Recombinant von Willebrand Factor (vonicog alfa) reduces platelet inhibition caused by antiplatelet drugs and has potential as an acute haemostatic agent

Patients taking antiplatelet drugs who experience major bleeding, such as intracerebral hemorrhage (ICH), have an increased risk of death or disability.¹ There are approximately 2.9 million deaths worldwide per year from (ICH)² and a quarter of these patients are taking antiplatelet drugs.³ Strategies to reduce the elevated risk of death, such as platelet transfusion⁴ and tranexamic acid,⁵ have so far proven ineffective. One potential strategy for reducing the antiplatelet drug effect is to increase plasma von Willebrand Factor (VWF) levels. VWF facilitates platelet-collagen and platelet-platelet interactions, especially under high shear conditions present in arteries.⁶

Desmopressin (1-deamino-8-D-arginine vasopressin; DDAVP) is a vasopressin analog that stimulates release of endogenous VWF from Weibel-Palade bodies present in vascular endothelial cells. It enhances platelet function and is under investigation in clinical trials for antiplatelet drug-associated ICH⁷ and to reduce perioperative bleeding risk.⁸ However, it takes 60 to 90 minutes (min) to take effect, produces highly variable changes in VWF levels, and is associated with side effects such as hyponatremia and hypotension.⁸

Figure 1.Recombinant Von Willebrand Factor improves platelet function after antiplatelet treatment in vitro. (Ai) Representative confocal z-stack images and (Aii) thrombus volumes from healthy donor blood pre-treated with cangrelor (1 μM), acetylsalicylic acid (aspirin, ASA) (100 μM), or both, and treated with 0, 0.5, 1, or 2 U/mL recombinant Von Willebrand Factor (rVWF). Magnification 20X. Perfusion was at 1000 s⁻¹ over type I collagen for 6 minutes. Red lines indicate mean thrombus volume of untreated control samples. 2-way ANOVA: *P<0.05, **P<0.01, NS: not significant.

Direct elevation of plasma VWF levels via infusion with vonicog alfa, a purified recombinant VWF (rVWF) product, may offer significant advantages due to its instantaneous and predictable effect. Vonicog alfa is approved in the US and Europe to treat von Willebrand disease (VWD)⁹ and contains a high proportion of ultra-high molecular weight multimers due to its synthesis in the absence of ADAMTS-13¹⁰ which may enhance its hemostatic activity. This study reports the in vitro efficacy and mechanism of action of rVWF as a platelet function-enhancing hemostatic agent in blood samples from patients receiving antiplatelet therapy.

The effect of rVWF was studied using an in vitro thrombus formation assay due to the shear-dependent nature of the contribution of VWF to primary hemostasis. Thrombus formation was measured by perfusing citrated whole blood through type I collagen-coated (100 μg/mL type I) microfluidic flow chips (Vena8, Cellix Ltd.) for 6 min at an arterial shear rate (1000s^-1), selected to reflect physiological shear conditions in small arteries and arterioles, where VWF-mediated platelet adhesion is most relevant. Samples were then fixed with 10% formyl saline (Sigma-Aldrich) and stained with 4 μg/mL DiOC₆ (Thermo Fisher Scientific). Blood samples were treated with rVWF (vonicog alfa, supplied by Takeda), with concentrations expressed as U/mL (VWF:RCo). The volume of thrombi was measured by acquiring z-stack image series using an A1R confocal fluorescence microscope (Nikon).

We initially investigated concentration dependence of rVWF in blood samples donated by healthy subjects. Healthy subjects aged 21 to 65 years were recruited to the study using procedures approved by the University of Reading Research Ethics Committee (UREC 20/20). Blood samples treated for 10 min with acetylsalicylic acid (aspirin, ASA), P2Y₁₂ antagonist cangrelor, or both, formed significantly smaller thrombi compared to vehicle-treated samples (Figure 1A). Addition of rVWF restored thrombus volume to vehicle-treated levels under all three conditions. rVWF was more potent at restoring platelet function of ASA-treated samples, with significant increase in thrombus volume even at the lowest concentration of rVWF tested (0.5 U/mL). Thrombus volumes in cangrelor only or ASA + cangrelor-treated samples were significantly increased only at higher concentrations of rVWF (2 and 5 U/mL).

