von Willebrand factor in hemodialysis patients. Does acquired von Willebrand syndrome occur?

INTRODUCTION AND AIMS: von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by endothelial cells and megakaryocytes that has an essential role in the primary hemostasis, in fact, the high-molecular-weight multimers (HMWMs) of vWF are the most effective in mediating platelet binding to the extracellular matrix. Acquired von Willebrand syndrome (AvWS) is a bleeding disorder characterized by the loss of HMWMs of vWF with consequent impaired vWF binding to platelets. Specific hemodynamic conditions of high vascular shear stress and immunologic diseases are linked to the development of this syndrome. Data on vWF function and its multimeric pattern in maintenance hemodialysis patients are scant and inconsistent. The aim of this study was to investigate the potential development of biochemical markers of AvWS and to preliminarily explore whether the reduction of HMWMs may be associated with an increased risk of bleeding.

METHODS: Sodium citrate plasma was collected from 53 hemodialysis patients (HP) and 25 healthy control subjects (C). Plasma levels of von Willebrand factor Antigen (vWF:Ag) were measured, and vWF functional activity was assessed by evaluating its Ristocetin cofactor activity (vWF:Rcof). A vWF:Rcof/vWF:Ag ratio of <0.7 was considered indicative of dysfunctional vWF. von Willebrand Multimers were separated using sodium dodecyl sulfate (SDS)-agarose low-resolution gels and blotted onto a nitrocellulose membrane. Multimer separation was performed based on electrophoretic mobility. Multimers were detected immunologically using rabbit anti-human-vWF antibodies. Densitometric analysis of multimers and quantification of the percentage of different electrophoretic areas were performed using ImageJ software (NIH, USA). Acquired von Willebrand syndrome (AvWS) was diagnosed if the vWF:Rcof/vWF:Ag ratio was below the normal range (<0.7) and densitometric analysis revealed a reduction HMWMs in hemodialysis patients (HP) compared to controls (C). To quantitatively estimate HMWMs loss, a control-derived threshold was established based on the 5th percentile of the control population (9%). Bleeding events in hemodialysis patients were retrospectively collected from the initiation of dialysis and classified according to ISTH criteria. Corresponding HMWMs levels and vWFRcof/vWF:Ag ratio values were analyzed by means of binary logistic regression. Statistical analysis was performed using MedCalc software (MedCalc Software Ltd., Ostend, Belgium). The alpha value for statistical significance was set at 0.05.

RESULTS: vWF:Ag levels were higher in HP compared to controls 169.3% (77.9-238.0) vs. 120.8% (82.3-141.6); p = 0.017. Hemodialysis patients (HP) exhibited also lower vWF:Rcof values to Controls, but this difference was not statistically significant 88.7% (30-148.2) vs. 105.2% (96.1-113.6); p = 0.167. The mean vWF:Rcof/vWF:Ag ratio was notably lower in HP than in controls 0.51 (0.38-0.70) vs. 0.88 (0.74-1.24); p < 0.0001, indicating a dysfunctional vWF profile in HP. vWF:Rcof/vWF:Ag ratio values were all above 0.7 in Controls. Densitometry analysis revealed a reduction in high molecular weight multimers, HMWMs 9.5% (3.1-14.1) vs. 12.5% (9.4-17.7); p = 0.016 and an increase in low molecular weight multimers (LMWMs) 49% (46.4-53.1) vs. 45.9% (38.7-49.2); p = 0.008 in HP compared to Controls. Among HP patients 47.2% exhibited HMWMs values below the control-derived cut-off. In contrast 8% of controls met this criterion; p < 0.001. Electrophoretic analysis confirmed a reduction in HMWMs and a greater representation of LMWMs in HP relative to Controls. In HP binary logistic regression showed that higher HMWMs levels were significantly associated with a reduced risk of bleeding OR = 0.71 [95% CI 0.57-0.89] p = 0.003, with a 29% decrease in bleeding odds per 1% increase in HMWMs. No significant association was observed between the vWF:Rcof/vWF:Ag ratio and bleeding risk.

CONCLUSIONS: Among hemodialysis patients, our data confirm the presence of the biochemical markers of AvWS which involve dysfunctional plasma vWF, as indicated by the low vWF:RCo/vWF:Ag ratio. Loss of HMWMs, defined using a control-derived cut-off, was identified in 47.2% of hemodialysis patients, and exploratory binary logistic regression suggests that this condition may be associated with an increased bleeding risk. Larger and prospective studies are needed to validate HMWMs as a potential biomarker of bleeding risk in this population.