Publications
68 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.
Dong Y, Xu Y, Lian C, et al., 2024, Safe and efficacious near superhydrophobic hemostat for reduced blood loss and easy detachment in traumatic wounds, ACS Applied Materials and Interfaces, Vol: 16, Pages: 4307-4320, ISSN: 1944-8244
Hemorrhage is the leading cause of trauma death, and innovation in hemostatic technology is important. The strongly hydrophobic carbon nanofiber (CNF) coating has previously been shown to have excellent hemostatic properties. However, the understanding of how CNF coating guides the coagulation cascade and the biosafety of CNF as hemostatic agents has yet to be explored. Here, our thrombin generation assay investigation showed that CNF induced fast blood coagulation via factor (F) XII activation of the intrinsic pathway. We further performed studies of a rat vein injury and demonstrated that the CNF gauze enabled a substantial reduction of blood loss compared to both the plain gauze and kaolin-imbued gauze (QuikClot). Analysis of blood samples from the model revealed no acute toxicity from the CNF gauze, with no detectable CNF deposition in any organ, suggesting that the immobilization of CNF on our gauze prevented the infiltration of CNF into the bloodstream. Direct injection of CNF into the rat vein was also investigated and found not to elicit overt acute toxicity or affect animal survival or behavior. Finally, toxicity assays with primary keratinocytes revealed minimal toxicity responses to CNF. Our studies thus supported the safety and efficacy of the CNF hemostatic gauze, highlighting its potential as a promising approach in the field of hemostatic control.
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.
Minns AF, Qi Y, Yamamoto K, et al., 2023, The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity, Journal of Biological Chemistry, Vol: 299, ISSN: 0021-9258
A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in β3-β4 (R756Q/R759Q/R762Q), β9-β10 (residues 828-835), and β6-β7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.
Prince RE, Schaeper U, Aretz J, et al., 2022, SLN140 a Small Interfering RNA Targeting Protein S Improves Hemostasis Potency in Hemophilia, 64th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, Pages: 1670-1671, ISSN: 0006-4971
Eladnani RP, Schaeper U, Aretz J, et al., 2022, A GalNAc conjugated Small Interfering RNA Targeting Protein S Improves Hemostasis Potency in Hemophilia, Publisher: E M H SWISS MEDICAL PUBLISHERS LTD, Pages: 2S-2S, ISSN: 1424-7860
Yamamoto K, Scavenius C, Meschis MM, et al., 2022, A top-down approach to uncover the hidden ligandome of low-density lipoprotein receptor-related protein 1 in cartilage, Matrix Biology, Vol: 112, Pages: 190-218, ISSN: 0174-173X
The low-density lipoprotein receptor-related protein 1 (LRP1) is a cell-surface receptor ubiquitously expressed in various tissues. It plays tissue-specific roles by mediating endocytosis of a diverse range of extracellular molecules. Dysregulation of LRP1 is involved in multiple conditions including osteoarthritis (OA) but little information is available about the specific profile of direct binding partners of LRP1 (ligandome) for each tissue, which would lead to a better understanding of its role in disease states. Here, we investigated adult articular cartilage where impaired LRP1-mediated endocytosis leads to tissue destruction. We used a top-down approach involving proteomic analysis of the LRP1 interactome in human chondrocytes, direct binding assays using purified LRP1 and ligand candidates, and validation in LRP1-deficient fibroblasts and human chondrocytes, as well as a novel Lrp1 conditional knockout (KO) mouse model. We found that inhibition of LRP1 and ligand interaction results in cell death, alteration of the entire secretome and transcriptional modulations in human chondrocytes. We identified a chondrocyte-specific LRP1 ligandome consisting of more than 50 novel ligand candidates. Surprisingly, 23 previously reported LRP1 ligands were not regulated by LRP1-mediated endocytosis in human chondrocytes. We confirmed direct LRP1 binding of HGFAC, HMGB1, HMGB2, CEMIP, SLIT2, ADAMTS1, TSG6, IGFBP7, SPARC and LIF, correlation between their affinity for LRP1 and the rate of endocytosis, and some of their intracellular localization. Moreover, a conditional LRP1 KO mouse model demonstrated a critical role of LRP1 in regulating the high-affinity ligands in cartilage in vivo. This systematic approach revealed the specificity and the extent of the chondrocyte LRP1 ligandome and identified potential novel therapeutic targets for OA.
