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Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19

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Science Translational Medicine  18 Nov 2020:
Vol. 12, Issue 570, eabd3876
DOI: 10.1126/scitranslmed.abd3876
  • Fig. 1 aPL antibodies, NET release, and renal function.

    Serum samples were obtained from 172 patients hospitalized with COVID-19. (A and B) Patients were divided into two groups on the basis of whether their serum samples were positive (+) or negative (−) for the presence of aPL antibodies (positivity was based on the manufacturer’s threshold). Shown is the amount of calprotectin in serum, a measure of neutrophil activation (A), and the clinical estimated glomerular filtration rate (eGFR) (B) for the two groups. (C and D) Patients were divided into two groups on the basis of whether their serum samples were positive (+) or negative (−) for the presence of aPS/PT antibodies (IgG and IgM considered together); the manufacturer’s thresholds were used to determine positivity. Shown is the amount of calprotectin (C) and the eGFR (D) for the two groups. Groups were analyzed by an unpaired t test: *P < 0.05, **P < 0.01, and ***P < 0.001. Horizontal black bars represent the mean. For patients who had serum samples available at multiple time points, only the first available serum sample was used in this analysis.

  • Fig. 2 COVID-19 patient IgG promotes NET release from normal neutrophils in vitro.

    (A) Control neutrophils were isolated from healthy individuals and cultured in the presence of human IgG (10 μg/ml) for 3 hours. IgG fractions were obtained from patients with COVID-19 who were or were not positive for aPL antibodies (aPS/PT or aβ2GPI as indicated), and from patients with antiphospholipid syndrome (APS) or catastrophic APS (CAPS). NET release was measured by the enzymatic activity of myeloperoxidase (MPO) after solubilization of NETs with micrococcal nuclease; fold increase is plotted relative to unstimulated neutrophils (no stim). Data are derived from four independent experiments. Comparisons were to the unstimulated group by one-way ANOVA with correction for multiple comparisons by Dunnett’s method: *P < 0.05, **P < 0.01, ***P < 0.001. (B) Representative images show released NETs, indicated by yellow arrows. DNA, blue; neutrophil elastase, green. Scale bars, 100 μm.

  • Fig. 3 IgG from patients with COVID-19 potentiates thrombosis in mice.

    (A) Schematic shows thrombus initiation in the inferior vena cava (IVC) of mice by local electrolysis leading to free radical generation and activation of the endothelium. (B and C) Mice were administered IgG from healthy individuals (control), from patients with COVID-19 who had high or low aPS/PT antibodies, or from patients with catastrophic APS (CAPS). Just before intravenous administration of IgG, mice were subjected to local electrolysis in the IVC. Thrombus length (B) and weight (C) were determined 24 hours after IgG injection. Scatter plots with individual data points (each point represents a single mouse) are presented. (D) Shown are photographs of representative thrombi from the experiments presented in (B) and (C). The rulers are measuring thrombi in millimeters. (E) Serum samples from mice in the experiments presented in (B) and (C) were tested for NET remnants measured by an ELISA that detected myeloperoxidase (MPO)–DNA complexes. Scatter plots with individual data points (each point represents a single mouse) are presented. OD, optical density. (F) Schematic shows thrombus initiation in the IVC of mice by a stenosis that was induced via placement of a fixed suture over a spacer that was subsequently removed. (G and H) Mice were treated intravenously with IgG from a healthy individual (control) or from a patient with COVID-19 with high aPS/PT antibodies. Just before intravenous administration of IgG, stenosis was induced. Twenty-four hours later, thrombus length (G) and weight (H) were determined. Scatter plots with individual data points (each point represents a single mouse) are presented. (I) Shown are photographs of representative thrombi from the experiments presented in (G) and (H). (J) Serum samples from mice in the experiments presented in (G) and (H) were tested for NET remnants measured by an ELISA that detected MPO-DNA complexes. Scatter plots with individual data points (each point represents a single mouse) are presented. Horizontal black bars represent the mean. Comparisons were by either one-way ANOVA with correction for multiple comparisons by Dunnett’s method (B, C, and E) or unpaired t test (G, H, and J): *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Table 1 Prevalence of antiphospholipid antibodies in serum from patients with COVID-19 (n = 172).

