98 results found
Petri A, Sasikumar P, Badia Folgado P, et al., 2024, TFPIα anticoagulant function is highly dependent upon protein S in vivo, Science Advances, Vol: 10, ISSN: 2375-2548
Tissue factor pathway inhibitor α (TFPIα) is the major physiological regulator of the initiation of blood coagulation. In vitro, TFPIα anticoagulant function is enhanced by its cofactor, protein S. To define the role of protein S enhancement in TFPIα anticoagulant function in vivo, we blocked endogenous TFPI in mice using a monoclonal antibody (14D1). This caused a profound increase in fibrin deposition using the laser injury thrombosis model. To explore the role of plasma TFPIα in regulating thrombus formation, increasing concentrations of human TFPIα were co-injected with 14D1, which dose-dependently reduced fibrin deposition. Inhibition of protein S cofactor function using recombinant C4b binding protein β-chain, significantly reduced the anticoagulant function of human TFPIα in controlling fibrin deposition. We report an in vivo model that is sensitive to the anticoagulant properties of the TFPIα-protein S pathway and show the importance of protein S as a cofactor in the anticoagulant function of TFPIα in vivo.
Underwood MI, Thomas MR, Scully MA, et al., 2023, ADAMTS13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura, Journal of Thrombosis and Haemostasis, ISSN: 1538-7836
BackgroundPatients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti–ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer–CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition.ObjectivesTo explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters.MethodsWe developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse.ResultsIn 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar.ConclusionADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.
Gierula M, Noakes VM, Salles-Crawley I, et al., 2023, The TFPIα C-terminal tail is essential for TFPIα-FV-short-protein S complex formation and synergistic enhancement of TFPIα, Journal of Thrombosis and Haemostasis, Vol: 21, Pages: 3568-3580, ISSN: 1538-7836
BackgroundFor maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear.ObjectivesTo determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement.MethodsTFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1–249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation.ResultsWhile WT TFPIα and TFPIα K3m bound FV-short with high affinity (Kd∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively.ConclusionFull TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.
Underwood MI, Alwan F, Thomas MR, et al., 2023, Autoantibodies enhance ADAMTS-13 clearance in patients with immune thrombotic thrombocytopenic purpura, Journal of Thrombosis and Haemostasis, Vol: 21, Pages: 1544-1552, ISSN: 1538-7836
BackgroundSevere deficiency in ADAMTS-13 (<10%) and the loss of von Willebrand factor–cleaving function can precipitate microvascular thrombosis associated with thrombotic thrombocytopenic purpura (TTP). Patients with immune-mediated TTP (iTTP) have anti-ADAMTS-13 immunoglobulin G antibodies that inhibit ADAMTS-13 function and/or increase ADAMTS-13 clearance. Patients with iTTP are treated primarily by plasma exchange (PEX), often in combination with adjunct therapies that target either the von Willebrand factor-dependent microvascular thrombotic processes (caplacizumab) or the autoimmune components (steroids or rituximab) of the disease.ObjectivesTo investigate the contributions of autoantibody-mediated ADAMTS-13 clearance and inhibition in patients with iTTP at presentation and through the course of the PEX therapy.Patients/MethodsAnti-ADAMTS-13 immunoglobulin G antibodies, ADAMTS-13 antigen, and activity were measured before and after each PEX in 17 patients with iTTP and 20 acute TTP episodes.ResultsAt presentation, 14 out of 15 patients with iTTP had ADAMTS-13 antigen levels of <10%, suggesting a major contribution of ADAMTS-13 clearance to the deficiency state. After the first PEX, both ADAMTS-13 antigen and activity levels increased similarly, and the anti-ADAMTS-13 autoantibody titer decreased in all patients, revealing ADAMTS-13 inhibition to be a modest modifier of the ADAMTS-13 function in iTTP. Analysis of ADAMTS-13 antigen levels between consecutive PEX treatments revealed that the rate of ADAMTS-13 clearance in 9 out of 14 patients analyzed was 4- to 10-fold faster than the estimated normal rate of clearance.ConclusionThese data reveal, both at presentation and during PEX treatment, that antibody-mediated clearance of ADAMTS-13 is the major pathogenic mechanism that causes ADAMTS-13 deficiency in iTTP. Understanding the kinetics of ADAMTS-13 clearance in iTTP may now enable further optimization of treatment of patients with iTTP.