Al Kafri N, Ahnstrom J, Teraz-Orosz A, et al., 2022, The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling, Biochemistry and Biophysics Reports, Vol: 30, ISSN: 2405-5808
The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.
Ariëns RA, Hunt BJ, Agbani EO, et al., 2022, Illustrated state-of-the-art capsules of the ISTH 2022 congress., Research and Practice in Thrombosis and Haemostasis, Vol: 6, Pages: 1-45, ISSN: 2475-0379
The ISTH London 2022 Congress is the first held (mostly) face-to-face again since the COVID-19 pandemic took the world by surprise in 2020. For 2 years we met virtually, but this year's in-person format will allow the ever-so-important and quintessential creativity and networking to flow again. What a pleasure and joy to be able to see everyone! Importantly, all conference proceedings are also streamed (and available recorded) online for those unable to travel on this occasion. This ensures no one misses out. The 2022 scientific program highlights new developments in hemophilia and its treatment, acquired and other inherited bleeding disorders, thromboinflammation, platelets and coagulation, clot structure and composition, fibrinolysis, vascular biology, venous thromboembolism, women's health, arterial thrombosis, pediatrics, COVID-related thrombosis, vaccine-induced thrombocytopenia with thrombosis, and omics and diagnostics. These areas are elegantly reviewed in this Illustrated Review article. The Illustrated Review is a highlight of the ISTH Congress. The format lends itself very well to explaining the science, and the collection of beautiful graphical summaries of recent developments in the field are stunning and self-explanatory. This clever and effective way to communicate research is revolutionary and different from traditional formats. We hope you enjoy this article and will be inspired by its content to generate new research ideas.
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.
Santamaria S, Martin DR, Dong X, et al., 2021, Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8, Journal of Biological Chemistry, Vol: 297, Pages: 1-17, ISSN: 0021-9258
A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.
Martin DR, Santamaria S, Koch CD, et al., 2021, Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach., Journal of Proteomics, Vol: 249, Pages: 1-8, ISSN: 0165-022X
The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control:ADAMTS digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SIGNIFICANCE: Versican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used label-free proteomics approach to identify versican cleavage sites
Ahnstrom J, 2021, FV/FVa revealed, BLOOD, Vol: 137, Pages: 3011-3013, ISSN: 0006-4971
Ahnström J, Gilbert GE, 2021, Factor V mutation illuminates the dominant anticoagulant role and importance of an unidentified platelet modifier, Journal of Thrombosis and Haemostasis, Vol: 19, Pages: 1168-1170, ISSN: 1538-7836
Kearney KJ, Butler J, Posada OM, et al., 2021, Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI, Proceedings of the National Academy of Sciences of the United States of America, Vol: 118, Pages: 1-9, ISSN: 0027-8424
Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.
Santamaria S, Cuffaro D, Nuti E, et al., 2021, Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide, Scientific Reports, Vol: 11, ISSN: 2045-2322
ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of β-N-acetyl-d-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.
Brinkman HJM, Ahnström J, Castoldi E, et al., 2021, Pleiotropic anticoagulant functions of protein S, consequences for the clinical laboratory. Communication from the SSC of the ISTH., Journal of Thrombosis and Haemostasis, Vol: 19, Pages: 281-286, ISSN: 1538-7836
Hereditary deficiencies of protein S (PS) increase the risk of thrombosis. However, assessing the plasma levels of PS is complicated by its manifold physiological interactions, while the large inter-individual variability makes it problematic to establish reliable cut-off values. PS has multiple physiological functions, with only two appearing to have significant anticoagulant properties: the activated protein C (APC) and tissue factor pathway inhibitor alpha (TFPIα) cofactor activities. Current clinical laboratory investigations for deficiency in PS function rely only on the APC-dependent activity. This communication presents an argument for reclassifying the qualitative PS deficiencies to differentiate the two major anticoagulant functions of PS. Reliable assays are necessary for accurate evaluation of PS function when making a specific diagnosis of PS deficiency based on the anticoagulant phenotype alone. This report emphasizes the pleiotropic anticoagulant functions of PS and presents evidence-based recommendations for their implementation in the clinical laboratory.
