Platelet-activating histone/antihistone IgG complexes in anti-PF4-negative thrombosis and thrombocytopenia syndrome

Abstract

Thrombosis and thrombocytopenia syndromes (TTS) describe immune-mediated thrombotic adverse reactions after vaccination against COVID-19. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a well-known subentity of TTS, caused by adenovirus vector-based vaccines. VITT is mediated by anti-platelet factor 4 (PF4) immunoglobulin G (IgG) antibodies, activating platelets via Fc-γ IIa receptors (FcγRIIa). We describe clinical and serological features of 18 patients with anti-PF4/heparin enzyme-linked immunosorbent assay (ELISA)-negative TTS in temporal relationship to messenger RNA (mRNA)-based COVID-19 vaccination. Symptoms began at a median of 7 (range 1 - 61) days after vaccination. Patients showed thrombocytopenia (platelet count 59 × 103/μL; range, 0 to 127 × 103/μL); petechiae (n = 7), venous thromboembolism (n = 11), arterial thrombosis (n = 6), disseminated intravascular coagulation (n = 1), and combined arterial and venous thromboses (n = 1). Twelve sera-induced FcγRIIa-dependent and caspase-independent procoagulant activation of platelets indicated by phosphatidylserine exposure and CD62P expression. We found histones precipitated with IgG fractions of TTS sera. Antibodies binding to histones were found in 8 of 12 platelet-activating sera. Ex vivo-generated histone/antihistone IgG complexes strongly activated platelets via FcγRIIa, whereas antihistone IgG alone did not. Platelet autoantibodies were detected in 7 of 12 sera targeting glycoprotein (GP) IIb/IIIa (n = 5), GPIb/IX (n = 5), and GPIa/IIa (n = 3). However, sera containing platelet anti-GPIIb/IIIa autoantibodies activated also platelets from a patient with Glanzmann thrombasthenia, making it unlikely that these autoantibodies are causative for platelet activation. Finally, 2 of 114 healthy vaccinees developed antihistone antibodies after mRNA-based COVID-19 vaccination. Our data indicate a new subentity of TTS associated with platelet-activating histone/antihistone IgG complexes. Further studies are warranted to characterize the biological and clinical role of post-mRNA-based vaccination antihistone antibodies. The SeCo trial was registered at www.ClinicalTrials.gov as #NCT04370119.

Graphical abstract

Figure 1.

Clinical course of TTSs. Platelet counts (black line) D-dimer levels (blue line) and the clinical course of 2 index patients with anti-PF4–negative TTS after mRNA-based vaccination against COVID-19. (A) Index case 1: 57-year-old male with a history of kidney transplantation in 2009 and a recent COVID-19 infection with progressive thrombosis of the right femoral vein. Despite antithrombotic triple therapy, immunosuppression, and high-dose IVIG, only immunoadsorption was able to control the situation and resulted in a sustained platelet count increase. (B) Index case 2: 37-year-old male with a history of Hodgkin lymphoma in remission and prior episodes of ITP with petechiae, headache, thrombocytopenia, and elevated D-dimer. IVIG was administered to stabilize platelet counts but the patient developed pulmonary embolism and splanchnic vein thrombosis. Therapeutic dose anticoagulation, immunosuppression, and plasma exchange increased platelet counts without further thromboembolic events. LMWH, low-molecular-weight heparin; UFH, unfractionated heparin; VKA, vitamin K antagonists.

Figure 2.

Platelet activation pattern induced by TTS sera. Platelet activation induced by sera from 18 patients with anti-PF4/heparin ELISA–negative TTS. Control sera (n = 6) were obtained from healthy blood donors. Platelets of healthy donors were washed and incubated with the patients’ sera. Each dot represents the mean of at least 3 experiments. (A) Procoagulant platelet formation and (B) CD62P expression was assessed in flow cytometry. Activation was blocked by the monoclonal antibody IV.3, which inhibits platelet activation via FcγRIIa and by RGDS (blocks signaling of GPIIb/IIIa). Cutoff was defined as mean + 3 standard deviations of negative control (shown in beige). Negative control: phosphate-buffered saline; positive control: 20 μM thrombin receptor activating peptide + 100 ng/mL convulxin. Statistics were performed using an ordinary 1-way analysis of variance followed by an uncorrected Fisher least significant difference test, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; TRAP/Conv, thrombin receptor activating peptide + convulxin.

Figure 3.

