The pivotal role of von Willebrand factor binding to platelet αIIbβ3 in stabilizing the formation of a platelet plug at sites of injury

Abstract

The interaction between von Willebrand factor (VWF) and platelet αIIbβ3 is thought to be essential for clot formation at injury sites, but its biological properties remain poorly understood due to the complexity and overlap with other αIIbβ3 ligands. Here, we developed a novel binding assay using a recombinant αIIbβ3 headpiece to evaluate VWF-αIIbβ3 binding in plasma from 441 Zimmerman Program participants. The VWF:αIIbβ3 to VWF:Ag ratio was significantly lower in patients with type-1, 2A, and 2B VWD than healthy controls. We identified five index cases with the p.R2464C variant in the VWF-C-domain, where affected family members displayed significantly reduced VWF:αIIbβ3/VWF:Ag ratios. To investigate the function of the VWF-αIIbβ3 interaction, we created a mouse model (VWF^RGES/RGES) by altering the VWF-RGDS motif to RGES, which abolished VWF-αIIbβ3 binding. VWF^RGES/RGES mice exhibited increased blood loss following lateral tail vein transection and reduced thrombus stability in a laser injury model, showing a 59-fold larger AUC for emboli compared to wild-type. However, initial bleeding times and outcomes of carotid artery injury were comparable. Overall, our VWF:αIIbβ3 binding assay is valuable for characterizing VWD, and the VWFRGES mouse model underscores the physiological significance of the VWF-αIIbβ3 interaction, highlighting that VWF-αIIbβ3 interaction is crucial for stabilizing platelet plug formation at injury sites.

Introduction

von Willebrand factor (VWF) is a large multimeric glycoprotein that plays multiple roles in hemostasis and thrombosis.¹ At the site of injury, after being exposed to the subendothelial matrix and bound to collagens, VWF serves as a central player interacting with its counterpart platelets through receptors glycoprotein Ibα (GPIbα) and GPIIb/IIIa (α_IIbβ₃ integrin).^2,3 VWF binds to GPIbα during vascular injury, enabling platelet tethering and starting primary hemostasis. This activates platelets, linking VWF to α_IIbβ₃ integrin and interacting with collagen in the subendothelial matrix during secondary hemostasis.^4,5 The interaction between VWF and the α_IIbβ₃ integrin is critical in maintaining hemostasis by supporting platelet adhesion and aggregation at vascular injury sites.⁶ While the importance of the interaction between VWF and α_IIbβ₃ is recognized, its biological properties remain poorly understood due to the complex and dynamic nature of VWF-α_IIbβ₃ interaction, influenced by factors like blood flow shear rates, platelet activation,⁷ and competition with other α_IIbβ₃ ligands, such as fibrinogen.⁸ Disentangling VWF’s specific contributions from other ligands that bind to the α_IIbβ₃ integrin is particularly challenging, making it difficult to fully understand how this interaction influences platelet plug formation and hemostatic function.^{9 ,10} A significant challenge in studying VWF-α_IIbβ₃ interactions lies in the transient, activation-dependent nature of the binding. VWF only binds to α_IIbβ₃ when platelets are activated, leading to necessary conformational changes.¹¹ This process is further complicated because fibrinogen, the most abundant coagulation protein in the blood, competes with VWF for binding to the same α_IIbβ₃ receptor.¹² VWF binds to platelet-GPIb and increases at injury sites attached to collagen, complicating this interaction. It also undergoes shear-dependent changes, unfolding to expose binding domains that interact with platelet receptors.¹³

In addition to these biochemical challenges, experimental systems often fail to capture the dynamic flow conditions under which VWF-α_IIbβ₃ interactions are most relevant.¹⁴ Flow-based in vitro assays or microfluidic devices are needed to simulate physiological conditions accurately, but they are technically demanding. A cell-based assay utilizing α_IIbβ₃-stably-expressed on HEK293 demonstrated impaired binding to recombinant VWF (rVWF) with certain variants in the C-domains; however, it was ineffective for plasma samples.¹⁵ Reliable assays to measure VWF’s binding to α_IIbβ₃ are essential for understanding how mutations in VWF affect this interaction and improving diagnostics for von Willebrand disease (VWD). In vivo models that isolate VWF’s role in this binding without fibrinogen interference can provide important tools to investigate the significance of this interaction in platelet plug formation and thrombus stability.

In this study, we developed a novel ELISA-based assay using recombinant α_IIbβ₃ headpiece to assess VWF binding in plasma samples from VWD subjects enrolled in the Zimmerman Program. We then created a mouse model with the VWF-RGES mutation to disrupt the RGD-dependent VWF-α_IIbβ₃ binding. Utilizing these tools, we systematically evaluated the impact of the VWF-α_IIbβ₃ interaction on hemostasis. Our results provide new insights into the role of VWF-α_IIbβ₃ binding in stabilizing platelet aggregates, contributing to a deeper understanding of hemostatic mechanisms and bleeding disorders.

Methods

The details of antibodies and reagents, as well as the methods and statistics used in this study, are provided in the Online Supplementary Appendix.