Marlar RA, Gausman JN, Tsuda H, et al., 2021, Recommendations for clinical laboratory testing for protein S deficiency: Communication from the SSC committee plasma coagulation inhibitors of the ISTH, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 19, Pages: 68-74, ISSN: 1538-7933
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- Citations: 14
Gierula M, Ahnstrom J, 2020, Anticoagulant protein S-New insights on interactions and functions, Journal of Thrombosis and Haemostasis, Vol: 18, Pages: 2801-2811, ISSN: 1538-7836
Protein S is a critical regulator of coagulation that functions as a cofactor for the activated protein C (APC) and tissue factor pathway inhibitor (TFPI) pathways. It also has direct anticoagulant functions, inhibiting the intrinsic tenase and prothrombinase complexes. Through these functions, protein S regulates coagulation during both its initiation and its propagation phases. The importance of protein S in hemostatic regulation is apparent from the strong association between protein S deficiencies and increased risk for venous thrombosis. This is most likely because both APC and TFPIα are inefficient anticoagulants in the absence of any cofactors. The detailed molecular mechanisms involved in protein S cofactor functions remain to be fully clarified. However, recent advances in the field have greatly improved our understanding of these functions. Evidence suggests that protein S anticoagulant properties often depend on the presence of synergistic cofactors and the formation of multicomponent complexes on negatively charged phospholipid surfaces. Their high affinity binding to negatively charged phospholipids helps bring the anticoagulant proteins to the membranes, resulting in efficient and targeted regulation of coagulation. In this review, we provide an update on protein S and how it functions as a critical hemostatic regulator.
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.
Ahnström J, Gierula M, Temenu J, et al., 2020, Partial rescue of naturally occurring active site factor X variants through decreased inhibition by tissue factor pathway inhibitor and antithrombin., Journal of Thrombosis and Haemostasis, Vol: 18, Pages: 136-150, ISSN: 1538-7836
BACKGROUND: Activated coagulation factor X (FXa) is the serine protease component of prothrombinase, the physiological activator of prothrombin. FX Nottingham (A404T) and Taunton (R405G) are two naturally occurring mutations, identified in families with a bleeding phenotype. OBJECTIVE: To functionally characterise these FX variants. METHODS: The activity and inhibition of recombinant FX variants was quantified in plasma based and pure component assays. RESULTS: The prothrombin times in FX-depleted plasma supplemented with FX Nottingham and Taunton were greatly increased compared to wild-type (WT) FX. Kinetic investigations of activated variants in the prothrombinase complex showed kcat /Km , reduced ~50-fold and ~5-fold, respectively, explaining the prolonged PT times. The substituted residues are located in the protease domain Na+ -binding loop, important for the activity of FXa, as well as its inhibition. Both FXa Nottingham and Taunton showed reduced affinity for Na+ . Plasma-based thrombin generation assays triggered with 1pM tissue factor (TF) demonstrated only small differences in activities compared to WT FX, but large reductions at 10pM TF. Severely reduced inhibition of both FXa Nottingham and Taunton by tissue factor pathway inhibitor (TFPI) and antithrombin (AT), was shown in pure-component FXa inhibition assays. FXa Nottingham and Taunton produced higher amounts of thrombin than WT FXa in pure-component prothrombinase assays in the presence of TFPI and AT, explaining the results from the plasma-based assay. CONCLUSIONS: FX Nottingham and Taunton both display decreased proteolytic activity. However, their reduced activity in plasma triggered by low TF can be rescued by decreased inhibition by the natural FXa inhibitors, TFPI and AT.
