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.

Keywords: AZD1222 vaccination; FcγRIIA; VITT; anti‐PF4 antibodies; thrombosis.

FIGURE 1

Laboratory parameters of seven patients with VITT 199 to 217 days following vaccination with AZD1222. Gray box denotes normal range for platelet count and fibrinogen, brown box denotes normal range for D‐dimer. A/C, anticoagulation; Dex, dexamethasone; IP, inpatient; IVIg, intravenous immunoglobulin; NR, normal range; OP, outpatient; PEX, plasma exchange; Pred, prednisolone. The patients are identified by their number (P1‐P7). Laboratory parameters and details of clinical information of the patients with VITT can be found in Supplementary Materials

FIGURE 2

Levels of anti‐PF4 IgG and PF4 level's in the serum of patients with VITT during follow‐up. (Ai) Serum anti‐PF4 levels of VITT patients (n = 7) at diagnosis and during follow‐up timepoints after vaccination. Samples measured in duplicate, mean ± SEM. OD 405, optical density at 405 nm. Ref, clinical control reference range <0.4 O.D. (Aii) Patient serum anti‐PF4 IgG levels at diagnosis and later follow‐up timepoint (nearest to 200 days) (n = 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

FIGURE 3

Measurement of patient serum‐mediated platelet aggregation in controls and patient platelets, patient platelet responsiveness, and PF4 serum levels. (A) Serum for three VITT patients (P1, P5, P7) activates platelets during follow‐up. Aggregation (area under the curve [AUC]) of healthy “responsive” donor platelets (mean + SD, n = 3) and VITT patient platelets (2 × 10⁸/ml) with diagnostic and follow‐up patient serum (14:1 v:v) in the absence and presence of 10 µg/ml PF4. (B) Decline in platelet aggregation with follow‐up serum for four VITT patients (P2, P3, P4, P6) with donor and patient platelets. (C) Median fluorescence intensity of CD32a (FcγRIIA)‐positive washed platelets (2 × 10⁷/ml) (n = 8) healthy controls (black), including the three responsive controls, and n = 7 VITT patients (P1; day 200, P2; day 221, P3; day 280, P4; day 157, P5; day 158, P6; day 109 and P7; day 112 after vaccination, VITT patient results shown in blue). Results shown as mean ± SD. (D) Aggregation (AUC) of patient (n = 7) and healthy “responsive” control platelets (n = 3) with 0.1 U/ml thrombin or 1 μg/ml collagen‐related‐peptide for 8 min. VITT patient results shown in blue