Anti-PF4 positivity and platelet activation after Ad26.COV2·S vaccination in Brazil.

INTRODUCTION: The Ad26.COV2·S (Janssen/Johnson & Johnson) COVID-19 vaccine, has been rarely associated with vaccine-induced immune thrombocytopenia and thrombosis (VITT). We investigated the prevalence of anti-PF4 antibody positivity, thrombocytopenia, D-dimer elevation, plasmatic thromboinflammatory markers, and platelet functional assays following Ad26.COV2·S vaccination in Rio de Janeiro, Brazil. METHODS: From July to September 2021, participants were assessed prior, 1, and 3 weeks post-vaccination. Platelet count and D-dimer were measured at each visit and anti-PF4 at week 3. A positive anti-PF4 prompted retrospective testing of the sample from week 0. Individuals with new thrombocytopenia or elevated D-dimer, positive anti-PF4, and 38 matched controls without laboratory abnormalities were evaluated for plasmatic p-selectin, tissue factor, and functional platelet activation assays. RESULTS: 630 individuals were included; 306 (48.57%) females, median age 28 years. Forty-two (6.67%) presented ≥1 laboratory abnormality in week 1 or 3. Five (0.79%) had thrombocytopenia, 31 (4.91%) elevated D-dimer, and 9 (1.57%) had positive anti-PF4 at week 3. Individuals with laboratory abnormalities and controls showed a slight increase in plasmatic p-selectin and tissue factor. Ten individuals with laboratory abnormalities yielded increased surface expression of p-selectin, and their ability to activate platelets in a FcγRIIa dependent manner was further evaluated. Two were partially inhibited by high concentrations of heparin and blockage of FcγRII with IV.3 antibody. Plasma obtained before vaccination produced similar results, suggesting a lack of association with vaccination. CONCLUSIONS: Vaccination with Ad26.COV2·S vaccine led to a very low frequency of low-titer positive anti-PF4 antibodies, elevation of D-dimer, and mild thrombocytopenia, with no associated clinically relevant increase in thromboinflammatory markers and platelet activation.

Heparin-induced thrombocytopenia with very high antibody titer is associated with slower platelet recovery and higher risk of thrombosis.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by platelet-activating antibodies targeting platelet factor 4 (PF4) and heparin complex. A higher antibody titer is reflected in a higher optical density (OD) by enzyme-linked immunosorbent assay for heparin-PF4 antibodies. This single-institution retrospective study of 116 HIT patients examined the effect of heparin-PF4 OD on time to platelet recovery, vascular thrombosis, and in-hospital mortality. Patients were divided into 3 cohorts based on heparin-PF4 OD: cohort 1 had an OD ≥ 2 and ≤ 2.4, cohort 2 had an OD > 2.4 and ≤ 2.8, and cohort 3 had an OD > 2.8. A higher OD titer was associated with significantly increased time to platelet recovery when compared between cohorts 1 versus 2 (HR = 0.599, p = 0.0221) and 1 versus 3 (HR = 0.515, p = 0.0014), as well as an increased risk of thrombosis (79.4%-cohort 3 vs 53.8%-cohort 2 vs 46.1%-cohort 1, p = 0.04), but had no impact on mortality (2.62-alive vs 2.65-deceased, p = 0.7432). A higher OD titer can inform risk assessment and support decision-making in HIT patients; however, prospective studies are needed to further clarify the impact of heparin-PF4 OD on outcomes.

Flow cytometry vs conventional methods for the evaluation of anti-PF4/heparin antibodies: a single center study.

Aim: Heparin-induced thrombocytopenia (HIT) is a rare, life-threatening, immune-mediated adverse effect of heparin administration. This study compares frequently used laboratory assays in terms of their effectiveness in HIT diagnosis. Materials & methods: Fifty patients with suspected HIT were tested by gel immunoassay and solid phase PF4/heparin antibody ELISA. On positive results, platelet activation markers P-selectin and Annexin V were assayed using flow cytometry. Results: Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2 IU/ml). Conclusion: The results are in accordance with the currently available literature and flow cytometry seems a promising alternative in HIT laboratory investigation.

[Box: see text].

Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units, and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody-negative patients. We observed statistical differences between antibody-positive and -negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody-positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

Anti-platelet factor 4 immunothrombotic syndromes.

Anti-platelet factor 4 immunothrombotic syndromes comprise a group of disorders that include heparin-induced thrombocytopenia and vaccine-induced immune thrombocytopenia and thrombosis. These are highly prothrombotic, immunological disorders characterised by specific clinical and pathological criteria which include thrombocytopenia and thrombosis. While they are predominantly triggered by heparin and the adenoviral vector vaccines, respectively, other provoking factors have been described, as well as spontaneous forms. The unexplained co-occurrence of thrombocytopenia with thrombosis should raise suspicion and prompt testing. This nutshell review discusses the pathophysiology, presenting features and diagnostic criteria for these conditions.

Serum Plasminogen Activator Inhibitor-1, α 1-Acid Glycoprotein, C-Reactive Protein, and Platelet Factor 4 Levels-Promising Molecules That Can Complete the "Puzzle" of the Biochemical Milieu in Severe Burns: Preliminary Results of a Cohort Prospective Study.

Background: Burns represent a serious health problem, associated with multiple-organ failure, prolonged hospitalization, septic complications, and increased rate of mortality. The main aim of our study was to evaluate the levels of various circulating molecules in children with severe burns (more than 25% TBSA), in three different moments: 48 h, day 10, and day 21 post-burn. Materials and Methods: This study included 32 children with burns produced by flame, hot liquid, and electric arc and 21 controls. Serum plasminogen activator inhibitor-1 (PAI-1), α 1-acid glycoprotein (AGP), C-reactive protein (CRP), and platelet factor 4 (PF4) were detected using the Multiplex technique. Several parameters, such as fibrinogen, leucocyte count, thrombocyte count, triiodothyronine, thyroxine, and thyroid-stimulating hormone were also determined for each patient during hospitalization. Results: Significant statistical differences were obtained for CRP, AGP, and PF4 compared to the control group, in different moments of measurements. Negative correlations between CRP, AGP, and PF4 serum levels and burned body surface, and also the hospitalization period, were observed. Discussions: CRP levels increased in the first 10 days after burn trauma and then decreased after day 21. Serum PAI-1 levels were higher immediately after the burn and started decreasing only after day 10 post-burn. AGP had elevated levels 48 h after the burn, then decreased at 7-10 days afterwards, and once again increased levels after 21 days. PF4 serum levels increased after day 10 since the burning event. Conclusions: Serum CRP, AGP, PAI-1, and PF4 seem to be promising molecules in monitoring patients with a burn within the first 21 days.

Anti-PF4 ELISA-Negative, SRA-Positive Heparin-Induced Thrombocytopenia.

Heparin products are frequently used in the inpatient setting to prevent and treat venous thromboembolism, but they simultaneously put patients at risk of developing heparin-induced thrombocytopenia (HIT). The 4Ts score determines the pretest probability of HIT. Diagnosis is made with a screening antiplatelet factor (PF4) immunoassay and the serotonin-release assay (SRA) as a confirmatory test. Anti-PF4 assays have high sensitivity (98%) but lower specificity (50%) and result in frequent false-positive tests. We present a rare case from our institution of a patient with anti-PF4-Polyanion ELISA-negative, SRA-positive HIT and describe the challenges in making a timely diagnosis in this case.

Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody negative patients. We observed statistical differences between antibody positive and negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.

Type II Heparin-Induced Thrombocytopenia Manifesting As Cardiac Arrest Following Intravenous Heparin Bolus During an Elective Procedure: A Case Report and Literature Review.

Heparin-induced thrombocytopenia (HIT) is a rare and life-threatening autoimmune-mediated adverse drug reaction seen in patients who are exposed to various forms of pharmacological heparin, including unfractionated heparin (UFH) and low molecular weight heparin (LMWH). Despite the presence of thrombocytopenia, these patients face the risk of clot formation and bleeding simultaneously. Prompt cessation of heparin and the initiation of non-heparin anticoagulants are important for the patient's survival. Typically, clinical diagnosis of HIT is necessary, and waiting for lab test results, which can take days, may not be always feasible. Here, we present a case of an unusual presentation of type II HIT, complicated by significant thrombocytopenia, pulmonary hemorrhage, and cardiac arrest after receiving intravenous (IV) heparin bolus during an elective cardiac ablation procedure for paroxysmal atrial fibrillation.