Patient populations and phenotype characterization

A total of 441 subjects, including VWD index cases and healthy controls (HC), were initially analyzed from clinical hematology centers as part of the Zimmerman Program for the Molecular and Clinical Biology of VWD¹⁶ (see Online Supplementary Appendix). Informed consent was obtained, and the study was approved by the Institutional Review Board. Bleeding scores were assessed using ISTH-BAT.¹⁷ Full exonic Sanger sequencing of the VWF gene identified variants in index cases, and targeted sequencing confirmed these variants in family members. Central laboratory VWF testing was also conducted to validate phenotypic diagnoses as reported.^18,19

VWF-αIIbβ3 binding activity assay

The biological function of VWF in binding to α_IIbβ₃ was quantified by our novel ELISA-based assay using an antibody-captured recombinant human α_IIbβ₃ (rh-α_IIbβ₃) headpiece²⁰ (Figure 1A). The anti-human GPIIIa monoclonal antibody (MoAb) AP-3 was coated on a 96-well plate to capture rh-α_IIbβ₃. Plasma samples were heated to 56°C for 15 minutes (min), centrifuged to remove fibrinogen, and diluted heat-defibrinated plasma (HDP) samples were incubated for one hour (hr).

Biotinylated anti-hVWF antibody AVW15 detected bound human VWF (hVWF). Two protocols determined mouse VW-F:α_IIbβ₃ binding: one with rh-α_IIbβ₃ (mVWF:hα_IIbβ₃) and another with recombinant mouse α_IIbβ₃ (mVWF:mα_IIbβ₃). A biotinylated anti-VWF antibody (Dako) detected bound mVWF

Generation of von Willebrand disease with RGES variant mouse models

All animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. The change of VWF c.7527T>A (p.Asp2509Glu), which converts the RGDS motif to RGES and impacts VWF binding to activated platelets as reported in an in vitro study,²¹ was introduced into cloned mVWF-cDNA using a CRISPR-Cas9 strategy²² to create an RGES-VWD mouse model. VWF^RGES/+ offspring were crossed to develop the VWF^RGES/RGES model. VWF^RGES/RGES mice were crossed with fibrinogen deficient (Fib-^/-)²³ or γ-chain variant (Fibγ^Δ5/γΔ5)²⁴ mice to generate Fib^-/-VWF^RGES/RGES and Fib^γΔ5/γΔ5 VWF^RGES/RGES mice.

von Willebrand factor antigen and von Willebrand factor-collagen binding activity assays

Mouse blood samples were collected and plasma was isolated as reported.²⁵ von Willebrand factor antigen (VWF:Ag) levels in mouse plasma were determined by ELISA.²⁶ For human VWF:Ag ELISA, anti-hVWF monoclonal antibodies (MoAb) were used. To determine the binding functions of RGES-VWF to collagen, we performed bindings assays for VWF with collagen-III (VWF:CB3) and collagen-IV (VWF:CB4) as reported^27-29 using samples from VWD subjects and VWF^RGES/RGES mice.

Phenotypic assessments

The functional phenotype in VWF-RGES mice was assessed by in vitro rotational thromboelastometry (ROTEM) assay native whole blood thrombin generation assay (nWB-TGA), and Venaflux following procedures described in our previous reports,³⁰-³² and by five In vivo injury models: 1) lateral tail vein transection (TVT) injury;³³ 2) tail-tip transection (TTT) by clipping 4 mm lengthwise for a 20 min bleeding test; 3) 6-hr tail bleeding test;³⁴ 4) FeCl₃-induced carotid artery injury; and 5) cremaster intravital laser injury^35,36

Results

The competency of a novel functional VWF/αIIbβ3, binding assay in characterizing subjects with various types of von Willebrand disease

To evaluate the binding capacity of VWF to the α_IIbβ₃ complex, we developed a novel functional assay using a recombinant α_IIbβ₃ headpiece: VWF:α_IIbβ₃ (Figure 1A). The rh-α_IIbβ₃ binds to HDP samples from human, rat, and mouse wild-type (WT) VWF but not to mutant VWF from RGES mice, demonstrating RGD-dependent binding (Online Supplementary Figure S1). The ratio of VWF:α_IIbβ₃ to VWF:Ag was used to characterize our VWD cohorts enrolled in the Zimmerman Program.¹⁶ The VWF:α_IIbβ₃/VWF:Ag ratio in the normal healthy control (HC) group was 0.87±0.25, which was significantly higher compared to those in the low VWF (LVWF), type-1, type-2A, type-2B, and type-2M-VWD groups (Table 1, Figure 1B). The 5-95% range of the VWF:α_IIbβ₃/VWF:Ag ratio from HC was between 0.53-1.39. Using only subjects with normal multimers (HC, LVWF, and type-1-VWD), we observed that reduced VWF:α_IIbβ₃/ VWF:Ag significantly correlated with lower VWF:Ag (Figure 1C). Using VWF:CB3 (VWF:Collagen-III [CB3] binding) as a surrogate for multimer size,³⁷ we found that low VWF:CB3/ VWF:Ag (reflecting loss of HMW multimers)³⁷ correlates with lower VWF:α_IIbβ₃ binding in Zimmerman Index Cases (LVWF, type-1-VWD, and type-2-VWD) (Figure 1D).

