key: cord-0710417-3nvi9crz authors: McFadyen, James D.; Sharma, Prerna; Moon, Mitchell J.; Noonan, Jonathon; Goodall, Elizabeth; Tran, Huyen A.; Peter, Karlheinz title: Activation of circulating platelets in vaccine‐induced thrombotic thrombocytopenia and its reversal by intravenous immunoglobulin date: 2021-08-16 journal: Br J Haematol DOI: 10.1111/bjh.17750 sha: b93b9458e832cb7e9aa42912a08a4e1d44a6b5f4 doc_id: 710417 cord_uid: 3nvi9crz nan We report a 55-year-old female with a background history of hypertrophic cardiomyopathy, ischaemic heart disease, obstructive sleep apnoea, epilepsy, and schizophrenia, who presented to hospital with chest pain 14 days after the first dose of the ChAdOx1 nCoV-19 vaccine. Consistent with a diagnosis of VITT, her platelet count was 45 9 10 9 /l with a D-dimer of 65Á94 µg/ml and strongly positive HIT enzyme-linked immunosorbent assay (ELISA, 143Á9%; normal cut-off 10Á5% -Stago Asserachrom, Diagnostica Stago S.A.S., Asni eres-sur-Seine, France) (Fig 1) . A subsequent computed tomography (CT) pulmonary angiogram demonstrated bilateral pulmonary emboli and venous doppler ultrasonography revealed a proximal deep vein thrombosis involving the popliteal vein. Therapeutic anticoagulation was commenced with fondaparinux 7Á5 mg subcutaneously daily and IVIg was administered at a dose of 90 g (1 g/kg). Informed consent was obtained, and blood samples were taken before and after the administration of IVIg for flow cytometry analysis of platelet activation. Over the following 5 days the platelet count steadily incremented, which was associated with a concordant fall in the D-dimer. However, 6 days after the initial dose of IVIg the patient developed chest pain and dyspnoea, in association with a reduction in platelet count to 108 9 10 9 /l. Subsequent imaging with a CT pulmonary angiogram revealed a reduction in thrombus burden of the left lung; however, demonstrated extension of thrombosis within the segmental and subsegmental vessels of the right lower lobe. Therefore, fondaparinux was changed to bivalirudin and a further dose of IVIg (1 g/kg) was administered. This was associated with an increment of the platelet count to 157 9 10 9 /l. Over the following days, the platelet count remained normal and the patient at the time of reporting is stably anticoagulated with bivalirudin and being transitioned to oral anticoagulation with a vitamin K antagonist, given the known interactions of carbamazepine with direct oral anticoagulants. Prior to IVIg administration a significant percentage of platelets were positive for markers of platelet activation. Indeed, 13% of platelets expressed activated glycoprotein IIb/IIIa (GPIIb/IIIa, CD41/CD61, a IIb b 3 ) with 19Á1% demonstrating P-selectin [CD62P, granule membrane protein-140 (GMP-140)] surface expression (Fig 2) . In keeping with the significant degree of platelet activation there was a significant increase in platelet-monocyte aggregates, with 26Á5% of monocytes displaying evidence of bound platelets. Strikingly, repeat testing after IVIg administration demonstrated that levels of PAC-1 binding, P-selectin expression, and plateletmonocyte aggregates had returned to a level comparable to a healthy donor. These data suggest that a significant percentage of circulating platelets in patients with VITT are highly activated. The finding of elevated levels of circulating correspondence platelet-monocyte aggregates is indicative of platelet activation; however, also suggests that monocyte activation is a feature of VITT. Overall, these findings are consistent with the pro-thrombotic presentation of our patient with VITT. To our knowledge, the present case provides the first direct evidence of a patient with VITT presenting with activated platelets in the circulation and the first direct evidence of the ability of IVIg to abrogate this enhanced platelet activation state in VITT. Recent reports have suggested that plasma/ serum of patients with VITT have the propensity to activate platelets from healthy donors. 