Platelet Factor 4 and Longevity of Patients with Essential Thromobocythemia: An Example of Antagonistic Pathogenic Pleiotropy.

This article presents the concept of Antagonistic Pathogenic Pleiotropy, in which an abnormality that causes a specific pathology can simultaneously reduce other morbidities through unrelated mechanisms, resulting in the pathology causing less morbidity or mortality than expected. The concept is illustrated by the case of essential thrombocythemia (ET). Patients with ET have substantially elevated platelets and are therefore expected to have increased thrombotic events leading to reduced life expectancy. However, patients with ET do not have reduced life expectancy. A possible explanation is that elevated platelets produce higher levels of platelet factor 4 (PF4), which has been found to reduce age-associated decline in immune and cognitive function in mice and has been suggested as a treatment for age-associated illness. The benefit of elevated PF4 is hypothesized to balance the increased morbidity from hematological causes. Searches for other indications where a well-defined pathology is not associated with concomitant reduction in overall mortality may be a route to identifying factors that could protect against, prevent, or treat chronic disease.

A career in solving clinical-pathological conundrums: Heyde syndrome, anti-platelet factor 4 disorders, and microvascular limb ischemic necrosis.

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical-pathological conundrums addressed during my 35-year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, anti-platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT- and cancer-associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

The Binding of the SARS-CoV-2 Spike Protein to Platelet Factor 4: A Proposed Mechanism for the Generation of Pathogenic Antibodies.

Pathogenic platelet factor 4 (PF4) antibodies contributed to the abnormal coagulation profiles in COVID-19 and vaccinated patients. However, the mechanism of what triggers the body to produce these antibodies has not yet been clarified. Similar patterns and many comparable features between the COVID-19 virus and heparin-induced thrombocytopenia (HIT) have been reported. Previously, we identified a new mechanism of autoimmunity in HIT in which PF4-antibodies self-clustered PF4 and exposed binding epitopes for other pathogenic PF4/eparin antibodies. Here, we first proved that the SARS-CoV-2 spike protein (SP) also binds to PF4. The binding was evidenced by the increase in mass and optical intensity as observed through quartz crystal microbalance and immunosorbent assay, while the switching of the surface zeta potential caused by protein interactions and binding affinity of PF4-SP were evaluated by dynamic light scattering and isothermal spectral shift analysis. Based on our results, we proposed a mechanism for the generation of PF4 antibodies in COVID-19 patients. We further validated the changes in zeta potential and interaction affinity between PF4 and SP and found that their binding mechanism differs from ACE2-SP binding. Importantly, the PF4/SP complexes facilitate the binding of anti-PF4/Heparin antibodies. Our findings offer a fresh perspective on PF4 engagement with the SARS-CoV-2 SP, illuminating the role of PF4/SP complexes in severe thrombotic events.

Vaccine-Induced Immune Thrombotic Thrombocytopenia: Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of the ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced thrombocytopenia (HIT), VITT arises with the development of platelet-activating anti-PF4 antibodies 4-42 days post-vaccination, typically featuring thrombocytopenia and thrombosis at unusual sites. AIM: To explore the unique properties, pathogenic mechanisms, and long-term persistence of VITT antibodies in patients, in comparison with other anti-PF4 antibody-mediated disorders. DISCUSSION: This review highlights the complexity of VITT as it differs in antibody behavior and clinical presentation from other anti-PF4-mediated disorders, including the high incidence rate of cerebral venous sinus thrombosis (CVST) and the persistence of anti-PF4 antibodies, necessitating a re-evaluation of long-term patient care strategies. The nature of VITT antibodies and the underlying mechanisms triggering their production remain largely unknown. CONCLUSION: The rise in awareness and subsequent prompt recognition of VITT is paramount in reducing mortality. As vaccination campaigns continue, understanding the role of adenoviral vector-based vaccines in VITT antibody production is crucial, not only for its immediate clinical implications, but also for developing safer vaccines in the future.

The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells.

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

Characterization of complexes of PF4 and heparins by size-exclusion chromatography coupled with multi-angle light scattering detector.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT.

