J. Arnoud Marquart

The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization and cross-linking

A proliferation‐inducing ligand (APRIL) (also known asTALL‐2 and TRDL‐1) is a member of the tumor necrosis factor (TNF) superfamily that has tumorigenic properties but is also important for the induction of humoral immune responses. APRIL binds two TNF receptors: transmembrane activator and cal‐cium modulator and cyclophilin ligand interactor (TACI) and B‐cell maturation antigen (BCMA) as well as heparan sulfate proteoglycans (HSPGs). The aim of this study was to clarify the role of the HSPG interaction in canonical APRIL signaling, because it has been proposed to act as a docking site and also to play a role in direct signaling. In this study, we generated point mutants of soluble APRIL that lack either the capacity to bind HSPGs or TACI and BCMA and then tested the function of these mutants in mouse B‐cell assays. In contrast to previous reports, we found that APRIL alone is sufficient to costimulate B‐cell proliferation and drive IgA production and does not require artificial antibody cross‐linking. We found no evidence that APRIL requires signaling through HSPGs but, notably, were able to show that binding of APRIL to HSPGs is crucial for mediating natural APRIL cross‐linking to allow for optimal activation of murine B cells.— Kimberley, F. C., Van Bostelen, L., Cameron, K., Hardenberg, G., Mar‐ quart, J. A., Hahne, M., Medema, J. P. The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization andcross‐linking. FASEB J. 23, 1584–1595 (2009)

Factor Xa Activation of Factor V is of Paramount Importance in Initiating the Coagulation System: Lessons from a Tick Salivary Protein

Background— Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results— Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Conclusions— Our data elucidate a unique molecular mechanism by which ticks inhibit the host’s coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.

β2-Glycoprotein I is incorrectly named apolipoprotein H

b2-Glycoprotein I (b2-GPI) is a highly abundant protein present in blood, but without a known physiological function. In 1990, b2-GPI became a protein of great interest as it was shown by different groups that the so-called antiphospholipid antibodies present in antiphospholipid syndrome (APS) are in fact directed against this plasma protein [1,2]. It has been acknowledged that b2-GPIplaysan important role in the thromboticandpregnancy complications observed in APS. Thus, the correct biochemical characterization of b2-GPI is of pivotal importance [3,4]. In 1979, Polz and Kostner [5] showed the distribution of b2GPI over different human lipoproteins. Based on these observations, Lee et al. [6] designated b2-GPI as apolipoprotein H (apoH) [6]. Since then, the names b2-GPI and apoH have both been used for the same protein, and the official designation for the b2-GPI gene has become APOH. We were interested in whether the localization of b2-GPI on lipoproteins was influenced by the presence of antiphospholipid antibodies, and so we decided to reinvestigate the distribution of b2-GPI over the different lipoproteins and plasma fractions. We observed that after this original observation no other publications have confirmed the observed association of b2GPI with lipoproteins. Blood was drawn from five healthy volunteers in a fasting state and 3 h after consuming a classic English breakfast (>1000 kcal), to repeat the original experiments by Polz and Kostner.Moreover, plasmas from two septic patients, twoAPS patients with antibodies against b2-GPI and pooled plasma from more than 200 healthy volunteers were also investigated. The Institutional Review Boards of the University Medical Centre Utrecht and Academic Medical Centre Amsterdam approved this study and informed consent was obtained from all patients or their caretakers. Citrated blood samples were centrifuged (15 min, 1200 · g) and plasma was collected. Three millilitres of plasma were brought toadensity (D) of 1.250withKBrand layeredwith three KBr densities; D = 1.225, D = 1.100, and D = 1.006. A single-stepultracentrifugation (XL-90Beckman,BeckmanCoulter, Fullerton, CA, USA) was performed (96.000 · g for 19 h at 10 C), and fractions of 200 lL were collected with a fraction collector. Collected fractions were diluted at least 1000-fold in Tris-buffered saline (TBS; 50 mmol L Tris, 150 mmol L NaCl, 0.1% Tween-20, pH 7.4). Determination of b2-GPI was carried out by a homemade sandwich enzyme-linked immunosorbent assay (ELISA), using amousemonoclonal antibody3B7 as the capturing antibody and a rabbit polyclonal a-b2-GPI as a secondary antibody. Serial dilutions of normal pooled plasma (2.000· to 256.000· in TBS) were used as a standard curve. Very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) andhigh-density lipoprotein (HDL) sampleswere determinedby PAP 250 cholesterol enzymatic methods. Cholesterol reagent (Biomerieux, Le Fontanille, France) was added to 10 lL of the sample and measured on a spectrophotometer. As can be observed inFig. 1A, nob2-GPI could be detected in the different lipoprotein fractions fromahealthy volunteer.These resultswere confirmed with an additional four healthy volunteers, two septic patients and normal pooled plasma (Fig. 1B). The consumption of a classical English breakfast did not change the distribution of b2-GPI (data not shown). All fractions were also measured with surface plasmon resonance using a Biacore 2000 (Life Sciences, GE Healthcare, Uppsala, Sweden). To determine the binding of b2-GPI to the lipoproteins, anti-b2-GPI antibodies were coupled to a CM5chip and the fractions were applied to the chip. In the fractions containing the different lipoproteins, no b2-GPI could be detected (data not shown). Antibodies directed against apoA1 and apoB were also coupled to a CM5-chip. The different lipoproteins were then directly captured from the ultracentrifugation fractions, followed by an injection of anti-b2-GPI antibodies to detect potentially formed complexes between b2GPI and VLDL, LDL or HDL. No complexes were detected (data not shown). Subsequently, reconstituted HDL (CSL-111, Parkville, Victoria, Australia) was bound to an anti-apoA1 coupled chip and purified b2-GPI, from human plasma as described by Oosting et al. [7], was injected over the chip. No complex formation between purified b2-GPI and purifiedHDL could be observed (data not shown). To exclude the possibility that the separation technique for the lipoproteins could influence the outcome, lipoproteins from plasmas of three volunteers were separated using gel filtration on a Superose 6 HR 10/30 (Pharmacia Biotech, Uppsala, Sweden) column with inline fluorescence and ultraviolet detection. Fractions were diluted in BSA/TBS (20 mmol L Correspondence: Ç. Ağar, Department of Experimental Vascular Medicine, G1-144, Academic Medical Center, Meilbergdreef 9, 1105 AZ Amsterdam, the Netherlands. Tel.: +31 20 5666118; fax: +31 20 6916972. E-mail: c.agar@amc.uva.nl

A novel mutation in the F5 gene (factor V Amsterdam) associated with bleeding independent of factor V procoagulant function

We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency.

The Design and Characterization of Receptor-selective APRIL Variants

Background: APRIL binds two receptors, BCMA and TACI, so separating signaling outcomes is difficult. Results: We used an algorithm to design a variant of APRIL that specifically binds BCMA and two variants that selectively bind TACI. Conclusion: TACI and BCMA signals differ in the context of B cell stimulation. Significance: These variants will help decipher APRIL signaling in physiology and disease settings. A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.

Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ~20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Factor Xa Activation of Factor V Is of Paramount Importance in Initiating the Coagulation SystemClinical Perspective: Lessons From a Tick Salivary Protein

Background— Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results— Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Conclusions— Our data elucidate a unique molecular mechanism by which ticks inhibit the host’s coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

Factor Xa Activation of Factor V Is of Paramount Importance in Initiating the Coagulation SystemClinical Perspective

Background— Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results— Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Conclusions— Our data elucidate a unique molecular mechanism by which ticks inhibit the host’s coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.