Zukunft der Gerinnungsdiagnostik Sabine Eichinger Univ. Klinik für Innere Medizin I Medizinische Universität Wien
Überblick Thrombingenerierungstests Microparticles Thrombozytenfunktionstests
Thrombingenerierungstests Balance des Gerinnungssystems Antikoagulatorische Kräfte Prokoagulatorische Kräfte
Thrombingenerierungstests Hypokoagulabilität Antikoagulatorische Kräfte Prokoagulatorische Kräfte
Thrombingenerierungstests Hypokoagulabilität Schwere Störungen meist durch Routinetests identifizierbar Limitationen milde hämorrhagische Diathesen antithrombotische Effekte komplexe Veränderungen Ausmaß der Befundänderung Blutungsneigung
Mann K, Chest 2003 Thrombingenerierungstests Thrombin Generation Clot Formation 4% Thrombin 96% Thrombin
Thrombingenerierungstests Thrombingenerierung Thrombin (nm) peak thrombin lag phase AUC = endogenous thrombin potential time to peak Time (minutes)
Thrombingenerierungstests Fluorogen Technothrombin TGA (Technoclone, Wien) Thrombinoscope TM Assay (Thrombinoscope BV, Holland) Chromogen ETP (Siemens, Deutschland)
Factor VII-Deficiency Al Dieri, Thromb Haemost 2002
Factor XI-Deficiency Al Dieri, Thromb Haemost 2002
Dargaud, Thromb Haemost 2005 Thrombingenerierungstests zur Erfassung der Hypokoagulabilität Schwere Blutungsneigung Mangel n ETP (%) Faktor:C (%) F VIII 25 3-47 <1-10 ETP < 50% bei allen Patienten mit schwerer Blutungsneigung F IX 6 14-48 <1-7
Al Dieri, Thromb Haemost 2002 Thrombingenerierungstests zur Erfassung der Hypokoagulabilität Schwere Blutungsneigung Mangel n ETP (%) Faktor:C (%) F II 7 5-15 1-8 Keine oder geringe F V 6 0-20 1-6 Blutungsneigung bei Patienten F X 2 0 1-2 mit ETP > 30%
Enoxaparin und Thrombingenerierung Traby & Eichinger, submitted
before 0.5 h 1 h 2 h 3 h 4 h Eichinger, EJCI 2009 Thrombingenerierung nach rviia bei Patienten mit Hämophilie A und Hemmstoff gg. FVIII 225 200 Peak thrombin nm 175 50 25 0
Thrombingenerierungstests Anwendungsmöglichkeiten Erfassung und Quantifizierung des individuellen Thromboserisikos Erfassung und Quantifizierung des individuellen Blutungsrisikos Monitoring prokoagulatorischer Therapien Monitoring konventioneller und neuer Antithrombotika Untersuchung der Plättchen-Plasma-Interaktion Pharmakologische Forschung
Thrombin generation tests Limitations Optimal amount of tissue factor and phospholipids unknown effects of platelets missed (platelet poor plasma) effects of whole blood missed effects of endothelium missed
Überblick Thrombingenerierungtests Microparticles Thrombozytenfunktionstests
Microparticles extracellular TF TF TF intracellular
Microparticles TF TF extracellular - TF intracellular
Microparticles TF TF 0.1 1μm Annexin V positiv (phosphatylserine)
Balance between proliferation, stimulation & death Hemostasis TF Intercellular crosstalk TF Vascular function Angiogenesis Inflammation Immunity
Measurable parameters of hemostatic activity TF TF Antigen exposure Tissue factor Von willebrand factor Receptors for factor V and VIII GP Ib, IIb-IIIa upa, upar Phospholipid composition Cellular source Size (0.1 to 1.0 µm)
Detection methods and limitations Methods Numbers Activity Antigen PS Size Procoag. activity assays - ++ - + - Flow cytometry + - + + + Immunoassays + - ++ - - Impedance flow cytom. ++ - ++ + ++ Confocal microscopy + - ++ - - Dynamic light scattering - - - - ++ Electron microscopy - - + - ++
Scott syndrome (Autosomal recessive) bleeding disorder Impaired migration of phosphatidylserine to the outer membrane of blood cells and an decreased shedding of microparticles Under discussion Reduced high affinity binding sites for factors V and VIII on platelets Sims PJ, J Biol Chem 1989; Rosing J, Blood 1985; Toti F, Blood 1996
Idiopathic thrombocytopenic purpura Case control study Parameter: Platelet derived microparticles (PMP) Study population: 62 patients with ITP, 33 controls Results PMP in ITP, but high variation among patients PMP in ITP without bleeding manifestations Limitation Data still need confirmation in a larger trial Jy W, J Lab Clin Med 1992
Hemophilia Case control study Parameter: Prothrombinase assay Study population: 79 hemophiliacs, 62 controls Results No significant difference Higher values in individuals <18 years Levels during acute bleeding episode Proulle V, Br J Haematol 2005
Hemophilia treatment Recombinant Factor VIIa Recombinant Factor VIII Recombinant Factor IX DDAVP Proulle V, Thromb Haemost 2004
Überblick Thrombingenerierungtests Microparticles Thrombozytenfunktionstests
For Some, Aspirin May Not Help Hearts By ANDREW POLLACK Published: July 20, 2004 More than 20 million Americans take aspirin regularly to help prevent heart attacks and strokes. But new evidence suggests that for many of them, the pills do little if any good. Recent studies have found that anywhere from 5 percent to more than 40 percent of aspirin users are ''nonresponsive'' or ''resistant'' to the medicine. That means that aspirin does not inhibit their blood from clotting, as it is supposed to. ''They are taking it for stroke and heart attack prevention, and it's not going to work,'' said Dr. Daniel I. Simon, the associate director of interventional cardiology at Brigham and Women's Hospital in Boston and an associate professor at Harvard.
