Modulation of thromboinflammation in hospitalized COVID-19 patients with aprotinin, low molecular weight heparin, and anakinra: the DAwn- AntiCo study

Background/Introduction: Thromboinflammation in severe COVID-19 is associated with disease severity and outcome. The kallikrein pathway is suggested to mediate thromboinflammation in COVID-19 by activating inflammatory pathways and contactmediated coagulation. Purpose(s): The DAWn-antico study investigates if a multitarget modulation of the thromboinflammatory response improves outcomes in hospitalized patients with severe COVID-19. Method(s): In this multicenter open-label randomized clinical trial (EudraCT 2020-001739-28), patients hospitalized with COVID- 19 were 1:2 randomized to receive standard of care (SOC) or SOC plus study intervention. The intervention consisted of aprotinin (2,000,000 IE IV four times daily) combined with low-molecular-weight heparin (LMWH;SC 50 IU/kg twice daily at the ward, 75 IU/kg twice daily at intensive care). Additionally, patients with predefined hyperinflammation received the interleukin-1- receptor antagonist anakinra (100mg IV four times daily). The primary outcome was time to a sustained 2-point improvement on the 7-point WHO ordinal scale for clinical status, or discharge. The trial was funded by Life Sciences Research Partners, Research Foundation Flanders (G0G4720N), and KU Leuven COVID-19 fund. Result(s): Between 24 June 2020 and 01 February 2021, 105 patients were randomized, and 102 patients were included in the full analysis set (intervention N=67 vs. SOC N=35). Twenty-five patients from the intervention group (37%) received anakinra. The intervention did not affect the primary outcome (HR 0.77 [CI 0.50;1.19], p=0.24) or mortality (intervention n=3 (4.6%) vs. SOC n=2 (5.7%), HR 0.82, [CI 0.14;4.94], p=0.83). There was one treatment-related adverse event in the intervention group (hematuria, 1.49%). There was one thrombotic event in the intervention group (1.49%) and one in the SOC group (2.86%), but no major bleedings. Conclusion(s): In hospitalized COVID-19 patients, modulation of thromboinflammation with high-dose aprotinin and LMWH with or without anakinra did not improve outcome in patients with moderate to severe COVID-19. (Disclosure: this RCT was presented at ISTH 2022 in London and will be published in Research and Practise in Thrombosis and Haemostasis).

Long-term persistence of anti-PF4- antibodies following vaccine-induced thrombotic thrombocytopenia after first ChAdOx1 vaccination

Background: Vaccine-induced Thrombotic Thrombocytopenia (VITT) is a rare but potentially life-threatening complication of the ChAdOx1 COVID-vaccine, which involves binding of IgG antibodies against platelet factor 4 (PF4) to the platelet Fc-gamma receptor. This causes platelet activation with thrombosis and thrombocytopenia. Because of the similarity with heparin-induced thrombocytopenia (HIT), heparin is avoided in the acute treatment of VITT. There is limited information about the long-term persistence of anti-PF4- antibodies and their clinical relevance. Aim(s): To describe long-term clinical and serological outcomes after VITT. Method(s): A case series of patients from Leuven University Hospitals with confirmed VITT and at least 6 months of follow-up. All patients provided informed consent. Anti-PF4 antibodies were measured via chemiluminescence (HemosIL AcuStar HIT-IgG( PF4-H), Werfen) and enzyme-linked immunosorbent assay (ELISA) with immobilised polyvinylsulfonate/PF4 complexes (PF4-IgG Immucor, GTI Diagnostics);with an optical density (OD) cut-off of 0.4. Aggregation of platelets after exposure to patient plasma with 0, 1 or 200 IU/ml of heparin was measured by whole-blood impedance aggregometry (HIMEA) (Roche multiplate analyser). Result(s): Three middle-aged women presented with thrombosis with thrombocytopenia and a positive anti-PF4 ELISA 9 to 16 days after first ChAdOx1 vaccination. Their clinical presentation, lab results and treatment are summarised in Table 1. All patients recovered rapidly after non-heparin anticoagulation with (case 2-3) or without (case 1) intravenous immunoglobulins. All patients received subsequent COVID-vaccination with an mRNA-based vaccine without thrombocytopenia or symptoms. Anti-PF4- antibodies remained elevated in two patients after 3 months and in one out of three after more than 6 months, but HIMEA results for all follow-up tests became negative (Figure 1). Conclusion(s): We report good short-and long-term outcomes of three cases of VITT, including successful subsequent vaccination with an mRNA vaccine. Anti-PF4- antibodies can persist for at least several months. In contrast with the initial presentation, these persistent anti-PF4- antibodies did not trigger platelet activation in our patients. (Table Presented).

