One of the missions of the Belgian Society on Thrombosis and Haemostasis (BSTH) is to support young researchers in the field of haematology. This support comes in the form of different prices granted during the annual BSTH meeting. In 2022, the Paul Capel price was granted to Sarah Vandelanotte, MBS (KU Leuven, Belgium) in the category of basic research, and to Dr. Matthias Engelen (UZ Leuven, Belgium) in the category of Clinical/Laboratory Research. The first presentation focussed on how to treat rt-PA resistant ischemic stroke thrombi. The second walked us through the results of theDAWn-Antico trial, which analysed if modulation of thrombo-inflammation with aprotinin, LMWH and anakinra could improve clinical outcomes in COVID patients. Finally, Sarah Vandelanotte was also awarded with the 2022 CSL Behring award for her work on the development of novel gene therapy strategies that in time may serve as a long-term treatment for von Willebrand disease (VWD).
After an ischemic stroke, the aim is to rapidly dissolve the thrombus and allow recirculation of the blood. Currently, the recombinant tissue plasminogen activator (rt-PA) alteplase is the only FDA-approved drug that can be used to lyse thrombi in this setting. However, due to its contraindications and the limited time window, rt-Pas can only be used in about 10-15% of patients. Furthermore, among these patients the treatment is effective in less than 50% due to rt-PA resistance. Hence, there is a need for new and more efficient thrombolytic therapies. Since 2015, the mechanical removal of ischemic stroke thrombi has been increasingly being used, creating an opportunity to study these thrombi.1–4 In this respect, the analysis of >500 thrombi revealed a huge heterogeneity in the composition of these thrombi, ranking from platelet-rich to red blood cell (RBC)-rich thrombi, as well as some mixed thrombi that contain both platelet- and RBC-rich areas at different quantities. RBC-rich areas consist of both packed RBCs and fibrin fibres. In contrast, platelet-rich areas are more complex and contain platelets, fibrin, VWF, DNA and neutrophil extracellular traps (NETs). Ms. Vandelanotte and colleagues studied the thrombolytic potential of rt-PA on different types of thrombi and tested if targeting other structural components of these thrombi could promote lysis in rt-PA-resistant thrombi. The first step in this research consisted of recreating platelet-rich, mixed and RBC-rich thrombi analogues in vitro. These thrombi were subjected to a lysis procedure without any thrombolytic agent (control) and with rt-PA (experiment). With rt-PA, RBC-thrombi were reduced by 44% compared to the control. In contrast, this reduction was only about 20% in mixed and platelet-rich thrombi. When the same experiment was performed using patients’ thrombi, an overall reduction of the thrombus weight of around 20% was observed when rt-PA was added. However, while rt-PA had a considerable effect on RBC-rich thrombi (>50% RBCs), there was no, or at best a very limited effect on RBC-poor thrombi (<50% RBCs). The presence of higher levels of extracellular DNA in platelet-rich (RBC-poor) thrombi formed the rationale to add a DNase to rt-PA. This resulted in a huge increase in the lysis of these thrombi, while there was no effect on RBC-rich thrombi.5
In summary, this investigation shows that ischemia stroke thrombi can be divided into different types depending on their composition (RBC-rich, mixed and platelet-rich thrombi). Rt-PA can reduce thrombi with a high RBC content but has no effect on platelet-rich thrombi. Adding DNase to rt-PA improves the lysis level of these RBC-poor thrombi. These findings could potentially improve the treatment approach for rt-PA-resistant ischemic stroke thrombi.
