key: cord-1032015-6reou0rx
authors: Nurmohamed, Nick S.; Collard, Didier; Reeskamp, Laurens F.; Kaiser, Yannick; Kroon, Jeffrey; Tromp, Tycho R.
title: Lipoprotein(a), venous thromboembolism and COVID-19: A pilot study
date: 2021-12-21
journal: Atherosclerosis
DOI: 10.1016/j.atherosclerosis.2021.12.008
sha: c5ac98059024fb38e119d081b418aeda0fd8e091
doc_id: 1032015
cord_uid: 6reou0rx

BACKGROUND AND AIMS: Thrombosis is a major driver of adverse outcome and mortality in patients with Coronavirus disease 2019 (COVID-19). Hypercoagulability may be related to the cytokine storm associated with COVID-19, which is mainly driven by interleukin (IL)-6. Plasma lipoprotein(a) [Lp(a)] levels increase following IL-6 upregulation and Lp(a) has anti-fibrinolytic properties. This study investigated whether Lp(a) elevation may contribute to the pro-thrombotic state hallmarking COVID-19 patients. METHODS: Lp(a), IL-6 and C-reactive protein (CRP) levels were measured in 219 hospitalized patients with COVID-19 and analyzed with linear mixed effects model. The baseline biomarkers and increases during admission were related to venous thromboembolism (VTE) incidence and clinical outcomes in a Kaplan-Meier and logistic regression analysis. RESULTS: Lp(a) levels increased significantly by a mean of 16.9 mg/dl in patients with COVID-19 during the first 21 days after admission. Serial Lp(a) measurements were available in 146 patients. In the top tertile of Lp(a) increase, 56.2% of COVID-19 patients experienced a VTE event compared to 18.4% in the lowest tertile (RR 3.06, 95% CI 1.61–5.81; p < 0.001). This association remained significant after adjusting for age, sex, IL-6 and CRP increase and number of measurements. Increases in IL-6 and CRP were not associated with VTE. Increase in Lp(a) was strongly correlated with increase in IL-6 (r = 0.44, 95% CI 0.30–0.56, p < 0.001). CONCLUSIONS: Increases in Lp(a) levels during the acute phase of COVID-19 were strongly associated with VTE incidence. The acute increase in anti-fibrinolytic Lp(a) may tilt the balance to VTE in patients hospitalized for COVID-19.

Almost two years after the first outbreak in Wuhan, China, Coronavirus disease 2019 is still driving one of the largest pandemics in the history of mankind. Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to a heterogeneous disease, varying from asymptomatic to acute respiratory distress syndrome requiring intensive care unit (ICU) admission. A hallmark of severe cases of COVID-19 is a cytokine storm, 1 with interleukin-6 (IL-6) mainly produced by macrophages and dendritic cells serving as a principal cytokine driving inflammation. 2 COVID-19 is also associated with an increased propensity for thrombotic complications. 3 Approximately 21% of all hospitalized COVID-19 patients are found to have symptomatic venous thromboembolism (VTE) despite the use of routine thrombosis prophylaxis, compared with 0.9-2.9% in highrisk non-COVID-19 patients using prophylaxis. [4] [5] [6] In severe COVID-19 patients admitted to the ICU, 31% of patients suffer from VTE, compared to 5% in COVID-19 patients not admitted to the ICU. 4 These observations imply that COVID-19 elicits hypercoagulability in hospitalized patients, the mechanism of which remains to be elucidated. Lipoprotein(a) [Lp(a)], a low-density lipoprotein (LDL) like particle covalently bound to an apolipoprotein(a) protein, has anti-fibrinolytic properties due to its homology with plasminogen while lacking the active protease domain, and has been linked to VTE incidence. 7 The LPA gene encoding for Lp(a) contains an IL-6 response element; 8 hence IL-6 can upregulate Lp(a) production, explaining why Lp(a) levels are increased in patients with elevated plasma IL-6. 9 In line, treatment with the IL-6 signaling inhibitor tocilizumab lowered Lp(a) levels by up to 30% in rheumatoid arthritis patients. 10 We hypothesized that Lp(a) elevation may act in concert with the acute inflammatory changes during COVID-19 to evoke a disproportionate increase in COVID-19 related J o u r n a l P r e -p r o o f thrombosis. To further evaluate this, we measured Lp(a), IL-6 and high-sensitivity C-reactive protein (CRP) in serial samples of 219 sequential COVID-19 patients admitted to the hospital.

