Heparin- and Vaccine-Independent Anti-Platelet Factor 4 Immunothrombosis

Background: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. It is unknown whether a VITT-like prothrombotic disorder exists in the absence of vaccination.Methods: We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or vaccination who tested strongly positive by PF4/polyanion EIA and negative/weakly-positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (i.e., VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL® AcuStar HIT-IgG(PF4-H)) as well as a newly-developed chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera also included an exploratory anti-PF4 antibody-positive but HIPA negative/weak cohort prior to 2020 (n=188).Findings: We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n=3; cerebral venous sinus thrombosis, n=4), thrombocytopenia (median platelet count nadir, 49×109/L), and hypercoagulability (greatly elevated D-dimer levels). All 9 patients showed strong VITT-like reactivity by platelet activation assay and tested positive in the novel anti-PF4 chemiluminescence assay; 3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay. Our exploratory cohort identified 13 additional patients from before 2020 with VITT-like anti-PF4 antibodies.Interpretation: VITT-like platelet-activating anti-PF4 antibodies can be associated with a severe prothrombotic disorder characterized by thrombocytopenia, thrombosis at unusual sites, and very high D-dimer levels, in the absence of preceding exposure to heparin or adenovirus vector-based vaccines. Such VITT-like antibodies can be identified by a new chemiluminescence assay developed to detect VITT antibodies. Further, such antibodies are found in sera obtained prior to 2020, and thus are not explained by Covid-19 vaccination.Trial Registration:Funding: DFG: 374031971-TRR240, GR2232/9-1, SCHO2052/1-1, TH2320/3-1. EMA contract No.EMA/2021/17/TDA. Gerhard-Domagk-Research-Program, Universitätsmedizin Greifswald (L.S.)Declaration of Interest: A. Greinacher reports grants and non-financial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, Werfen, nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V. outside the submitted work. O. Esteban, P. Dobosz, M. Broto, S. Rovira Puig, R. Torres, J. Serra, R. Llevadot and M. Palicio are employees of Werfen. O. Esteban, P. Dobosz, M. Broto, R. Llevadot, and A. Greinacher have filed a patent application (PCT/EP2023/062615) covering the design and use of the new rapid anti- PF4 assay. E. Lindhoff-Last has received lecture honoraria and advisory fees from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb/Pfizer, Daiichi-Sankyo, Portola, CSL Behring, Viatris, Werfen, Norgine and Aspen and institutional research support from Bayer AG, Bristol-Myers Squibb/Pfizer, Daiichi-Sankyo and CSL Behring for a different research project. F. Langer has received personal fees for lectures or consultancy from Alexion, AstraZeneca, Bayer, BioMarin, BioNTech, Bristol Myers Squibb, Chugai, CSL Behring, Daiichi Sankyo, Grifols, Janssen-Cilag, LEO Pharma, Mitsubishi Tanabe Pharma, Novo Nordisk, Pfizer, Roche, Swedish Orphan Biovitrum, Takeda, Viatris, Werfen and institutional research support from Bayer, Chugai, CSL Behring, Intersero, Novo Nordisk, Pfizer, Swedish Orphan Biovitrum. L. Alberio reports institutional research support from Bayer, CSL Behring, Novartis, Novo Nordisk, Octapharma, Roche, Sobi, Takeda/Shire, Werfen as well as fees for lectures or consultancy from Bayer, Biotest, Boehringer-Ingelheim, CSL Behring, Daiichi Sankyo, Novo Nordisk, OrPha Swiss, Roche, Sanofi-Aventis, Sanofi-Genzyme, Siemens, Sobi, Takeda/Shire, Viatris, Werfen. M. Endres reports grants from Bayer and fees paid to the Charité from Abbot, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, Sanofi, Novartis, Pfizer, all are all outside the submitted work. T. Thiele reports personal fees and other from Bristol Myers Squibb, personal fees and other from Pfizer, personal fees from Bayer, personal fees and other from Chugai Pharma, other from Novo Nordisk, personal fees from Novartis, other from Daichii Sankyo, other from LFB Pharma, all of which are outside the submitted work. L. Schönborn receives a young investigator grant of the medical faculty of the Universitätsmedizin Greifswald. T. Hoffmann has received lecture honoraria and advisory fees from CSL Behring, Werfen and Takeda. C. Boehme has received lecture honoraria from Abbvie and travel grants from Pfizer and Sanofi outside the submitted work. T.E. Warkentin has received lecture honoraria from Werfen (Instrumentation Laboratory), and royalties from Informa (Taylor & Francis); has provided consulting services to Ergomed, Paradigm Pharmaceuticals, Octapharma, and Veralox Therapeutics; has received research funding from Werfen (Instrumentation Laboratory); and has provided expert witness testimony relating to heparin-induced thrombocytopenia (HIT) and non‐HIT thrombocytopenic and coagulopathic disorders. J. Wesche, J. Fuhrmann, J. J. Wang, T. P. Gordon, L. Grosse, E. Biguzzi, and T. Liman declare no conflict of interest.Ethical Approval: The study was approved by the ethics board of the Universitätsmedizin Greifswald (BB 052/21a). Permission to describe patient cases has been obtained. To respect confidentiality, patient ages are given as ranges. For the exploratory cohort the ethics commission permitted retesting of de-identified repository serum samples from before 2020, selected by the original test result.