Anti‐platelet factor 4 immunoglobulin G levels in vaccine‐induced immune thrombocytopenia and thrombosis: Persistent positivity through 7 months

Abstract Background Anti‐platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine‐induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown. Objectives To assess how clinical and platelet parameters, anti‐PF4 antibody levels, and patient serum reactivity changes during follow‐up after VITT presentation. Methods We describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199–280) after vaccination. We measured anti‐PF4 antibodies and PF4 levels in patient serum during follow‐up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time. Results Anti‐PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9–2.6 OD) and remain relatively high (>1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow‐up and no patients had recurrent thrombosis. Patients’ platelets during follow‐up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow‐up serum alone was weaker at stimulating donor and patient platelets. However, follow‐up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50. Conclusions Explanations for reduced platelet activation during follow‐up, despite similar total anti‐PF4 antibody levels, remains unclear. Clinical implications of persistent anti‐PF4 antibodies in VITT require further study.

• We measured lab tests for VITT patients after recovery in affected patients.
• Anti-Platelet Factor-4 antibody levels were high after 7-months but platelet activation reduced.
• Implications of reduced platelet activation despite high anti-PF4 antibody levels remain unclear.

| INTRODUC TI ON
Vaccination is the major defense against the SARS-CoV-2 pandemic.
In April 2021, a new syndrome of vaccine-induced immune thrombocytopenia and thrombosis (VITT) was described. in the United States. 5,6 VITT is characterized by thrombocytopenia, high D-dimer levels, and aggressive thrombosis at unusual venous sites, including cerebral venous sinuses. Arterial thromboses have also been described. 7,8 Serum from patients with VITT has been shown to activate platelets via the FcγRIIA receptor. 1,2,9,10 Patients with VITT have very high levels of antibodies to platelet factor 4 (PF4) 3 that bind with high affinity to a single site on PF4. 9 Vaccine-induced immune thrombocytopenia and thrombosis treatment modalities have evolved rapidly and include non-heparin anticoagulation, corticosteroids, intravenous immunoglobulin (IVIg), thrombectomy, and plasma exchange. 7,11 Rituximab has also been used in patients who are nonresponsive to first-line therapies. 7 Despite treatment, mortality remains around 20% to 50% depending on presence of intracerebral bleeding, geographical region, and ability to deliver early treatment. 4,12,13 Those that respond do so quickly and are discharged on non-heparin anticoagulation.
Here, we describe the clinical course, laboratory results, anti-PF4 IgG, PF4 serum levels, and the ability of serum to activate healthy donor and patient platelets in seven patients with VITT who survived their initial presentation and were followed for up to 280 days after vaccination with AZD1222.

| Patients and ethical approval
All patients presenting with VITT at our center who survived to discharge were recruited. Blood was collected at diagnosis and each time the patients arrived for clinical follow-up. Informed consent was provided by the patients or next of kin in those who lacked ca-

| PF4 antibody and PF4 serum level measurements
Patient serum samples were collected at diagnosis and multiple follow-up timepoints. Healthy control serum was taken from multiple donors, including AZD1222-vaccinated healthy controls (n = 4, from the COCO study) and the same-day controls (n = 3, healthy non-agematched controls processed alongside follow-up serum). Serum was collected following centrifugation (2000 g, 10 min, room temperature [RT]) of clotted blood. Anti-PF4 antibodies were measured using an anti-PF4/heparin enzymatic immunoassay (EIA, LIFECODES PF4 IgG assay; Immucor GTI Diagnostics) for IgG PF4-heparin antibodies. Anti-PF4 antibodies in patient serum (diluted 1:50) from all time points were measured on the same day using a Molecular Devices platelet reader (wavelength 405 nm). Serum PF4 levels were measured using the PF4 human ELISA kit (Invitrogen). Serum was diluted 1:500 in assay buffer and performed according to the manufacturer's protocol. Patient samples were measured in duplicate.