The Amsterdam University Medical Centers (UMC) COVID-19 biobank is a prospective cohort study containing clinical data and material from patients admitted with COVID-19 in both academic hospitals of Amsterdam UMC, between March 24 and May 23 2020. All hospitalized patients ≥18 years with a positive SARS-CoV-2 polymerase chain reaction (PCR) test as well as those with a high clinical suspicion and unequivocal findings on CT-imaging without an alternative diagnosis were eligible for inclusion. 11 For the current analysis, COVID-19 patients were selected when a blood sample was available within 2 days of ward-and/or ICU-admission. Patients were treated according to the local protocol that included thromboprophylaxis, but did not include the use of remdesivir, hydroxychloroquine, azithromycin, convalescent plasma, corticosteroids or any other immunomodulatory therapy.

Thromboprophylaxis consisted of low-molecular weight heparin (fraxiparin 5700 IE), once daily for patients <100 kg and twice daily for patients >100 kg. As of April 3 2020, all ICUadmitted COVID-19 patients were also given the twice daily dose. Comprehensive clinical and outcome data were acquired from the CovidPredict cohort, as previously reported. 12 In short, this included daily reports, laboratory results and detailed clinical outcome data.

J o u r n a l P r e -p r o o f Patients or their legal representatives received written information about the study and were asked to give written informed consent for participation. If direct informed consent of patients was not feasible, patients could be included with a deferred consent procedure. In case of deferred consent, patients or their legal representatives were informed about the biobank as soon as possible. To ensure all patients willfully participated in the biobank and provide a possibility to opt out from the biobank, comprehensive information and an opt-out form was sent to the patients three months after discharge. This study was approved by the Medical Ethical Committees of both Amsterdam UMC hospitals.

Blood samples were routinely collected at the emergency department, ICU and hospital ward following hospitalization. Since we used biobank samples obtained in clinical practice, the number of blood withdrawal and time points of blood withdrawal varied across participants. The analysis was restricted to patients who had a first sample available within two days after admission. Plasma samples were stored at -80°C after centrifugation. After thawing, Lp(a) levels were determined using an immunoturbidimetric (mass) assay (Quantia Lp(a), Architect, Abbott). This assay is calibrated for 5-points of apo(a) isoforms, which minimizes isoform sensitivity. IL-6 and CRP levels were determined using multiplex immunoassays (Bio-Plex 200, Biorad).

The primary outcome was incidence of VTE during hospitalization. VTE was defined as an objectively confirmed diagnosis of distal or proximal extremity DVT, pulmonary embolism (PE), or venous thrombosis at other sites including catheter-related thrombosis.

Secondary outcomes were disease adverse outcomes defined as ICU admission and all-cause mortality during the first 21 days following hospital admission. All-cause mortality was defined as either mortality during admission or discharge for palliative care, either at home or at a palliative care facility. If the patient was discharged home from the hospital and no further follow-up data was available, we considered the patient to be event-free for the remaining study period.

Baseline characteristics are reported as mean ± standard deviation (SD) for normally distributed data, as medians with interquartile range (IQR) for non-normally distributed data, and as number with proportion for categorical data. Between-group differences were evaluated using the appropriate tests (Mann-Whitney U, Kruskal-Wallis, Chi-squared, respectively).

Baseline samples were defined as samples taken on the first or second day after admission. Baseline Lp(a), IL-6 and CRP levels were related to COVID-19 disease outcomes in a logistic regression analysis adjusted for age (continuous) and sex in COVID-19 patients. (Table 1) . One or more VTE events occurred in 67 (31%) patients admitted for COVID-19. Categorized by type of thrombosis, 31 (46%) of patients with a VTE developed an extremity DVT during admission, 42 (62%) patients experienced a pulmonary embolism and 11 (5%) patients had a VTE located elsewhere (Table 2) . During hospitalization, 121 of the 219 (56%) COVID-19 patients were admitted to the ICU and 54 (25%) patients died ( Table 2 ). 

