Multiple Electrode Aggregometry (Multiplate): Functional Assay for Vaccine-Induced (Immune) Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) was first described in 2021 and represents an adverse reaction to adenoviral vector COVID-19 vaccines AstraZeneca ChAdOx1 nCoV-19 (AZD1222) and Johnson & Johnson Ad26.COV2.S vaccine. VITT is a severe immune platelet activation syndrome with an incidence of 1-2 per 100,000 vaccinations. The features of VITT include thrombocytopenia and thrombosis within 4-42 days of first dose of vaccine. Affected individuals develop platelet-activating antibodies against platelet factor 4 (PF4). The International Society on Thrombosis and Haemostasis recommends both an antigen-binding assay (enzyme-linked immunosorbent assay, ELISA) and a functional platelet activation assay for the diagnostic workup of VITT. Here, the application of multiple electrode aggregometry (Multiplate) is presented as a functional assay for VITT.

Serotonin Release Assay: Functional Assay for Heparin- and Vaccine-Induced (Immune) Thrombotic Thrombocytopenia.

The serotonin release assay (SRA) has been the gold-standard assay for detection of heparin-dependent platelet-activating antibodies and integral for the diagnosis for heparin-induced thrombotic thrombocytopenia (HIT). In 2021, a thrombotic thrombocytopenic syndrome was reported after adenoviral vector COVID-19 vaccination. This vaccine-induced thrombotic thrombocytopenic syndrome (VITT) proved to be a severe immune platelet activation syndrome manifested by unusual thrombosis, thrombocytopenia, very elevated plasma D-dimer, and a high mortality even with aggressive therapy (anticoagulation and plasma exchange). While the platelet-activating antibodies in both HIT and VITT are directed toward platelet factor 4 (PF4), important differences have been found. These differences have required modifications to the SRA to improve detection of functional VITT antibodies. Functional platelet activation assays remain essential in the diagnostic workup of HIT and VITT. Here we detail the application of SRA for the assessment of HIT and VITT antibodies.

Anti-PF4 antibodies after SARS-CoV-2 vaccines: Long term follow-up

Introduction A subgroup of anti-platelet factor 4 (PF4) antibodies can activate platelets via Fcgamma RIIA and cause thrombotic and thrombocytopenic diseases such as heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia (VITT). Nonpathological anti-PF4 antibodies are detected in 1-7 % of healthy blood donors and in 2-8 % of SARS-CoV-2 vaccinated individuals. In this study, we investigated the long-term course of anti- PF4 antibodies detected after the first SARS-CoV-2 vaccination in healthy subjects and in patients with VITT. Method Five healthy subjects (all female, median age (range): 40 years (29-62) ) who had anti-PF4 antibodies after the first vaccination with ChadOx1 nCov19 (Vaxzevria, AstraZeneca-Oxford) were included. None of the subjects developed VITT. Blood samples were collected as part of a longitudinal study (TuSeRe:exact) evaluating the immune response to SARS-CoV-2 vaccines among employees of an University Hospital. In addition, data from 4 patients with VITT (3 female, median age (range): 44 years (22 -62 years)) were included for long-term follow-up of anti-PF4 antibodies. Anti-PF4/heparin antibodies were measured using a commercially available ELISA assay (Zymutest HIA IgG, Hyphen BioMed, France). Platelet activation was tested with a modified heparin- induced platelet aggregation assay (HIPA). Results In the non-VITT group, the median (range) OD for IgG anti-PF4/heparin antibodies was 0.69 (0.60-1.83) after the first vaccination. Blood samples were available up to 16 months after the first vaccination (range: 5-16 months). Anti-PF4 antibody levels decreased in all subjects despite further vaccination. However, antibody levels returned to pre-vaccination levels in only one subject. In one subject who had received two doses of ChadOx1 nCov19, anti-PF4 antibodies remained above OD 1.0 at the last follow-up. All samples were negative in the modified HIPA assay. Patients with VITT received mRNA-based vaccine as second vaccination against SARS CoV2. No significant drop in platelet count or new thromboembolic complication was observed. Conclusion Nonpathological anti-PF4 antibodies can be detected even several months after the first vaccination. The clinical significance of these antibodies in case of subsequent exposure to a vector vaccine or heparin is not yet clear. Furthermore, subsequent vaccination seems safe in VITT patietns.