Single-cell RNA-seq analysis decodes the kidney microenvironment induced by polystyrene microplastics in mice receiving a high-fat diet.

In recent years, the environmental health issue of microplastics has aroused an increasingly significant concern. Some studies suggested that exposure to polystyrene microplastics (PS-MPs) may lead to renal inflammation and oxidative stress in animals. However, little is known about the essential effects of PS-MPs with high-fat diet (HFD) on renal development and microenvironment. In this study, we provided the single-cell transcriptomic landscape of the kidney microenvironment induced by PS-MPs and HFD in mouse models by unbiased single-cell RNA sequencing (scRNA-seq). The kidney injury cell atlases in mice were evaluated after continued PS-MPs exposure, or HFD treated for 35 days. Results showed that PS-MPs plus HFD treatment aggravated the kidney injury and profibrotic microenvironment, reshaping mouse kidney cellular components. First, we found that PS-MPs plus HFD treatment acted on extracellular matrix organization of renal epithelial cells, specifically the proximal and distal convoluted tubule cells, to inhibit renal development and induce ROS-driven carcinogenesis. Second, PS-MPs plus HFD treatment induced activated PI3K-Akt, MAPK, and IL-17 signaling pathways in endothelial cells. Besides, PS-MPs plus HFD treatment markedly increased the proportions of CD8+ effector T cells and proliferating T cells. Notably, mononuclear phagocytes exhibited substantial remodeling and enriched in oxidative phosphorylation and chemical carcinogenesis pathways after PS-MPs plus HFD treatment, typified by alterations tissue-resident M2-like PF4+ macrophages. Multispectral immunofluorescence and immunohistochemistry identified PF4+ macrophages in clear cell renal cell carcinoma (ccRCC) and adjacent normal tissues, indicating that activate PF4+ macrophages might regulate the profibrotic and pro-tumorigenic microenvironment after renal injury. In conclusion, this study first systematically revealed molecular variation of renal cells and immune cells in mice kidney microenvironment induced by PS-MPs and HFD with the scRNA-seq approach, which provided a molecular basis for decoding the effects of PS-MPs on genitourinary injury and understanding their potential profibrotic and carcinogenesis in mammals.

NCOA5 Haploinsufficiency in Myeloid-Lineage Cells Sufficiently Causes Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

BACKGROUND & AIMS: The nuclear receptor coactivator 5 (NCOA5) is a putative type 2 diabetes susceptibility gene. NCOA5 haploinsufficiency results in the spontaneous development of nonalcoholic fatty liver disease (NAFLD), insulin resistance, and hepatocellular carcinoma (HCC) in male mice; however, the cell-specific effect of NCOA5 haploinsufficiency in various types of cells, including macrophages, on the development of NAFLD and HCC remains unknown. METHODS: Control and myeloid-lineage-specific Ncoa5 deletion (Ncoa5ΔM/+) mice fed a normal diet were examined for the development of NAFLD, nonalcoholic steatohepatitis (NASH), and HCC. Altered genes and signaling pathways in the intrahepatic macrophages of Ncoa5ΔM/+ male mice were analyzed and compared with those of obese human individuals. The role of platelet factor 4 (PF4) in macrophages and the underlying mechanism by which PF4 affects NAFLD/NASH were explored in vitro and in vivo. PF4 expression in HCC patient specimens and prognosis was examined. RESULTS: Myeloid-lineage-specific Ncoa5 deletion sufficiently causes spontaneous NASH and HCC development in male mice fed a normal diet. PF4 overexpression in Ncoa5ΔM/+ intrahepatic macrophages is identified as a potent mediator to trigger lipid accumulation in hepatocytes by inducing lipogenesis-promoting gene expression. The transcriptome of intrahepatic macrophages from Ncoa5ΔM/+ male mice resembles that of obese human individuals. High PF4 expression correlated with poor prognosis of HCC patients and increased infiltrations of M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells in HCCs. CONCLUSIONS: Our findings reveal a novel mechanism for the onset of NAFLD/NASH and HCC initiated by NCOA5-deficient macrophages, suggesting the NCOA5-PF4 axis in macrophages as a potential target for developing preventive and therapeutic interventions against NAFLD/NASH and HCC.