In the entire cohort, baseline Lp(a), IL-6 and CRP levels were not associated with VTE incidence. Baseline IL-6 levels were associated with ICU admission (Supplemental Figure   3 ). The Kaplan Meier analysis showed a significant association between delta Lp(a) tertiles and VTE event free survival in the first 21 days after admission (p <0.001, Figure 3 ).

In addition, we compared COVID-19 patients who were admitted to the ICU or died following hospitalization to those who were not admitted to the ICU or who survived. As expected, higher delta Lp(a) levels were observed in the patients admitted to the ICU ( (Table 3 ). In all models, additional adjustment for delta CRP did not materially change the findings. There were no signs of multicollinearity; the VIFs were 1.20 and 1.38 for delta Lp(a) and delta IL-6, respectively.

J o u r n a l P r e -p r o o f

Lastly, we compared associations between the delta values of the three biomarkers during admission. Delta IL-6 was strongly correlated with delta Lp(a) (r = 0.44, 95% CI 0.30-0.56, p <0.001) and with delta CRP (r = 0.18, 95% CI 0.01-0.34, p=0.040). Conversely, delta CRP was not correlated with delta Lp(a) (p=0.580). In addition, there was a modest association between delta Lp(a) and delta D-dimer (r = 0.19; 95% CI 0.01-0.37; p=0.04).

This study demonstrates that in patients hospitalized with COVID-19 Lp(a) levels rise threefold during the hospitalization. Although Lp(a) levels upon admission were not predictive of VTE, the patients in the highest tertile of Lp(a) increase during admission, representing absolute increases of 20 mg/dL, experience a 56% incidence of VTE despite the use of VTE prophylaxis. In contrast, the change in the inflammatory biomarkers IL-6 and CRP was not associated with VTE incidence. Lp(a) is an anti-fibrinolytic particle, it is proinflammatory and also a known acute phase reactant. 13 Collectively, these data imply an integrated impact of Lp(a) in conjunction with the heightened inflammatory and prothrombotic state on VTE incidence in hospitalized COVID-19 patients.

Lp(a) levels are primarily genetically determined with more than 80% of plasma levels mediated by variations in kringle IV type 2 (KIV2) isoform number and various single nucleotide polymorphisms in the coding region of the LPA gene. 13 However, during acute inflammatory episodes, Lp(a) has been found to be transiently elevated and persisting for 3-4 months. This increase has been partly attributed to an IL-6 response element in the LPA gene. 14 Indeed, we substantiate that Lp(a) levels are upregulated in the course of a SARS-CoV-2 infection, with an up to three-fold increase of Lp(a) levels in 21 days following J o u r n a l P r e -p r o o f hospital admission. In line with this, we show that the day 4 peak of IL-6 levels, which likely already increase from baseline before admission, precedes the Lp(a) increase, supporting Lp(a) changes following the IL-6 increase (r = 0.44).

We also observed that patients with the highest Lp(a) increase during COVID-19 have a striking increase in VTE incidence (Central Illustration). In contrast, CRP increase, a wellknown acute phase reactant also regulated by IL-6, 15 was not associated with VTE incidence.

