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.

LDL receptor-related protein 1 contributes to the clearance of the activated factor VII-antithrombin complex.

Essentials Factor VIIa is cleared principally as a complex with antithrombin. Enzyme/serpin complexes are preferred ligands for the scavenger-receptor LRP1. Factor VIIa/antithrombin but not factor VIIa alone is a ligand for LRP1. Macrophage-expressed LRP1 contributes to the clearance of factor VIIa/antithrombin. SUMMARY: Background Recent findings point to activated factor VII (FVIIa) being cleared predominantly (± 65% of the injected protein) as part of a complex with the serpin antithrombin. FVIIa-antithrombin complexes are targeted to hepatocytes and liver macrophages. Both cells lines abundantly express LDL receptor-related protein 1 (LRP1), a scavenger receptor mediating the clearance of protease-serpin complexes. Objectives To investigate whether FVIIa-antithrombin is a ligand for LRP1. Methods Binding of FVIIa and pre-formed FVIIa-antithrombin to purified LRP1 Fc-tagged cluster IV (rLRP1-cIV/Fc) and to human and murine macrophages was analyzed. FVIIa clearance was determined in macrophage LRP1 (macLRP1)-deficient mice. Results Solid-phase binding assays showed that FVIIa-antithrombin bound in a specific, dose-dependent and saturable manner to rLRP1-cIV/Fc. Competition experiments with human THP1 macrophages indicated that binding of FVIIa but not of FVIIa-antithrombin was reduced in the presence of annexin-V or anti-tissue factor antibodies, whereas binding of FVIIa-antithrombin but not FVIIa was inhibited by the LRP1-antagonist GST-RAP. Additional experiments revealed binding of both FVIIa and FVIIa-antithrombin to murine control macrophages. In contrast, no binding of FVIIa-antithrombin to macrophages derived from macLRP1-deficient mice could be detected. Clearance of FVIIa-antithrombin but not of active site-blocked FVIIa was delayed 1.5-fold (mean residence time of 3.3 ± 0.1 h versus 2.4 ± 0.2 h) in macLRP1-deficient mice. The circulatory presence of FVIIa was prolonged to a similar extent in macLRP1-deficient mice and in control mice. Conclusions Our data show that FVIIa-antithrombin but not FVIIa is a ligand for LRP1, and that LRP1 contributes to the clearance of FVIIa-antithrombin in vivo.

Circulatory support devices: fundamental aspects and clinical management of bleeding and thrombosis.

Circulatory support devices are increasingly being used to overcome cardiac or respiratory failure. Long-term devices are used either as a 'bridge to transplant' to support patients who are unable to wait any longer for a heart transplant, or, more recently, as 'destination therapy' for older patients suffering from end-stage heart failure and who have contraindications to heart transplantation. Short-term support devices for high-risk percutaneous coronary intervention, or as a 'bridge for decision' for patients suffering from refractory cardiogenic shock, have also been developed. The clinical benefit of such assist devices has been demonstrated in several important studies, but, unfortunately, thrombotic and bleeding complications are two major clinical issues in patients requiring these devices. Overcoming these issues is of major importance to allow the safe and broad use of these devices, and to consider them as true alternatives to heart transplantation. The present review focuses on thrombotic and bleeding complications, and describes how the risk of thrombosis and bleeding may vary according to the clinical indication, but also according to the type of device. We describe the current knowledge of the mechanisms underlying the occurrence of these complications, provide some guidance for choosing the most appropriate anticoagulation regimen to prevent their occurrence for each type of device and indication, and provide some recommendations for the management of patients when the complication occurs.

Shear stress-independent binding of von Willebrand factor-type 2B mutants p.R1306Q & p.V1316M to LRP1 explains their increased clearance.