We then used the VWF:α_IIbβ₃ assay to test the binding ability of VWF from VWD subjects with a variant in the α_IIbβ₃-binding region (VWF-C-domain). We identified five index cases with p.R2464C in the VWF-C-domain; four were phenotypically diagnosed as LVWF and one as type-1-VWD. All p.R2464C subjects had a decreased VWF:α_IIbβ₃/VWF:Ag ratio ≤0.5 but had normal VWF multimers. Since our p.R2464C Index cases were clinically diagnosed as either LVWF or type-1-VWD, we compared the characteristics of subjects with the p.R2464C variant to type-1-VWD/LVWF. There was no difference in VWF activity as determined by VWF:GPIbM binding or VWF:Rco³⁸ between subjects with p.R2464C and type-1-VWD/LVWF cohort (Table 2). Compared to subjects with type-1-VWD/ LVWF, the VWF:Ag level in the group with p.R2464C variant was similar (Figure 2A). However, the functional VWF:α_IIbβ₃ binding level and VWF:α_IIbβ₃/VWF:Ag ratio in the p.R2464C group were significantly lower than in the type-1-VWD/LVWF group, including subjects with other C-domain variants (Figure 2A). We then compared unaffected (N=6) versus affected (N=5) family members and index cases (N=4) within the four families with only the p.R2464C variant (one family had another VWF variant in addition to p.R2464C). In these families, we found the VWF:α_IIbβ₃/VWF:Ag ratio in members with the R2464C variant was significantly lower than in unaffected family members (0.37±0.06 vs. 0.88±0.57, P<0.01) (Figure 2B). The bleeding score in affected members was significantly higher than in family members without the R2464C variant (6±5.3 vs. 0.67±1.63, P<0.05) (Figure 2C).

These data demonstrated that the functional VWF-α_IIbβ₃ binding assay we established is valuable for the characterization of patients with VWD and for studying VWF:α_IIbβ₃ interactions in vivo in a mouse model.

Establishing a VWF-RGES model

To investigate the functional properties of the interaction between VWF and α_IIbβ₃, we developed a mouse model that directly alters the VWF-RGDS binding motif to VWF-RGES using a CRISPR/Cas9 strategy (Online Supplementary Figure S2A). We introduced variant RGES into cloned mouse VWF (mVWF) cDNA for mouse model development. Three lines of VWF^RGES mice carrying the c.7527T>A change with a similar phenotype were generated. Animals were genotyped using PCR and a diagnostic SacII site introduced in the RGES-VWF allele (Online Supplementary Figure S2B, C) from which the WT-VWF allele produced a 783-bp band, while the RGES-VWF mutation resulted in 579-bp and 204-bp bands. Thus, VWF^+/+ mice showed only the 783-bp fragment, homozygous VWF^{RGES/ RGES} mice had both the 579-bp and 204-bp fragments, and heterozygous VWF^RGES/+ mice displayed all three fragments (Online Supplementary Figure S2C).

Table 1.Comparison of the ratio of the von Willebrand factor’s α_IIbβ₃ binding capacity to von Willebrand factor total antigen among various types of von Willebrand disease subjects.

Table 2.The characteristics of family members with and without the p.R2464C variant in the Zimmerman Program.

Figure 1.Developing the functional assay to assess von Willebrand factor binding to platelet α_IIbβ₃ integrin in human plasma from subjects with von Willebrand disease. For the functional von Willebrand factor (VWF) binding to platelet α_IIbβ₃ integrin (VWF:α_IIbβ₃) binding assay, an anti-human GPIIIa (β₃) monoclonal antibody (AP-3) was coated on a 96-well ELISA plate and recombinant human α_IIbβ₃ headpiece was captured from a 0.25 µg/mL solution. Plasma samples from individuals enrolled in the Zimmerman Program were heated, defibrinated, and incubated with the antibody captured α_IIbβ₃. The unbound VWF was washed off, and the remaining α_IIbβ₃-bound human VWF was detected using biotinylated mouse anti-human VWF monoclonal antibody, AVW15, and p-nitrophenyl phosphate (PNPP) was used as the substrate. The standard curve was constructed by measuring VWF binding from serially diluted heat-defibrinated plasma from SSC/ISTH Lot#5. The levels of VWF antigen (VWF:Ag) and VWF binding to collagen III (VW-F:CB3) in plasmas were determined by ELISA. Human plasma from SSC/ISTH lot#5 was used as the standard. (A) Schematic diagram of the VWF:α_IIbβ₃ binding activity assay. (B) The ratio of functional VWF:α_IIbβ₃ binding activity to VWF:Ag level [VWF:α_IIbβ₃/ VWF:Ag] in subjects with various types of von Willebrand disease (VWD). (C) The correlation between the VWF:α_IIbβ₃/VWF:Ag ratio and the VWF:Ag level in samples from index cases of patients with type-1-VWD, and low-VWF levels (LVWF), as well as the healthy controls (HC); N=321. (D) The correlation between the VWF:α_IIbβ₃/VWF:Ag ratio and the VWF:CB3/VWF:Ag ratio in samples from the VWD Index patients; N=357. (B-D) Each data point represents one subject. These results demonstrate that our novel functional VWF:α_IIbβ₃ binding activity assay utilizing the mobilized recombinant human α_IIbβ₃ headpiece-mediated ELISA is valuable for char acterizing various types of VWD.

To characterize VWF^RGES animals, we used ELISA^25,26,39 to determine plasma VWF:Ag levels and our new VWF:α_IIbβ₃ binding assay. The plasma VWF:Ag level in VWF^RGES/RGES mice was 119.32±37.96U/dL, comparable to VWF^RGES/+ and VWF^+/+ mice (Figure 3A). The VWF^RGES/RGES mice had normal levels of plasma FVIII (data not shown). Notably, no mVWF bound to rh-α_IIbβ₃ in HDP samples from the VWF^RGES/RGES group, while the VWF^RGES/+ group showed a binding level of 50.57±17.82U/dL, significantly lower than in the VWF^+/+ group (98.14±40.64U/dL) (Figure 3B). Despite impaired binding to α_IIbβ₃, VWF^RGES mice displayed similar binding to collagen-III and collagen-IV compared to VWF^+/+ mice (Online Supplementary Figure S3A-D). VWF^RGES mice had normal VWF multimers (Figure 3C) and FVIII:C levels (Online Supplementary Figure S3E). To eliminate fibrinogen’s interference with VWF binding to α_IIbβ₃, we crossed VWF^RGES mice onto a Fib-^/- background. The mVWF:ma_IIbP₃ binding activity in non-defibrinated plasma was 91.84±45.45U/dL in the Fib-^/- group (Figure 3D). In the Fib-^/-VWF^RGES/+ group, it was 18.96±7.94U/dL, while no binding was detected in the Fib-^/-VWF^RGES/RGES and Fib-^/-VWF-^/-groups. Overall, the VWF^RGES model has normal plasma VWF levels and multimers but lacks a_IIb|3₃ binding activity while still effectively binding to collagen-III and IV.