The discordant impact of Lp(a) versus IL-6 and CRP on VTE incidence may imply a direct pathophysiological role of Lp(a) elevation in eliciting a thrombogenic potential. A likely explanation for this effect is the high homology of apolipoprotein(a) to plasminogen, and this explanation was further supported by an association between Lp(a) increase and D-dimer increase, which has also been previously documented. 16 Apolipoprotein(a) lacks protease activity but has similar lysine binding activity to plasminogen allowing accumulation of Lp(a) to the same sites of exposed lysines, such as in denuded endothelium, where it may interfere with plasminogen activation. 17 In some but not all studies, Lp(a) has been shown to attenuate fibrinolysis, thereby promoting thrombus growth. 14 The pro-thrombotic effect of Lp(a) in clinical studies has been controversial, with substantiation in some observational studies with a preponderance of elevated Lp(a) levels in VTE patients compared with controls (Lp[a] elevation in 20% of VTE patients compared with 7% in controls; 95% CI 1.9-5.3, p <0.001). 18 However, genetic studies 19 and in vitro studies failed to support a causal role. 20 Lp(a) concentrations in the UK biobank were also not associated with thromboembolic events in COVID-19 patients. 21 Part of these inconsistent results may relate to the fact that Lp(a) by itself is not a pro-thrombotic factor; it is anti-fibrinolytic and thus predominantly may cause clot-propagation in pre-existing thrombi as 'second hit' agent. This 'second hit' mechanism could already be activated in relatively low Lp(a) levels, even below the ASCVD risk threshold of 50 mg/dl from the 2019 ESC/EAS guidelines. In the case of COVID-19, J o u r n a l P r e -p r o o f severe endothelial injury and ongoing active coagulation may be particularly sensitive to Lp(a) tipping the balance to clot propagation and clinical expression of VTE. Besides an antifibrinolytic effect, Lp(a) also activates pro-inflammatory pathways in endothelial cells and monocytes, which may further enhance the pro-thrombotic state. 22 Recent findings emphasized that oxidized phospholipids carried by Lp(a) may be the main cause of this proinflammatory effect. 23 Collectively, these data lend further support to a direct effect of Lp(a) on thrombogenesis, contributing to the disproportionately enhanced VTE incidence in COVID-19 patients hallmarked by Lp(a) increase during the infectious episode.

In absence of available interventions to reduce Lp(a), multiple studies have investigated the effect of tocilizumab, a monoclonal antibody inhibiting IL-6, in COVID-19

patients. To date, the majority of these studies have not shown a reduction in disease severity or complication rates. [24] [25] [26] [27] However, randomized administration of tocilizumab in 389 earlydisease COVID-19 patients showed a 44% reduction of the composite endpoint of mechanical ventilation and death. 28 Unfortunately, this study did not report the effect of either Lp(a) levels or VTE incidence in COVID-19 patients.

Current guidelines recommend routine VTE prophylaxis with low-molecular weight heparin (LMWH) in hospitalized patients with COVID-19, which we adhered to in the present study. 29 Our findings lend support to a marked contribution of Lp(a) increase in augmenting the pro-thrombotic state in COVID-19 patients. In view of the more than threefold increase in VTE incidence in the upper tertile compared to the lowest tertile of Lp(a) increase, a switch towards therapeutically dosed low-molecular weight heparin may be advised in patients in whom marked increases in Lp(a) are observed during hospitalization.

J o u r n a l P r e -p r o o f standard use of therapeutic anticoagulation is associated with increased risk of bleeding complications, without an overall reduction of VTE. 30 However, future studies are required to prospectively evaluate Lp(a) and VTE incidence in COVID-19 patients before using this regimen. It is tempting to speculate whether a single administration of the apo(a) antisense oligonucleotide pelacarsen, which reduces Lp(a) levels by more than 80%, 31 or lipid apheresis that can acutely remove Lp(a) and oxidized phospholipids, 32 may also be of clinical value to reduce VTE incidence in severe COVID-19 patients.

The number and timing of samples differed between patients and there were no pre-or post-admission Lp(a) levels available. Included patients were already in a disease state requiring hospital admission, and no Lp(a) levels in the healthy state nor long-term Lp(a) changes could be assessed in this study. Since every patient was in a different disease stage both at and during admission, we were unable to analyze these measurements using traditional statistical methods. These differences in disease stage also complicated analysis of baseline Lp(a) values. Therefore, we analyzed delta Lp(a) levels after log transformation, presuming maximal change in Lp(a) levels reflects the disease-mediated increase in Lp(a).

