Characterization of the interaction between von Willebrand factor and osteoprotegerin

BACKGROUND AND OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. METHODS AND RESULTS: In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. CONCLUSIONS: Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.     

Fifty years of Weibel–Palade bodies: the discovery and early history of an enigmatic organelle of endothelial cells1

Summary. In 1962, a rod‐shaped cytoplasmic organelle of endothelial cells, later called the Weibel–Palade body, was serendipitously discovered by electron microscopy. It contains a set of parallel tubules and is wrapped in a membrane. Subsequent studies in the following decades established the unique localization of this organelle in endothelial cells of all vertebrates studied, meaning that it could serve as a marker of endothelial cells in tissue cultures. However, these studies did not reveal its functional significance, except for an indication that it could be related to an undefined thromboplastic substance. Twenty years after its discovery as a structural entity, it was shown by others that it houses von Willebrand factor and is thus clearly related to the coagulation system. In this review, I provide a personal historical account of the discovery and the subsequent limited work that I carried out on the organelle, putting it in the perspective of the current state of knowledge after half a century of research by many scientists.     

Genetic alteration of the D2 domain abolishes von Willebrand factor multimerization and trafficking into storage

Summary.  Background: The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. Patients/Methods: We identified a heterozygous 18 base pair, in-frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer-intense multimer pattern. This deletion results in loss of amino acids 436–442 of VWFpp, which include one cysteine. Results: Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild-type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. Conclusions: The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.     

Requirements for cellular co-trafficking of factor VIII and von Willebrand factor to Weibel–Palade bodies

Background:  von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. Objectives:  In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. Methods:  Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. Results:  Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding ( K _d = 0.065 ± 0.008 n m ) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel–Palade bodies (WPBs). Conclusions:  Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.     

von Willebrand factor assembly and secretion

Summary.  During its life history, von Willebrand factor (VWF) experiences a remarkable sequence of conformational changes that are triggered by differences in pH between the endoplasmic reticulum (ER), Golgi and extracellular environments. VWF subunits dimerize in the ER and assemble into disulfide-linked multimers in the trans-Golgi, which lacks known chaperones and has an acidic pH that inhibits disulfide rearrangement. VWF has circumvented these problems by evolving N-terminal domains that function as an oxidoreductase at the low pH of the Golgi. VWF multimers also condense into tightly packed, tubular arrays for storage in the Weibel–Palade bodies of endothelial cells. Like multimer assembly, tubular packing depends on low pH and Ca²⁺. Upon secretion, exposure to the neutral pH of the extracellular environment allows enormous VWF multimers to uncoil without tangling, which is crucial for hemostasis. Recent studies have identified some of the biochemical and structural properties that underlie these self-organizing behaviors.     

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

Essentials

• Endothelial activation initiates multiple processes, including hemostasis and inflammation.
• The molecules that contribute to these processes are co‐stored in secretory granules.
• How can the cells control release of granule content to allow differentiated responses?
• Selected agonists recruit an exocytosis‐linked actin ring to boost release of a subset of cargo.

Summary

Background

Endothelial cells harbor specialized storage organelles, Weibel‐Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P‐selectin. During full fusion, secretion of this large hemostatic protein and smaller pro‐inflammatory proteins are thought to be inextricably linked.

Objective

To determine if secretagogue‐dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis.

Methods

We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high‐throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins.

Results

Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms.

Conclusions

Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo‐content release and the treatment of patients with von Willebrand disease.

     

von Willebrand factor and inflammation

Von Willebrand factor (VWF) is a plasma glycoprotein best known for its crucial hemostatic role in serving as a molecular bridge linking platelets to subendothelial components following vascular injury. In addition, VWF functions as chaperone for coagulation factor VIII. In pathological settings, VWF is recognized as a risk factor for both arterial and venous thrombosis, as well as a molecular player that directly promotes the thrombotic process. Recent years have seen the emergence of the concept of immuno‐thrombosis by which inflammatory cells participate in thrombotic processes. In return, reports about the involvement of hemostatic proteins or cells (such as platelets) in inflammatory responses have become increasingly common, emphasizing the intricate link between hemostasis and inflammation. However, evidence of a link between VWF and inflammation arose much earlier than these recent developments. At first, VWF was considered only as a marker of inflammation in various pathologies, due to its acute release by the activated endothelium. Later on, a more complex role of VWF in inflammation was uncovered, owing to its capacity to direct the biogenesis of specific endothelial organelles, the Weibel–Palade bodies that contain known inflammation players such as P‐selectin. Finally, a more direct link between VWF and inflammation has become apparent with the discovery that VWF is able to recruit leukocytes, either via direct leukocyte binding or by recruiting platelets which in turn will attract leukocytes. This review will focus on these different aspects of the connection between VWF and inflammation, with particular emphasis on VWF‐leukocyte interactions.     