Figure 2.Using functional von Willebrand factor binding to platelet αIIbβ3 integrin activity assay to characterize von Willebrand disease subjects with the R2464C variant. For the functional VWF:α_IIbβ₃ binding assay, an anti-human GPIIIa (β3) monoclonal antibody (AP-3) was coated on a 96-well plate, and recombinant human α_IIbβ₃ headpiece was captured. Plasma samples from the Zimmerman Program were heat-defibrinated and incubated with the antibody-captured α_IIbβ₃. After washing away the unbound von Willebrand factor (VWF), the remaining bound VWF was detected using a biotinylated mouse anti-human VWF antibody (AVW15) and p-nitrophenyl phosphate (PNPP) as the substrate. The standard curve was established with VWF binding from serially diluted heat-defibrinated plasma from SSC/ISTH Lot #5. VWF antigen (VWF:Ag) levels were quantitated by ELISA and human plasma from SSC/ISTH lot#5 was used as the standard. (A) The levels of VWF:Ag and VWF:α_IIbβ₃, and the ratio of VWF:α_IIbβ₃/VWF:Ag in patients with p.R2464C variant compared to those with type-1-VWD and LVWF. (B) The ratio of VWF:α_IIbβ₃/VWF:Ag in the affected family members from our Index Cases with p.R2464C variant compared to those in unaffected members. (C) The bleeding score of the affected family members from our Index Cases with p.R2464C variant compared to those in unaffected members. *P<0.05; **P<0.01; ****P<0.0001; NS: no significant difference between the two groups. (A-C) Each data point represents one subject. These results demonstrate that our functional VWF:α_IIbβ₃ binding activity assay is viable in quantitating the capacity of VWF in binding to α_IIbβ₃ integrin, and the ratio of VWF:α_IIbβ₃/VWF:Ag is a valuable parameter for clinical diagnosis of von Willebrand disease (VWD).

Assessment of the functional properties VWF-RGES in whole blood assays of VWFRGES model mice

To determine how VWF-RGES affects hemostatic properties in whole blood, we used native ROTEM³⁰ and nWB-TGA.³¹ There are no significant differences in any parameters from ROTEM and nWB-TGA between the VWF^RGES/ RGES group and the VWF^+/+ and VWF^RGES/+ groups (Online Supplementary Figures S4 and S5). To assess the effect of VWF-RGES on platelet adhesion to collagen under flow conditions, we used the VenaFlux microfluidic platform on the collagen-III-coated chip²⁷ to analyze whole blood from various mouse models: Fib^γΔ5/γΔ5VWF^RGES/RGES, Fib^-/-VWF^RGES/RGES, Fib^-/-VWF^-/-, Fib^-/-, and C57BL/6J-WT. Under shear stress of 67.5dyn/cm², platelet coverage on the collagen-III-coated chip increased over time, reaching 40-50% by the study’s end, except for the Fib^/-VWF^-/- group, which showed negligible adhesion (Online Supplementary Figure S6A, B).

We tested the anti-GPIIb/IIIa antibody LeoH4, blocking the α_IIbβ₃ receptor, to determine if collagen adhesion occurred. No adhesion was found in the VWF^-/- group, but platelet coverage in the WT/Leo.H4 group matched the WT/IgG and WT/saline controls, as well as the Fib^-/- group. Interestingly, platelets in the WT/Leo.H4 group adhered as single entities rather than aggregates, unlike the WT/IgG and WT/saline groups that showed platelet aggregates (Online Supplementary Figure S6C, D).

Together, these data demonstrate that even though the VWF-α_IIbβ₃ interaction is impaired in RGES-VWD blood, the hemostatic parameters determined by ROTEM and nWB-TGA are unaffected, and platelets can still effectively adhere to collagen for thrombus formation through another VWF-mediated pathway in the Venaflux model.

Assessment of the in vivo bleeding phenotypes in VWFRGES model mice

To investigate how RGES-VWF affects the bleeding phenotype in mice, we used five In vivo injury models: 1) TVT;³³ 2) TTT;²² 3) 6-hr tail bleeding test;³⁴ 4) FeCl₃-induced injury;⁴⁰ and 5) cremaster laser injury.³⁵ In the blinded TVT injury model, no significant differences in primary, rechallenge, or total bleeding time were found between the VWF^RGES/RGES and C57BL/6J-WT groups (Figure 4A, B). However, the VWF^{RGES/ RGES} group experienced significantly higher blood loss during the rechallenge and total tests than the WT group, while the primary bleeding phase loss remained similar (Figure 4C). The VWF^-/- group had a significantly longer bleeding time than the VWF^RGES/RGES group, but no significant difference in blood loss was noted, regardless of phase.