K E Y W O R D S
anti-PF4 antibodies, AZD1222 vaccination, FcγRIIA, thrombosis, VITT

| Statistical analysis
All data are presented as mean ± standard deviation (SD) unless stated. Statistical comparisons performed by one-way ANOVA with Tukey multiple comparisons after normality tests. All analysis performed with GraphPad Prism 9.0 (La Jolla, CA). We next compared the ability of diagnostic and follow-up serum to activate platelets in the absence or presence of PF4. Patient serum taken at diagnosis or during follow-up was tested on washed platelets from three healthy donors who were previously found to be responsive to VITT serum. 10 The same three donors were used for all patient serum at all timepoints. Serum taken at or soon after diagnosis from all seven patients induced powerful aggregation, which in three cases (P2, P3, P4) did not require addition of PF4 ( Figure 3A,B). Immediately following initial treatment for VITT (steroids, IVIg, and plasma exchange), serum from P2, P3, and P4 no longer/weakly activated healthy control platelets without additional PF4, with the PF4 enhancement also diminishing over time ( Figure 3B). We previously showed IVIg and plasma exchange reduced patient serum-mediated platelet activation. 10 However, it is interesting that in some patients, exogenous PF4 rescued this.

| RE SULTS AND D ISCUSS I ON
Aggregation to P3, P6, and P7 serum in the presence of PF4 also decreased over time, although mild and robust aggregation could be observed to P6 serum at day 157 and P7 serum at day 112, respectively ( Figure 3). In stark contrast, the latest follow-up serum We wanted to assess platelet characteristics and function of patient platelets. FcγRIIA (CD32a) levels were measured on all patients' platelets at latest follow-up timepoint, and most were found to be similar levels to healthy controls ( Figure 3C). P3's platelet FcγRIIA levels were low compared with controls and other VITT patients.
However, the responsiveness of all patients' platelets to thrombin (0.1 unit/ml) and collagen-related-peptide (1 μg/ml), measured at latest follow-up timepoint, was similar to controls ( Figure 3D).  7) compared with healthy control serum levels (n = 5, including serum from the three responsive donors). Individual patient symbol corresponds to patient labels in (i). Results reported as mean ± SD. One-way ANOVA with Tukey's multiple comparison test, **p < 0.01, ****p < 0.0001. (Bi) Serum PF4 levels of all patients with VITT (n = 7) at diagnosis and follow-up timepoints after vaccination. (Bii) Patient serum PF4 levels at diagnosis and latest follow-up timepoint (n = 7) compared with healthy controls (n = 10), including AZD1222-vaccinated individuals (n = 4, open circle). Individual patient symbol corresponds to patient labels in (i). Results reported as mean ± SD. One-way ANOVA with Tukey multiple comparison test, ***p = 0.0005, ****p < 0.0001 This study shows that although anti-PF4 IgG antibody levels are high at diagnosis and, although they decline during follow-up, they still remain above control reference range 7 or more months following AZD1222 vaccination. In our cohort, there is only an average 38% reduction in anti-PF4 IgG levels (range −10% to 69%) during follow-up. These patients still do not relapse, however. Two recent studies have also showed a decline in anti-PF4 IgG levels in VITT patients in a shorter follow-up period (median 11-12 weeks). 16,17 Interestingly only 3/35 patients 16  having the potential to activate patient and donor platelets. We consider this particularly important given that light transmission aggregation is considered a lower sensitivity assay than the gold standard serotonin release assay in a similar condition, heparininduced thrombocytopenia (HIT). 18 Further, we show that serum PF4 levels reduce at VITT presentation, but return to normal over time and that the responsiveness of the patients' own platelets and FcγRIIA levels are similar to controls. The absence of relapse may be due to ongoing treatment with DOACs. It may also be due to consumption of a subset of high affinity or platelet-activating anti-PF4 IgG antibodies that are required to generate immune complexes at initial diagnosis that are no longer present during follow-up.
One limitation in the present study is the reliance on ELISAs to measure anti-PF4 IgG antibodies. These do not quantitatively