Thrombosis Research 134 (2014) 1167–1168

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Editorial

Evaluating the interaction of von Willebrand factor and ADAMTS13 - and perhaps also beyond ADAMTS13 A R T IC L E

IN F O

Article history: Received 29 August 2014 Accepted 13 September 2014 Available online 30 September 2014 Keywords: von Willebrand factor ADAMTS13 Thrombosis

von Willebrand factor (VWF) is a large adhesive protein that forms large multimers that each express a number of key hemostasis activities, including binding to platelets via several receptors, most notable glycoprotein Ib (GPIb), binding to sub-endothelial matrix components (most notably collagen), and binding and protection of factor VIII (FVIII) function [1]. As the number of multimers increase, so to does the overall VWF activity, and its ability to capture and arrest platelets to sites of vascular injury, and thereby arrest bleeding. Defects in VWF, or deficiency of VWF, or the larger VWF forms as represented by so-called high molecular weight (HMW) multimers, leads to a common bleeding disorder called von Willebrand disease (VWD) [2]. Although six different defined types of VWD have been defined, depending on the presenting phenotype [3], the pathophysiology of VWD is most often represented by a failure of platelet attachment to sites of damaged tissue and consequent excessive bleeding, particularly at sites of high vascular shear. In some patients, overall levels of VWF may be in the normal range, but there is an absence of the larger molecules, or the HMW forms, and representing a qualitative defect, and caused by either faulty VWF multimer assembly or elevated proteolysis. In contrast, excessive levels of VWF, particularly the larger HMW forms, are associated with increasing thrombotic risk. This appears to be true in general, where otherwise normal patients expressing elevated levels of VWF are at risk [4], as well as in certain patients where the ability to control the expression or level of HMW forms of VWF becomes compromised. This is certainly the case for patients with thrombotic thrombocytopenic purpura (TTP), which is otherwise characterized by a severe deficiency of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), an enzyme responsible for physiological cleavage of VWF [5,6]. This deficiency state can arise as an acquired disorder (due to autoantibodies against ADAMTS13), or as a congenital disorder, also called Upshaw Schulman syndrome. Lack of ADAMTS13 leads to the persistence of ultra large (UL) VWF multimers in the circulation, which in the presence of additional triggers leads to shear stress and unfolding of VWF, and enhanced

http://dx.doi.org/10.1016/j.thromres.2014.09.029 0049-3848/Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved.

platelet aggregation. These platelet aggregates in particular affect the blood flow in the microcirculation and cause organ damage and the clinical symptoms of TTP. Investigation of VWF for evaluation of VWD, and also for potential thrombosis risk when in excess, is usually carried out using a large number of assays, including total VWF level as reflected by an ‘antigen’ assay (VWF:Ag) as well as various activity assays that reflect surrogates for the various VWF functions [2]. In normal laboratory practice, the most common application for VWD diagnostics is of ‘static’ assays performed using standard laboratory instrumentation. The term ‘static’ here defines test systems that utilize test tubes, cuvettes, wells, or other chambers, where test reactions take place that may or may not employ mixing steps, but which do not employ any shear stress or flow-based assessments that characterize the true in vivo activity of VWF. In general, assays may include the following technologies: platelet agglutination or aggregation, latex particle agglutination, enzyme linked immunosorbent assay (ELISA), immunofluroescence, and/or flow cytometry [2]. Standard VWF activity assays include ristocetin cofactor (VWF:RCo) as a surrogate for VWF-platelet binding activity and collagen binding (VWF:CB) as a surrogate for VWF-subendothelial tissue binding activity. The VWF:RCo assay utilizes an antibiotic (ristocetin) to cause unfolding of VWF that then permits adhesion of this VWF to platelets (in vivo, the unfolding of the VWF required to promote platelet binding is facilitated by vascular blood flow stresses). Given well recognized problems with the VWF:RCo assay, including high variability and poor sensitivity to low levels of VWF that compromises diagnostic utility [2,7], a number of new assays have emerged, including the so called INNOVANCE activity assay (VWF:Ac; Siemens) that uses latex particles instead of platelets, and recombinant GPIb expressing two gain of function mutations that permits VWF binding without the need for ristocetin. This assay will in theory be able to replace the problematic VWF:RCo assay for routine diagnostic assessment of VWF-platelet-GPIb-binding activity [8]. Nevertheless, diagnostics in VWD remains an imperfect science [2,7]. Assessment of ADAMTS-13 levels and activity, as well as antibodies to ADAMTS-13, is carried out using a different set of assays, although these are also ‘static’ in nature. Again, these assays are imperfect, and results do not always predict disease or outcome in patients with TTP [9]. In general, the static laboratory assays currently utilized to assess VWF level and activity are generally adequate for identification of deficiencies or excess in VWF, as well as major defects in VWF, and thus for the identification of VWD, as well as determination of VWD type, which is important in terms of applied therapy. Similarly, the static laboratory assays currently utilized to assess ADAMTS13 level and activity, or antibodies to ADAMTS13, are generally adequate for identification of ADAMTS13 deficiencies, and thus for the identification of TTP. However, these assays are simply tools for in vitro assessment of surrogates to the

