A case report of vaccine-induced immune thrombotic thrombocytopenia (VITT) with genetic analysis.

The emergence of the rare syndrome called vaccine-induced immune thrombocytopenia and thrombosis (VITT) after adenoviral vector vaccines, including ChAdOx1 nCov-19, raises concern about one's predisposing risk factors. Here we report the case of a 56-year-old white man who developed VITT leading to death within 9 days of symptom onset. He presented with superior sagittal sinus thrombosis, right frontal intraparenchymal hematoma, frontoparietal subarachnoid and massive ventricular hemorrhage, and right lower extremity arterial and venous thrombosis. His laboratory results showed elevated D-dimer, C-reactive protein, tissue factor, P-selectin (CD62p), and positive anti-platelet factor 4. The patient's plasma promoted higher CD62p expression in healthy donors' platelets than the controls. Genetic investigation on coagulation, thrombophilia, inflammation, and type I interferon-related genes was performed. From rare variants in European or African genomic databases, 68 single-nucleotide polymorphisms (SNPs) in one allele and 11 in two alleles from common SNPs were found in the patient genome. This report highlights the possible relationship between VITT and genetic variants. Additional investigations regarding the genetic predisposition of VITT are needed.

Anti-PF4 antibodies associated with disease severity in COVID-19.

Severe COVID-19 is characterized by a prothrombotic state associated with thrombocytopenia, with microvascular thrombosis being almost invariably present in the lung and other organs at postmortem examination. We evaluated the presence of antibodies to platelet factor 4 (PF4)-polyanion complexes using a clinically validated immunoassay in 100 hospitalized patients with COVID-19 with moderate or severe disease (World Health Organization score, 4 to 10), 25 patients with acute COVID-19 visiting the emergency department, and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 hospitalized patients with COVID-19 (95.0%) irrespective of prior heparin treatment, with a mean optical density value of 0.871 ± 0.405 SD (range, 0.177 to 2.706). In contrast, patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels of the antibodies. In a high proportion of patients with COVID-19, levels of all three immunoglobulin (Ig) isotypes tested (IgG, IgM, and IgA) were simultaneously elevated. Antibody levels were higher in male than in female patients and higher in African Americans and Hispanics than in White patients. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from patients with COVID-19 induced higher levels of platelet activation than did sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19.

No changes in hemostasis after COVID-19-heterologous vaccination schedule: A subanalysis of the phase 2 CombiVacS study.

Background: Several cases of unusual thrombotic events and thrombocytopenia were described after vaccination with recombinant adenoviral vectors encoding the spike protein antigen of SARS-CoV-2. Objectives: The objective of this study was to elucidate the impact of a COVID-19 heterologous vaccination schedule, including priming with adenovirus vaccine, on hemostasis profiles. Methods: The present study is a subanalysis of the CombiVacS clinical trial initiated in April 2021 that included adult participants previously vaccinated with a single dose of ChAdOx1-S. Between 8 and 12 weeks after vaccination, they were randomly assigned (2:1) to receive either BNT162b2 vaccine (intervention group, n = 99) or continue observation (control group, n = 50). Samples drawn before and 28 days after a vaccination with BNT162b2 were analyzed for platelet count and markers of hemostasis (D-dimer, anti-PF4 antibodies, cfDNA, PAI-1, thrombin generation, and serum capacity to activate platelets). Results: Platelet count from all participants after receiving BNT162b2 was within the normal range. Anti-PF4 antibodies were present in 26% and 18% of the subjects from the control and intervention groups, respectively, at day 28. In most cases, the levels of anti-PF4 antibodies were high before receiving BNT162b2. Serum from these participants did not activate platelets from healthy controls. There were no differences between the groups in PAI-1 and cfDNA plasma levels. According to the D-dimer plasma concentration, the thrombin generation test showed that none of the participants had a procoagulant profile. Conclusion: Our data suggest that the heterologous vaccination against COVID-19 with ChAdOx1-S and a second dose with BNT162b2 might be safe in terms of haemostasis.

Central retinal vein occlusion post ChAdOx1 nCoV-19 vaccination - can it be explained by the two-hit hypothesis?