Decreased DNase activity in severe COVID-19 correlates with increased circulating NET burden C. Martens1

Background: COVID-19 represents a perfect storm for release of neutrophil extracellular traps (NETs) and resultant pathology from immunothrombosis. Levels of NETs in circulation are regulated by endogenous plasma DNases, which have been shown to be reduced in various diseases including myocardial infarction and sepsis. We previously reported elevated NET biomarkers in admission samples from our first wave study cohort. Aim(s): To characterize DNase activity and biomarkers of released NETs in the context of COVID-19 immunothrombosis. Method(s): With ethical permission and informed consent, we prospectively collected citrated platelet-poor plasma samples from patients admitted to the COVID ward (55 patients) or intensive care unit (216 patients) from March 2020-December 2021 as part of the COntAGIouS trial at UZ Leuven in Belgium (NCT04327750), with special attention paid to sample preparation and storage to preserve NET fragments and DNase activity. Consecutive samples were obtained within 48 hours of admission, between days 6-8, and upon hospital or ICU discharge. Analysis was batch-performed for MPO, MPO-DNA, PF4, sP-selectin, citrullinated histones, DNase activity, VWF:Ag, and FVIII:Ag levels. Result(s): In ICU patients, MPO, VWF, sP-selectin, and NET biomarkers were elevated throughout hospitalization, peaking at day 6-8 after admission, whereas PF4 and FVIII remained highly elevated through the time of ICU discharge. DNase activity was decreased in admission samples, normalized at day 6-8, and strongly increased at the time of discharge, indicating a potential compensatory mechanism. DNase activity was negatively correlated with MPO-DNA values (r = -0.29, p = 0.0013). sPselectin and NET levels were significantly higher in admission samples for patients who experienced a thrombotic event in the period during hospitalization, including pulmonary embolism, DVT, myocardial infarction, and/or stroke. Conclusion(s): Elevated NET levels and decreased DNase activity in plasma are correlated in severe COVID-19, together with elevated markers of thrombotic risk. Approaches to restore DNase activity in plasma may be beneficial in COVID-19-associated immunothrombosis.

A multicenter randomized clinical trial of modulation of host thromboinflammatory response in hospitalized patients with COVID-19: The DAWn-Antico study

Background: Thromboinflammation in severe COVID-19 is associated with disease severity and inferior outcome. Evidence suggests that the kallikrein pathway potentially plays a vital role in COVID-19 associated thromboinflammation as it both activates downstream inflammatory pathways and contact-mediated coagulation. Aim(s): To investigate whether modulation of this pronounced thromboinflammatory response can improve outcomes in hospitalized patients with severe COVID-19. Method(s): This multicenter randomized clinical trial was approved by the ethics committee and supported by the KU Leuven COVID-19 fund and Research Foundation Flanders (FWO). After informed consent, eligible patients were 1:2 randomized to receive standard of care (SOC) or SOC plus study intervention (figure 1). The intervention consisted of off-label -kallikrein-inhibiting -aprotinin combined with low molecular weight heparin (LMWH). Additionally, patients with predefined hyperinflammation were treated with the interleukin-1 receptor antagonist anakinra. The primary endpoint was time to sustain a 2-point improvement in the WHO ordinal scale for clinical status. Result(s): Three hospitals in Belgium included 102 patients (35 SOC vs. 67 intervention). Twenty-five patients from the intervention group (37%) were treated with anakinra. Patients had elevated D-dimers (mean 1012.4 mug/L;SD 991.9 mug/L) and C-reactive protein (mean 81.4 mg/L;SD 59.6 mg/L) at admission confirming baseline activation of coagulation and inflammatory pathways. During hospitalization, 37% of patients were admitted to the ICU (29% SOC vs. 42% intervention), and 20% needed invasive ventilation (12% SOC vs. 25% intervention). The intervention did not affect the time to sustained clinical improvement or hospital discharge (figure 2), nor secondary clinical endpoints. Except for D-dimers at day 3, there was no significant C-reactive protein or D-dimer reductions. There were no differences in treatment-related adverse events. Conclusion(s): In hospitalized COVID-19 patients, additional modulation of thromboinflammation with high-dose aprotinin and LMWH with or without anakinra was feasible and safe but did not improve clinical nor biochemical outcomes.