Figure 3.Characterization of von Willebrand factor and its functional binding activity to αIIbb3 in RGES variant von Willebrand factor model mice. (A) Plasma von Willebrand factor (VWF) antigen (VWF:Ag) levels. Blood samples were collected from three lines of RGES variant von Willebrand disease (VWD) model (VWF^RGES) mice by tail bleeds using 3.8% sodium citrate as an anticoagulant, and plasmas were isolated for VWF assays. Plasmas from VWF^+/+ and VWF^RGES/+ littermates were used as parallel controls. Mouse VWF antigen (VWF:Ag) levels were determined by ELISA assay using anti-mouse VWF monoclonal antibody 344.2 for capture and biotin-conjugated rabbit anti-human VWF polyclonal antibody, Dako, that is known to cross-react with mouse VWF for detection. Plasma pooled from our wild-type C57BL/6J colony was used as the standard. Plasma from VWF^/- mice were used as a negative control. (B) The capacity of mouse VWF binding to recombinant human α_IIbβ₃ headpiece (mVWF:hα_IIbβ₃). Anti-human β₃ monoclonal antibody AP3 was coated onto a 96-well plate to capture recombinant human α_IIbβ₃ from a 0.25 µg/mL solution. Plasma from VW-FRGES mice was heat-defibrinated and incubated with the antibody-bound α_IIbβ₃. After washing away unbound mouse VWF, the a_IIbP₃-bound mVWF was detected using ELISA reagents. A standard curve was created by measuring mVWF binding from serially diluted heat-defibrinated plasma from wild-type C57BL/6J mice. (C) The ability of RGES-VWF in multimerization. To examine if VWF in VWF^RGES/RGES mice can fully multimerize, we ran VWF multimers on plasma samples from RGES mice. Plasma from C57BL/6 mice were run in parallel. (D) The capacity of mouse VWF binding to recombinant murine α_IIbβ₃ (mVWF:mα_IIbβ₃). To eliminate fibrinogen interference, some VWF^RGES mice were crossed onto a fibrinogen-deficient (Fib^-/-) background. Anti-mouse CD41 (aIIb) monoclonal antibody was coated on a 96-well plate to capture recombinant murine α_IIbβ₃ (mα_IIbβ₃) at 5 µg/mL. Undefibrinated mouse plasma samples from Fib^-/-VWF^RGES were added, allowing bound mVWF to be detected via ELISA after washing away unbound VWF. A standard curve was created from serially diluted pooled plasma from Fib^/- mice. Plasmas from C57BL/6, Fib^+/+, and Fib^+/- mice were assayed as controls. (A, B, D) Each data point represents one mouse. ****P<0.0001. These results demonstrate that VWF-RGES mice have normal levels of plasma VWF but are incapable of binding mouse α_IIbβ₃ integrin.

In the TTT model, there were no significant differences in bleeding time and blood loss between the VWF^RGES/RGES and WT groups. In contrast, the bleeding time and blood loss in the VWF^-/- group were significantly different from those in the VWF^RGES/RGES and WT groups (Figure 5A). In the 6-hr tail bleeding test, the percentage of remaining hemoglobin in the VWF^RGES/RGES group was comparable to that in the WT group. Re-bleeds occurred in 2 of 6 VWF^RGES/RGES mice but not in the WT animals during the test (Figure 6B). In the FeCl₃-induced carotid artery injury model, there was no significant difference in the occlusion time between the VWF^RGES/RGES group and the C57BL/6J-WT group (Figure 5C).

Together, these data demonstrate that the VWF-RGES variant does not result in prolonged bleeding times regardless of arterial or venous injury models in mice. However, VWF^{RGES/ RGES} animals lost more blood in the TVT injury model than WT mice.

Figure 4.Assessment of the bleeding phenotype in RGES variant von Willebrand disease model mice using lateral tail vein transection injury model. RGES variant von Willebrand disease (VWF^RGES/RGES) model mice were anesthetized with isoflurane, and a 1-mm deep transection was performed in the left lateral tail vein at a 2.5 mm diameter, using an aluminum template to assess the bleeding phenotype. The tail was submerged in 14 mL of pre-warmed saline for 15 minutes (min). If bleeding stopped, the tail was removed from the saline; if not, it remained submerged for the full duration. Clotting was tested three times, and bleeding times were recorded. Blood loss was quantified by lysing red blood cells in 10 mL of distilled water and measuring hemoglobin at OD575 nm, with calculations based on a standard curve from the pooled blood of wild-type C57BL/6J mice. Total bleeding times and blood losses across the three re-challenges were combined. Mice from the wild-type C57BL/6J colony and VWF^-/-served as controls. (Ai-Aii) Bleeding time (AI) and blood loss (Aii) during primary challenge. (Bi-Bii) Bleeding time (Bi) and blood loss (Bii) during re-challenges. (Ci-Cii) Total bleeding time (Ci) and blood loss (Cii) from primary and re-challenges. (A-C) Each data point represents one mouse. *P<0.005; **P<0.01; ***P<0.001; NS: no significant difference between the two groups. These results demonstrate that von Willebrand factor (VWF) with the RGES variant significantly affects blood loss during rechallenge bleeding tests, but it does not impact bleeding time, nor does it affect blood loss or bleeding time in the primary challenge. TVT: tail vein transection.

