Relevance of anti-platelet factor 4/heparin antibodies and platelet activation in systemic inflammatory diseases and thrombosis disorders: insight from the COVID-19 pandemic

Abstract

Background: The increased interest in anti-platelet factor 4 (PF4)-heparin complex (anti-PF4/H) antibodies following the COVID-19 pandemic has established them as crucial players in immunothrombosis.

Objectives: We aimed to investigate the involvement of anti-PF4/H antibodies during COVID-19 and after vaccination, particularly in patients with systemic inflammatory disease (SID).

Methods: This retrospective study analyzed the presence of anti-PF4/H antibodies and their ability to induce platelet activation in COVID-19 patients with and without suspected heparin-induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT) patients, and in controls and SID patients following COVID-19 vaccination.

Results: No significant increase in anti-PF4/H antibody levels was observed during COVID-19 regardless of disease severity. Despite a 2-fold increase in HIT suspicion observed during the pandemic, there was no corresponding increase in HIT diagnoses. Additionally, no significant increase in anti-PF4/H levels was noted after vaccination, even in SID patients. None of the positive anti-PF4/H antibodies detected in COVID-19 or vaccination cohorts induced platelet activation, measured by soluble P-selectin levels and flow cytometry-based on platelet microvesicle generation. Finally, in VITT patients, unlike in HIT patients, anti-PF4/H levels were strongly associated with platelet microvesicle assay and moderately with soluble P-selectin levels.

Conclusion: Our study found no significant increase in anti-PF4/H antibodies in COVID-19 or after vaccination, including in SID patients. However, in VITT patients, but not in HIT patients, these antibodies were correlated with platelet activation. This finding suggests that anti-PF4/H antibodies play a different role in the pathophysiology of VITT but that their interest is limited outside clear contexts of HIT/VITT suspicion.

Keywords: COVID-19; antibodies; connectivite tissue disease; thrombocytopenia; vaccine; vasculitis.

Figure 1

Frequency of positive immunoglobulin G (IgG) heparin–platelet factor 4 complex antibodies (anti-PF4/H) in patients with COVID-19 and association to circulating soluble P-selectin (sP-selectin). IgG anti-PF4/H (optical density; OD, 450 nm) results are considered positive with an OD > 0.50 (dashed red line: weakly positive when 0.30 < OD ≤ 0.50 (dashed black line); or negative if OD < 0.30. For platelet functional testing analysis by flow cytometry (FC), the platelet activation index, expressed as a percentage, is represented in the upper left plot of each graph.(A) IgG anti-PF4/H results in COVID-19 patients at admission, according to the highest level of care (ward only, ward then intensive care unit (ICU), ICU, or ICU directly). (B) Correlation between sP-sel and IgG anti-PF4/H levels in COVID-19 patients at admission. (C) IgG anti-PF4/H results in COVID-19 patients on the day of intubation (T0) and 7 days after intubation (T1). (D) Correlation between sP-sel and IgG anti-PF4/H levels in COVID-19 patients at T0 and T1. (E) Platelet functional testing by flow cytometry (FC) of platelet microvesicle assay (PMA) in plasma from COVID-19 patient #1 with weak positive IgG anti-PF4/H. (F) Platelet functional testing by FC of PMA in plasma from COVID-19 patient #2 with positive IgG anti-PF4/H at T1. (G) Platelet functional testing by FC of PMA in plasma from COVID-19 patient #3 with weak positive IgG anti-PF4/H at T1. (H) Platelet functional testing by FC of PMA in plasma from patient with confirmed heparin-induced thrombocytopenia (HIT) with positive IgG anti-PF4/H and a positive serotonin release assay; used as a positive control. (I) Platelet functional testing by FC of PMA in plasma from patient with confirmed vaccine-induced immune thrombotic thrombocytopenia (VITT) with positive IgG anti-PF4/H and positive serotonin release assay; used as a positive control. FS INT, forward scatter intensity; UFH, unfractionated heparin.

Figure 2

Flowchart of patients with heparin-induced thrombocytopenia (HIT) suspicion during COVID-19 outbreak and in the same period in 2019. anti-PF4/H, heparin-platelet factor 4 complex antibodies; SRA, serotonin release assay.