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Editorial

in vivo activities, which in situ actually occur in high shear stress, flowbased, blood systems. Static assays are therefore limiting for evaluation of true in vivo activities, and thus assessment of the interaction of ADAMTS13 with VWF, and thus failures in this process that lead to TTP and the devastating clinical consequences of this thrombotic microangiopathy. In vivo, stimulated endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream. Elongation of VWF strings due to flow shear on the endothelial cells' surface leads to accessibility of VWF binding sites for proteins, including platelet membrane GPIb. The prothrombotic VWF strings are then size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. Therefore, the report by Kraus and coworkers [10] of an evaluation of the ADAMTS13/VWF relationship using several novel and interesting approaches should be of major interest. In this study, VWF string formation was induced by histamine stimulation of human umbilical vein endothelial cells (HUVEC) under unidirectional shear flow and VWF strings were detected, and then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. In one interesting twist to the ADAMTS-13/VWF story, these researchers have been very clever to use the components of one static but arguably clever assay (the Siemens INNOVANCE activity assay) in an ‘off-label’ manner to assess this interaction. Although the ADAMTS-13/VWF interaction has previously been successfully investigated using platelets plus VWF and ADAMTS-13 (example [11]), the incorporation and inclusion of platelets in such studies is often technically challenging – requiring fresh blood collections, fresh platelet preparation, and meticulous sample handling to avoid artifactual platelet activation. The use of latex beads (in place of platelets) in the novel test approach avoids these problems and provides a stable test system that can be stored refrigerated until needed. Naturally, the use of GPIb with gain of function mutations might be considered less physiologically relevant for some investigational applications, so the authors provided additional evidence for utility of this approach using latex beads coupled with wild-type GPIb. No increase in shear rate was necessary for binding of the wild-type GPIb-beads to VWF strings. For even further benefit, the authors also provided evidence for utility of an approach that omitted the beads entirely, and using VWF identified with fluorescence labeled antibodies. The authors [10] summarised their work as follows: “we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13” to “investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13 … without the need for isolated platelets.” “Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semiautomated, quantitative assessment of recombinant and plasma ADAMTS13 activity.” “Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of VWD and TTP as it allows detection of shear flow-dependent dysfunction of VWDassociated VWF mutants as well as TTP-associated ADAMTS13 mutants.” My own belief is that this undersells the potential utility of this technology. Although ADAMTS represents a major moderator of VWF activity, the study of haemostasis teaches us that many redundancies and additional safeguards often become incorporated into various haemostatic mechanisms. For VWF, the VWF gene is an obvious and major moderator of VWF activity, and mutations in the VWF gene cause VWD [12]. However, additional factors moderate VWF level and activity. The best well known is the ABO-blood group, with O group

