key: cord-0735936-pxvc3k1k
authors: Nazy, Ishac; Jevtic, Stefan D.; Moore, Jane C.; Huynh, Angela; Smith, James W.; Kelton, John G.; Arnold, Donald M.
title: Platelet‐activating immune complexes identified in critically ill COVID‐19 patients suspected of heparin‐induced thrombocytopenia
date: 2021-03-14
journal: J Thromb Haemost
DOI: 10.1111/jth.15283
sha: da33d2d3320d9f331ae90c2067469868ff9f1066
doc_id: 735936
cord_uid: pxvc3k1k

BACKGROUND: Thrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID‐19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID‐19 patients may be suspected of heparin‐induced thrombocytopenia (HIT), given similar clinical features. OBJECTIVES: We investigated the presence of platelet‐activating anti‐platelet‐factor 4 (PF4)/heparin antibodies in critically ill COVID‐19 patients suspected of HIT. PATIENTS/METHODS: We tested 10 critically ill COVID‐19 patients suspected of HIT for anti‐PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti‐human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS‐CoV‐2 antibodies were measured using an in‐house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin‐13 Domain (ADAMTS13) activity and the presence of anti‐ADAMTS13 antibodies. RESULTS: Heparin‐induced thrombocytopenia was excluded in all samples based on anti‐PF4/heparin antibody and SRA results. Notably, six COVID‐19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)‐mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS‐CoV‐2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID‐19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy. CONCLUSIONS: Our study identifies platelet‐activating ICs as a novel mechanism that contributes to critically ill COVID‐19.

ured along with A Disintegrin And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) activity and the presence of anti-ADAMTS13 antibodies.

Results: Heparin-induced thrombocytopenia was excluded in all samples based on anti-PF4/heparin antibody and SRA results. Notably, six COVID-19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)-mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID-19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy.

Our study identifies platelet-activating ICs as a novel mechanism that contributes to critically ill COVID-19.

Coronavirus disease 2019 (COVID-19) is a highly transmissible viral infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a global pandemic. 1, 2 Critically ill patients with COVID-19 can have prominent coagulation abnormalities, including mild thrombocytopenia and diffuse arterial and venous thrombosis. [3] [4] [5] [6] While the mechanism is unclear, the clinical presentation shares many features of heparin-induced thrombocytopenia (HIT), namely thrombocytopenia and thrombosis during critical illness. 7 Heparin-induced thrombocytopenia is a prothrombotic disorder that typically presents as thrombocytopenia related to heparin treatment and is associated with a high risk of thrombosis. HIT is caused by IgG-specific antibodies targeting platelet-factor 4 (PF4) that form immune complexes (IC) and cause platelet activation through the FcγRIIA receptor. 8, 9 Recent reports have speculated that ICs also contribute to the pathobiology of severe COVID-19. 10 Retrospective analysis has shown that anti-PF4/heparin antibodies are identified more frequently in COVID-19 hospitalized patients compared to the general population. 11 However, it is unclear if these contribute to platelet activation. In this report, we describe platelet-activating ICs in critically ill COVID-19 patients suspected of HIT. These ICs are not formed from IgG-specific anti-PF4/heparin antibodies and occur in conjunction with elevated von Willebrand factor (VWF) and anti-COVID-19 antibodies. Platelet-activating ICs may thus be an additional mechanism contributing to severe COVID-19. Testing for anti-PF4/heparin antibodies was done using an anti-PF4/heparin enzymatic immunoassay (EIA, LIFECODES PF4 enhanced assay; Immucor GTI Diagnostics) for IgG, IgM, and IgA PF4-heparin antibodies. If positive, an in-house, IgG-specific anti-PF4/heparin EIA was performed. 12 All samples were then tested for functional platelet activation in the serotonin release assay (SRA) with heparin (0, 0.1, 0.3, and 100 U/ml) as previously described. An anti-human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA engagement. 13 Testing for IgG-, IgA-, and IgM-specific antibodies against the receptor-binding domain (RBD) and spike protein of SARS-CoV-2 virus was done using our in-house ELISA, which has a sensitivity of 97.1% and a specificity of 98%. 14 VWF antigen levels were assessed by the HemosIL von Willebrand Factor antigen automated chemiluminescent immunoassay (Instrumentation Laboratory). VWF activity was measured using the Innovance VWF Ac Assay (OPHL03; Siemens). A Disintigren And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) metalloproteinase activity and anti-ADAMTS13 antibody were tested for all patients, as previously described, to determine whether VWF changes were related to ADAMTS13 activity. 15 All testing was done using plasma from blood collected in sodium citrate. Data represent mean ± standard error of the mean and are analyzed by Student's t test. Results are considered significant at P < .05.

Ethics Board.

Blood samples were referred to the MPIL for HIT testing from 10 critically ill COVID-19 patients ( Table 1 ). All patients developed thrombocytopenia and had been exposed to heparin. Four patients developed thrombosis, one did not, and data were unavailable for the remaining five. Initial testing for IgG, IgA, and

antigen-antibody complex, COVID-19, heparin-induced thrombocytopenia, thrombocytopenia, thrombosis Essentials • Severe coronavirus disease 19 (COVID-19) can resemble heparin-induced thrombocytopenia (HIT).

• COVID-19 patients referred for HIT testing were found to be negative for HIT.

• Severe COVID-19 sera demonstrate immune complexes that activate platelets through FcγRIIA signalling.

• Severe COVID-19 patients feature increased von Willebrand factor antigen and activity unrelated to ADAMTS13 antibodies.

IgM anti-PF4/heparin antibodies was negative in five samples (OD 405 nm < 0.4), thus excluding HIT (Table 2) . IgG-specific anti- Immune complexes are known to trigger endothelial cell activation in diseases such as HIT and lupus vasculitis with subsequent VWF release. 17, 18 Similarly, increased VWF release is a hallmark of COVID-19 coagulopathy and has been correlated with mortality. 19, 20 We therefore tested VWF antigen in our samples, and found markedly elevated levels (range 2.1-10.1 U/ml; mean = 5.9 U/ml; normal range 0.5-1.5 U/ml) compared to healthy controls (range 0.9-1.5 U/ml; mean = 1.2 U/ml; Figure 2A ). Ristocetin activity, as a marker of VWF activity, was also significantly increased (range 2.2-8.52 U/ml; mean 4.6 U/ml; normal range 0.8-1.8 U/ml) compared to healthy controls (range 0.53-1.67 U/ ml, mean = 1.18 U/ml; Figure 2B ). These increases were not significantly different between COVID-19 patients with or without immune complexes. These data confirm elevated VWF in our samples, as has previously been shown.

To determine if this VWF increase was related to ADAMTS13 metalloproteinase function, we tested for ADAMTS13 activity and the presence of anti-ADAMTS13 antibody. ADAMTS13 activity was moderately reduced (<40%) in all COVID-19 samples but only severely deficient (<10%) in one ( Figure 2C ). Only one sample contained anti-ADAMTS13 antibody, which did not correspond to severe ADAMTS13 deficiency ( Figure 2D ). The elevated VWF is therefore not secondary to severe ADAMTS13 reduction and also confirms that anti-ADAMTS13 antibodies are not responsible for the platelet activation.

We have described our findings in critically ill COVID-19 samples ill COVID-19 patients. 21 It is important to note that the plateletactivating ICs in our cohort were identified using a buffer control (i.e., no addition of heparin). This step should thus be included in all SRA testing, as it can both identify atypical HIT and non-HIT platelet-activating ICs. 22 Together, these observations highlight a novel mechanism for COVID-19 ICs in severe illness.

We also confirm that this mechanism is separate from previously noted coagulopathic changes, namely involving the VWF--ADAMTS13 axis. 19 Prior studies have shown that VWF antigen levels and reduction of ADAMTS-13 activity correlate with disease severity. 23 These changes are also associated with increased mortality, likely secondary to microthrombotic complications. We demonstrate that our observations are not secondary to severe ADAMTS13 activity reduction (<10%) or the presence of anti-ADAMTS13 antibody, as described in thrombotic thrombocytopenic purpura (TTP).

This pattern is more consistent with a secondary thrombotic microangiopathy and is likely compounded by the presence of plateletactivating ICs. 24, 25 One limitation of this study is the inability to characterize the specificity of these platelet-activating ICs. It is possible that the ICs are composed of COVID-19 virus--antibody complexes, similar to that seen with H1 N1 viral infection. 26 We thus propose a model whereby certain critically ill COVID-19

patients feature a novel IC-mediated thrombotic microangiopathy that is characterized by significant platelet activation. These ICs can produce a highly prothrombotic state resembling HIT but with unique platelet-activating properties.

Funding support for this work was provided by a grant from the 

The authors declare no competing financial interests. ICs. C, ADAMTS13 activity is moderately reduced (10%-40%) in COVID-19 compared to healthy controls without an associated increase in (D) anti-ADAMTS13 antibody. One COVID-19 sample was positive for presence of an anti-ADAMTS13 antibody, but this sample did not correspond to a severe reduction in ADAMTS13 activity. Therefore, none of the COVID-19 samples meet criteria for TTP and are more in keeping with enhanced endothelial activation secondary to a secondary thrombotic microangiopathy. *p < .05, **p < .01, 2-tailed, unpaired Student's t-test

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