Figure 3

Frequency of positive immunoglobulin G (IgG) heparin–platelet factor 4 complex antibodies (anti-PF4/H) and related platelet activation from different cohorts of individuals before and after COVID-19 vaccination. Results of IgG anti-PF4/H (optical density, OD, 450 nm) are positive with an OD > 0.50 (dashed red line), weakly positive when 0.30 < OD ≤ 0.50 (dashed black line); or negative if OD < 0.30. For platelet functional testing analysis by flow cytometry (FC), the platelet activation index, expressed as a percentage, is represented in the upper left plot of each graph. (A) Frequency of positive IgG anti-PF4/H in individuals from different cohort before and after COVID-19 vaccination. (B) Frequency of positive IgG anti-PF4/H in patients with systemic inflammatory diseases (SID) before and 28 days and 3 months after COVID-19 vaccination. Yellow dots represent to patients who received their first dose of the ChAdOx1 nCoV-19 vaccine (Oxford–AstraZeneca). (C) Correlation between soluble P-selectin (sP-selectin) and IgG anti-PF4/H levels in patients evaluated after thrombotic event (TE) following COVID-19 vaccination. (D) Platelet functional testing by FC of platelet microvesicle assay (PMA) in serum of the only individual from the control₂₀₂₁ cohort with positive IgG anti-PF4/H at day 28, with unfractionated heparin (UFH) or platelet factor 4 (PF4) addition. (D) Platelet functional testing by FC of PMA in the serum from the only patient with SID with positive IgG anti-PF4/H, with UFH or PF4 addition. (E) Platelet functional testing by FC of PMA in the plasma from the only patient addressed after TE following COVID-19 vaccination and positive IgG anti-PF4/H, with UFH or PF4 addition. FS INT: forward scatter intensity; HCW, health care worker.

Figure 4

Platelet activation in confirmed heparin-induced thrombocytopenia (HIT) and confirmed vaccine-induced immune thrombotic thrombocytopenia (VITT) with heparin or platelet factor 4 addition. For platelet functional testing analysis by flow cytometry (FC), the platelet activation index, expressed as a percentage, is represented in the upper left plot of each graph. (A) Platelet functional testing by FC of platelet microvesicle assay (PMA) in the plasma from a patient with confirmed HIT with positive immunoglobulin G heparin–platelet factor 4 (PF4) complex antibodies and positive serotonin release assay. (B) Platelet functional testing by FC of PMA in the serum from a patient with confirmed VITT with positive immunoglobulin G immunoglobulin G heparin-PF4 complex antibodies and positive serotonin release assay; used as a positive control. (C)Truncated violin plots showing the index of platelet activation by PMA in 20 serum samples from 4 confirmed VITT patients at diagnosis and during follow-up. FS INT: forward scatter intensity; UFH, unfractionated heparin.

Figure 5

Different platelet activation profiles between patients with confirmed heparin-induced thrombocytopenia (HIT) and with vaccine-induced immune thrombotic thrombocytopenia (VITT). (A) Correlation between the index of platelet activation by flow cytometry (FC) of platelet microvesicle assay (PMA) in buffer (unfractionated heparin [UFH], 0.0 U/mL) and immunoglobulin G (IgG) heparin–platelet factor 4 complex antibodies (anti-PF4/H) levels in samples from confirmed heparin-induced thrombocytopenia (HIT) patients. (B) Correlation between the index of platelet activation by FC of PMA with UFH 0.1 U/mL and IgG anti-PF4/H levels in samples from confirmed HIT patients. (C) Correlation between the index of platelet activation by FC of PMA with UFH 100.0 U/mL and IgG anti-PF4/H levels in samples from confirmed HIT patients. (D) Correlation between soluble P-selectin (sP-selectin) and IgG anti-PF4/H levels in samples from confirmed HIT patients. (E) Correlation between the index of platelet activation by FC of PMA in buffer (UFH 0.0 U/mL) and IgG anti-PF4/H levels in samples from VITT patients. (F) Correlation between the index of platelet activation by FC of PMA with UFH 0.1 U/mL and IgG anti-PF4/H levels in samples from VITT patients. (G) Correlation between the index of platelet activation by FC of PMA with UFH 100.0 U/mL and IgG anti-PF4/H levels in samples from VITT patients. (H) Correlation between sP-selectin and IgG anti-PF4/H levels in samples from VITT patients. OD, optical density.