Malik A, Sayed AA, Han P, et al., 2023, The role of CD8+ T cell clones in immune thrombocytopenia, Blood, Vol: 141, Pages: 2417-2429, ISSN: 0006-4971
Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.
Teraz-Orosz A, Gierula M, Petri A, et al., 2022, Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP., Blood Advances, Vol: 6, Pages: 704-715, ISSN: 2473-9529
Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction, involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement, nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S, are known. To screen for functionally important regions within protein S LG1 we generated seven variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T, displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed four protein S variants in which 4-6 surface exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high affinity C4BP-binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423 and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function.
Kim H-J, Xu Y, Petri A, et al., 2021, Crystal structure of ADAMTS13 CUB domains reveals their role in global latency, Science Advances, Vol: 7, ISSN: 2375-2548
ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.
Constantinescu-Bercu A, Wang YA, Woollard K, et al., 2020, The GPIbα intracellular tail - role in transducing VWF- and Collagen/GPVI-mediated signaling, Publisher: Cold Spring Harbor Laboratory
Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GPIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail important for VWF-mediated signaling. GPIbαΔsig/Δsig platelets bound VWF normally under flow but formed fewer filopodia on VWF/botrocetin, demonstrating that the deleted region does not affect ligand binding but appreciably impairs VWF-dependent signaling. Notably, while haemostasis was normal in GPIbαΔsig/Δsig mice, GPIbαΔsig/Δsig platelets exhibited defective responses after collagen-related-peptide stimulation and formed smaller aggregates on collagen-coated microchannels at low and high shears. Flow assays performed with plasma-free blood or in the presence of αIIbβ3-or GPVI-blockers suggested reduced αIIbβ3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.
Ferreira PM, Bozbas E, Tannetta SD, et al., 2020, Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function, Scientific Reports, Vol: 10, ISSN: 2045-2322
Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.
Constantinescu-Bercu A, Salles-Crawley II, Crawley JTB, 2020, SLC44A2-A novel therapeutic target for venous thrombosis?, Journal of Thrombosis and Haemostasis, Vol: 18, Pages: 1556-1558, ISSN: 1538-7836
Constantinescu-Bercu A, Grassi L, Frontini M, et al., 2020, Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2, eLife, Vol: 9, ISSN: 2050-084X
Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Iba-dependent platelet 'priming' induces integrin aIIbb3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet aIIbb3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated aIIbb3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.
Schelpe A-S, Petri A, Roose E, et al., 2020, Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function, Blood Advances, Vol: 4, Pages: 1072-1080, ISSN: 2473-9529
Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.
Petri A, Kim HJ, Xu Y, et al., 2019, Crystal structure and substrate-induced activation of ADAMTS13, Nature Communications, Vol: 10, ISSN: 2041-1723
Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor(VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13.Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domaincleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich andspacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of theADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloproteasedomain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacerdomains reveals that the metalloprotease domain exhibits a latent conformation in which the active-sitecleft is occluded supporting the requirement for an allosteric change to enable accommodation of thesubstrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate forADAMTS13.
