Anti-PF4 VITT antibodies are oligoclonal and variably inhibited by heparin

Abstract

Background COVID-19 vaccines have been associated with a rare thrombotic and thrombocytopenic reaction, Vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by platelet-activating anti-PF4 antibodies. This study sought to assess clonality of VITT antibodies and evaluate their characteristics in antigen-based and functional platelet studies. Methods Anti-PF4 antibodies were isolated from five patients with VITT secondary to ChAdOx1 nCoV-19 (n=1) or Ad26.COV2.S (n=4) vaccination. For comparative studies with heparin-induced thrombocytopenia (HIT), anti-PF4 antibodies were isolated from one patient with spontaneous HIT, another with classical HIT, and two patients with non-pathogenic (non-platelet activating) anti-PF4 antibodies. Isolated antibodies were subject to ELISA and functional testing, and mass spectrometric evaluation for clonality determination. Results All five VITT patients had oligoclonal anti-PF4 antibodies (3 monoclonal, one bi- and one tri-clonal antibodies), while HIT anti-PF4 antibodies were polyclonal. Notably, like VITT antibodies, anti-PF4 antibodies from a spontaneous HIT patient were monoclonal. The techniques employed did not detect non-pathogenic anti-PF4 antibodies. The ChAdOx1 nCoV-19-associated VITT patient made an excellent recovery with heparin treatment. In vitro studies demonstrated strong inhibition of VITT antibody-induced platelet activation with therapeutic concentrations of heparin in this and one Ad26.COV2.S-associated VITT patient. Oligoclonal VITT antibodies with persistent platelet-activating potential were detected at 6 and 10 weeks after acute presentation in two patients tested. Two of the 5 VITT patients had recurrence of thrombocytopenia and one patient had focal seizures several weeks after acute presentation. Conclusion Persistent oligoclonal anti-PF4 antibodies mediate VITT. Current guidance to avoid heparin in all VITT patients may need to be reassessed.

Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS

“…For VITT, only ELISA-based immunological assays are consistently sensitive to anti-PF4/X antibodies, with rapid assays, otherwise sensitive to anti-PF4/H antibodies in HIT, being mostly negative in VITT [ 23 , 28 , 37 ]. Moreover, the addition of therapeutic levels of heparin augments the detection of anti-PF4/H antibodies in HIT/HITT, by both immunological and functional assays, whereas for VITT, therapeutic levels of heparin may reduce the detection of anti-PF4/X antibodies by immunological (ELISA) testing and inhibit platelet activation in functional assays [ 30 , 35 ]. This is probably due to the fact that anti-PF4 antibodies in HIT vs. VITT target different epitopes on PF4 and thus may compete for PF4 binding, as recently shown by Huynh and colleagues [ 38 ].…”

“…Some additional insights into the differences between the antibodies formed in different pathologies have recently emerged. Singh et al [ 35 ], in an interesting study, albeit including only a small number of cases, showed that antibodies from HITT patients tended to be polyclonal, whereas those from patients with VITT and from one case of ‘spontaneous HIT’ were oligoclonal. Moreover, immunological antibody detection by ELISA for VITT cases was the most accurate when using noncomplexed PF4, followed by PF4 in complex with polyvinyl sulfonate (PVS), and the least accurate with PF4 in complex with heparin (PF4/H).…”

“…An example of this for two patients from the Westmead Hospital laboratory is provided in Figure 5 . However, therapeutic heparin does not inhibit platelet activation in all VITT patients [ 34 , 35 ], and so this does not provide an infallible distinction from HITT.…”

Cerebral Venous Sinus Thrombosis due to Thrombosis with Thrombocytopenia Syndrome Following Ad26.COV2.S: A First Real-World Case Report of a Male Subject

“…At the time of the last report, 3 deaths were reported, and only 4 patients were discharged. Though there is concern that heparin exposure could potentially worsen the thrombosis and affect the overall outcome, emerging evidence shows that exposure to heparin did not lead to significant harm 11,12 with mortality rate of 20% vs 16% in those who did and did not receive heparin, respectively. More data is needed to address this question.…”

The aetiopathogenesis of vaccine-induced immune thrombotic thrombocytopenia

“…> In blood samples from most VITT patients, PF4 and high titers of anti-PF4 have been detected. [48][49][50] PF4 circulates at higher concentrations in younger than in older people with enhancement during inflammatory situations. 51 Anti-PF4 antibodies are not normally detectable but high titers are present in VITT.…”