Clopidogrel ADP-Rezeptor Arachidonsäure Aspirin COX-1 Thromboxan A 2 Thromboxan A 2 -Rezeptor
Aspirinresistenz vs. Therapieversagen Im Labor: Aspirinresistenz keine oder inkomplette Hemmung der TxA 2 Produktion und/oder der TxA 2 -abhängigen Thrombozytenfunktionstests In der Klinik: Therapieversagen Arterielle Thrombose unter ASS
Aspirinresistenz - Labortests Blutungszeit Thrombozytenaggregometrie Platelet function analyzer (PFA)-100 Verify-now Cone platelet analyzer Durchflusszytometrie Thrombelastographie Bestimmung von TxB 2 im Serum Bestimmung von 11-dehydro TxB 2 im Harn
Aspirinresistenz - Labortests Blutungszeit unspezifisch Thrombozytenaggregationstest nicht ausreichend sensitiv Platelet schlecht function reproduzierbar analyzer (PFA)-100 Verify kaum nowevaluiert ConeBediener-abhängig platelet analyzer Durchflusszytometrie teuer Thrombelastographie zeitaufwendig Bestimmung von TxB 2 im Serum Bestimmung von 11-dehydro TxB 2 im Harn
Gibt es eine Aspirinresistenz? Aspirinresistenz - Labortests Bestimmung von TxB 2 im Serum
Fontana, JTH 2006 Gibt es eine Aspirinresistenz?! ASS-Resistenz bei normalen Probanden 96 Probanden, 100 mg ASS/d über 1 Woche ASS-resistent (%) 100 80 60 40 20 Serum TxB2 PFA 100 0
Krasopoulos G, BMJ 2008 Aspirinresistenz <-> Therapieversagen? Risk of any cardiovascular event in aspirin resistant patients OR 3.85; 95% CI 3.08-4.8
Aspirin-resistant thromboxane biosynthesis and the risk of MI, stroke, and cardiovascular death Eikelboom, Circulation 2002
Clopidogrelresistenz <-> Therapieversagen The CYP2C19 genetic variant is a major determinant of prognosis in young patients who are receiving clopidogrel treatment after myocardial infarction (Collet, Lancet 2009) Among patients with an acute myocardial infarction who were receiving clopidogrel, those carrying CYP2C19 loss-of-function alleles had a higher rate of subsequent cardiovascular events than those who were not (Simon, N Engl J Med 2009) Among persons treated with clopidogrel, carriers of a reducedfunction CYP2C19 allele had significantly lower levels of the active metabolite of clopidogrel, diminished platelet inhibition, and a higher rate of major adverse cardiovascular events, including stent thrombosis, than did noncarriers (Mega, N Engl J Med 2009)
Aspirin/Clopidogrelresistenz Compliance nicht berücksichtigt publication bias inadäquate Testsysteme inadäquate Studienbedingungen... kleine Serien wenige prospektive Studien
Kuliczkowski, Eur Heart J 2009 Position paper of the Working Group on Antiplatelet Drugs Resistance of the PSC/ESC For research purposes only: For the assessment of ASA-specific effects, the proposed test is the use of aggregation induced by arachidonic acid and of TXB2 concentrations in serum. For the assessment of clopidogrel-specific effects, the proposed test is Aggregation induced with ADP or flow cytometric analysis of vasodilatator Stimulated phosphoprotein phosphorylation.
Kuliczkowski, Eur Heart J 2009 Aspirin/Clopidogrelresistenz - gibt es eine klinische Relevanz? NEIN! Position paper of the Working Group on Antiplatelet Drugs Resistance of the PSC/ESC There are no clinical data obtained from prospective trials in sufficiently large numbers of patients, showing that the routine or even the occasional determination/ monitoring of platelet function while on therapy with antiplatelet drugs and consequent therapeutic decisions leads to any practical clinically relevant advantage. Any such recommendation, even in the setting of potentially lethal situations, Appears premature and de facto impossible to implement in practical terms at the current stage of knowledge.