Subsequent vaccinations in VITT patients other than SARS-CoV2 vaccination

Introduction Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare, but severe side effect after vaccination with adenovirus vector-based vaccines (ChAdOx1 nCoV-19, AstraZeneca and Ad26.COV2.S, Johnson & Johnson/ Janssen) in which platelet activating anti-platelet factor 4 (PF4) antibodies cause thrombocytopenia and thrombosis at unusual sites. Patients and treating physicians are concerned about whether other vaccinations can also trigger thrombosis in patients with a history of VITT. We showed that VITT patients can safely receive their second and third vaccination against Covid-19 with an mRNA-based vaccine. [1] However, there is limited information on whether other vaccines than against Covid-19 could booster platelet activating anti-PF4 antibodies. Uncertainty increased after a report of VITT caused by human papilloma vaccination. [2] Method In our follow-up study of patients with laboratory confirmed VITT (EUPAS45098), an anti-PF4/heparin IgG enzyme immune assay (EIA) and a PF4-dependent platelet activation assay (PIPA) were performed at regular intervals and after each vaccination reported to us. Results Seventy-one VITT patients (43 female, median age at VITT diagnosis 48, range 18-80) were followed for a median of 56 weeks (range: 13-77 weeks). During the follow-up period, eight vaccinations other than against Covid-19 were reported: Six vaccinations against influenza (three Influvac, two Vaxigrip Tetra, one Influsplit Tetra) and two consecutive vaccinations against tick-borne encephalitis (TBE) in one patient. In six patients who received vaccination against influenza, all patients showed decreasing or stable EIA optical density (OD) levels. None of them showed a reactivation of platelet-activating anti- PF4-antibodies in the PIPA. The patient who was vaccinated against TBE twice showed stable EIA OD levels and remained negative in the PIPA throughout. No new thrombosis or recurrent thrombocytopenia were observed after any vac- cination. Five out of six patients still received therapeutic anticoagulation, one patient did not receive any anticoagulative drug (Fig. 1). Conclusion Similar to observations after consecutive mRNA-vaccinations against Covid-19 in VITT patients, vaccinations against influenza and TBE very unlikely reactivate platelet-activating anti-PF4-antibodies. Further follow up of the VITT patient cohort is performed to detect any new safety signal related to recurrence of VITT. (Table Presented).

Challenges and Strategies for Veno Venous (VV) Extracorporeal Membrane Oxygenation (ECMO) in COVID-19 Acute Respiratory Distress Syndrome (ARDS) During Second Wave of Pandemic: A Case Series

Case series: Extracorporeal membrane oxygenation (ECMO) use for severe acute respiratory distress syndrome due to coronavirus disease 2019 (COVID-19) patients has increased during the second wave of the pandemic. However, there are many complications associated with the management of ECMO in critically ill COVID- 19 patients. We report a case series of challenges and strategies for managing critically ill COVID-19 patients on ECMO support for severe ARDS. Seven COVID-19 patients required VV ECMO of which three were women and four were men of median age of 43 years. Among seven, three cases (42%) recovered. We experienced multiple challenges and complications in the management of the patients, being a non-ECMO centre with limited resources, in heavy workload during the second wave of the pandemic. All the patients required multiple invasive procedures like placement of invasive lines, frequent bronchoscopies for bronchial toileting. Displacement of both ECMO cannulas required repositioning under ultrasound guidance, four patients underwent percutaneous tracheostomy on ECMO. Three patients had ECMO-oxygenator failure that required the exchange of a new ECMO circuit. ACT was monitored for the management of anticoagulation. A challenging task is to achieve a balance between bleeding and thrombotic events, for which anticoagulation had to be stopped for the acceptable ACT, required transfusion of multiple blood products for correcting coagulopathy. One patient developed HIT antibodies and managed with bivalirudin for the management of anticoagulation which was challenging in titrating the drug dose and ACT. Two patients had an intracranial haemorrhage on ECMO support, managed conservatively despite anticoagulation. Pseudoaneurysm of femoral vein diagnosed and managed with ultrasound-guided thrombin injection. Four patients got decannulated from ECMO. One patient had unexplained severe haemolysis immediately after initiation of ECMO, unfortunately, he could not recover. Management of VV ECMO in resource-limited, non-ECMO centre in a pandemic is challenging. Mortality depends on various factors, despite expertise, advanced critical care management in COVID- 19 ARDS and ECMO. Increased use of VV ECMO during the second wave of pandemic reported significant changes in strategies for management of challenges, though further studies are still required for the best outcome.