Figure 2.Recombinant Von Willebrand Factor improves platelet function after antiplatelet treatment ex vivo. (Ai) Von Willebrand Factor (VWF) antigen levels measured by ELISA in plasma samples spiked with recombinant VWF (rVWF). (Aii) VWF multimer gel and (Aiii) densitometry profiles showing increased high molecular weight multimers with rVWF. (B) Thrombus volumes from patient samples obtained under treatment with acetylsalicylic acid (aspirin, ASA) (Bi), clopidogrel (Bii), or dual antiplatelet therapy (DAPT) (Biii) at 1000 s⁻¹. 2-way ANOVA: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3.Recombinant Von Willebrand Factor enhances platelet aggregation by reducing dependence on intracellular signaling. (Ai) Confocal fluorescence images of fixed and stained platelets (green) and Von Willebrand Factor (VWF) (red) on type I collagen after perfusion of whole blood with vehicle or 0.5, 2, or 5 U/mL recombinant VWF (rVWF) at 1000 s-¹ for 6 minutes. Magnification 20X. (Aii) Bar charts of fluorescence intensity of VWF staining within platelet aggregates after subtracting background fluorescence. (Bi) Representative images of thrombi formed after pretreatment with vehicle, 20 μg/mL AK2 or 10 µM eptifibatide in the presence or absence of 1 U/mL rVWF and (Bii) mean thrombus volumes. (C) A schematic model illustrating normal primary hemostasis whereby platelet activation is initiated by GPVI signaling, stimulating release of ADP and thromboxane A₂ (TxA₂) signaling, leading to activation of intracellular signaling pathways and integrin α_IIbβ₃-mediated fibrinogen bridging. (Left) Antiplatelet drug inhibition: P2Y₁₂ antagonists and aspirin inhibit ADP and TxA₂ pathways, reducing activatory signaling and impairing platelet aggregation. Collagen-evoked GPVI signaling remains intact, but aggregate growth is limited (center), and enhancement by rVWF: rVWF facilitates platelet-platelet interactions via a GPIb and integrin α_IIbβ₃-dependent mechanism. This enables aggregate growth despite reduced activatory signaling, supporting thrombus formation in the presence of antiplatelet effects (right). Bars represent the mean thrombus volume ± standard error of mean. 2-way ANOVA: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

We hypothesized that this enhancement might be due to the high proportion of high and ultra-high molecular weight multimers present in rVWF. High molecular weight multimers have high affinity for platelet GPIb and collagen, and consequently make the greatest contribution to primary hemostasis.¹¹ VWF multimer gels (Figure 2Ai) indicated that rVWF increased VWF multimer levels in the high and ultra-high molecular weight range (Figure 2Aii-iii). We compared the effect of rVWF to a non-recombinant VWF product with lower ultra-high molecular weight von Willebrand Factor multimer (UHMWM) content¹² and found that, although thrombus volume was enhanced by both products, and the effect of aspirin-treated platelets appeared similar, thrombus volume remained significantly inhibited in the presence of P2Y₁₂ blockade (Online Supplementary Figure S1).

We then investigated the efficacy of rVWF in a cohort of patients receiving oral antiplatelet therapy with aspirin, a P2Y₁₂ antagonist or both (dual antiplatelet therapy, DAPT) (Figure 2B). Patients were recruited under Oxford Radcliffe Biobank research tissue bank ethics (HTA License Number 12217, Oxfordshire C REC: 09/H0606/5+5, project approval code SC/0173). All subjects provided informed consent in accordance with the principles of the Declaration of Helsinki. Patients were receiving antiplatelet therapy for ischemic heart disease (17/22), suspected ischemic heart disease (2/22; one with coronary artery spasm, one with atrial fibrillation and normal coronary arteries), or ischemic stroke (3/22) (Online Supplementary Table S1). Blood samples were collected into vacutainers containing 3.2% (w/v) sodium citrate. To define the therapeutic range for rVWF, we focused our experiments on a narrower set of concentrations (0.5, 1, and 2 U/mL). Thrombus volumes were significantly increased after treating with 0.5 U/mL (100%), 1 U/mL (177%) and 2 U/mL (217%) rVWF in samples from patients treated with aspirin only (Figure 2Bi). For patients receiving a P2Y₁₂ antagonist alone (Figure 2Bii) or DAPT (Figure 2Biii), rVWF caused a significant increase in thrombus volume at 1 U/mL (252%) and 2 U/mL (211%) but not 0.5 U/mL. The magnitude of the improvements in platelet function compare favorably to those of a previous study in which DDAVP responses in patients with post-operative bleeding was assessed using a similar thrombus formation assay, in which a modest but significant increase in thrombus surface coverage of 15% was observed after DDAVP infusion.¹³

