Johannes Jacobus Voorberg

Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V

Abnormal coagulation factor V may underlie the thrombotic events associated with resistance to activated protein C (APC). We analysed 27 consecutive patients with documented idiopathic (recurrent) thromboembolism for the occurrence of point mutations within the APC sensitive regions of blood coagulation factor V. In 10 patients we observed a single basepair mutation resulting in a substitution of Arg506 to Gln. This mutation was significantly linked to in-vitro resistance to APC in these subjects. This mutation at Arg506 of factor V may form the molecular basis for the thrombotic events associated with APC resistance.

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro‐vWF) that consists of four repeated domains, denoted D1‐D2‐D′‐D3‐A1‐A2‐A3‐D4‐B1‐B2‐B3‐C1‐C2. Previously, we have defined the domains on the pro‐vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel‐Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full‐length vWF cDNA, vWF‐containing organelles are encountered in monkey kidney CV‐1 cells that are morphologically similar to the endothelial‐specific Weibel‐Palade bodies. Expression in CV‐1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense‐cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a ‘condensed’, multimeric vWF is required for targeting to the Weibel‐Palade body.

Factor V Arg506-->Gln mutation in patients with antiphospholipid antibodies.

Antiphospholipid antibodies (APA) have thought to be implicated in the pathogenesis of both arterial and venous thrombosis. Because of heterogeneity of APA, direct evidence of their involvement in a thrombotic event is not yet available. Development of thrombosis in the antiphospholipid antibody syndrome (APS) may occur because of the presence of addi tional risk factors. Here we have analysed 60 patients with APA for the presence of the Arg506 → Gln mutation in factor V. Among them 26 suffered from deep venous thrombosis, 13 from arterial thrombosis and 21 had no history of arterial or venous thrombosis. In the first group four patients were found to be heterozygous and one homozygous for the factor V Arg506 → Gln mutation. None of the patients with the factor V mutation was found in the second and third group. The incidence of factor V mutation was significantly elevated in the group of patients with venous thrombosis. These data suggest that in patients with anti phospholipid antibodies the factor V Arg506 → Gln mutation may play a major role in the occurrence of venous thrombosis.

Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles.

Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF.

Patient-derived monoclonal antibodies directed towards beta2 glycoprotein-1 display lupus anticoagulant activity

Summary.  Background: Patients with antiphospholipid syndrome (APS) display a heterogeneous population of antibodies with beta2 glycoprotein‐1 (β2GP1) as the major antigen. Objectives: We isolated and characterized human mAbs directed against β2GP1 from the immune repertoire of APS patients. Methods: Variable heavy chain repertoires from B cells from two APS patients with anti‐β2GP1 antibodies were cloned into the pHEN1‐VLrep vector. Constructed full‐length IgG antibodies were tested for lupus anticoagulant (LAC) activity and binding to β2GP1 and its domains. Results: Two clones of each patient were selected on the basis of the reactivity of single chain Fv (scFv) fragments displayed on phages towards full‐length β2GP1 and its isolated domain I. The affinity of selected antibodies for β2GP1 was lost when transforming from phages to monovalent scFvs, and was regained when antibodies were constructed as complete IgG, indicating a role for bivalency in binding to β2GP1. Both selected clones from patient 2 recognized domain I of β2GP1, and for both clones selected from patient 1, binding required the presence of both domain I and domain II. All mAbs displayed LAC activity in both activated partial thromboplastin time‐based and dilute Russell’s viper venom test‐based clotting assays and in thrombin generation. Conclusions: In this study, we show successful cloning of patient‐derived mAbs that require domain I of β2GP1 for binding, and that display LAC activity that is dependent on their affinity for β2GP1. These antibodies can help us to gain more insights into the pathogenesis of APS, and may facilitate standardization of APS diagnosis.