BACKGROUND: von Willebrand factor (VWF) is cleared in a shear stress- and macrophage-dependent manner by LRP1. von Willebrand disease (VWD)-type 2B mutants are endocytosed more efficiently than wild-type (wt)-VWF by macrophages. OBJECTIVE: To investigate if VWD-type 2B mutations in the VWF A1-domain affect LRP1 binding and LRP1-dependent clearance. METHODS: Recombinant Fc-tagged A1 domain (A1-Fc, A2-Fc, A3-Fc) and full-length VWF (wt or mutants thereof) were tested for binding to LRP1 or a recombinant fragment thereof in a static immunosorbent assay. Mutant and wt-VWF were also compared for clearance in mice lacking macrophage LRP1 (macLRP1(-) ) and control mice (macLRP1(+) ). RESULTS: We found that A1-Fc but not A2-Fc or A3-Fc binds dose-dependently to LRP1. Binding of A1-Fc to LRP1 was markedly enhanced by the VWD-type 2B mutation p.V1316M. As expected, full-length wt-VWF was unable to bind LRP1 under static conditions unless ristocetin was added. In contrast, the presence of the p.V1316M or p.R1306Q mutation induced spontaneous binding to LRP1 without the need for ristocetin or shear stress. Both mutants were cleared more rapidly than wt-VWF in control macLRP1(+) mice. Surprisingly, deletion of macrophage LRP1 abrogated the increased clearance of the VWF/p.R1306Q and VWF/p.V1316M mutant. CONCLUSION: The VWF A1-domain contains a binding site for LRP1. Certain VWD-type 2B mutations relieve the need for shear stress to induce LRP1 binding. Enhanced LRP1 binding coincides with a reduced survival of VWF/p.R1306Q and VWF/p.V1316M. Our data provide a rationale for reduced VWF levels in at least some VWD-type 2B patients.

Clearance of von Willebrand factor.

Quantitative deficiencies in von Willebrand factor (VWF) are associated with abnormal hemostasis that can manifest in bleeding or thrombotic complications. Consequently, many studies have endeavored to elucidate the mechanisms underlying the regulation of VWF plasma levels. This review focuses on the role of VWF clearance pathways. A summary of recent developments are provided, including results from genetic studies, the relationship between glycosylation and VWF clearance, the contribution of increased VWF clearance to the pathogenesis of von Willebrand disease and the identification of VWF clearance receptors. These different studies converge in their conclusion that VWF clearance is a complex phenomenon that involves multiple mechanisms. Deciphering how such different mechanisms coordinate their role in this process is but one of the remaining challenges. Nevertheless, a better insight into the complex clearance pathways of VWF may help us to better understand the clinical implications of aberrant clearance in the pathogenesis of von Willebrand disease and perhaps other disorders as well as aid in developing alternative therapeutic approaches.

von Willebrand factor: the old, the new and the unknown.

von Willebrand factor (VWF) is a protein best known from its critical role in hemostasis. Indeed, any dysfunction of VWF is associated with a severe bleeding tendency known as von Willebrand disease (VWD). Since the first description of the disease by Erich von Willebrand in 1926, remarkable progress has been made with regard to our understanding of the pathogenesis of this disease. The cloning of the gene encoding VWF has allowed numerous breakthroughs, and our knowledge of the epidemiology, genetics and molecular basis of VWD has been rapidly expanding since then. These studies have taught us that VWF is rather unique in terms of its multimeric structure and the unusual mechanisms regulating its participation in the hemostatic process. Moreover, it has become increasingly clear that VWF is a more all-round protein than originally thought, given its involvement in several pathologic processes beyond hemostasis. These include angiogenesis, cell proliferation, inflammation, and tumor cell survival. In the present article, an overview of advances concerning the various structural and functional aspects of VWF will be provided.

Decreased active von Willebrand factor level owing to shear stress in aortic stenosis patients.

BACKGROUND: Aortic stenosis patients often show bleeding complications. Previously, a prolonged platelet function analyzer (PFA-100) closure time was observed with plasma of severe aortic stenosis patients. To elucidate a possible role of circulating preactivated von Willebrand factor (VWF), we determined the level of VWF in its active, platelet-binding conformation in plasma of patients with aortic stenosis. PATIENTS/METHODS: Sixty-two aortic stenosis patients were included in this study. VWF and related parameters were measured, and the results were related to severity of aortic stenosis. RESULTS: VWF activation factor, indicating the proportion of circulating VWF able to bind to platelets, correlated negatively with peak transvalvular gradient and PFA-100 closure time. No correlation was observed between ADAMTS13 activity and peak transvalvular gradient or PFA-100 closure time. Both VWF antigen and VWF propeptide levels were significantly higher in patients with mild and moderate aortic stenosis, but not in those with severe stenosis. CONCLUSIONS: Our data demonstrate that the aortic pressure gradient is inversely associated with VWF activation factor, but not with VWF antigen or VWF multimerization in patients with aortic stenosis. These findings might have implications for the bleeding observed in patients with aortic stenosis.

Factor VIII and von Willebrand factor--too sweet for their own good.

