Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thrombotic disorders include myocardial infarction (MI), acute ischemic stroke (AIS) and venous thromboembolism (VTE), which encompasses pulmonary embolism (PE), and deep vein thrombosis (DVT). To prevent further complications or mortality in patients with MI and AIS, rapid restoration of blood flow is needed to minimize organ damage. Such treatment also is needed in patients with massive PE. Blood flow can be restored mechanically via percutaneous coronary intervention with stent implantation for MI and by thrombectomy in patients with AIS or PE. Alternatively, pharmacological reperfusion can be achieved by systemic administration of plasminogen activators (PAs). PAs convert plasminogen to the fibrinolytic enzyme, plasmin. Plasmin then degrades the clot into soluble fragments. Streptokinase (SK) and urokinase (UK) were the first therapeutic clot dissolving drugs but both lead to excessive bleeding complications because of non-specific effects. Current therapy focuses on clot specific agents such as recombinant tissue-PA (rt-PA) or tenecteplase (TNK), a rt-PA variant. However, there is a risk of intracranial bleeding in at least 1% of patients, which can be fatal or disabling. Thus, a need exists for new strategies to enable safer reperfusion that are not associated with potentially fatal side effects. This study focuses on the therapeutic role of alpha2-antiplasmin (α2AP). α2AP is the primary inhibitor of plasmin. One approach to thrombolysis is to attenuate α2AP with an inhibitory antibody (A2AP IgG). Inhibition of α2AP would enable clot lysis with lower doses of PAs, thereby reducing the risk of bleeding and serving as a safer approach to thrombolytic therapy. We aimed to characterize A2AP IgG and evaluate its effect on fibrinolysis in vitro and in vivo. A2AP IgG1 was selected and developed using phage display and an antibody gene library with human and rabbit α2AP as the antigen. Affinity maturation was performed and the Fc portion of the A2AP IgG1 was subsequently changed to the IgG4 isotype which yielded A2AP IgG4. A2AP IgG4 binds α2AP with 63-fold higher affinity than A2AP IgG1 as determined using surface plasmon resonance (SPR). SDS-PAGE and western blot analysis reveals that both antibodies bind to the plasmin-α2AP (PAP) complex, fibrinogen, and fragment X but not to α2AP; results confirmed by ELISA. In functional studies, A2AP IgG1 significantly reduced plasmin inhibition by α2AP by 5.5-fold. Both A2AP IgG1 and A2AP IgG4 shortened tissue-PA (t-PA)-mediated clot lysis in a concentration dependent manner. A2AP IgG4 was 2.2-fold more potent than A2AP IgG1 in human plasma and 1.4-fold more potent in rabbit plasma. Compared with t-PA or TNK alone, addition of either antibody enhanced the lysis of preformed plasma clots. Combining A2AP IgG4 with 10% of the highest t-PA or TNK dose produced more clot lysis than the highest dose of t-PA or TNK alone. In a rabbit jugular vein thrombosis model, A2AP IgG4 alone produced 20% lysis. When combined with a low dose of TNK, 40% clot lysis resulted, which was significantly greater than the 30% clot lysis observed with a higher dose of TNK. A2AP IgG4 alone or in combination with a lower dose of TNK did not cause significantly more bleeding than the higher dose of TNK alone and did not degrade circulating fibrinogen. Thus, we have shown that by inactivating α2AP, A2AP IgG attenuates α2AP activity, and accelerates clot lysis in vitro and in vivo. This demonstrates that antibody-mediated inhibition of α2AP, enhances thrombolysis and enables use of lower doses of PAs. / Thesis / Master of Science in Medical Sciences (MSMS)

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 17:04:50.447

L'étude systématique de l'hémostase post-thrombolytique a été peu étudiée. Chez 80 malades thrombolysés consécutifs, une étude prospective a comporté l'étude – aux heures 0, 2 et 24 – des facteurs de l'hémostase suivants: fibrinogène, plasminogène, PDF (produits de dégradation de la fibrine et du fibrinogène), D-dimères, alpha2-antiplasmine et facteur XIII, ainsi que l'hématocrite et la numération plaquettaire. Des calculs statistiques approfondis ont exploré les corrélations des variations des facteurs hémostatiques entre eux et avec 37 paramètres cliniques et étiologiques. Processus moléculaires post-thrombolytiques. Le rt-PA induit deux processus, indépendants statistiquement à la 2ème heure: d'une part une élévation des PDF et des D-dimères; d'autre part, une baisse du fibrinogène, corrélée à une baisse du plasminogène (r=0,48, p=0.01), de l'alpha2-antiplasmine (r=0.48, p =0.004) et du facteur XIII (r=0.44, p=0.01). La baisse du plasminogène est corrélée significativement avec celle de l'alpha2-antiplasmine (r=0.77, p<0.001), et du facteur XIII (r=0.47, p=0.02). La mise en jeu de facteurs anti-fibrinolytiques, qui n'avait jamais été décrite précédemment, peut jouer un rôle dans une limitation de la fibrinolyse et dans la rethrombose. Des corrélations sont notées entre la baisse précoce du plasminogène et l'étiologie cardioembolique (p=0.04), et un mauvais pronostic final (p=0.03), possiblement en rapport la thrombolyse intense de gros caillots. Les hématomes intra-cérébraux parenchymateux (HP) sont liés significativement à la baisse du fibrinogène (p=0.01) et à l'augmentation des PDF (p=0.01). Une baisse du fibrinogène au-dessous de 2g/L multiplie la probabilité de HP précoce par un facteur 12,82. Ainsi est confirmé le modèle d'une “coagulopathie précoce avec dégradation du fibrinogène”», prédictive de l'hématome, proposé par l'équipe lyonnaise de thrombolyse en 2004 / A systematic study of post-thrombolytic haemostasis has rarely been performed. In 80 consecutive patients, we have prospectively studied at hours 0, 2 and 24 the following parameters: fibrinogen, plasminogen, alpha2-antiplasmin, factor XIII, fibrin(ogen) Degradation Products (FDP), D-dimers, haematocrit and platelet count. Comprehensive statistical studies calculated correlations of the haemostatic values betwen themselves and with 38 etiological and clinical parameters. Molecular dynamics. Two changes between h0 and h2 were statistically independent: an increase in FDP and D-Dimers; a decrease in fibrinogen, plasminogen, alpha2-antiplasmin and factor XIII. At h2, the decrease in fibrinogen was significantly correlated with that of plasminogen (0.48, p = 0.01), alpha2-antiplasmin (0.48, p = 0.004), and factor XIII (0.44, p = 0.01). The decrease in plasminogen was significantly correlated with those of antifibrinolytic components, alpha2-antiplasmin (r=0.77, p<0.001) and factor XIII (0.47, p=0.02). To our knowledge, such an activation of antifibrinolytic components had not hitherto been mentioned. The h2 decrease of plasminogen was correlated with cardioembolic etiology (p=0.04) and final poor oucome (p=0.03), a fact possibly due to intense thrombolysis of large clots. Patients having early parenchymal hematomas (PH) showed h2 haemostasis disturbances: high FDP (p=0.01), and low fibrinogen (p=0.01). The decrease in fibrinogen less than 2g/L multiplies the odds of early PH by a factor 12.82. Thus, we confirm the model of an “early fibrinogen degradation coagulopathy” predictive of hematomas, which had been coined by the Lyon thrombolysis team in 2004