Identification of histones as a target protein of immune complexes. Proteomic analysis of PEG-precipitated sera. Three sera from patients with anti-PF4–negative TTS (patients 2, 4, and 11; Table 1) were compared with 3 sera from healthy control donors, each subjected to PEG precipitation. Proteins were determined and quantified using LC electron-spray ionization tandem MS and results depicted in a volcano plot. Histones H2B type 1B (H2A1B), type 1-A H2B type 1-K (H2B1K); and H4 were found with the highest ratios (y-axis) among the significantly enriched proteins. ∗Highest ranked identification in LC electron-spray ionization tandem MS. adj., adjusted.

Figure 4.

Signaling pathways involved in immune complex–mediated procoagulant platelet activation. Platelet activation depends on extracellular calcium and is partially mediated through Syk signaling. (A) Platelets from n = 4 healthy donors were exposed to TTS sera after preincubation with the indicated inhibitors and blocking agents. (B) Platelets from n = 4 healthy donors were exposed to 2 μg/mL histone octamer and a mix of the corresponding mouse antihistone antibodies (0.1 μg/mL each) after preincubation with the indicated inhibitors and blocking agents. Statistics were performed using an ordinary 1-way analysis of variance followed by an uncorrected Fisher least significant difference test. Controls are the uninhibited conditions with TTS sera (A) and generated histone/antihistone complexes (B) ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (C) Quantification of cleaved caspase 3/7 activity after treatment with platelet activation–inducing or noninducing plasma samples (n = 4 per group) as determined by flow cytometry. Viable platelets were assessed without paraformaldehyde fixation after incubation with TTS sera inducing PS exposure (PS+) compared with sera from patients with TTS and/or immune thrombocytopenia that do not induce PS exposure (PS−). Gating and analyses of MFIs were performed using FlowJo version 10 (BD Biosciences). AB, antibody; MFI, mean fluorescence intensity.

Figure 5.

Platelet activation induced by histones, antihistone antibodies, and histone/antihistone immune complexes. PS exposure and CD62P expression of washed platelets incubated with high concentrations of histone octamers and antihistone antibodies alone (A-B); with various concentrations of histone octamers mixed with antihistone antibodies (B-C); and histone dimers H2A/H2B with a mix of the corresponding antihistone antibodies (D-E). PS exposure was assessed after annexin-V staining and CD62P expression in flow cytometry. Immune complexes were produced by incubating human histones and mouse antihistone antibodies dose dependently. High concentrations of histone octamers were able to activate platelets. This was independent of FcγRIIa because platelet activation could not be blocked by the monoclonal antibody IV.3 (which inhibits platelet activation via FcγRIIa) (A-B). In contrast, 10-fold lower concentrations of histone octamers were only able to activate platelets after incubation with the corresponding antihistone antibodies. This could be blocked by the monoclonal antibody IV.3 showing that activation by immune complexes was FcγRIIa dependent (B-C). The same was observed for histone dimers H2A/H2B incubated with the corresponding antihistone antibodies ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; TRAP/Conv, thrombin receptor activating peptide + convulxin.

Figure 6.

Activation response of GPIIbIIIa-deficient platelets to TTS sera. CD41 expression and platelet activation of Glanzmann platelets in response to sera from patients with TTS. (A) CD41 measured on normal platelets and platelets from a patient with Glanzmann thrombasthenia. (B) Patients’ sera containing or lacking platelet autoantibodies with GPIIb/IIIa specificity were incubated with normal platelets and platelets from a patient with Glanzmann thrombasthenia. Platelet activation was assessed by testing PS exposure after annexin-V staining in flow cytometry, cutoff was defined as mean of negative sera (shown in beige).

Figure 7.

Development of antihistone IgG in temporal relationship to mRNA vaccination. (A) Antihistone IgG titers in 2 of 144 vaccinated participants of the SeCo study, who received 2 shots of mRNA-based COVID-19 vaccines. (B) Platelet activation analysis with the sera of vaccinees who tested positive for antihistone IgG. Platelets of healthy donors were washed and incubated with the vaccinees’ sera. Each dot represents the mean of at least 3 tests. No procoagulant platelet formation was detected by testing PS exposure after annexin-V staining in flow cytometry. Negative control: phosphate buffered saline; positive control: 20 μM thrombin receptor activating peptide + 100 ng/mL convulxin. PBS, phosphate-buffered saline; TRAP/Conv, thrombin receptor activating peptide + convulxin; vacc., vaccination.