Peghaire C, Dufton N, Lang M, et al., 2019, The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature, Nature Communications, Vol: 10, Pages: 1-17, ISSN: 2041-1723
Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anti-coagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed- specific anti-coagulant pathway in microvasculature exposed to low SS.
Ahnström J, 2019, The potential of serpins for future treatment for haemophilia, Journal of Thrombosis and Haemostasis, Vol: 17, Pages: 1629-1631, ISSN: 1538-7836
Khan TZ, Gorog DA, Arachchillage DJ, et al., 2019, Impact of lipoprotein apheresis on thrombotic parameters in patients with refractory angina and raised lipoprotein(a): Findings from a randomized controlled cross-over trial, Journal of Clinical Lipidology, Vol: 13, Pages: 788-796, ISSN: 1876-4789
BACKGROUND: Raised lipoprotein(a) [Lp(a)] is a cardiovascular risk factor common in patients with refractory angina. The apolipoprotein(a) component of Lp(a) exhibits structural homology with plasminogen and can enhance thrombosis and impair fibrinolysis. OBJECTIVES: The objective of the study was to assess the effect of lipoprotein apheresis on markers of thrombosis and fibrinolysis in patients with high Lp(a). METHODS: In a prospective, single-blind, crossover trial, 20 patients with refractory angina and raised Lp(a) > 50 mg/dL were randomized to three months of weekly lipoprotein apheresis or sham. Blood taken before and after apheresis/sham was assessed using the Global Thrombosis Test, to assess time taken for in vitro thrombus formation (occlusion time) and endogenous fibrinolysis (lysis time), as well as von Willebrand Factor, fibrinogen, D-dimer, thrombin/anti-thrombin III complex, prothrombin fragments 1 + 2, and thrombin generation assays. RESULTS: Lp(a) was significantly reduced by apheresis (100.2 [interquartile range {IQR}, 69.6143.0] vs 24.8 [17.2,34.0] mg/dL, P = .0001) but not by sham (P = .0001 between treatment arms). Apheresis prolonged occlusion time (576 ± 116 s vs 723 ± 142 s, P < .0001) reflecting reduced platelet reactivity and reduced lysis time (1340 [1128, 1682] s vs 847 [685,1302] s, P = .0006) reflecting enhanced fibrinolysis, without corresponding changes with sham. Apheresis, but not sham, reduced von Willebrand Factor (149 [89.0, 164] vs 64.2 [48.5, 89.8] IU/dL, P = .0001), and fibrinogen (3.12 ± 0.68 vs 2.20 ± 0.53 g/L, P < .0001), and increased prothrombin fragments 1 + 2 (158.16 [128.77, 232.09] vs 795.12 [272.55, 1201.00] pmol/L, P = .0006). There was no change in D-dimer, thrombin/anti-thrombin III complex, or thrombin generation assay with
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.
Santamaria S, Yamamoto Y, Teraz-Orosz A, et al., 2019, Exosites in hypervariable loops of ADAMTS dpacer domains control substrate recognition and proteolysis, Scientific Reports, Vol: 9, ISSN: 2045-2322
ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739–744 and 837–844) and ADAMTS-4 (residues 717–724 and 788–795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.
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.
Khan TZ, Gorog DA, Arachchillage D, et al., 2018, Impact of lipoprotein apheresis on thrombotic parameters in patients with refractory angina and raised lipoprotein(a), European-Society-of-Cardiology Congress, Publisher: OXFORD UNIV PRESS, Pages: 1305-1306, ISSN: 0195-668X
Thwaites RS, Gunawardana NC, Broich V, et al., 2018, Biphasic activation of complement and fibrinolysis during the human nasal allergic response, Journal of Allergy and Clinical Immunology, Vol: 141, Pages: 1892-1895.e6, ISSN: 0091-6749
Complement, coagulation and fibrinolysis contribute to the pathology of many respiratory diseases. Here we detail the biphasic activation of these pathways following nasal allergen challenge. Understanding these mechanisms may lead to therapeutic insight in common respiratory diseases.
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