Optimization of laboratory diagnosis of heparin-induced thrombocytopenia using HemosIL-AcuStar-HIT-IgG assay.

OBJECTIVE: The aim of this study was to determine an optimal cutoff value for the newly available HemosIL-AcuStar-HIT-IgG assay (AcuStar) for the diagnosis of heparin-induced thrombocytopenia (HIT). METHOD: We evaluated the performance of AcuStar using serotonin release assay (SRA) as the gold standard and incorporated 4T score calculation in a cohort of suspected HIT cases. Statistical analysis was performed to determine optimal cutoff value for the diagnosis of HIT. RESULT: A diagnosis of HIT can be excluded with a platelet factor 4 (PF4) value of <0.4 U/mL by AcuStar and 4T score in the low-risk category (≤3). All other cases will require confirmation with a functional test. CONCLUSION: Our study resulted in the implementation of a diagnostic algorithm for laboratory diagnosis of HIT, which incorporates pretest calculation of 4T score and AcuStar as a screening test, with reflex confirmation by SRA. This new algorithm resulted in extended hours of test availability and a more rapid turnaround time in reporting PF4 results.

Hematology in the post-COVID era: spotlight on vaccine-induced immune thrombotic thrombocytopenia and a conceptual framework (the 4P's) for anti-PF4 diseases.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a life-threatening prothrombotic disorder first identified following the introduction of adenoviral vector vaccines for COVID-19. The condition is characterized by anti-PF4 antibodies and clinically presents with thrombocytopenia and thrombosis often in unusual anatomical sites. AREAS COVERED: In this review, we discuss the clinical presentation, diagnostic testing, and treatment of VITT. We also review VITT-like syndromes that have been described in patients without previous vaccination. We propose a conceptual framework for the mechanism of anti-PF4 diseases that includes sufficiently high levels of PF4, the presence of a Polyanion that can form immune complexes with PF4, a Pro-inflammatory milieu, and an immunological Predisposition - the 4Ps. EXPERT OPINION: Significant progress has been made in understanding the characteristics of the VITT antibody and in testing methods that can confirm that diagnosis. Future work should be directed at understanding long-term outcomes, mechanisms of thrombosis, and individual risk factors for this rare but dangerous immune-thrombotic disease.

Biophysical studies do not reveal direct interactions between human PF4 and Ad26.COV2.S vaccine.

BACKGROUND: COVID-19 vaccines have been widely used to control the SARS-CoV-2 pandemic. In individuals receiving replication-incompetent, adenovirus vector-based COVID-19 vaccines (eg, ChAdOx1 nCoV-19 [AstraZeneca] or Ad26.COV2.S [Johnson & Johnson/Janssen] vaccines), a very rare but serious adverse reaction has been reported and described as vaccine-induced immune thrombotic thrombocytopenia (VITT). The exact mechanism of VITT following Ad26.COV2.S vaccination is under investigation. Antibodies directed against human platelet factor 4 (PF4) are considered critical in the pathogenesis of VITT, suggesting similarities with heparin-induced thrombocytopenia. It has been postulated that components of these vaccines mimic the role of heparin by binding to PF4, triggering production of these anti-PF4 antibodies. OBJECTIVES: This study aimed to investigate the potential interaction between human PF4 and Ad26.COV2.S vaccine using several biophysical techniques. METHODS: Direct interaction of PF4 with Ad26.COV2.S vaccine was investigated using dynamic light scattering, biolayer interferometry, and surface plasmon resonance. For both biosensing methods, the Ad26.COV2.S vaccine was immobilized to the sensor surface and PF4 was used as analyte. RESULTS: No direct interactions between PF4 and Ad26.COV2.S vaccine could be detected using dynamic light scattering and biolayer interferometry. Surface plasmon resonance technology was shown to be unsuitable to investigate these types of interactions. CONCLUSION: Our findings make it very unlikely that direct binding of PF4 to Ad26.COV2.S vaccine or components thereof is driving the onset of VITT, although the occurrence of such interactions after immunization (potentially facilitated by unknown plasma or cellular factors) cannot be excluded. Further research is warranted to improve the understanding of the full mechanism of this adverse reaction.