The kallikrein-kinin system in bronchoalveolar lavage fluid from patients with COVID-19

Background : Markers of both inflammation and coagulation are linked to clinical outcome in coronavirus disease 2019 (COVID-19). Binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the angiotensin-converting enzyme 2 receptor, which is involved in kinin breakdown, interferes with the kallikreinkinin pathway. This could result in increased vascular permeability, fluid excess in the lungs and pulmonary edema. Furthermore, the kallikrein-kinin pathway links coagulation and inflammation through its interactions with the contact activation pathway of coagulation via factor XII and with neutrophil extracellular traps (NETs). These insights could help to explain the clinical presentation of COVID-19 pneumonia with pulmonary coagulopathy and the high incidence of thromboembolic complications in COVID-19. Aims : Given the lack of clinical evidence to support this hypothesis, we studied the kallikrein-kinin system in bronchoalveolar lavage (BAL) fluid. Methods : In BAL fluid samples from patients with or without COVID-19, we performed in-depth analyses of kinin peptides (bradykinin, Lys-bradykinin, Lys-bradykinin-(1-8), bradykinin-(1-8), bradykinin-(1-7), and bradykinin-(1-5)) using a liquid chromatography with tandem mass spectrometry assay, along with measurements of plasma and tissue kallikrein hydrolytic activity and myeloperoxidase (MPO)-DNA complexes as a biomarker for NETs. Informed consent and ethical approval were obtained. Results : We observed higher levels of the most downstream kinin peptide bradykinin-(1-5) (Figure 1), higher tissue kallikrein activity (Figure 2), and higher levels of MPO-DNA complexes (699.0 ng/mL [66.0-142621.0], median [range], n = 21 vs 70.5 [9.9-960.0], n = 19;P < 0.001) in BAL fluid from patients with COVID-19 compared to those in BAL fluid from patients without COVID-19. Conclusions : Our data support the hypothesis that SARS-CoV-2 induces dysregulation of the kallikrein-kinin system, which contributes to thromboinflammation in COVID-19. These findings encourage the investigation of drugs that target the kallikrein-kinin system as a potential treatment option for patients with COVID-19.

Weight adjusted prophylactic to intermediate dosed thromboprophylaxis in COVID-19

Background : COVID-19 is frequently associated with venous thromboembolism (VTE), and the use of thromboprophylaxis has been suggested to improve hospitalized patients ' outcomes. We, therefore, intensified our thromboprophylactic protocol starting March 31st. Aims : We aimed to validate the implementation of an intensified thromboprophylactic protocol by reporting VTE incidence and safety while awaiting randomized controlled trials. Methods : On March 31st, 2020, we implemented an intensified thromboprophylactic protocol based on weight and disease severity (50 IU anti-Xa LMWH/kg, once daily at the ward, twice daily at the intensive care unit (ICU)). ICU patients were monitored daily with anti-Xa serum levels. Full therapeutic doses were restricted to patients with a prior indication for therapeutic anticoagulation or confirmed VTE. As early reports demonstrated high VTE incidence, screening with duplex ultrasound became standard of care in our center as soon as logistically possible. We excluded patients with a prior indication for therapeutic anticoagulation and incidental findings of COVID-19 for analysis. The ethical committee has approved this observational study. Results : We analyzed 412 symptomatic and confirmed Covid-19 cases, of which 116 were admitted to the ICU. All symptomatic VTE cases were reported, and 20% of all patients (38% of ICU patients) received screening with venous ultrasound. In 219 patients who received the standard dose of LMWH, 16 patients (7.3%) had VTE, 10 of which were symptomatic (4.6%) (Figure 1). In 193 patients who received intensified thromboprophylaxis, there were no symptomatic VTE cases, three incidental DVT cases (1.6%), and one incidental pulmonary embolism (0.5%). Interestingly, rates of major bleeding were low (Figure 2). Conclusions : In a large cohort of hospitalized patients with COVID-19, we report no symptomatic VTE after implementing systematic thromboprophylaxis with weight-adjusted prophylactic (ward) to intermediate (ICU), but not therapeutic doses of LMWH. This strategy was associated with a low risk of major bleeding.