associated with lower levels of plasma VWF, with this thought to occur as a result of increased VWF clearance. Additional moderators include various proteases and reductases, including thrombospondin, as well as plasmin, neutrophil elastase and thrombin [13]. Recently, another VWF moderator, complement factor H, has emerged and its deficiency has been implicated in thrombotic microangiopathies that are not clearly related to ADAMTS-13 deficiency/antibodies [14]. Finally, ADAMTS13 activity, or lack thereof, has implications way beyond TTP [15]. Accordingly, the novel approaches reported by Kraus and colleagues [10] have implications beyond ADAMTS13/VWF/TTP, and I look forward to future uses of these assays to further evaluate the physiological and pathophysiological moderators of VWF.

References [1] Yee A, Kretz CA. Von Willebrand factor: form for function. Semin Thromb Hemost 2014;40:17–27. [2] Favaloro EJ. Diagnosing von Willebrand disease: a short history of laboratory milestones and innovations, plus current status, challenges, and solutions. Semin Thromb Hemost 2014;40:551–70. [3] Sadler JE, Budde U, Eikenboom JCJ, Favaloro EJ, Hill FG, Holmberg L, et al. Working Party on von Willebrand Disease Classification. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006;4:2103–14. [4] Lenting PJ, Denis CV, Wohner N. Von Willebrand factor and thrombosis: risk factor, actor and pharmacological target. Curr Vasc Pharmacol 2013 Jul;11(4):448–56. [5] Turner N, Nolasco L, Moake J. Generation and breakdown of soluble ultralarge von Willebrand factor multimers. Semin Thromb Hemost 2012;38:38–46. [6] Chapman K, Seldon M, Richards R. Thrombotic microangiopathies, thrombotic thrombocytopenic purpura, and ADAMTS-13. Semin Thromb Hemost 2012;38: 47–54. [7] Favaloro EJ, Bonar RA, Meiring M, Duncan E, Mohammed S, Sioufi J, et al. Evaluating errors in the laboratory identification of von Willebrand disease in the real world. Thromb Res 2014;134:393–403. [8] Geisen U, Zieger B, Nakamura L, Weis A, Heinz J, Michiels JJ, et al. Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF:RCo. Thromb Res 2014;134:246–50. [9] Rock G, Clark WF, Anderson D, Benny B, Sutton D, Leblond P, et al. Members of the Canadian Apheresis Group. ADAMTS-13 may not predict disease or outcome in patients with Thrombotic Thrombocytopenic Purpura. Thromb Res 2013;131:308–12. [10] Kraus E, Kraus K, Obser T, Oyen F, Klemm U, Schneppenheim R, et al. Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13. Thromb Res 2014;134:1285–91. [11] Kragh T, Napoleone M, Fallah MA, Gritsch H, Schneider MF, Reininger AJ. High shear dependent von Willebrand factor self-assembly fostered by platelet interaction and controlled by ADAMTS13. Thromb Res 2014;133:1079–87. [12] Favaloro EJ, Krigstein M, Koutts J, Brighton T, Lindeman R. Genetic testing for the diagnosis of von Willebrand Disease: benefits and limitations. J Coag Disord 2010; 2:37–47. [13] Wohner N, Kovács A, Machovich R, Kolev K. Modulation of the von Willebrand factor-dependent platelet adhesion through alternative proteolytic pathways. Thromb Res 2012;129:e41–6. [14] Turner N, Nolasco L, Nolasco J, Sartain S, Moake J. Thrombotic Microangiopathies and the Linkage between von Willebrand Factor and the Alternative Complement Pathway. Semin Thromb Hemost 2014;40:544–50. [15] Eerenberg ES, Levi M. The Potential Therapeutic Benefit of Targeting ADAMTS13 Activity. Semin Thromb Hemost 2014;40:28–33.

Emmanuel J. Favaloro Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, NSW, 2145, Australia Tel.: +1 612 9845 6618; fax: +1 612 9689 2331. E-mail address: [email protected]. 29 August 2014