Shear stress is required for the endocytic uptake of the factor VIII-von Willebrand factor complex by macrophages.

BACKGROUND: Low-density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. OBJECTIVES: It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)-von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte-derived macrophages. METHODS: A confocal microscopy study was employed to study the uptake by monocyte-derived macrophages of a functional green fluorescent FVIII-GFP derivative in the presence and absence of VWF. RESULTS: The results revealed that FVIII-GFP is internalized by macrophages. We found that FVIII-GFP co-localizes with LDL receptor-related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII-GFP. However, FVIII-GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII-VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII-GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII-GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII-GFP and VWF was inhibited. CONCLUSION: The results show that shear stress is required for macrophages to internalize both constituents of the FVIII-VWF complex.

Tissue factor pathway inhibitor is an inhibitor of factor VII-activating protease.

BACKGROUND: Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α(2) -antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied. OBJECTIVES: To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α(2) -antiplasmin. METHODS: Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition. RESULTS AND CONCLUSIONS: We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α(2) -antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.

Factor seven activating protease (FSAP): does it activate factor VII?

BACKGROUND: Factor seven activating protease (FSAP) was initially reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources. OBJECTIVES: Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis. RESULTS: Expression of wild-type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg(313) at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAP(R313Q) in its intact form. Thermolysin-activated FSAP displayed the same reactivity toward the substrate S-2288 as plasma-derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased V(max) by 2-3-fold, without affecting K(m) (62 nm) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation. CONCLUSIONS: While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.

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

BACKGROUND: Patients with antiphospholipid syndrome (APS) display a heterogeneous population of antibodies with beta(2) glycoprotein-1 (ß(2)GP1) as the major antigen. OBJECTIVES: We isolated and characterized human mAbs directed against ß(2)GP1 from the immune repertoire of APS patients. METHODS: Variable heavy chain repertoires from B cells from two APS patients with anti-ß(2)GP1 antibodies were cloned into the pHEN1-VLrep vector. Constructed full-length IgG antibodies were tested for lupus anticoagulant (LAC) activity and binding to ß(2)GP1 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 ß(2)GP1 and its isolated domain I. The affinity of selected antibodies for ß(2)GP1 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 ß(2)GP1. Both selected clones from patient 2 recognized domain I of ß(2)GP1, 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 ß(2)GP1 for binding, and that display LAC activity that is dependent on their affinity for ß(2)GP1. These antibodies can help us to gain more insights into the pathogenesis of APS, and may facilitate standardization of APS diagnosis.