A Case Report of Thrombotic Thrombocytopenia After ChAdOx1 nCov-19 Vaccination and Heparin Use During Hemodialysis.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but life-threatening complication. VITT strongly mimics heparin-induced thrombocytopenia (HIT) and shares clinical features. Heparin is commonly used to prevent coagulation during hemodialysis. Therefore, nephrologists might encounter patients needing dialysis with a history of heparin exposure who developed thrombotic thrombocytopenia after vaccination. A 70-year-old male presented with acute kidney injury and altered mental status due to lithium intoxication. He needed consecutive hemodialysis using heparin. Deep vein thrombosis of left lower extremity and accompanying severe thrombocytopenia of 15,000/µL on 24 days after vaccination and at the same time, nine days after heparin use. Anti-platelet factor 4 antibody test was positive. Anticoagulation with apixaban and intravenous immunoglobulin (IVIG) infusion resolved swelling of his left calf and thrombocytopenia. There were no definitive diagnostic tools capable of differentiating between VITT and HIT in this patient. Although VITT and HIT share treatment with IVIG and non-heparin anticoagulation, distinguishing between VITT and HIT will make it possible to establish a follow-up vaccination plan in a person who has had a thrombocytopenic thrombotic event. Further research is needed to develop the tools to make a clear distinction between the clinical syndromes.

Mimickers of Hit Antibodies in Critically Ill COVID Patients

Introduction: Infection with SARS CoV2 leads to respiratory failureand can lead to support of extracorporeal oxygenation (ECMO)leading to exposure to heparin. Exposure to heparin and developmentof thrombocytopenia raises the suspicion of HIT. The strong positiveIgG PF4-heparin antigen test by immunochromatography is followedby platelet aggregation test in our center. We present a case of covid-19 in which HIT was strongly positive on gel agglutination butfunctional assay was negative. Review of literature shows that this could be due to circulating platelet immune complexes in critically illcovid patients which simulate HIT antibodies.Aims &Objectives: Materials &Methods: 7 months pregnantfemale, non-vaccinated, asthmatic, COVID 19 positive patient wasadmitted to our hospital. On admission her fetal ultrasound wasnormal and was started on non-invasive ventilation along with supportive care. However she deteriorated and shifted to veno-arterialextracorporeal membrane oxygenation support (ECMO). Her plateletcount dropped by>50% at day 6 of heparin exposure with anintermediate probability for HIT (4Tscore 5). HIT gel agglutinationtest was positive for IgG antibodies for antiheparin/PF4 antibodies butfunctional assay based on heparin-induced platelet aggregation(HIPA) revealed no increase in aggregation of patient serum with0.5U/ml and 1U/ml heparin dosage. Heparin induced thrombocytopenia was ruled out due to PAT and patient continued on ECMO.This could be due to increased platelet activating immune complexesor anti-PF4 antibodies mimicking antiheparin/PF4 antibodies. However, patient deteriorated and succumbed to the disease.Result: Heparin induced thrombocytopenia was ruled out due to PATand patient continued on ECMO. This could be due to increasedplatelet activating immune complexes or anti-PF4 antibodies mimicking antiheparin/PF4 antibodies. However, patient deteriorated andsuccumbed to the disease.Conclusions: Pathophysiology of Covid 19 disease in critical caseshas shown exacerbated immune reactions, increased endothelialinjury, which causes increased release of PF4 leading to plateletactivation.3 A recent work has also shown significantly increasedplatelet apoptosis, secondary to IgG-mediated FccRIIA signaling, incritically ill COVID-19 patients.4 It is also possible that the circulating immune complexes may be formed by corona virus-antibodycomplexes (as seen in H1N1 viral infection) 0.5 A high titre of antiPF4/heparin antibody test may not strongly predict the presence ofclinically relevant HIT antibodies, thus a confirmatory functional testshould be performed.

Evaluation of Heparin-Induced Thrombocytopenia in Severe COVID-19 (Case Series)

