Interpreting High Levels of Unfolded Von Willebrand Factor in Patients with the Antiphospholipid Syndrome

Von Willebrand factor (VWF) is a multimeric plasma protein that regulates the adhesion of platelets to subendothelial collagen at sites of vascular injury. Unfolding of VWF exposes the binding site for platelet VWF receptor glycoprotein Ib and is necessary for VWF-platelet binding. β2-glycoprotein I (β2GPI) binds to the A1 domain of VWF, making β2-GPI a biologically relevant inhibitor of the platelet-VWF interaction. The antiphospholipid syndrome (APS) is an auto-immune disease associated with thrombosis and/or recurrent pregnancy loss in combination with the persistent presence of auto-antibodies directed to β2GPI, amongst others. Especially anti-β2-GPI antibodies that express lupus anticoagulant (LAC) activity are associated with thrombosis in APS. Subsequently, auto-antibodies against β2-GPI that counteract the inhibitory action of β2-GPI on platelet-VWF interaction are proposed to contribute to the increased risk of thrombosis in APS. In this study, we investigated whether high plasma levels of unfolded VWF are associated with thrombotic APS, non-APS thrombosis and non-APS auto-immune diseases (AID).

We measured the levels of total VWF and unfolded VWF in a sub-cohort of the APS multicenter study, consisting of 93 normal controls, 54 thrombotic APS patients, 39 non-APS thrombosis patients and 49 non-APS AID patients. Subjects were enrolled at multiple medical European centers. The study was approved by the local institutional review boards, and performed in accordance with the Declaration of Helsinki. Ninety-six percent of APS patients suffered from primary APS and 4% from secondary APS. LAC was present in 70% of the APS patients, whereas 59%, 63%, and 59% respectively, were positive for anti-cardiolipin, anti-β2GP, or anti-PS/PT antibodies. In 74% of APS patients, thrombosis presented as venous thrombosis and in 41% as arterial thrombosis. In APS negative thrombosis patients, 82% experienced venous thrombosis and 18% arterial thrombosis.

Total VWF antigen levels were determined using the Liatest VWF assay on an automated STA-R Max coagulation analyser (Diagnostica Stago, France). Unfolded VWF levels were measured using a llama-derived single-domain antibody (VHH) directed against a cryptic epitope in the A1 domain of VWF, which is only exposed upon unfolding of VWF. Total VWF and unfolded VWF levels were significantly higher in APS patients (+41% and +53%), thrombosis patients (+49%, and +50%) and AID patients (+31% and +36%) compared to normal controls. A cut-off value was calculated as the 90^th percentile of the normal controls for total VWF (138%) and unfolded VWF (136%). Patients were classified based on the determined cut-off values. For total VWF, 33% of APS patients, 36% of thrombosis patients, and 20% of AID patients were above the cut-off value. For unfolded VWF, 46% of APS patients, 36% of thrombosis patients, and 29% of AID patients were above the cut-off value. Multinomial logistic regression showed that total VWF values above the 90^th percentile cut-off resulted in an odds ratio (OR) of 3.82 (CI: CI: 1.44-11.3, p<0.001) for thrombotic APS, and an OR of 4.65 (CI: 1.64-13.2, p<0.001) for non-APS thrombosis, after adjustment for age and sex. No significant association was detected between high total VWF levels and AID. Unfolded VWF levels above the 90^th percentile were significantly associated with thrombotic APS (OR: 8.51; CI:3.26 - 22.2; p<0.001), non-APS thrombosis (OR:5.87; CI:2.07 - 16.7, p=0.001) and AID (OR:3.71; CI:1.40 – 9.87; p=0.009) in the age- and sex-adjusted model.

In conclusion, we found a remarkable association of high unfolded VWF levels and thrombotic APS, non-APS thrombosis and non-APS AID. Subjects with unfolded VWF levels above the 90^th percentile cut-off value had a 8.5-fold higher OR for APS. Interestingly, high unfolded VWF levels were significantly associated with non-APS thrombosis as well, although with a lower OR of 5.9. For non-APS, non-thrombosis AID a 3.7-fold higher OR was found. These data suggest an additive effect of the auto-immune disease component and the thrombosis component of the APS pathology, resulting in a stronger association of high unfolded VWF levels and APS, although this needs to be confirmed in other populations.