PURPOSE: To report a case of central retinal vein occlusion (CRVO) seven days following the first dose of ChAdOx1 nCoV-19 vaccine and propose a hypothesis for the possible underlying pathogenesis. OBSERVATION: A 31-year-old male presented with CRVO with cystoid macular edema, one week after receiving his first ChAdOx1 nCoV-19 vaccine dose. Apart from mild hyperhomocysteinemia, no major thrombophilic or systemic risk factors were found. Anti-platelet factor 4 antibodies, specific for vaccine-induced immune thrombotic thrombocytopenia, were also negative. However, he tested strongly positive (> 250 U/mL) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG spike antibodies, 2 weeks post the first dose - suggestive of a prior subclinical infection. CONCLUSION: COVID-19 is known to be associated with an altered host one-carbon metabolism resulting in hyperhomocysteinemia. We hypothesize that a prior subclinical infection with COVID-19, the first hit, may have led to hyperhomocysteinemia in our patient and vaccination must have been the second hit that triggered the thrombotic event. Further studies, including correlation of thrombotic complications with IgG antibody titres post-vaccination, are essential in order to better understand the pathogenesis of such events.

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies. OBJECTIVES: A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing. METHODS: Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays. RESULTS: A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods. CONCLUSION: ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies.

Anti-platelet factor 4 immunoglobulin G levels in vaccine-induced immune thrombocytopenia and thrombosis: Persistent positivity through 7 months.

Background: Anti-platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown. Objectives: To assess how clinical and platelet parameters, anti-PF4 antibody levels, and patient serum reactivity changes during follow-up after VITT presentation. Methods: We describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199-280) after vaccination. We measured anti-PF4 antibodies and PF4 levels in patient serum during follow-up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time. Results: Anti-PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9-2.6 OD) and remain relatively high (>1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow-up and no patients had recurrent thrombosis. Patients' platelets during follow-up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow-up serum alone was weaker at stimulating donor and patient platelets. However, follow-up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50. Conclusions: Explanations for reduced platelet activation during follow-up, despite similar total anti-PF4 antibody levels, remains unclear. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.

The Relevance of Anti-PF4 Antibody Isotypes and Endogenous Glycosaminoglycans and their Relationship with Inflammatory Biomarkers in Pulmonary Embolism Patients.

INTRODUCTION: Previous studies have shown that inflammation may contribute to the interplay of endogenous glycosaminoglycans (GAGs) and anti-PF4 antibodies. In this study, we quantified the levels of anti-PF4 antibody isotypes and endogenous GAGs together with inflammatory biomarkers in pulmonary embolism (PE) patients to determine whether there is a relationship in between. Identification of this relationship may provide insight to the complex pathophysiology of PE and HIT and may also be useful for development of potential prognostic, diagnostic and therapeutic interventions. MATERIALS AND METHODS: Plasma samples from PE patients (n: 210) were analyzed for anti-PF4 antibody isotypes and various thrombo-inflammatory cytokines utilizing commercially available biochip array and ELISA methods. The endogenous GAG levels in PE patients' plasma were quantified using a fluorescence quenching method. The collected data analyzed to demonstrate the relationship between various parameters. RESULTS: The endogenous GAG levels were increased in the PE group (P < .05). The levels of anti-PF4 antibody isotypes were higher in varying levels in comparison to the normal group (P < .05). Inflammatory cytokines have shown varying levels of increase with IL-6, IL-8 and IL-10 showing the most pronounced values. Mortality outcome was related to increased GAGs and some of the cytokines. CONCLUSION: In this study, we demonstrated increased levels of anti-PF4 antibody isotypes, endogenous GAGs, and inflammatory biomarkers in a large patient cohort in PE. The levels of the endogenous GAGs and inflammatory biomarkers were associated with PE severity and mortality. More studies are needed to understand this complex pathophysiology.

COVID-19 adenovirus vaccine triggers antibodies against PF4 complexes to activate complement and platelets.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. OBJECTIVE: To assess antibodies in interaction with the activation of platelets and complement triggered by VITT. METHODS: Antibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. RESULTS: None of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. CONCLUSIONS: Our findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.

Consumption of complement in a 26-year-old woman with severe thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination.

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.