Ferreira PM, Tannetta DS, Bozbas E, et al., 2019, Platelet-derived extracellular vesicles have different phenotypes and functional activities depending on their triggered stimuli, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3070-3070, ISSN: 0007-1188
Gierula M, SallesCrawley II, Santamaria S, et al., 2019, The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex, Journal of Thrombosis and Haemostasis, ISSN: 1538-7933
Background: Activated protein C (APC)-mediated inactivation of factor (F)Va is greatlyenhanced by protein S. For inactivation to occur, a trimolecular complex between FVa,APC and protein S must form on the phospholipid membrane. However, directdemonstration of complex formation has proven elusive.Objectives:To elucidate the nature of the phospholipid-dependent interactions betweenAPC, protein S and FVa.Methods:We evaluated binding of active site blocked APC to phospholipid-coatedmagnetic beads in the presence and absence of protein S and/or FVa. The importanceof protein S and FV residues were evaluated functionally.Results: APC alone bound weakly to phospholipids. Protein S mildly enhanced APCbinding to phospholipid surfaces, whereas FVa did not. However, FVa together withproteinS enhanced APC binding(>14-fold), demonstrating formation of an APC/proteinS/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding,agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A)with reduced APC cofactor function exhibited essentially normal augmentation of APCbinding to phospholipids, but diminished APC/protein S/FVa complex formation,suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara(W1920R), an APC resistant FV variant, also did not efficiently incorporate into thetrimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggestedthat the mutation impairs its affinity for phospholipid membranes and with protein Swithin the complex. Conclusions: FVa plays a central role in the formation of its inactivation complex.Furthermore, membrane proximal interactions between FVa, APC and protein S areessential for its cofactor function.
Crawley JTB, Zalli A, Monkman JH, et al., 2019, Defective fibrin deposition and thrombus stability in Bambi‐/‐ mice is mediated by elevated anticoagulant function, Journal of Thrombosis and Haemostasis, ISSN: 1538-7933
BackgroundBAMBI is a transmembrane protein related to the type I TGF‐β receptor family that is present on both platelets and endothelial cells (EC). Bambi‐deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization.ObjectiveWe aimed to delineate how BAMBI influences endothelial function and thrombus stability.MethodsBambi‐deficient mice were subjected to the laser‐induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and TFPI was also assessed in these mice.ResultsThrombus instability in Bambi‐/‐ mice was associated with a profound defect in fibrin deposition. Injection of hirudin into Bambi+/+ mice prior to thrombus formation recapitulated the Bambi‐/‐ thrombus instability phenotype. In contrast, hirudin had no additional effect upon thrombus formation in Bambi‐/‐ mice. Deletion of Bambi in EC resulted in mice with defective thrombus stability caused by decreased fibrin accumulation. Increased levels of the anticoagulant proteins TFPI and thrombomodulin, were detected in Bambi‐/‐ mouse lung homogenates. EC isolated from Bambi‐/‐ mouse lungs exhibited enhanced ability to activate protein C due to elevated thrombomodulin levels. Blocking thrombomodulin and TFPI in vivo fully restored fibrin accumulation and thrombus stability in Bambi‐/‐ mice.ConclusionsWe demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium, and also highlight the importance of these anticoagulant proteins in the laser‐induced thrombosis model.
Colige A, Monseur C, Crawley J, et al., 2019, Proteomic discovery of substrates of the cardiovascular protease ADAMTS7, Journal of Biological Chemistry, Vol: 294, Pages: 8037-8045, ISSN: 0021-9258
The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.
Teraz-Orosz A, Nichola C, Crawley J, et al., 2019, Detection of anti-platelet antibodies in immune thrombocytopenia by flow cytometry, British Journal of Haematology, Vol: 184, Pages: 844-847, ISSN: 1365-2141
Thielemans L, Hanif M, Crawley J, 2019, The Coagulation Cascade and its Therapeutic Modulation, HEART OF THE MATTER: KEY CONCEPTS IN CARDIOVASCULAR SCIENCE, Editors: Terracciano, Guymer, Publisher: SPRINGER INTERNATIONAL PUBLISHING AG, Pages: 193-206, ISBN: 978-3-030-24218-3
de Groot R, Monseur C, Colige A, et al., 2018, ADAMTS7 substrate and cleavage site specificity, Matrix Biology Europe Meeting, Publisher: WILEY, Pages: A50-A51, ISSN: 0959-9673