    The manufacturer’s cutoff: aCL IgG, IgM, IgA = 20 IgG, IgM, IgA phospholipid units; aβ2GPI IgG, IgM, IgA = 20 standard IgG, IgM, IgA units; aPS/PT IgG, IgM = 30 IgG, IgM phosphatidylserine units; aPL antibody, antiphospholipid autoantibodies; aCL, anticardiolipin antibodies; aβ2GPI, anti–β2 glycoprotein I antibodies; aPS/PT, anti-phosphatidylserine/prothrombin antibodies.

    aPL antibodyNumber of positive
    (manufacturer’s cutoff)
    %Number of positive
    (titer ≥40 units)
    %
    aCL IgG84.721.2
    aCL IgM3923137.6
    aCL IgA63.510.58
    2GPI IgG52.931.7
    2GPI IgM95.274.1
    2GPI IgA74.131.7
    aPS/PT IgG42242112
    aPS/PT IgM31182112
    Any positive aPL89525230
  • Table 2 Correlation of antiphospholipid antibodies with clinical and laboratory variables in patients with COVID-19.

    Thiry-six patients had serum samples from multiple time points; for those patients, only the first available serum sample was used for determining correlations. ns, not significant; NETs, neutrophil extracellular traps; MPO, myeloperoxidase.

    aPL score
    (modified)
    aCL IgGaCL IgM2GPI IgG2GPI IgMaPS/PT IgGaPS/PT IgM
    SpearmanrPrPrPrPrPrPrP
    Clinical and laboratory variables
    SpO2/FiO2−0.051ns−0.16*−0.19*−0.10ns−0.022ns−0.11ns−0.16*
    C-reactive
    protein
    0.031ns0.15ns0.17*0.075ns−0.040ns0.058ns0.16*
    D-dimer0.087ns0.092ns0.24**0.041ns0.000ns0.005ns0.037ns
    Platelet
    count
    0.17*0.095ns0.29****0.17*0.11ns−0.009ns0.23**
    Neutrophil
    count
    0.10ns0.13ns0.19*0.047ns0.041ns−0.008ns0.096ns
    Calprotectin0.26***0.29****0.28***0.11ns0.090ns0.25***0.23**
    NETs (MPO/
    DNA)
    0.18*0.16*0.25***0.20**0.13ns0.033ns0.23**

    *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

    Supplementary Materials

    • stm.sciencemag.org/cgi/content/full/scitranslmed.abd3876/DC1

      Table S1. Demographic and clinical characteristics of the COVID-19 patient cohort.

      Table S2. Prevalence of aPL antibodies in serum from patients with COVID-19 (n = 172) based on the first available sample.

      Fig. S1. Testing for various aPL antibodies according to day of hospitalization.

      Fig. S2. Lack of association between aPS/PT antibodies and aPS antibodies in serum from patients with COVID-19.

      Fig. S3. A positive aPL antibody test is associated with greater neutrophil activation and worse kidney function.

      Fig. S4. Nadir eGFR in patients with COVID-19 with or without a prior history of renal disease.

      Fig. S5. aPL antibody status as a predictor of oxygenation efficiency.

      Fig. S6. aPL antibody status as a predictor of peak troponin and D-dimer.

      Fig. S7. D-dimer plasma concentrations in patients with or without obesity.

      Fig. S8. Determination of purity of IgG fractions from patients with COVID-19.

      Fig. S9. Dipyridamole suppresses IgG-induced NET release.

      Fig. S10. IgG from patients with COVID-19 does not potentiate thrombin generation in cell-free pooled normal plasma.

      Fig. S11. Measurement of citrullinated histone H3 in mouse thrombi.

      Data file S1. NET release measured by MPO activity.

      Data file S2. Thrombus length and weight in mice.

      Data file S3. MPO-DNA in mice (stenosis and electrolysis models).

      Data file S4. Thrombus length and weight in stenosis mouse model.

    • The PDF file includes:

      • Table S1: Demographic and clinical characteristics of COVID-19 patient cohort
      • Table S2: Prevalence of aPL antibodies in serum from COVID-19 patients (n=172) based on the first-available sample.
      • Fig S1: Testing for various aPL antibodies according to day of hospitalization.
      • Fig S2: Lack of association between aPS/PT antibodies and aPS antibodies in serum from COVID-19 patients.
      • Fig S3: A positive aPL antibody test is associated with greater neutrophil activation and worse kidney function.
      • Fig S4: Nadir eGFR in COVID-19 patients with or without a prior history of renal disease.
      • Fig S5: aPL antibody status as a predictor of oxygenation efficiency.
      • Fig S6: aPL antibody status as a predictor of peak troponin and D-dimer.
      • Fig S7: D-dimer plasma concentrations in patients with or without obesity.
      • Fig S8: Determination of purity of IgG fractions from COVID-19 patients.
      • Fig S9: Dipyridamole suppresses IgG-induced NET release.
      • Fig S10: IgG from COVID-19 patients does not potentiate thrombin generation in cell-free pooled normal plasma.
      • Fig S11: Measurement of citrullinated histone H3 in mouse thrombi.

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      Other Supplementary Material for this manuscript includes the following:

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