Due to the limited power of this study, we could not account for the exact temporality of changes in Lp(a). To adjust for the potential influence of duration of admission on the number of Lp(a) measurements and possible immortal time bias, we adjusted for the number of measurements in the logistic regression model. Second, the limited sample size could also explain the fact that we did not find a significant correlation between baseline Lp(a) levels and adverse outcomes. Larger studies routinely measuring Lp(a) serially in hospitalized J o u r n a l P r e -p r o o f patients will be required to substantiate these findings. Third, a relatively large proportion of the hospitalized study population was admitted to the ICU and almost half of patients were from non-Caucasian descent, which makes it difficult to extrapolate our results to the overall COVID-19 population and could explain the relatively low baseline Lp(a) levels.

Furthermore, the change in Lp(a) was limited to VTE and not COVID-related mortality, likely because VTE was diagnosed and treated before complications related to it developed.

Lastly, it has been appreciated that endothelial function and thrombosis are intimately involved in COVID-19 and clinicians are more attentive to anti-coagulation relative to the early phases of the pandemic when this study was performed. Whether this has reduced the incidence of Lp(a)-associated VTE remains to be determined.

In conclusion, the increase of Lp(a) levels during COVID-19 hospitalization is associated with a high incidence of VTE. Further studies are needed to determine whether interventions reducing Lp(a) elevation will be able to reduce the VTE incidence in severe COVID-19 patients. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

Cytokine release syndrome in severe COVID-19

COVID-19 cytokine storm: the interplay between inflammation and coagulation

Thromboembolism risk of COVID-19 is high and associated with a higher risk of mortality: A systematic review and meta-analysis

Antithrombotic Therapy for Patients With COVID-19: JACC State-of-the-Art Review

Es N van. Incidence of venous thromboembolism in hospitalized patients with COVID-19

Lipoprotein (a) as a cause of cardiovascular disease: Insights from epidemiology, genetics, and biology

5' Control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family

IL-6 blockade by monoclonal antibodies inhibits apolipoprotein (a) expression and lipoprotein (a) synthesis in humans

Effects of inhibition of interleukin-6 signalling on insulin sensitivity and lipoprotein (A) levels in human subjects with rheumatoid diseases

Society C-19 SRWG of the DR. CO-RADS: A Categorical CT Assessment Scheme for Patients Suspected of Having COVID-19-Definition and Evaluation

Cardiovascular risk factors and COVID-19 outcomes in hospitalised patients : a prospective cohort study

A Test in Context: Lipoprotein(a): Diagnosis, Prognosis, Controversies, J o u r n a l P r e -p r o o f 18 and Emerging Therapies

Its Potential Association with Thrombosis and Inflammation in COVID-19: a Testable Hypothesis

Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19

Association of D-dimer with Plaque Characteristics and Plasma Biomarkers of Oxidation-Specific Epitopes in Stable Subjects with Coronary Artery Disease

Lipoprotein(a) metabolism: potential sites for therapeutic targets

Increased lipoprotein (a) levels as an independent risk factor for venous thromboembolism

Potent reduction of plasma lipoprotein (a) with an antisense oligonucleotide in human subjects does not affect ex vivo fibrinolysis

SARS-CoV-2 infections: susceptibility to infections, ischemic heart disease and thromboembolic events

Atherogenic Lipoprotein(a) Increases Vascular Glycolysis, Thereby Facilitating Inflammation and Leukocyte Extravasation

Oxidized phospholipids on Lipoprotein(a) elicit arterial wall inflammation and an inflammatory monocyte response in humans

Efficacy of Tocilizumab in Patients Hospitalized with Covid-19

Group C-19 C. Effect of Tocilizumab vs Usual Care in Adults Hospitalized With COVID-19 and Moderate or Severe Pneumonia: A Randomized Clinical Trial

Effect of Tocilizumab vs Standard Care on Clinical Worsening in Patients Hospitalized With COVID-19 Pneumonia: A Randomized Clinical Trial

Tocilizumab in Hospitalized Patients with Severe Covid-19

Tocilizumab in Patients Hospitalized with Covid-19 Pneumonia

VTE in Patients With Coronavirus Disease 2019: CHEST Guideline and Expert Panel Report

AntiCoagulaTIon cOroNavirus -ACTION

Lipoprotein(a) Reduction in Persons with Cardiovascular Disease

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia1