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Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations.

To determine the optimal timing for imaging brain tumours and other brain lesions with 18F-labelled fluoromethylcholine (18F-FCho) PET.

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33scFv, and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 → Ala/Ala (fVIIIYFP 2092/93) and 2 single mutants Lys2092 → Ala and Phe2093 → Ala. KM33scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIIIYFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33scFv inhibited binding to platelets and fVIIIYFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIIIYFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIIIYFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIIIYFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.

von Willebrand factor (VWF) serves as adhesive surface for platelets to adhere to the vessel wall. We have recently found that beta2-glycoprotein I is able to inhibit platelet binding to VWF, indicating a role in the pathophysiology of arterial thrombosis. In the present study, we investigated whether differences in beta2-glycoprotein I plasma levels influence the risk of myocardial infarction. We have measured beta2-glycoprotein I and VWF antigen levels in 539 men with a first myocardial infarction and in 611 control subjects. Although we did not find a profound effect of beta2-glycoprotein I plasma levels on myocardial infarction in the overall population, we found a dose-dependent protective effect of increasing beta2-glycoprotein I plasma levels on myocardial infarction in men 60 years and older. In this age group, we found an odds ratio of 0.41 (95% confidence interval, 0.22-0.74) for high beta2-glycoprotein I levels compared with low levels. High plasma levels of beta2-glycoprotein I remained protective for myocardial infarction despite high levels of VWF. To conclude, high circulating levels of beta2-glycoprotein I appeared to be associated with a reduced risk of myocardial infarction in elderly men. In vivo experiments are needed to investigate the exact contribution of beta2-glycoprotein I on the pathophysiology of myocardial infarction.

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.

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.

The discovery that antiphospholipid antibodies recognize plasma proteins that bind to phospholipids rather than recognizing phospholipids themselves has been a major advance in research into antiphospholipid syndrome (APS). It is now established that ß2-glycoprotein I (ß2GPI) is the most important antigen for antiphospholipid antibodies. However, the possible pathologic mechanism is still much debated. This is mainly because not all patients with anti-ß2GPI antibodies show clinical symptoms that are related to APS. Several reports indicate that anti-ß2GPI antibodies with lupus anticoagulant (LA) activity are clinically of much importance. Most patients with LA caused by anti-ß2GPI antibodies suffer from thrombosis as a result of recognition of the first domain of ß2GPI by these antibodies. In the search for a pathologic mechanism that might explain the high occurrence of thrombosis in patients with anti-domain I antibodies (LA-causing anti-ß2GPI antibodies), it was found that these antibodies show increased resistance to the anticoagulant activity of annexin A5. We have shown that the same population of antibodies also displays increased resistance to activated protein C. Owing to the diversity of clinical symptoms related to APS, it is likely that other pathologic mechanisms also contribute to the occurrence of APS-related symptoms.

Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII–LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.

It has been established that low-density lipoprotein receptor-related protein (LRP) is involved in the cellular uptake and degradation of coagulation factor VIII (FVIII) in vitro. To address the physiologic role of LRP in regulating plasma FVIII in vivo, we used cre/loxP–mediated conditional LRP- deficient mice (MX1cre+LRPflox/flox). Upon inactivation of the LRP gene, MX1cre+LRPflox/floxmice had significantly higher plasma FVIII as compared with control LRPflox/floxmice (3.4 and 2.0 U/mL, respectively; P< .001). Elevated plasma FVIII levels in MX1cre+LRPflox/floxmice coincided with increased plasma von Willebrand factor (VWF) (2.0 and 1.6 U/mL for MX1cre+LRPflox/floxand control LRPflox/floxmice, respectively; P< .05). Elevation of plasma FVIII and VWF persisted for at least 6 weeks after inactivation of the LRP gene. Upon comparing plasma FVIII and VWF in individual mice, we observed an increase of the FVIII/VWF ratio in MX1cre+LRPflox/floxmice as compared with control LRPflox/floxmice. Administration of either a vasopressin analog or an endotoxin resulted in increased plasma VWF, but not FVIII. In clearance experiments, MX1cre+LRPflox/floxmice displayed a 1.5-fold prolongation of FVIII mean residence time. Adenovirus-mediated overexpression of the 39-kDa receptor–associated protein (RAP) in normal mice resulted in a 3.5-fold increase of plasma FVIII. These data confirm that the regulation of plasma FVIII in vivo involves a RAP-sensitive mechanism. Surprisingly, plasma FVIII in MX1cre+LRPflox/floxmice increased 2-fold after RAP gene transfer. We propose that RAP-sensitive determinants other than hepatic LRP contribute to the regulation of plasma FVIII in vivo.

Today, there is a trend towards integration of complex RF systems on a single die. The interaction between the different building blocks will cause a performance degradation of each individual building block. So, optimizing the performance of the total system is under discussion. When combining a CMOS power amplifier together with an up-conversion mixer, some pre-amplifier stages have to be placed between the mixer and the CMOS power transistor. How this stages have to be designed (sized) and how they affect the performance of the power amplifier, will be investigated in this paper.

Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (VH) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the VH1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from VH gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10–, DP-14–, and DP-88–encoded antibodies. In contrast, binding of the DP-5–encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5–encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from VH gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII.

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo–3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm2. Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the γ-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor–like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

A well-known complication of factor VIII replacement therapy in patients with hemophilia A is the development of inhibitory antibodies. Several studies have demonstrated the presence of a binding site for factor VIII inhibitors in the A3 domain. Six different human monoclonal single-chain variable domain antibody fragments (scFv) directed toward the A3-C1 domains of factor VIII have been isolated, using phage display technology. Sequence analysis revealed that the VH domains of 2 scFv were encoded by germline gene segments from the VH1 gene family and 4 by germline gene segments belonging to the VH3 gene family. Epitope mapping of the scFv was performed, using a series of hybrid factor VIII/factor V light chain fragments. This analysis revealed that 5 of 6 scFv were directed against a region encompassing amino acid sequence Q1778-D1840 in the A3 domain, a previously identified binding site for factor VIII inhibitors. Only 2 of 5 scFv directed against amino acid sequence Q1778-D1840 inhibited the procoagulant activity of factor VIII. Our results define the properties of human antibodies directed against region Q1778-D1840 in the A3 domain. Binding of one, noninhibitory scFv was independent of the region Q1778-D1840, suggesting the presence of an additional binding site for anti–factor VIII antibodies in the A3-C1 domains of factor VIII.

The interaction between the endocytic receptor low density lipoprotein receptor–related protein (LRP) and either coagulation factor IX or its active derivative factor IXa was studied. Purified factor IX was unable to associate with LRP when analyzed by surface plasmon resonance. By contrast, factor XIa–mediated conversion of factor IX into factor IXa resulted in reversible dose- and calcium-dependent binding to LRP. Active-site blocking of factor IXa did not affect binding to LRP, whereas LRP binding was efficiently inhibited in the presence of heparin or antibodies against factor IX or LRP. The factor IXa–LRP interaction could be described by a 2-site binding model with equilibrium dissociation constants of 27 nmol/L and 69 nmol/L. Consistent with this model, it was observed that factor IXa binds to 2 different recombinant receptor fragments of LRP (denoted cluster II and cluster IV) with equilibrium dissociation constants of 227 nmol/L and 53 nmol/L, respectively. The amount of factor IXa degraded by LRP-deficient cells was 35% lower than by LRP-expressing cells, demonstrating that LRP contributes to the transport of factor IXa to the intracellular degradation pathway. Because ligand binding to LRP is often preceded by binding to proteoglycans, the contribution of proteoglycans to the catabolism of factor IXa was addressed by employing proteoglycan-deficient cells. Degradation of factor IXa by proteoglycan-deficient cells proceeded at a 83% lower rate than wild-type cells. In conclusion, the data presented here indicate that both LRP and proteoglycans have the potential to contribute to the catabolism of factor IXa.

Ca2+binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+binding to the first EGF-like domain, due to a replacement of Asp64by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+by Ca2+further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km≈ 1, 2, and 4 x 103M-1s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent (≈700- and 200-fold, respectively), indicating that Asp64replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+binding to the first EGF-like domain of factor IX.

Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J., Christophe, O. D., ter Maat, H., Rees, D. J. G., and Mertens, K. (1996)J. Biol. Chem.271, 25332–25337). In the present study, we have investigated the functional role of residue Glu78, which is not involved in calcium binding. Glu78is also located in the first EGF-like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu78is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor IX-specific function. In this study, we addressed the hypothesis that Glu78exerts its biological activity by interacting with another residue. One candidate was found to be the second EGF-like domain residue, Arg94, which is also associated with severe hemophilia B when mutated. We constructed a series of mutants that included mutations at position 78 alone (Glu78to Lys/Glu78to Asp) or at both positions 78 and 94 (Glu78to Lys and Arg94to Asp). The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor IXa in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa. In contrast, in the presence of factor VIIIa, factor IXa Glu78to Asp and factor IXa Glu78to Lys/Arg94to Asp were stimulated to the same extent as normal factor IXa, whereas factor IXa Glu78to Lys was markedly less stimulated (140-foldversus2,000-fold). This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor IXa to factor VIIIa. This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy (Ser558–Gln565) or light chain (Glu1811–Lys1818) and in direct binding studies. We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.

Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor Xlla (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3‘ residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2} Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3} Apart from the important role of the PI residue, no consensus can be presented on the nature of other residues within the P3 to P3‘ region with regard to target protease specificity.