SUMMARY: Although factor VIII (FVIII) and von Willebrand factor (VWF) are products of two distinct genes, they circulate in plasma as a tight non-covalent complex. Moreover, they both play a critical role in the haemostatic process, a fact that is illustrated by the severe bleeding tendency associated with the functional absence of either protein. FVIII is an essential cofactor for coagulation factor IX, while VWF is pertinent to the recruitment of platelets to the injured vessel wall under conditions of rapid flow. FVIII and VWF have in common that they are heavily glycosylated: full-length FVIII contains 20 N-linked and at least seven O-linked glycans, while VWF contains 12 N-linked and 10 O-linked glycans. Three decades of research have revealed that the carbohydrate structures of FVIII and VWF contribute to many of the steps that can be distinguished in the life-cycle of these proteins, including biosynthesis/secretion, function and clearance. In this review, several of these aspects will be discussed. In addition, the interaction of the FVIII/VWF complex with two families of carbohydrate-binding proteins, i.e. Galectins and Siglecs, and their potential physiological relevance will be discussed.

von Willebrand factor activation, granzyme-B and thrombocytopenia in meningococcal disease.

SUMMARY BACKGROUND: During invasive meningococcal disease, severe thrombocytopenia is strongly associated with a poor outcome. OBJECTIVES: In order to elucidate the pathophysiological mechanism behind the development of thrombocytopenia, we studied the role of von Willebrand factor (VWF) in meningococcal disease. PATIENTS/METHODS: Thirty-two children with severe meningococcal disease admitted to our university hospital were included in this study. VWF and related parameters were measured and results were correlated with the development of shock and thrombocytopenia. RESULTS: At admission, all patients had increased levels of (active) VWF and VWF propeptide. The highest VWF propeptide levels were observed in patients with shock, indicating acute endothelial activation. Although VWF propeptide levels in patients with shock, with or without thrombocytopenia, were similar, increased active VWF was significantly lower in patients with thrombocytopenia as compared with patients without thrombocytopenia. ADAMTS13 was moderately decreased. However, the VWF multimeric pattern was minimally increased. We assume that these findings are explained by VWF consumption and perhaps by granzyme B (GrB). In vitro experiments showed that GrB is able to cleave VWF multimers in plasma, whereas GrB was high in patients with shock, who developed thrombocytopenia. CONCLUSIONS: Our results demonstrate that consumption of VWF, derived from endothelial cells, could be a key feature of meningococcal disease and primary to the development of thrombocytopenia during shock.

The disappearing act of factor VIII.

Factor VIII (FVIII) is a plasma protein critical to the haemostatic system. This notion is illustrated by the severe bleeding disorder that is associated with its functional absence, known as haemophilia A. In addition, several epidemiological studies have revealed an association between the presence of elevated levels of FVIII and thrombotic complications. In view of its relation to thrombotic and haemorrhagic disorders, it is not surprising that FVIII has gained wide attention from the research community in the previous decades. This research has led to a better understanding of not only the structural, functional and physiological aspects of this intriguing protein, but also of the pathogenesis of haemostatic defects associated with FVIII. In the present review, focus will be on the interaction between FVIII and surface receptors that are able to capture FVIII. These interactions are of importance for FVIII, as they may affect both function and survival of FVIII.

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission.

BACKGROUND: Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. OBJECTIVE: To study ADAMTS13 and VWF-related features in TTP patients in remission. METHODS: ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibalpha-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. RESULTS: ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. CONCLUSION: ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.

The effect of exercise on von Willebrand factor and ADAMTS-13 in individuals with type 1 and type 2B von Willebrand disease.

BACKGROUND: The effect of exercise on von Willebrand factor (VWF) and ADAMTS-13 levels in individuals with von Willebrand disease (VWD) has never been reported. OBJECTIVES: The aim was to quantify the effect of a standardized exercise protocol on individuals with type 1 and type 2B VWD. PATIENTS/METHODS: Thirty individuals from three groups (10 controls, 11 with type 1 VWD and 9 with type 2B VWD) completed the Standard Bruce Protocol Treadmill Test. A bleeding questionnaire was administered and blood tests were performed pre- and immediately postexercise. The groups were well matched for age, gender and body mass index (BMI). RESULTS: There was a correlation in all groups between the metabolic equivalents (METS) achieved and the degree of change of VWF and FVIII:C levels (P < 0.002, Pearson's correlation). There was a significant postexercise increase in VWF:Ag, VWF:RCo, FVIII:C and activated VWF levels in both the control group and in the type 2B VWD group, but not in the type 1 VWD group. Specific to the type 2B VWD group was an increase in the percentage of high molecular weight multimers (P = 0.022), a decrease in the mean platelet count compared with the other groups (P < 0.001) and an increase in the ADAMTS-13 level (P = 0.001). CONCLUSIONS: There are significant differences in the effects of exercise on individuals with type 1 and type 2B VWD compared with controls. Further clinical studies are necessary to evaluate exercise as a therapeutic option in VWD.

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.