Repolarization abnormalities at admission predict 30-day outcome in COVID-19

Introduction: COVID-19 can be related with a poor clinical outcome. ECG abnormalities in COVID-19 have been widely described, but literature on the predictive value of a 12-lead ECG at hospital admission and normalization of these abnormalities after infection is limited. Purpose: To describe the predictive value of ECG abnormalities on admission and after recovery of COVID-19. Methods: After informed consent patients older than 18 years admitted with COVID-19 between March and July 2020 were included in a prospective registry. Diagnosis was confirmed by PCR-assay or based on suggestive clinical and radiological presentation. Demographic and clinical data were collected by review of the electronic medical record. All ECGs from admission until last follow-up were assessed lead by kead for repolarization abnormalities. The index ECG was defined as first ECG available after admission, a post-COVID ECG was obtained after hospital discharge in the absence of acute pathology. Minor abnormalities included iso-electric T-waves and ST-depression ≤2 mm. Major abnormalities were ST-depression >2 mm, ST-elevation, biphasic T-waves and T-wave inversion. Myocardial regions were defined as anterior (V1-V4), lateral (I, aVL, V5, V6) and inferior (II, III, aVF). Patients with a ventricular pacemaker were excluded. Results: A total of 283 patients were included, median age 65 years and 64.7% were male. The 30-day mortality rate was 20.5%. In 96.8% of patients an ECG was available within 48 hours after admission. Repolarization abnormalities were observed in 48.8% of patients. In 27.2% this was limited to minor abnormalities. Abnormal repolarization was related to age, cardiovascular medical history, renal function, high-sensitive troponin-T and NT-proBNP levels. There were no significant differences in clinical presentation, ICU admission, need for ventilation or ECMO. On Kaplan-Meier analysis (figure) the presence (p < 0.001) and the extent of repolarization abnormalities (p < 0.001) were associated with 30-day mortality. Forward Cox regression modelling identiefied age (per year, HR 1.07, 95% CI 1.05-1.09), history of heart failure (HR 2.12, 95% CI 1.08-4.52), neurological disorders (HR 2.47, 95% CI 1.36-4.51), active oncological disease (HR 2.13, 95% CI 1.01-4.50) and the extent of repolarization abnormalities (per region, HR 1.37, 95% CI 1.05-1.79) as independent predictors. A post-COVID ECG was available in 172 patients (60.8%), the median time between index and post-COVID ECG was 63.3 days. There was 1 new first-degree AV-block and 1 new RBBB. Repolarization abnormalities were present in 32 patients (11.3%);however, only 3 patients (1.7%) had new abnormalities, 2 of whom died during further follow-up. Conclusions: The extent of repolarization abnormalities on an ECG at admission for COVID-19 is an independent predictor of 30-day mortality. New ECG abnormalities after COVID-19 infection are uncommon but may be associated with adverse outcome.

A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study.

BACKGROUND: The peak of the global COVID-19 pandemic has not yet been reached, and many countries face the prospect of a second wave of infections before effective vaccinations will be available. After an initial phase of viral replication, some patients develop a second illness phase in which the host thrombotic and inflammatory responses seem to drive complications. Severe COVID-19 disease is linked to high mortality, hyperinflammation, and a remarkably high incidence of thrombotic events. We hypothesize a crucial pathophysiological role for the contact pathway of coagulation and the kallikrein-bradykinin pathway. Therefore, drugs that modulate this excessive thromboinflammatory response should be investigated in severe COVID-19. METHODS: In this adaptive, open-label multicenter randomized clinical trial, we compare low molecular weight heparins at 50 IU anti-Xa/kg twice daily-or 75 IU anti-Xa twice daily for intensive care (ICU) patients-in combination with aprotinin to standard thromboprophylaxis in hospitalized COVID-19 patients. In the case of hyperinflammation, the interleukin-1 receptor antagonist anakinra will be added on top of the drugs in the interventional arm. In a pilot phase, the effect of the intervention on thrombotic markers (D-dimer) will be assessed. In the full trial, the primary outcome is defined as the effect of the interventional drugs on clinical status as defined by the WHO ordinal scale for clinical improvement. DISCUSSION: In this trial, we target the thromboinflammatory response at multiple levels. We intensify the dose of low molecular weight heparins to reduce thrombotic complications. Aprotinin is a potent kallikrein pathway inhibitor that reduces fibrinolysis, activation of the contact pathway of coagulation, and local inflammatory response. Additionally, aprotinin has shown in vitro inhibitory effects on SARS-CoV-2 cellular entry. Because the excessive thromboinflammatory response is one of the most adverse prognostic factors in COVID-19, we will add anakinra, a recombinant interleukin-1 receptor antagonist, to the regimen in case of severely increased inflammatory parameters. This way, we hope to modulate the systemic response to SARS-CoV-2 and avoid disease progressions with a potentially fatal outcome. TRIAL REGISTRATION: The EU Clinical Trials Register 2020-001739-28 . Registered on April 10, 2020.

Anticoagulant therapy in COVID-19 critically ill: Should we go for more?

Critically ill COVID-19 patients often develop a severe pro-thrombotic milieu, as reflected by the markedly increased d-dimer levels. Several cohort studies have reported high rates of thrombotic complications, including deep venous thrombosis (DVT) and pulmonary embolism (PE), myocardial infarction, stroke and microvascular thrombosis. Accordingly, COVID-19 patients who are hospitalized either at a normal, non-intensive care unit (ICU) or at the ICU need to receive appropriate dosages of anticoagulant therapy to prevent or treat these thrombotic complications. This manuscript summarizes the institutional guidance for the antithrombotic prophylaxis and treatment of VTE as outlined by a multidisciplinary team of experts during the first weeks of the COVID-19 pandemic in Europe. Controlled studies are needed to verify the optimal anticoagulation for both prophylaxis and treatment.