To investigate the mechanism of action of rVWF, we first imaged rVWF localization within thrombi. A concentration-dependent increase in VWF staining within platelet aggregates formed on collagen was observed following addition of rVWF to whole blood from healthy donors (Figure 3Ai and Aii). This suggests that rVWF promotes platelet-platelet interactions required for aggregation. This finding agrees with a previous study reporting that elevated plasma VWF increases thrombus formation on collagen via enhancement of aggregate formation rather than by facilitating adhesion to collagen.¹⁴

As both GPIb and integrin α_IIBβ₃ serve as receptors for VWF, we investigated the dependence of the rVWF effect on these receptors by inhibiting the interaction with GPIb and integrin α_IIBβ₃ using the blocking antibody AK2 and eptifibatide, respectively. Both inhibitors ablated the rVWF-mediated increase in thrombus volume, indicating a dependence on both GPIb and integrin α_IIBβ₃ in mediating the interaction with rVWF. Integrin α_IIBβ₃ must adopt a high affinity confirmation that enables ligand binding, which can either be induced by activation of intracellular signaling processes, such as activation of PI3K, PKC and RAP1b,¹⁵ or via ‘priming’ in which binding of GPIb with VWF under high shear conditions initiates partial activation and inside-out activation of α_IIBβ₃.¹⁶ As antiplatelet drugs inhibit TxA2 and P2Y₁₂ receptor-mediated platelet signaling pathways, we hypothesized that the efficacy of rVWF might depend upon GPIb-mediated priming to induce integrin α_IIBβ₃ activation. We investigated activation of platelet signaling during thrombus formation by fixing thrombi formed on collagen after 60 seconds. Thrombi were then permeabilized and stained using an antibody raised against phosphorylated PKC substrates which can serve as marker of activatory platelet signaling (Online Supplementary Figure S2). This indicated that rVWF increased the proportion of platelets within the thrombi with low levels of platelet activation and staining negative for PKC activity, suggesting that rVWF-mediated platelet priming of integrin α_IIBβ₃ may contribute to the efficacy of rVWF. This is consistent with previous findings that exogenously added VWF can restore normal patterns of platelet aggregation on collagen surfaces by reinforcing integrin α_IIBβ₃-dependent platelet–platelet interactions.¹⁷ A schematic model summarising this mechanism is shown in Figure 3C.

Reversal or reduction of antiplatelet effects in major hemorrhage remains an area of unmet need. A concentration of 1 U/mL (100 IU/dL) rVWF (vonicog alfa) was required to achieve significant correction of DAPT-associated inhibition in vitro, which would correspond to an infusion of approximately 50 IU/kg, a range already used clinically in VWD for major bleeding or peri-operative management.¹⁸ Clinical trials of rVWF for patients with major bleeding who are taking anti-platelet drugs will be needed to determine whether the in vitro efficacy of rVWF can be translated into clinical benefits by improving hemostatic efficacy without significant increase in thrombotic events.

Footnotes

• Received September 16, 2025
• Accepted January 16, 2026

Correspondence

*MJRD and JLM contributed equally as first authors

A.P. BYE - a.bye@reading.ac.uk

Disclosures

This work was partially funded by an Investigator Initiated Research Grant from Takeda. The study was proposed by the authors and the design, data collection, analysis, interpretation and write up were conducted independently. MJRD has received speakers’ fees and advisory board fees from Takeda that are unrelated to this work. All of the other authors have no conflicts of interest to disclose.

Contributions

Funding

This study was supported by a British Heart Foundation programme grant (RG/20/7/34866), a Rosetrees Trust Translation Research Fellowship Award (StGeorges-21\1), a Takeda Investigator Initiated Research Grant (IIR-GBR-001882), and an Academy of Medical Sciences Springboard Award (SBF0010\1194). The study drug (vonicog alpha) was supplied by Takeda. The schematic diagram was created with BioRender.com.

Acknowledgments

The authors acknowledge the contribution to this study made by the Oxford Centre for Histopathology Research and the Oxford Radcliffe Biobank, which are supported by the National Institutes of Health Research Oxford Biomedical Research Centre.