Introduction: Thrombocytopenia is associated with severe coronavirus disease-2019 (COVID-19) with the reported incidence rate to be between 5-41.7%. Heparin-induced thrombocytopenia (HIT) is typically considered a minor contributor with low incidence of 0.2-3%. However, one study noted an 8% incidence of HIT in patients with severe COVID-19. Due to the potential higher risk of HIT and the baseline higher risk of thrombosis in severe COVID-19 infection, it is important to evaluate HIT prevalence in severe COVID-19 patients. We reviewed seven potential HIT cases. Case: All seven patients with positive heparin-PF4 antibodies had severe acute respiratory distress syndrome. D-dimer was elevated in four. Median duration of heparin and/or low-molecular-weight heparin exposure was 16 days. In five cases, HIT diagnosis was made greater than 10 days post exposure. All patients had intermediate to high pretest probability for HIT. Three patients had confirmed thrombosis, and one experienced multiple clotted lines (despite negative imaging for thrombosis). Argatroban was initiated in all patients. Serotonin release assay (SRA) was obtained for two patients-one resulted positive. Only two patients survived to be discharged from the intensive care unit/hospital. Discussion: HIT may be a larger contributor to thrombocytopenia in severe COVID-19 patients. When reviewing data from pre-COVID-19 years, the incidence rate of those screened for HIT ranged between 0-4.8%. During our study period, the incidence rate of those screened for HIT was 12.9%. 78% of those with positive antibodies had COVID-19. This potential increased incidence may be attributed to patient/disease specific factors or to increased doses of heparin treatment of possible thrombosis. Until it is further characterized, it is important to screen thrombocytopenic patients with severe COVID-19 for HIT. Conclusion: True incidence of HIT in severe COVID-19 is unclear, but it may be an important contributor to thrombocytopenia that can affect patients' thrombosis risk and anticoagulation choice which merits further review.

The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

[Formula presented] Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in “autoimmune” heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics “autoimmune” HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures: T iele: Bristol Myers Squibb: Honoraria, Other;Pfizer: Honoraria, Other;Bayer: Honoraria;Chugai Pharma: Honoraria, Other;Novo Nordisk: Other;Novartis: Honoraria;Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria;Biomarin/Prosensa: Other, Research Funding;Sagent: Other, Research Funding;Rovi: Other, Research Funding;Gore inc.: Other, Research Funding;Bayer Healthcare: Other, Research Funding;Paringenix: Other, Research Funding;BMS: Honoraria, Other, Research Funding;MSD: Honoraria, Other, Research Funding;Boehringer Ingelheim: Honoraria, Other, Research Funding;Aspen: Honoraria, Other, Research Funding;Portola: Other;Ergomed: Other;Instrument Laboratory: Honoraria;Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

The Role of PF4 Antibodies in Pediatric Sars-Cov-2 Infections

Background The ChAdOx1 nCoV-19 vaccine has been shown to induce Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT), a syndrome that shares clinical features with heparin-induced thrombocytopenia (HIT). The mechanism of thrombocytopenia and thrombosis in these disorders appears to be related to the development of pathologic anti-PF4/heparin antibodies, some of which could activate complement. Interestingly, we and others have found that complement activation is vital when both pediatric and adult patients have severe respiratory illness from SARS-CoV-2 virus (COVID-19) or in the post-infectious multisystem inflammatory syndrome in children (MIS-C). We hypothesized that patients with severe COVID-19 or MIS-C develop similar anti-PF4/heparin antibodies, which lead to endothelial complement activation that drive the inflammatory responses seen in these diseases. Methods Our cohort included 30 pediatric patients with positive SARS-CoV-2 RT-PCRs: 10 each of severe COVID-19 (“Severe”, MIS-C, and mild/asymptomatic (“Mild”) infection. Using ELISA, we evaluated the levels of antibodies to various platelet-related proteins including PF4, PF4-heparin, and NAP2;in addition, we examined the ability of plasma from each patient to activate complement. The antibody levels were compared to control samples including samples from adult patients with VITT and HIT. Statistical analyses with ANOVA were performed to evaluate differences. Results Patients with MIS-C have a significantly higher anti-PF4 antibody concentration (as measured by mean optical density [OD]) than patients with either mild/asymptomatic disease, or severe COVID-19: Severe 0.5 +/- 0.14;Mild 0.3 +/- 0.12;MIS-C 0.77 +/- 0.35, p=0.003 MIS-C vs. Mild);Similar results were seen for anti-PF4/heparin antibodies: Severe 0.4 +/- 0.14;Mild 0.35 +/- 0.12;MIS-C 0.64 +/- 0.3, p=0.003 MIS-C vs. Mild;p=0.034 MIS-C vs. Severe). These were similar to values obtained for the HIT sample (Figure). Conclusion Patients with MIS-C and severe COVID19 have significant detectable anti-PF4 and PF4/heparin antibodies in contrast to those patients with mild/asymptomatic disease. Our previous studies have shown that patients with MIS-C and COVID-19 have evidence of endovascular complement activation in the form of elevated soluble membrane attack complex (sC5-b9). We have also previously demonstrated that VITT anti-PF4 and anti-PF4/heparin antibodies activate complement and result in endothelial cell activation. These antibodies in pediatric SARS-CoV-2 infection may be involved in the development of more severe disease manifestations. Ongoing investigations will identify if this is due to endothelial complement activation and inflammatory responses that accompany severe disease. This is the first demonstration of the role of anti-PF4 and PF4/heparin antibodies in pediatric SARS-CoV-2. [Formula presented] Disclosures: Bassiri: Guidepoint Global: Consultancy;Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company. Teachey: Janssen: Consultancy;NeoImmune Tech: Research Funding;Sobi: Consultancy;BEAM Therapeutics: Consultancy, Research Funding. Lambert: ClinGen, ISTH, ASH, GW University: Honoraria;Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Bayer: Consultancy;Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Astra Zeneca: Research Funding;Novartis: Consultancy, Honoraria, Research Funding;Sysmex: Research Funding;PDSA: Research Funding;Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Fundi g.