Schelpe A-S, Petri A, Vandeputte N, et al., 2018, Anti-CUB1 or Anti-Spacer Antibodies That Increase ADAMTS13 Activity Act By Allosterically Enhancing Metalloprotease Domain Function, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Santamaria S, Crawley JTB, Yamamoto K, et al., 2017, A comparison of COMP (TSP5) proteolysis by ADAMTS7 and ADAMTS4, Autumn Meeting of the British-Society-for-Matrix-Biology (BSMB), Publisher: WILEY, Pages: A3-A4, ISSN: 0959-9673
Sandhu B, Bishop H, Lioja D, et al., 2017, A NEW EX VIVO MODEL OF REPERFUSION INJURY IN HUMAN ORGANS, Publisher: WILEY, Pages: 505-505, ISSN: 0934-0874
Sandhu B, Ahnstroem J, Crawley J, et al., 2017, CYTOTOPIC THROMBIN INHIBITION ATTENUATES MICROVASCULAR ENDOTHELIAL ISCHAEMIA-REPERFUSION INJURY, Publisher: WILEY, Pages: 48-48, ISSN: 0934-0874
Sandhu B, Prendecki M, Crawley J, et al., 2017, Cytotopic thrombin inhibition prior to cold ischaemia attenuates microvascular endothelial ischaemia-reperfusion injury, American Transplant Congress, Publisher: Wiley, Pages: 731-731, ISSN: 1600-6135
Sandhu B, Bishop H, Loja D, et al., 2017, Human organs allocated for research - maximising use of a precious resource using a new ex vivo model of reperfusion injury, American Transplant Congress, Publisher: Wiley, Pages: 498-498, ISSN: 1600-6135
Santamaria S, Reglińska-Matveyev N, Gierula M, et al., 2017, Factor V anticoagulant cofactor activity that targets the early phase of coagulation, Journal of Biological Chemistry, Vol: 292, Pages: 9335-9344, ISSN: 0021-9258
Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa (FVIIa). Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV) which has been activated by FXa. Since full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that while FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ~8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa, also enhanced TFPI-mediated inhibition of FXa ~12-fold in the presence of protein S. In contrast, neither activated FV (FVa) nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not FVa) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.
Thomas MR, de Groot R, Scully MA, et al., 2015, Pathogenicity of anti-ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura, EBioMedicine, Vol: 2, Pages: 942-952, ISSN: 2352-3964
BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated. METHODS: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG. RESULTS: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increa
Maino A, Siegerink B, Lotta LA, et al., 2015, Plasma ADAMTS-13 levels and the risk of myocardial infarction: an individual patient data meta-analysis, Journal of Thrombosis and Haemostasis, Vol: 13, Pages: 1396-1404, ISSN: 1538-7933
BackgroundLow ADAMTS-13 levels have been repeatedly associated with an increased risk of ischemic stroke, but results concerning the risk of myocardial infarction are inconclusive.ObjectivesTo perform an individual patient data meta-analysis from observational studies investigating the association between ADAMTS-13 levels and myocardial infarction.MethodsA one-step meta-analytic approach with random treatment effects was used to estimate pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for confounding. Analyses were based on dichotomous exposures, with the 5th and 1st percentiles of ADAMTS-13 antigen levels as cut-off values. Quartile analyses, with the highest quartile as a reference category, were used to assess a graded association between levels and risk (‘dose’ relationship). Additionally, we assessed the risk of the combined presence of low ADAMTS-13 and high von Willebrand factor (VWF) levels.ResultsFive studies were included, yielding individual data on 1501 cases and 2258 controls (mean age of 49 years). Low ADAMTS-13 levels were associated with myocardial infarction risk, with an OR of 1.89 (95% CI 1.15–3.12) for values below the 5th percentile versus above, and an OR of 4.21 (95% CI 1.73–10.21) for values below the 1st percentile versus above. Risk appeared to be restricted to these extreme levels, as there was no graded association between ADAMTS-13 levels and myocardial infarction risk over quartiles. Finally, there was only a minor synergistic effect for the combination of low ADAMTS-13 and high VWF levels.ConclusionsLow ADAMTS-13 levels are associated with an increased risk of myocardial infarction.
Maino A, Siegerink B, Lotta LA, et al., 2015, ADAMTS13 and the risk of myocardial infarction: an individual patient data meta-analysis, Publisher: Wiley, Pages: 288-289, ISSN: 1538-7933
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