Monitoring the Hemostatic Effects of Inhibitory Alloantibodies in Von Willebrand Disease Using Global Coagulation Assays

Introduction

Repeated exposure to von Willebrand factor (VWF) concentrates is associated with the development of anti-VWF alloantibodies in 5-9 % of patients with type 3 von Willebrand disease (VWD). These antibodies are implicated in the loss of hemostatic response to VWF replacement therapies due to rapid clearance or inhibition of this factor. We evaluated the efficacy of various global coagulation tests and thrombus formation assays to monitor the effect of anti-VWF inhibitors on the response to plasma-derived (pd) VWF/FVIII treatment in a case of VWD that developed alloantibodies.

Methods

A 23-year-old man with type3 VWD, who started on prophylaxis with pdVWF/FVIII concentrates (Fanhdi®) at the age of 3 years, has been followed up since 2021. In 2023, the patient had several hemorrhagic episodes that were attributed to an increase in his physical activity.

VWF antigen (VWF:Ag), VWF activity by GPIb binding assays (VWF:GPIbR), and FVIII coagulation activity (FVIII:C) were analyzed before and after administration of the prophylactic dose (45U/Kg) of pdVWF/FVIII in plasma samples collected between 2021 and 2024. Inhibitor titration was performed using an adapted Bethesda assay.

Different assays were used to evaluate the basal hemostatic profile and ex vivo effect of pdVWF/FVIII: (a) Rotational thromboelastometry (ROTEM®) was performed to monitor clot formation with recalcification (NATEM) or with ellagic acid (INTEM) as activator. (b) Thrombin generation was measured by calibrated automated thrombography (CAT) using plasma activated with a low concentration of tissue factor (1 pM TF). (c) Coagulation- or platelet-dependent thrombus formation was evaluated under flow conditions using the Total Thrombus Formation Analysis System (T-TAS®) with microchips coated with collagen/thromboplastin (AR chip) or collagen (PL chip). The area under the flow pressure curve (AUC) was obtained.

Results

In samples collected in 2021, an almost complete lack of VWF (0% VWF:GPIbR) and FVIII activity (<1% FVIII:C) was obtained in the VWD patient before treatment, but the administration of pdFVW/FVIII (45U/kg) resulted in a good recovery of both VWF (115% VWF:GPIbR) and FVIII (91% FVIII:C) activity. In 2023, we observed a reduced recovery of VWF:Ag (33%), VWF:GPIbR (74%) and FVIII:C (6.8%) after treatment with the same dose of pdFVW/FVIII. These results suggested the presence of alloantibodies and further testing of samples collected in 2023 allowed to detect anti-VWF inhibitors (1.3-0.7 BU). In 2024, inhibitor titers decreased (0.3 BU) and recovery after treatment was improved (145% VWF:Ag , 118% VWF:GPIbR and 78% FVIII:C).

Global assays performed in 2021, prior to inhibitor development, showed that VWF/FVIII deficiency in type 3 VWD resulted in prolonged clotting time (CT) (1173 s NATEM_CT [reference range: 300-1000s]; 374 s INTEM_CT [reference range: 100-240 s]), decreased thrombin generation (46 nM thrombin peak [reference range: 131-254 nM]), and very reduced coagulation-dependent (AR_AUC: 209 [reference range: 1050-1600]) and platelet-dependent (PL_AUC: 12.6 [reference range >260]) thrombus formation. Analysis of pre-dose samples with inhibitors collected in 2023-2024 showed similar altered values.

ROTEM and CAT monitoring of the ex vivo effect of pdVWF/FVIII in all samples, with (w) or without (wo) inhibitors, showed a comparable response, with similar CT reduction (NATEM_CT wo: 799s vs w: 699s; INTEM_CT wo: 374s vs w: 343s) and thrombin peak increase (wo: 366 nM vs w: 397 nM). However, the total recovery of T-TAS AUC values is only obtained using blood samples without inhibitor (0 BU) (AR_AUC wo: 1100 vs w: 201-878; PL_AUC wo: 273 vs w: 53-164), and a high inverse correlation (r:-0.95; p<0.05) was observed between sample inhibitor levels and AUC values obtained after addition of increasing doses of pdVWF/FVIII.

Conclusion

The flow chamber-based T-TAS system proved to be a reliable point-of-care device for monitoring the effect of alloantibodies on the hemostatic response to replacement therapies in VWD. Further studies are needed to validate our findings.

Funding: Fundación Rodríguez Pascual; Cátedra UAM-ROCHE; ISCIII-FEDER (PI22/01461).

G Arias-Salgado:Novo Nordisck: Speakers Bureau. Butta:Novo Nordisck: Speakers Bureau; Novartis: Speakers Bureau; Sobi: Speakers Bureau; Grifols: Speakers Bureau. Martin Salces:Novo Nordisck: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Octapharma: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Rivas Pollmar:Roche: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Octapharma: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Novo Nordisck: Consultancy, Speakers Bureau. Jiménez-Yuste:Pfizer, Takeda, Novo Nordisk, Sobi, Octapharma, Roche, Grifols, CSL Behring, Bayer: Research Funding; Novo Nordisk, Pfizer, Takeda, Sobi, Roche, CSL Behring, Bayer, Octapharma, BioMarin, Spark: Honoraria; Novo Nordisk, Pfizer, Takeda, Sobi, Roche, CSL Behring, Bayer, Octapharma, BioMarin, Spark: Consultancy. Alvarez-Román:Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Research Funding, Speakers Bureau; Sobi: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau; Grifols: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Other: Sponsored symposia , Speakers Bureau.