Assessment of the in vivo thromboembolism phenotypes in VWFRGES model mice

We performed laser injury on cremaster arterioles to explore how the RGES variant of VWF affects clot stability. We quantified thrombus formation by measuring the fluorescence intensity of accumulated platelets and fibrin and monitored emboli downstream of the thrombus. The total fluorescence intensity of accumulated platelets in thrombi formed in the laser-injured cremaster arterioles was similar between the VWF^RGES/RGES and WT groups (Figure 6A, B). However, the VWF^RGES/RGES group displayed more fluctuations, with more numbers of drop-offs. The area under the curve (AUC) for platelet accumulation was comparable between the groups (Figure 6C). Additionally, the total fluorescence intensity of fibrin in the thrombus showed no significant difference between the groups, although the median AUC for VWF^{RGES/ RGES} appeared lower than that for WT (Figure 6D-F).

Figure 5.Assessment of the bleeding phenotype in RGES variant von Willebrand disease model mice using tail tip transaction and FeCl3-induced carotid artery injury models. (Ai-Aii) The tail bleeding test was performed by transecting a length of 4 mm of the tail tip. Animals were anesthetized with isoflurane, and 4 mm of the tail tip was clipped using a scalpel. The injured tail was submerged in 14 mL of pre-warmed saline for 20 minutes (min), during which bleeding times were recorded, and the results are shown in (Ai). Blood loss was measured by lysing red blood cells in 10 mL of dH₂O and analyzing hemoglobin at OD575 nm, using a standard curve from the pooled blood of wild-type C57BL/6J mice, and the results are shown in (Aii). Wild-type C57BL/6J and VWF^-/- mice served as controls. (Bi-Bii). The 6-hour (hr) tail bleeding test was performed by transecting the tail tip at a diameter of 1.6 mm. Animals were anesthetized with isoflurane, and the tail tip was clipped by a scalpel. Animals were monitored hourly, and bleeding time was recorded and shown in (Bi). Fifty microliters of blood were collected before and after the test for blood counts, and the percentage of the remaining hemoglobin after the test was calculated; results are shown in (Bii). (C) FeCl₃-induced carotid artery injury model. The right carotid artery of ketamine-anesthetized mice was exposed. A 1x2 mm filter paper soaked in 15% ferric chloride was applied to the carotid artery for 3 minutes, then the surface of the artery was washed with PBS. Blood flow was monitored using a Doppler ultrasound flow probe. Time to occlusion of the carotid artery was recorded. (A-C) Each data point represents one mouse. *P<0.05; ***P<0.001; NS: no significant difference between the two groups. These results demonstrate that RGES variant von Willebrand disease (VWF-RGES) variant does not impact the bleeding phenotype in tail tip clipping tests and FeCl₃-induced carotid artery injury in VWF^RGES mice.

Figure 6.Assessment of the bleeding and embolism phenotype in RGES variant von Willebrand disease model mice using the cremaster intravital laser injury model. Male mice were anesthetized with a mixture of ketamine (75 mg/kg) and dexmedetomidine (0.5 mg/kg). The arterioles in the cremaster muscle were exposed. Fluorophore-labeled antibodies were injected retro-orbitally to label fibrin and platelets. Fibrin was labeled with an anti-fibrin monoclonal antibody conjugated to Alexa Fluor 647, while platelets were labeled with a rat anti-mouse GPIbβ monoclonal antibody conjugated to DyLight488. The vascular injury was induced using an enclosed pulsed laser, and the arteriolar thrombus formation and embolization were monitored and captured. The thrombus size was measured, and embolic events downstream were evaluated by creating an embolism mask that captured the full diameter of the vessel unaffected by the thrombus. Emboli were quantified by detecting labeled platelet aggregates, measuring the fluorescent signal over time as an area under the curve (AUC). (A) The fluorescent intensity of accumulated platelets in the thrombus formed in injured arterioles from C57BL/6J wild-type (WT) mice. (B) The fluorescent intensity of accumulated platelets in the thrombus formed in injured arterioles from RGES variant von Willebrand disease (VWF^RGES) mice. (C) A comparison of the AUC of platelet accumulation intensity in the thrombi formed after laser injury in VWF^RGES and WT mice. (D) The fluorescent intensity of accumulated fibrin in the thrombus formed in the injured arterioles from WT mice. (E) The fluorescent intensity of accumulated fibrin in the thrombus formed in the injured arterioles from VWF^RGES mice. (F) A comparison of the AUC of fibrin accumulation intensity in the thrombi formed after laser injury in VWF^RGES and WT mice. (G) The fluorescent intensity of embolized platelet plugs in WT mice. (H) The fluorescent intensity of embolized platelet plugs in VWF^RGES mice. (I) A comparison of the AUC of platelet accumulated intensity in emboli in VWF^RGES and WT mice. (J) A comparison of the drop-offs with a clear, sharp drop-off of platelet accumulation in thrombus during 300 seconds (s) after laser injury in VWF^RGES and WT mice. RFU: relative fluorescence units. **P<0.01; NS: no significant difference between the two groups.

In contrast, the accumulated platelets in the thrombi/clots formed in the cremaster laser injury model exhibited striking instability, with frequent fragmentation and embolization of thrombus material (platelet-plugs) observed downstream in VWF^RGES/RGES, but not WT mice (see Online Supplementary Videos). The rate of embolic platelet plugs downstream of the thrombus formed at the injury sites in the VWF^RGES/RGES group was markedly higher than in the WT group (Figure 6G, H). The median AUC of emboli in the VWF^RGES/RGES group was 1.39x10⁶ RFU, 59-fold higher than in the WT group (2.34x10⁴ RFU) (Figure 6I). The number of drop-offs (peaks) during thrombus formation per injury of the cremaster arterioles in the VWF^RGES/RGES group was 7.25±3.49, which was significantly higher than in the WT group (2±1.29, P<0.01) (Figure 6J). Collectively, these observations reveal significant thrombus instability in VWF^RGES/RGES mice, confirming that VWF’s binding to platelet α_IIbβ₃ is crucial in stabilizing the formation of a platelet plug at sites of vascular injury.

