Cerebral venous sinus thrombosis and thrombocytopenia due to heparin-independent anti-PF4 antibodies after adenovirus infection

Figures

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  Figure 1.. Radiological images and course of the platelet counts throughout the hospitalization. (A) Digital subtraction angiography was performed showing thrombosis of the superior sagittal sinus (black arrows). (B) Angiography after mechanical recanalization shows the recanalized cerebral superior sagittal sinus (black arrows). (C) Platelet count and other laboratory findings throughout hospital admission labeled with relevant interventions. Abbreviations and normal range of laboratory parameters: platelet count (normal range: 150-450x103/µL), D-dimer (normal range: 0-0.5 μg/mL fibrinogen equivalent units), fibrinogen (normal range: 170-410 mg/dL), activated partial thromboplastin time (aPTT; normal range: <40 seconds), factor XIII (normal range: 70-140%). IVIG: intravenous immunoglobulin; EIA: enzyme immunosorbent assay; Ig: immunglobulin; HIPA: heparin-induced platelet activation; PF4: platelet factor 4.

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  Figure 2.. Patient sera induces platelet activation and procoagulant platelet phenotype. (A) Heparin-induced platelet activation (HIPA): patient serum activated platelets with exogenous platelet factor 4 (PF4) in HIPA assay, however, patient’s sera did not activate platelets in the presence of low (0.2 IU/mL) or high concentration (100 IU/mL) of heparin. Historical representative samples from a heparin-induced thrombocytopenia (HIT) and a vaccine-induced immune thrombotic thrombocytopenia (VITT), patient are also depicted in the figure. (B) Flow cytometer: procoagulant platelet phenotype, determined by co-expression of P-selectin and phosphatidylserine (PS) on platelet surface, was analyzed after incubation with patient’s sera. Where indicated, platelets were pretreated with IV.3 (Fcγ receptor IIA blocking monoclonal antibody) or immunoglobulin (IVIG). Patient’s serum was tested with washed platelets from 4 healthy donors. Historical samples of patients with HIT (N=3) and VITT (N=3) was also shown on the figure. HC: healthy control; **P<0.01, ****P<0.0001; IVIG: intravenous immunoglobulin.

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  Figure 3.. Patient sera induces thrombus formation in vitro. Platelet-rich plasma (PRP) obtained from healthy individuals in a volume of 37.5 µL was subjected to a 60-minute incubation with serum (5 µL) from the patient, all while under rotation. Following this incubation period, the samples were labeled using 3,3’-dihexyloxacarbocyaniniodid (DiOC6, 2.5 µM; Sigma Aldrich, Saint Louis, USA), Alexa Fluor (AF) 647-Annexin A5 (at a 1:200 dilution), AF 546-labeled human fibrinogen (at a concentration of 8.5 µg/mL), and Hoechst 33342 (at a concentration of 3 µg/mL; Thermo Scientific, Carlsbad, USA). When specified, the PRP was preincubated with IV.3 (20 µg/mL) or intravenous immunoglobulin (IVIG) (30 µg/mL). After the labeling procedure, the samples were reconstituted into autologous whole blood. Subsequently, the samples were recalcified and subjected to perfusion through microfluidic channels (BioFlux 200, Fluxion Biosciences, Alameda, USA) at a venous shear rate set at 250 s-1 (equivalent to 10 dyne/cm2) for a duration of 10 minutes. Images were acquired at x40 magnification in different fluorescence channels using a Zeiss Axio Observer 7 microscope. The acquired images were uniformly…