Background

Von Willebrand factor (VWF) is the largest soluble plasma protein, with essential functions in blood coagulation, control of bleeding, and angiogenesis. It is a complex glycoprotein with a mature subunit of 2,050 amino acids (Turecek et al., 2017). Approximately 19% of its molecular size are N- and O-linked carbohydrates, some of which resembling important blood group antigens. VWF is synthesized in endothelial cells and megakaryocytes. Intracellular polymerization starts with the formation of disulfide bridged dimers, which subsequently assemble into multimeric structures via additional disulfide bonds, creating multimers with apparent molecular masses between 500 kDa and 20,000 kDa. VWF is released into the circulation from its storage granules as ultra-large VWF with ultra-high molecular weight (UHMW). These UHMW multimers are potent mediators of platelet adhesion to other platelets and to subendothelial structures exposed upon vessel damage. However, under normal circumstances, UHMW VWF multimers are rapidly cleaved by the plasma protease ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 repeats) to smaller sized multimers with lower hemostatic and, thus, lower thrombogenic potential. ADAMTS13 specifically cleaves VWF at the bond between Tyr1605 and Met1606, generating 140-kDa N-terminal and 176-kDa C-terminal fragments, thus, regulating multimer size. The number of VWF multimers is generally seen as an indicator of functionality because more binding sites (e.g., for collagen and platelets) are exposed from larger multimers when the molecules become elongated under shear stress in the vasculature (Favaloro et al., 2021).

The main function of VWF is to mediate platelet binding and aggregation at sites of vascular injury. This process is known as primary hemostasis. VWF is critical for the formation of the primary hemostatic platelet plug by serving as glue between platelets through binding to platelet receptors glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury, and promoting binding of platelet aggregates to subendothelial matrix proteins such as collagens, which become exposed to flowing blood when lesions occur in vessels (Bryckaert et al., 2015). Shear forces in blood vessels promote folding and unfolding of VWF, essentially regulating its binding properties to receptors, which are further controlled by limited proteolytic cleavage by ADAMTS13. Furthermore, VWF serves as chaperone protein for coagulation factor VIII (FVIII) through tight binding, maintaining a highly controlled equilibrium between free and bound FVIII, which is also dependent on multimer size. Additionally, VWF determines the circulatory half-life of FVIII. All these functions of VWF depend on multimer size, and the degree of VWF multimerization correlates with the ability to promote platelet aggregation, as an essential step in blood coagulation.

Defects in VWF and ADAMTS13 are involved in hemostatic and thrombotic disorders. Deficiencies in VWF result in von Willebrand disease (VWD). VWD is the most common inherited bleeding disorder in the world, with an estimated prevalence of symptomatic disease of approximately 1 in 1000 individuals. VWD is a heterogeneous disease with different genetic subtypes, called type 1, type 2 (with subtypes 2A, 2B, 2M, and 2N), and type 3 (Peyvandi et al., 2019). In addition to genetic analysis, differential diagnosis of VWD and its subtypes requires determination of VWF activity by different functional assays and determination of the multimer pattern. Phenotypic von VWD classification largely relies on analysis of multimeric distributions of VWF and evaluation of its structure. Examples of typical multimer patterns, as seen in VWF subtype patients, can be found in textbooks and review articles (e.g., Schneppenheim and Budde, 2011). Although VWF multimer analysis is labor intensive, non-standardized, and limited to specialized laboratories, efforts have been made recently to establish ranges for VWF multimer distribution (Vangenechten and Gadisseur, 2020).

Inherited or immune mediated deficiencies of ADAMTS13 impair VWF cleavage, resulting in an increased portion of ultra-large multimers, which can cause thrombosis in the microvasculature. This disease is called thrombotic thrombocytopenic purpura (TTP), and its differential diagnosis requires VWF multimer analysis. Most recently, an impairment of the ratio of VWF and ADAMTS13 was identified as the main determinant of severity of COVID-19, with highly elevated levels of VWF and reduced ADAMTS13 resulting in thrombosis of the microvasculature, particularly in the lungs (Turecek et al., 2021; Seth et al., 2022).

Since the development of the first electrophoretic methods to separate and visualize multimers of VWF, the principle of these methods has not changed (Ruggeri and Zimmerman, 1981). Despite some modest improvements, the quality of test results largely depends on the quality and concentration of the agarose gel and the equipment used. Thus, the most demanding diagnostic test for VWD is still the VWF multimer analysis, an investigation that challenges even the most seasoned laboratory technologist (Lillicrap, 2013). Therefore, it is a limitation that methods are often not fully described with all the experimental details required to make the analytical system robust and reproducible. While multimer analysis of VWF is now performed for more than 40 years (Meyer et al., 1980), only a few publications describe the experimental methods, and even these do not give precise indications on how to perform the analysis. Here, we describe a validated method for electrophoretic separation of VWF multimers in low resolution SDS-agarose gels, with subsequent visualization by immunostaining (Aihara et al., 1986), allowing densitometric analysis. The method equally qualifies for analysis of VWF in blood from humans and other species, diagnosis of VWD subtypes, and characterization of VWF preparations derived from human or animal blood, produced by expression in cell culture systems, or by vectors used in gene therapy (Turecek et al., 2010). This method has been successfully used in quality control of recombinant and plasma-derived VWF drug products, and in preclinical and clinical studies with VWF drug candidates (Turecek et al., 1997).