Discussion

In this study, we developed a novel ELISA-based assay using the recombinant α_IIbβ₃ headpiece to determine VWF binding capacity to the α_IIbβ₃ integrin in VWD subjects’ HDP samples. Furthermore, we generated the first mouse model with a defect at the RGD motif of VWF, resulting in a complete lack of binding capacity to the α_IIbβ₃ integrin. Our study provides critical insights into the biological properties of the interaction between VWF and the α_IIbβ₃ integrin with important applications in understanding and clinical diagnosis of VWD. Using our novel VWF-α_IIbβ₃ binding assay and the VWF-RGES mouse model, we elucidated the functional role of the VWF-α_IIbβ₃ interaction in hemostasis and thromboembolism. Our findings highlight that VWF-α_IIbβ₃ binding is essential for stabilizing platelet aggregates at injury sites in mice, complementing the well-characterized VWF-GPIbα pathway. von Willebrand factor has a multidomain structure that interacts with various proteins, enabling its biological functions to encompass coagulation and a range of other important non-hemostatic processes.^1,13 While the contributions of other VWF domains, such as A1, A2, A3, and D, are well documented, variants in the C-domains remain poorly understood despite the presence of the important RGD motif known to interact with platelet α_IIbβ₃.⁴¹ One of the major barriers to understanding the functional relevance of the C-domains has been the absence of a reliable assay to measure VWF-α_IIbβ₃ interactions.⁴² The VWF:α_IIbβ₃ binding assay we developed demonstrated its viability by revealing significant reductions in VWF:α_IIbβ₃/VWF:Ag ratios in several VWD subtypes, including type-2A and type-1 individuals with the p.R2464C variant in the C-domains. It has been reported that among 12 known variants in the VWF-C-domain, 4 of them [p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala], but not p.Arg2464Cys (p.R2464C), exhibit reduced α_IIbβ₃ binding in a cell-based assay using GPIIb/IIIa-transfected-HEK293 cells and recombinant VWF.¹⁵ Interestingly, none of those four variants with reduced α_IIbβ₃ binding were found among our Zimmerman Program subjects.

Using the recombinant α_IIbβ₃ headpiece, our ELISA-based assay effectively detects the binding capacity of VWF from both human and mouse plasma samples to integrin α_IIbβ₃. Previous studies have shown that employing α_IIbβ₃ purified from human platelets in ELISA or α_IIbβ₃-stably-expressed HEK293 cell-based assays is only effective for assessing VWF derived from cell culture supernatants (rVWF), but not for plasma samples.^15,43,44 Importantly, the impairment in p.R2464C was not evident by VWF multimer analysis and VWF:Ag in our Zimmerman Program subjects, highlighting our assay’s ability to detect functional defects that may go undiagnosed by conventional methods. The significance of the interaction between VWF and the α_IIbβ₃ receptor, especially regarding variants in the C-domain, has been understudied in relevant pathophysiological contexts. This is due to the complexity of the α_IIbβ₃ receptor, which comprises two proteins with multiple competitive binding ligands and whose binding affinity is influenced by inside-out signaling during platelet activation.⁴⁵ Our VWF:α_IIbβ₃ binding assay fills a valuable role in characterizing VWD, enabling the detection of subclinical VWF defects that could affect bleeding or thrombosis risk. Patients with VWD variants not identified by other functional tests, or those with a positive BAT score but normal VWF testing, should be considered for the VWF:α_IIbβ₃ test to rule out this type of VWD.

The clinical implication of this assay extends beyond diagnostics. It provides a framework for assessing the impact of variants in the C-domains, helping to refine genotype-phenotype correlations in VWD. This assay could also be valuable for personalized treatment strategies in clinical settings by identifying patients whose bleeding tendencies stem from impaired VWF-α_IIbβ₃ interactions. Furthermore, understanding how these variants influence thrombus formation can guide the development of novel therapies targeting VWF-integrin interactions, especially in cases where standard treatments are less effective. It has been reported that a gain-of-function variant of VWF affecting the VWF-C-domain can enhance platelet aggregation, thereby increasing the thromboembolism risk.^44,46 Our study suggests that measuring VWF-α_IIbβ₃ binding could improve diagnostic precision in VWD in patients, especially for patients with variants within the C-domain and other thrombotic disorders, by identifying patients with subclinical platelet function defects.

We found that the ratio of VWF:α_IIbβ₃/VWF:Ag in the type-1, 2A, and 2B VWD groups diagnosed using traditional VWF assays was significantly lower than in the healthy-control group from subjects recruited in the Zimmerman Program, indicating the capacity of VWF binding to platelet α_IIbβ₃ integrin is impacted in VWD with a variety of variants. These findings emphasize the clinical relevance of VWF-α_IIbβ₃ interaction in bleeding disorders. While the impact of impaired binding may be masked under normal conditions, it becomes apparent under certain stresses, such as microvascular injuries. We speculate that VWF variants may affect VWF-α_IIbβ₃ interaction by other mechanisms besides directly impairing the RGD motif. These may include: 1) altered VWF multimer structure; 2) changes to the glycan profile of VWF; and 3) VWF clearance variants. Clearly, both the quality and quantity of VWF impact the functional properties of VWF-α_IIbβ₃ interaction. Further studies on the applications of our VWF-α_IIbβ₃ binding assay in understanding how VWF variants in other domains besides the C4-domain where the binding motif is located affect the VWF-α_IIbβ₃ interactions are warranted.

