Unfolded Von Willebrand Factor Interacts with Protein S and Limits Its Anticoagulant Activity

Background: The critical plasma anticoagulant protein S (PS) circulates in two pools: free (anticoagulant) or bound to complement component 4-binding protein (C4BP) (antiinflammatory). Acquired free PS deficiency occurs in patients with severe viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is associated with increased risk of thrombosis. von Willebrand Factor (VWF) is a large multimeric protein , which unfolds under shear, exposing protein binding sites. Elevation of VWF antigen and activity are associated with infection-induced vascular endothelial activation, including during SARS-CoV-2 infection. Here, we identified a shear-dependent association between VWF and PS, and assessed the effect of VWF on PS anticoagulant activity.

Methods: This study was approved by the University of Kentucky Institutional Review Board. Citrated plasma was collected from consenting adults, including 28 SARS-CoV-2+ inpatients, 49 SARS-CoV-2+ outpatients, and 31 healthy controls.

Results: Initial studies in our SARS-CoV-2+ patient cohort revealed that free PS, a measure of the anticoagulant pool, was similarly reduced in both inpatients (p=0.037) and outpatients (p=0.008) compared to controls, while total PS (p=0.964) was unchanged. This indicated an increase in a plasma PS-binding protein. However, known abundant PS-binding proteins C4BPβ and protein C, an anticoagulant that functions with PS, were unchanged (p=0.647 and p=0.185, respectively), and Mer, a macrophage receptor that binds PS, was only mildly increased (p=0.042).

Mass spectrometric and immunoblotting analyses revealed that PS interacts with VWF in pooled donor plasmas and that this interaction is enhanced >10,000,000-fold by shear-induced unfolding of VWF. Plasma VWF was elevated 3.8-fold in inpatients (p<0.0001) compared to outpatients and controls, suggesting endothelial activation, though no change in multimer distribution was apparent (n=4). We hypothesize that shear-induced unfolding of VWF exposes a binding site, which interacts either directly or indirectly with PS and contributes to acquired free PS deficiency. Consistent with this hypothesis, sheared-VWF dose-dependently inhibited free, but not total, PS antigen measurements, when measuring either purified proteins or plasma.

We next assessed the functional consequences of the VWF/PS interaction using purified proteins. Sheared-VWF dose dependently reversed FXa inhibition by the PS/Tissue Factor Pathway Inhibitor-α complex, in a chromogenic FXa activity assay. Conversely, Activated Protein C (APC) cofactor function was unaffected, either on phospholipid vesicles or on the surface of activated platelets, as measured either by the cleavage of factor Va heavy chain by western blot and by the rate of thrombin activation. Finally, to assess PS function in patients, we measured plasma thrombin generation. Despite anticoagulation, plasma thrombin generation in inpatient samples was comparable to controls (Endogenous Thrombin Potential (ETP): p=0.257; Lag Time: p=0.095), suggesting a significant hypercoagulability, possibly exacerbated by PS deficiency. Consistent with this, free PS negatively correlated with plasma thrombin generation parameters measured with thrombomodulin (TM) supplementation (r=-0.443, p=0.0008), which is required for sensitivity to the APC/PS pathway; ETP ratio (ETP with TM/ ETP without TM) in inpatients was higher compared to controls indicating less APC/PS activity (p=0.009); and these patients had profoundly elevated D-dimer (p<0.0001).

Conclusion: We propose a novel mechanism of acquired free PS deficiency, by which pathological shear forces unfold VWF, which interacts with PS, and at least partially blocks its anticoagulant function. It is possible that unfolded VWF further limits PS anticoagulant activity in vivo by sequestering it from APC, factor IXa, or other interacting partners. We anticipate that this mechanism also contributes to acquired free PS deficiency in other inflammatory conditions in which VWF is similarly unfolded.

Wood:Pfizer Inc: Research Funding.