Imperial College London

Dr Josefin Ahnström

Faculty of MedicineDepartment of Immunology and Inflammation

Lecturer
 
 
 
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Contact

 

+44 (0)20 3313 2298j.ahnstrom

 
 
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Location

 

5S5Commonwealth BuildingHammersmith Campus

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Summary

 

Publications

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59 results found

Teraz-Orosz A, Gierula M, Petri A, Jones DA, Keniyopoullos R, Badia Folgado P, Santamaria S, Crawley JTB, Lane DA, Ahnstrom Jet al., 2021, Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP., Blood Adv

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.

Journal article

Santamaria S, Martin DR, Dong X, Yamamoto K, Apte SS, Ahnstroem Jet 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.

Journal article

Martin DR, Santamaria S, Koch CD, Ahnström J, Apte SSet 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

Journal article

Ahnstrom J, 2021, FV/FVa revealed, BLOOD, Vol: 137, Pages: 3011-3013, ISSN: 0006-4971

Journal article

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

Journal article

Kearney KJ, Butler J, Posada OM, Wilson C, Heal S, Ali M, Hardy L, Ahnstrom J, Gailani D, Foster R, Hethershaw E, Longstaff C, Philippou Het 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.

Journal article

Santamaria S, Cuffaro D, Nuti E, Ciccone L, Tuccinardi T, Liva F, D'Andrea F, de Groot R, Rossello A, Ahnström Jet 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.

Journal article

Brinkman HJM, Ahnström J, Castoldi E, Dahlbäck B, Marlar RAet 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.

Journal article

Marlar RA, Gausman JN, Tsuda H, Rollins-Raval MA, Brinkman HJMet 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

Journal article

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.

Journal article

Ferreira PM, Bozbas E, Tannetta SD, Alroqaiba N, Zhou R, Crawley JTB, Gibbins JM, Jones CI, Ahnström J, Yaqoob Pet 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.

Journal article

Ahnström J, Gierula M, Temenu J, Laffan MA, Lane DAet 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.

Journal article

Peghaire C, Dufton N, Lang M, Salles I, Ahnstroem J, Kalna V, Raimondi C, Pericleous C, Inuabasi L, Kiseleva R, Muzykantov V, Mason J, Birdsey G, Randi Aet 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.

Journal article

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

Journal article

Khan TZ, Gorog DA, Arachchillage DJ, Ahnström J, Rhodes S, Donovan J, Banya W, Pottle A, Barbir M, Pennell DJet 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

Journal article

Ferreira PM, Tannetta DS, Bozbas E, Zhou R, Alroqaiba NA, Jones CI, Crawley JT, Gibbins JM, Ahnstrom J, Yaqoob Pet 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

Conference paper

Gierula M, SallesCrawley II, Santamaria S, TerazOrosz A, Crawley JTB, Lane DA, Ahnström Jet 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.

Journal article

Santamaria S, Yamamoto Y, Teraz-Orosz A, Koch C, Apte SS, de Groot R, Lane DA, Ahnstroem Jet 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.

Journal article

Crawley JTB, Zalli A, Monkman JH, Petri A, Lane DA, Ahnstrӧm J, SallesCrawley IIet 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.

Journal article

Khan TZ, Gorog DA, Arachchillage D, Ahnstrom J, Donovan J, Barbir M, Pennell DJet 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

Conference paper

Thwaites RS, Gunawardana NC, Broich V, Mann EH, Ahnström J, Campbell GA, Lindsley S, Singh N, Tunstall T, Lane DA, Openshaw PJ, Hawrylowicz CM, Hansel TTet 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.

Journal article

Santamaria S, Crawley JTB, Yamamoto K, Ahnstrom J, de Groot Ret 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

Conference paper

Santamaria S, Reglińska-Matveyev N, Gierula M, Camire RM, Crawley JTB, Lane DA, Ahnström Jet 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.

Journal article

Khan E, Ambrose N, Ahnstrom J, Kiprianos A, Stanford M, Eleftheriou D, Brogan P, Mason J, Johns M, Laffan M, Haskard Det al., 2016, A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet’s Syndrome, Clinical and Experimental Rheumatology, Vol: 6, ISSN: 1593-098X

Thrombosis is common in Behçet’s Syndrome (BS), and there is a need for better biomarkers for risk assessment. As microparticles expressing Tissue Factor (TF) can contribute to thrombosis in preclinical models, we investigated whether plasma microparticles expressing Tissue Factor (TF) are increased in BS. We compared blood plasma from 72 healthy controls with that from 88 BS patients (21 with a history of thrombosis (Th+) and 67 without (Th−). Using flow cytometry, we found that the total plasma MP numbers were increased in BS compared to HC, as were MPs expressing TF and Tissue Factor Pathway Inhibitor (TFPI) (all p < 0.0001). Amongst BS patients, the Th+ group had increased total and TF positive MP numbers (both p ≤ 0.0002) compared to the Th- group, but had a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI positive to TF positive MP counts (TFPI/TF) was significantly lower in Th+ versus Th− BS patients (p = 0.0002), and no patient with a TFPI/TF MP ratio >0.7 had a history of clinical thrombosis. We conclude that TF-expressing MP are increased in BS and that an imbalance between microparticulate TF and TFPI may predispose to thrombosis.

Journal article

Khan E, Ambrose N, Ahnstrom J, Kiprianos A, Stanford M, Eleftheriou D, Brogan P, Mason J, Johns M, Laffan M, Haskard Det al., 2016, A LOW BALANCE BETWEEN MICROPARTICLES EXPRESSING TISSUE FACTOR PATHWAY INHIBITOR AND TISSUE FACTOR IS ASSOCIATED WITH THROMBOSIS IN BEHCET'S SYNDROME, Publisher: CLINICAL & EXPER RHEUMATOLOGY, Pages: S143-S143, ISSN: 0392-856X

Conference paper

Ogbechi J, Ruf M-T, Hall BS, Bodman-Smith K, Vogel M, Wu H-L, Stainer A, Esmon CT, Ahnstroem J, Pluschke G, Simmonds REet al., 2015, Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions, PLOS PATHOGENS, Vol: 11, ISSN: 1553-7366

Journal article

Gierula M, Salles-Crawley II, Crawley JTB, Lane DA, Ahnstroem Jet al., 2015, Factor VA in synergy with protein s enhances activated protein C binding to phospholipids, Publisher: WILEY-BLACKWELL, Pages: 313-313, ISSN: 1538-7933

Conference paper

Reglinska-Matveyev N, Crawley JT, Camire R, Lane DA, Ahnstroem Jet al., 2015, FV enhances protein S cofactor function for TFPI in the inhibition of FXa, Publisher: WILEY-BLACKWELL, Pages: 54-54, ISSN: 1538-7933

Conference paper

Somajo S, Ahnstroem J, Fernandez-Recio J, Gierula M, Villoutreix BO, Dahlback Bet al., 2015, Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction, THROMBOSIS AND HAEMOSTASIS, Vol: 113, Pages: 976-987, ISSN: 0340-6245

Journal article

Somajo S, Ahnstroem J, Gierula M, Fernandez-Recio J, Villoutreix BO, Dahlbaeck Bet al., 2015, Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction, Journal of Thrombosis and Haemostasis, Vol: 113, Pages: 976-987, ISSN: 1538-7933

Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

Journal article

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