To rigorously investigate the functional properties of the VWF-α_IIbβ₃ interactions, we developed a novel mouse model carrying a VWF mutation that completely abrogates binding to the α_IIbβ₃ integrin while exhibiting otherwise normal expression, multimerization, and functions. This model allows us to explore the contribution of this interaction in hemostasis and thromboembolism. These mice exhibited markedly increased platelet plug instability, as evidenced by frequent embolization in cremaster muscle arterioles following laser injury. In contrast, other bleeding models, like FeCl₃-induced carotid artery injury and tail tip amputation tests, did not reveal significant differences between the VWF^RGES and WT groups. Our results demonstrate that the binding of VWF to α_IIbβ₃ is essential for stabilizing aggregated platelets. This function is not redundant and cannot be compensated for by fibrinogen, even though fibrinogen is known to crosslink platelets by binding to the same α_IIbβ₃ receptor during thrombus formation at the injury site.

Platelets play fundamental roles in hemostasis following all types of vessel injury, but they are more critical in arterial than venous environments.^47-49 Interestingly, VWF^RGES mice lost significantly more blood than WT mice during the rechallenge TVT bleeding test. However, there were no significant differences in blood loss during the primary bleeding test nor in bleeding times between the two groups. Thus, the interaction between VWF and platelet α_IIbβ₃ integrin plays a more important role in stabilizing the clot formation in secondary hemostasis than platelet adherence in primary hemostasis. The results from various In vivo arterial and venous injury models demonstrate that VWF-α_IIbβ₃ interaction is crucial for thrombus stability under specific conditions, such as dynamic vascular flow or microvascular injury, but may not uniformly affect bleeding across all hemostatic scenarios. Therefore, we speculate that any variant impairing this interaction may increase the risk of thromboembolism, even with normal VWF expression levels. Indeed, it has been reported that the transient expression of VWF with a mutation in RGD (D2509G) (RGG-VWF), achieved under a liver-specific promoter through hydrodynamic injection of cDNA in VWF^-/- mice, resulted in high plasma VWF levels. However, thrombus growth was delayed, and continuous embolization and occlusion times were significantly increased compared to WT-VWF transfected animals in the FeCl₃-induced injury model.⁵⁰ It remains unclear, though, to what extent these effects were caused by the loss of high molecular weight multimers of VWF introduced by hydrodynamic injection and ectopic hepatocyte expression in that study. Further research on the effect of VWF-α_IIbβ₃ interaction on thrombosis is necessary.

Our results also shed light on the redundant or complementary roles of fibrinogen and VWF in platelet aggregation and blood coagulation. Crossing the VWF variant mice onto a Fib^-/- background confirmed that VWF binding to platelets becomes critical when fibrinogen is absent. No platelets from Fib^-/-VWF^-/- whole blood could adhere to collagen. However, the binding of VWF to platelets and adherence onto collagen could still effectively occur in Fib^-/-VWF^RGES/RGES mice, even though the VWF-α_IIbβ₃ interaction was impaired, as platelets can still bind to VWF through the GPIα receptor. Our findings align with previous studies that demonstrate the complementary roles of fibrinogen and VWF, as well as the complexity of the intersecting pathways involving VWF and platelet integrin receptors in mediating platelet aggregation, especially under varying hemodynamic conditions.

In conclusion, our study shows that VWF-α_IIbβ₃ interactions are essential for platelet plug stability, especially under flow conditions affecting thrombus integrity. Our VWF-RGES mouse model combined with the VWF:α_IIbβ₃ binding assay provides new tools to advance research on VWF-platelet biology and VWD. These tools could enhance the diagnosis and treatment of bleeding disorders by improving our understanding of platelet function and VWF’s role in hemostasis and thrombosis. Future studies will investigate the therapeutic potential of targeting VWF-α_IIbβ₃ interactions in bleeding and thrombotic disorders.

Footnotes

• Received February 26, 2025
• Accepted May 9, 2025

Correspondence

Q. Shi qshi@versiti.org

Disclosures

No conflicts of interest to disclose.

Contributions

QS designed experiments, analyzed data, and wrote the manuscript. JGM, PAM, PAC, JAS and SAF designed and performed experiments, analyzed data, and edited the manuscript. JR maintained mouse colonies and performed genotyping. MLS helped with the laser injury model study. JZ provided critical reagents and edited the manuscript. HW helped to generate VWF-RGES mouse model. SLH helped in designing research. VHF helped in designing research and edited the manuscript. RRM designed and supervised research, and edited the manuscript.

Funding

This work was supported by the National Heart, Lung, and Blood Institute, National Institutes of Health grants HL139847 (to RRM), HL081588 (to RRM), HL144457 (to RRM), HL112614 (to RRM), and HL102035 (to QS), and support from the Versiti Blood Research Foundation, the Children’s Hospital of Wisconsin Foundation (to QS), the Midwest Athletes Against Childhood Cancer and Bleeding Disorders Fund (to QS).

Acknowledgments

We thank Dr. Mathew Flick at the University of North Carolina at Chapel Hill for providing fibrinogen knockout and γΔ5 variant mice. We thank Dr. Peter Newman at the Versiti Blood Research Institute for providing HEK293F cells stably expressing murine