Reduced von Willebrand factor secretion is associated with loss of Weibel–Palade body formation

Background:  von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. Objectives:  We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel–Palade body (WPB)‐like granules. Methods:  Transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild‐type VWF. Expression was also examined in HEK293 cells that form WPB‐like granules when transfected with wild‐type VWF. Results:  Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB‐like granules, while R2663C was capable of WPB‐like granules. Heterozygous expression of VWF variants had a negative impact on wild‐type VWF with a reduction in elongated WPB‐like granules in co‐transfected cells. Conclusions:  Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB‐like granules.     

Formation of platelet‐binding von Willebrand factor strings on non‐endothelial cells

Summary. Background and Objective: Von Willebrand factor (VWF) forms strings on activated vascular endothelial cells that recruit platelets and initiate clot formation. Alterations in VWF strings may disturb hemostasis. This study was aimed at developing a flexible model system for structure–function studies of VWF strings. Methods: VWF strings were generated by inducing exocytosis of pseudo‐Weibel–Palade bodies from VWF‐transfected HEK293 cells, and the properties of these strings under static conditions and under flow were characterized. Results: Upon exocytosis, VWF unfurled into strings several hundred micrometers in length. These strings could form bundles and networks, and bound platelets under flow, resembling authentic endothelial VWF strings. Anchorage of the platelet‐decorated VWF strings was independent of P‐selectin and integrin α_Vβ₃. Translocation of platelets along the strings, elongation and fragmentation of the strings frequently occurred under flow. Furthermore, VWF variants with the p.Tyr87Ser and p.Cys2773Ser mutations, which are defective in multimer assembly, did not give rise to VWF strings. Also, insertion of the green fluorescent protein into VWF inhibited string formation. Conclusions: HEK293 cells provide a flexible and useful model system for the study of VWF string formation. Our results suggest that structural changes in VWF may modulate string formation and function, and contribute to hemostatic disorders.     

Treatment of severe von Willebrand disease with a high-purity von Willebrand factor concentrate (Wilfactin®): a prospective study of 50 patients

BACKGROUND AND OBJECTIVES: A plasma-derived von Willebrand factor (VWF) concentrate with low factor VIII (FVIII) content was specifically developed to treat von Willebrand disease (VWD). Efficacy and safety were investigated by merging the results of two comparable protocols conducted prospectively in 5 European and 12 French centers. METHODS AND RESULTS: Fifty patients with clinically severe VWD (72% had VWF ristocetin cofactor activity less than 10 IU dL(-1) and 46% had FVIII < 20 IU dL(-1)) were treated with the concentrate as the only therapy, except for clinical situations requiring a priming dose of FVIII to rapidly correct an intrinsic coagulation defect. A total of 139 spontaneous bleeding episodes were treated; only 53 (38%) needed a concomitant FVIII dose. Outcome was excellent or good in 89% of the episodes. Forty-four patients underwent 108 surgical or invasive procedures. Outcome was excellent or good in 95 scheduled procedures (only VWF was infused) and 13 emergency procedures (a priming FVIII dose was co-administered with the first VWF infusion). There were no thrombotic complications and none of the 18 patients with type 3 VWD developed anti-VWF or anti-FVIII antibodies. CONCLUSIONS: This concentrate safely and effectively provides hemostasis in patients with clinically severe VWD.     

Laboratory diagnosis of von Willebrand disease

Von Willebrand disease is the most-common inherited bleeding disorder, including both quantitative (types 1 and 3) and qualitative (type 2) defects of von Willebrand factor. Among patients with suspected von Willebrand disease, the laboratory diagnosis requires three levels of testing: screening tests, specific assays for von Willebrand factor to establish the diagnosis, and discriminating tests to allow accurate characterization of the numerous types and subtypes of the disease. Because of their poor sensitivity, normal screening tests do not exclude the diagnosis. In most cases, specific measurements of von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity, and factor VIII levels in plasma allow differentiation of quantitative (proportionately decreased levels) and qualitative (discrepant levels) deficiencies of von Willebrand factor. Among the latter, a decreased von Willebrand factor ristocetin cofactor activity/von Willebrand factor antigen ratio is in favor of the three subtypes (2A, 2M, and 2B) defined by an abnormal interaction between von Willebrand factor and platelet glycoprotein Ib, whereas a decreased factor VIII/von Willebrand factor antigen ratio suggests subtype 2N, defined by a defective binding of von Willebrand factor to factor VIII. Several discriminating tests are available to definitively characterize each subtype. Moreover, for all variants, the link between phenotype and genotype is established using DNA analysis. In all cases, the precise characterization of type and subtype of von Willebrand disease remains essential for the choice of optimal therapeutic monitoring of each patient. Presented at the Joint Meeting of the World Health Organization and the International Society for Thrombosis and Hemostasis “Impact, Prevention and Control of von Willebrand’s disease” London, October 12–14, 1998