder (Acanthophis sp) and 4 patients bitten by the Papuan Black snake (Pseudechis papuanus) were studied. A wide range of haemostatic analytes were measured, comparing envenomed patients to 59 healthy Papuan controls. 68 patients bitten by the taipan suffered a consumptive coagulopathy, caused by the presence of a prothrombin activator in the venom. All 68 patients were severely defibrinated (120, APTT>180). A range of factor levels were reduced, including factors II, V, VIII, IX, XI and XII, the most markedly reduced were factors V and VIII (median 5.0u/dl and 7.0u/dl respectively). Of the physiological inhibitors measured. Protein C was consumed to a median level of 44u/dl. Plasminogen and ?2-antiplasmin were consumed to median levels of 53.5 and 41.0u/dl respectively, which in addition to markedly elevated FDPs, indicated active fibrin(ogen)lysis. As expected from a prothrombin activating component, markers of thrombin generation (TAT and F1+2) were elevated. Recovery of haemostatic proteins were measured in eight patients post antivenom treatment at two hourly intervals. Most factors returned to within the normal range by 48 hours. In-vitro studies were performed with crude venom from the Papuan taipan to further elucidate the mechanism of the coagulopathy. There was no evidence found of any direct activity of the venom on the fibrinolytic system, platelets or the coagulation system, other then secondary activation to thrombin generation. A confirmatory test for the detection of lupus anticoagulants was established using the unique properties of the procoagulant component

2021 Update of the International Council for Standardization in Haematology Recommendations for Laboratory Measurement of Direct Oral Anticoagulants

International audienceIn 2018, the International Council for Standardization in Haematology (ICSH) published a consensus document providing guidance for laboratories on measuring direct oral anticoagulants (DOACs). Since that publication, several significant changes related to DOACs have occurred, including the approval of a new DOAC by the Food and Drug Administration, betrixaban, and a specific DOAC reversal agent intended for use when the reversal of anticoagulation with apixaban or rivaroxaban is needed due to life-threatening or uncontrolled bleeding, andexanet alfa. In addition, this ICSH Working Party recognized areas where additional information was warranted, including patient population considerations and updates in point-of-care testing. The information in this manuscript supplements our previous ICSH DOAC laboratory guidance document. The recommendations provided are based on (1) information from peer-reviewed publications about laboratory measurement of DOACs, (2) contributing author's personal experience/expert opinion and (3) good laboratory practice

Effects of direct oral anticoagulant treatment on hemostasis in patients with atrial fibrillation

The direct oral anticoagulants (DOAC) – dabigatran, rivaroxaban, apixaban and edoxaban – were approved without the need for routine monitoring. However, in parallel with clinical implementation, studies on how to measure the effect of DOACs have become a research area of increasing interest during the last decade. The obvious need to assess the anticoagulant effect of DOACs in emergency situations such as bleeding and before emergency surgery has driven this research. Further characterization of the effects of DOACs on different parts of hemostasis has also been in focus of research. The aims of this thesis were to evaluate and improve the use of point-of-care (POC) tests to rapidly determine DOAC effects, and to further increase knowledge of different DOACs’ effect on hemostasis by analyzing their effect on both global and more specific coagulation assays. In study I, we evaluated two whole blood-based tests, the viscoelastic test rotational thromboelastometry (ROTEM), and the flow-based Total Thrombus-Formation analysis system (T-TAS) in patients with atrial fibrillation (AF) on dabigatran treatment. We found that dabigatran concentration correlated strongly with ROTEM clotting time (CT) when activated with two commercial triggers (r-values 0.92 and 0.93), but CT could still be within normal reference interval at significant dabigatran concentrations. There were weak correlations however, between dabigatran concentration and T-TAS data, both time-related thrombus formation and total thrombus formation (r-values 0.39-0.41). In study II, we used ROTEM-CT to show that a thrombin-based trigger was more sensitive than commercial triggers for detection of dabigatran, both in vitro and in samples from patients with AF. The thrombin-based trigger accurately discriminated between samples with dabigatran concentrations above or below 20 ng/mL (100 % sensitivity and specificity). In study III, we went on to investigate the most commonly used DOAC, i.e apixaban. Using ROTEMCT we showed that a factor Xa (FXa)-based trigger was more sensitive than a snake venom-based or commercial trigger for detection of apixaban, both in vitro and in samples from AF patients. The FXa-based trigger accurately discriminated between samples with apixaban concentration above or below 20 ng/mL (100 % sensitivity and specificity). Results were available within 20 min, considerably faster than emergency analyses of drugconcentrations determined at the chemistry lab of the hospital. In study IV, we investigated the effects of DOACs by analyzing both direct and global methods of hemostasis. For comparison, plasma from warfarin-treated patients were also analyzed. Dabigatran treatment was associated with a more permeable fibrin network, delayed thrombin generation and fibrin formation and a lower fibrin turn-over compared to rivaroxaban and apixaban. Warfarin tended to have stronger effects than the FXa-inhibitors. There were no significant differences between apixaban and rivaroxaban in any of the tests performed. In conclusion, ROTEM with DOAC-specific triggers is a promising method to rapidly detect DOAC effect in emergency situations. In patients treated with OACs according to clinical routine, dabigatran treatment was the drug associated with the broadest anticoagulating effects