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)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25487 |
| Date | January 2020 |
| Creators | Lindo, Carl Jr |
| Contributors | Weitz, Jeffrey, Health Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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