10 Our present report is the first to show that a large number of activated platelets are present in the circulating blood of a patient with VITT. The flow cytometric data demonstrating platelet activation is very robust: (i) Binding of PAC-1 identifies platelets with the activated GPIIb/IIIa receptor. The conformational change of this integrin from a low-to a high-affinity state for its ligand is reflecting the final common pathway of platelet activation. (ii) P-selectin expression on the platelet surface represents platelet degranulation, as P-selectin is 'stored' in a-granules, which are fused with the membrane upon platelet stimulation. (iii) Platelet-monocyte aggregate formation is a sensitive marker of platelet activation and has previously been found to be elevated in various thrombotic disorders such as acute coronary syndromes. Moreover, GPIIb/IIIa activation and P-selectin expression have previously been used to demonstrate platelet activation associated with HIT and GPIIb/IIIa inhibitor-induced thrombocytopenia. 10, 11 The reported data are also unique in respect to the high percentage of platelets circulating in an activated state, further highlighting the acuteness and severity of VITT. The finding of increased numbers of platelet-monocyte aggregates is important as they are not only a sensitive marker of platelet activation but are also likely reflective of enhanced monocyte activation as a feature of VITT. Activated monocytes have been implicated in the pathogenesis of HIT, given they express the FccRIIa receptor and therefore are also liable to activation by pathogenic antibodies. 12 Whilst platelet-monocyte aggregates have previously been associated with venous thromboembolic disease, HIT, and severe COVID-19 infection, 13 the number of platelet-monocyte aggregates detected, and the finding that this number is reduced after IVIg administration is a novel and important finding. This also strongly suggests that these changes are the result of pathological VITT antibodies. 14 Similar trends were seen in our present patient with platelet-neutrophil aggregates, which is of particular relevance as neutrophil extracellular trap formation has been proposed to be involved in VITT. 15 Although not elucidated in the present report, previous reports from both patients with VITT and HIT suggest the beneficial effects of IVIg in modulating platelet activation are likely due to the ability to competitively inhibit pathological antibody binding to platelet FccRIIa receptors. 2, 7 Notably, the clinical course of our present patient experiencing thrombus extension 6 days after IVIg likely suggests that high levels of Ig are required to competitively inhibit platelet FccRIIa receptor binding. This raises the interesting possibility of whether changes in platelet activation markers can be utilised to assess the response to IVIg treatment and/or potentially help predict treatment relapse. Collectively, these findings support the role of platelet activation as a central mechanism of VITT and confirm the beneficial role of IVIg in preventing platelet activation. SARS-CoV-2 vaccine-induced immune thrombotic thrombocytopenia Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination US case reports of cerebral venous sinus thrombosis with thrombocytopenia after Ad26.COV2.S vaccination Pathologic antibodies to platelet factor 4 after ChAdOx1 nCoV-19 vaccination Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination Adjunct immune globulin for vaccine-induced thrombotic thrombocytopenia The use of intravenous immunoglobulin in the treatment of vaccine-induced immune thrombotic thrombocytopenia A flow cytometric assay to detect platelet-activating antibodies in VITT after ChAdOx1 nCov-19 vaccination Antibodymediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4-heparin antibodies to platelets and the resultant platelet activation Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia The emerging threat of (micro)thrombosis in COVID-19 and its therapeutic implications Intravenous immune globulin to prevent heparin-induced thrombocytopenia Towards understanding ChAdOx1 nCov-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) James D. McFadyen designed research, analysed data and wrote the paper. Prerna Sharma performed research and analysed data. Mitchell J. Moon performed research and analysed data. Jonathon Noonan analysed data. Elizabeth Goodall analysed data. Huyen A. Tran analysed data and wrote the paper. Karlheinz Peter designed research, analysed data and wrote the paper. The study was funded by the National Health and Medical Research Council of Australia.