Vaccine-Induced Thrombocytopenia and Thrombosis (VITT) Antibodies Recognize Neutrophil-Activating Peptide 2 (NAP2) As Well As Platelet Factor 4 (PF4): Mechanistic and Clinical Implications

VITT is an immune-based complication of adenoviral-based vaccines used to immunize against SARS_CoV2. The antibodies in VITT have been described as directed at the platelet-specific chemokine PF4 (CXCL4). While the clinical course and target chemokine in VITT has much in common with the better-known thrombocytopenic/prothrombotic disorder, heparin-induced thrombocytopenia (HIT), which involves antibodies directed against PF4 bound to the polyanion heparin, the specific loci where VITT and PF4/polyanion HIT antibodies bind appear to differ in studies using alanine-scanning mutations of PF4 (Nature, 2021. DOI: 10.1038/s41586-021-03744-4). The VITT antigenic site localizes to a heparin-binding domain. Unlike the dominant HIT locus, the VITT locus is conserved not only between human and mouse PF4, but also between PF4 and the related platelet-specific chemokine NAP2 (CXCL7). NAP2 is also expressed and stored in platelet alpha-granules and is present in equimolar concentrations to PF4. Unlike PF4, NAP2 avidly binds the chemokine receptor CXCR2 and strongly activates neutrophils. We now show that antibodies from patients who developed VITT after both AstraZeneca (AZ) or Johnson and Johnson (JJ) adenoviral vaccines, unlike HIT antibodies, recognize mouse PF4 (Figure 1A). More importantly, both AZ and JJ VITT antibodies bound NAP2, while none of the HIT antibodies tested bound PF4 or NAP2 in the absence of heparin (Figure 1A). These results are consistent with the alanine-scanning studies that distinguish the HIT and VITT binding sites. Dynamic light scattering (DLS) showed that NAP2 and PF4 bind to the adenoviral vectors, including Ad5 and the AZ vector ChAdOx5, which leads to expression of SARS_CoV2 spike protein. ChAdOx2 vaccine and CsCl 2-purified ChAdOx2 bound to both proteins, but form larger complexes with NAP2 than with PF4 even at lower concentrations of this chemokine (Figure 1C). Removal of anti-PF4 antibodies by hPF4-Sepharose abrogated PF4-dependent binding, but did not significantly reduce binding to NAP2 (not shown), indicating that VITT plasma contains discrete pools of anti-PF4 and anti-NAP2 antibodies that may have distinct functional properties. Sandwich ELISA (not shown) and Western blot analysis of purified VITT IgG demonstrates the presence of hPF4-IgG and NAP2-IgG immune complexes in purified patient's IgG (Figure 2A). Functional studies show that both PF4 and NAP2 can activate platelets in the presence of VITT antibodies. Anti-PF4-depleted VITT IgG fraction retains the ability to activate platelets in the presence of NAP2 (Figure 2B). Thus, unlike HIT, VITT appears to target a shared antigenic site on the related chemokines PF4 and NAP2. This raises the question as to whether NAP2, as one the most abundant platelet chemokines released from activated platelets, is involved in the initiation and propagation of the immunothrombotic response. Additional studies are needed to see whether NAP2, which can potently and specifically activate neutrophils via CXCLR2, contributes to the specific thromboinflammatory phenotype seen in VITT. We propose using FcgammaRIIA+ mice that concurrently express human PF4 and NAP2 and specific knockout of each chemokine, available in our group, to further understand the pathogenesis of VITT and its thrombocytopenic/ prothrombotic phenotype. [Formula presented] Disclosures: Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees. Cines: Dova: Consultancy;Rigel: Consultancy;Treeline: Consultancy;Arch Oncol: Consultancy;Jannsen: Consultancy;Taventa: Consultancy;Principia: Other: Data Safety Monitoring Board.