COVID-19 disease is associated with a higher risk of thrombo-inflammation events and poor clinical outcomes. High-risk patients are normally treated with heparin to prevent thrombosis and with anti-inflammatory drugs. Additionally, the kallikrein pathway has been suggested to mediate thrombo-inflammation in COVID-19 patients by acting on the intrinsic coagulation pathway and on the bradykinin-associated inflammatory system.6–9 Aprotinin seems to be a perfect drug to target kallikrein as it strongly suppresses the kallikrein-kinin system (KKS), has a safe profile and may have an anti-viral effect according to in vitro experiments.6,10 The rationale to start the DAWn-Antico study was based on the hypothesis that multilevel modulation of thrombo-inflammation with aprotinin (upstream inhibition of thrombo-inflammation), low molecular weight heparin (LMWH) and the interleukin-1 receptor antagonist anakinra (downstream inhibition) could improve clinical outcomes in hospitalised patients with COVID-19.11 This trial included male or female (non-pregnant) patients with a proven SARS-CoV-2 infection (positive SARS-CoV-2 PCR test not older than 72 hours, or clinical radiographic signs and confirmation afterwards by PCR). The study included 102 patients with moderate to severe COVID-19 disease, who were randomly assigned (1:2) to standard-of-care (SOC, N= 35) or SOC + intervention (N= 67). The study intervention consisted of aprotinin (2.000.000 IE IV four times per day) combined with LMWH (50 IU/kg twice daily at the ward, 75 IU/kg twice daily at intensive care). Additionally, patients with predefined hyperinflammation received anakinra (100 mg IV four times per day). The primary endpoint of the study was the time to a sustained 2-point improvement on the 7-point WHO ordinal scale for clinical status, or discharge.
Unfortunately, the study did not meet its primary endpoint. In fact, after 28 days, the intervention did not affect the time to sustained clinical improvement or hospital discharge. In total, 82% and 86% of the patients showed clinical improvement in the intervention and SOC groups (p=0.24). No significant difference was observed in the cumulative incidence of intensive care unit (ICU) admission (42.2 vs. 28.6% at 28 days; p=0.197). The mortality rate was remarkably low and did not differ between the two study arms (4.6 vs. 5.7%; p=0.84). Also in terms of biochemical markers, such as D-dimers or C-reactive protein, there was no difference between the two groups. There was one thrombotic event in each group, but no cases of major bleeding were reported (1.49 vs. 2.86% in the intervention and SOC groups). Only one serious adverse event was observed in the intervention group (haematuria, 1.49%). These findings show that modulation of thrombo-inflammation with high-dose aprotinin and LMWH with or without anakinra is feasible and safe for patients with moderate to severe COVID-19 infection. However, it does not seem to improve clinical or biochemical outcomes in these patients compared to SOC.11
VWD is the most common inherited bleeding disorder. It is caused by a lack of functioning of high molecular weight (HMW) von Willebrand factor (VWF) multimers, which results in excessive bleeding. Unfortunately, the effect of the currently available treatments for VWD, such as desmopressin and recombinant or plasma derived VWF, is short-lived. Hence, there is an unmet clinical need for long-term treatment options for these patients. Previously, Portier et al. developed a sleeping beauty transposon-mediated gene therapy that could theoretically cure VWD in Vwf-knockout mice. This was a two-component system consisting of a transposon plasmid containing the murine Vwf gene and a plasmid encoding a transposase. These components were delivered to mice by hydrodynamic injection, resulting in a high and long-term production of VWF in the liver, as well as in the integration of Vwf in the liver genome. However, no HMW multimers were observed and, as a result, there was no long-term correction of the bleeding phenotype. Besides that, hydrodynamic injection is not clinically relevant since it cannot be used in humans.12 Consequently, the goal is to develop novel strategies that provides clinically relevant long-term correction of severe VWD in mice. As in previous experiments, this approach includes the sleeping beauty transposon to achieve long-term VWF expression. In contrast to previous attempts, the new method uses adenoviral vectors, which renders the technique clinically relevant. Adenovirus 5 (Ad5) has high transduction efficiency but targets hepatocytes in the liver, which does not result in the formation of HMW multimers, as previously shown. Consequently, the second variation of this method consists in redirecting the therapy to the endothelial cells. Another adenovirus, Ad17, transduces endothelial cells more efficiently than Ad5. However, no existing high-capacity vector can contain it due to its large size. As a solution, a chimer adenoviral vector will be constructed, composed of the Ad5 backbone, for which there are high-capacity vectors available, and the fibres of Ad17 to target endothelial cells.13,14 Additionally, an endothelial-specific promotor (TIE2) will be located in front of the Vwf gene instead of the liver promotor.
In short, a high-capacity adenovirus/transposase hybrid vector will be created, composed of the sleeping beauty transposon, the murine Vwf and the TIE2 promotor, all contained in an Ad5 vector with the Ad17 fibres. In vitro endothelial Vwf-knockout cells will be transduced and corrected using this construct, which afterwards will be tested in Vwf-knockout mice.14 This novel gene therapy could potentially serve as a long-term treatment and eventually a cure for VWD in humans.
Results from these new experiments are eagerly awaited.
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