Imperial College London

ProfessorJimCrawley

Faculty of MedicineDepartment of Immunology and Inflammation

Professor of Haemostasis
 
 
 
//

Contact

 

+44 (0)20 3313 2297j.crawley Website

 
 
//

Location

 

5S5aCommonwealth BuildingHammersmith Campus

//

Summary

 

Publications

Publication Type
Year
to

85 results found

Kim H-J, Xu Y, Petri A, Vanhoorelbeke K, Crawley J, Emsley Jet 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.

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

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

Journal article

Constantinescu-Bercu A, Grassi L, Frontini M, Salles II, Woollard K, Crawley JTet 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.

Journal article

Schelpe A-S, Petri A, Roose E, Pareyn I, Deckmyn H, De Meyer SF, Crawley JTB, Vanhoorelbeke Ket 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.

Journal article

Petri A, Kim HJ, Xu Y, de Groot R, Li C, Vandenbulcke A, Vanhoorelbeke K, Emsley J, Crawley Jet 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.

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

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

Colige A, Monseur C, Crawley J, Santamaria S, de Groot Ret 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.

Journal article

Teraz-Orosz A, Nichola C, Crawley J, Salles IIet 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

Journal article

de Groot R, Monseur C, Colige A, Santamaria S, Crawley JTBet al., 2018, ADAMTS7 substrate and cleavage site specificity, Matrix Biology Europe Meeting, Publisher: WILEY, Pages: A50-A51, ISSN: 0959-9673

Conference paper

Schelpe A-S, Petri A, Vandeputte N, Deckmyn H, De Meyer SF, Crawley JTB, Vanhoorelbeke Ket 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

Conference paper

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

Sandhu B, Bishop H, Lioja D, Galloway-Phillips N, Crawley J, Pusey C, Papalois Vet al., 2017, A NEW EX VIVO MODEL OF REPERFUSION INJURY IN HUMAN ORGANS, Publisher: WILEY, Pages: 505-505, ISSN: 0934-0874

Conference paper

Sandhu B, Ahnstroem J, Crawley J, Prendecki M, Galloway-Phillips N, Mason J, Dorling T, Smith R, Pusey C, Papalois Vet al., 2017, CYTOTOPIC THROMBIN INHIBITION ATTENUATES MICROVASCULAR ENDOTHELIAL ISCHAEMIA-REPERFUSION INJURY, Publisher: WILEY, Pages: 48-48, ISSN: 0934-0874

Conference paper

Sandhu B, Bishop H, Loja D, Galloway-Phillips N, Crawley J, Pusey C, Papalois Vet 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

Conference paper

Sandhu B, Prendecki M, Crawley J, Galloway-Phillips N, Vallant N, Mason J, Dorling A, Smith R, Pusey C, Papalois Vet 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

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

Thomas MR, de Groot R, Scully MA, Crawley JTet 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

Journal article

Maino A, Siegerink B, Lotta LA, Crawley JTB, Le Cessie S, Leebeek FWG, Lane DA, Lowe GDO, Peyvandi F, Rosendaal FRet 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.

Journal article

Maino A, Siegerink B, Lotta LA, Crawley JTB, le Cessie S, Leebeek FW, Lane DA, Lowe GD, Peyvandi F, Rosendaal FRet al., 2015, ADAMTS13 and the risk of myocardial infarction: an individual patient data meta-analysis, Publisher: Wiley, Pages: 288-289, 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

Salles-Crawley I, Monkman JH, Lane DA, Crawley JTet al., 2015, Endothelial BAMBI (BMP and activin membrane bound inhibitor) is important for fibrin generation and thrombus stability, Publisher: WILEY-BLACKWELL, Pages: 110-110, ISSN: 1538-7933

Conference paper

Thomas M, Groot RD, Crawley J, Scully Met al., 2015, Pathogenicity of anti-ADAMTS13 autoantibodies in acquired TTP, Publisher: WILEY-BLACKWELL, Pages: 473-473, ISSN: 1538-7933

Conference paper

South K, Salles-Crawley I, Crawley JT, Lane DAet al., 2015, The importance of conformational activation of ADAMTS13 for control of platelet deposition under flow, Publisher: WILEY-BLACKWELL, Pages: 766-767, ISSN: 1538-7933

Conference paper

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

Andreou AP, Efthymiou M, Yu Y, Watts HR, Noormohamed FH, Ma D, Lane DA, Crawley JTBet al., 2015, Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke, PLOS One, Vol: 10, ISSN: 1932-6203

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogenactivator (tPA) can be administered as thrombolytic therapy, it has majorlimitations, which include disruption of the blood-brain barrier and an increased risk ofbleeding. Treatments that prevent or limit such deleterious effects could be of major clinicalimportance. Activated protein C (APC) is a natural anticoagulant that regulates thrombingeneration, but also confers endothelial cytoprotective effects and improved endothelialbarrier function mediated through its cell signalling properties. In murine models of stroke,although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulantactions can further elevate the risk of bleeding. Thus, APC variants such asAPC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signallingfunction may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally,but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers.Thrombin generation assays suggested reductions in anticoagulant function of 50- to57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly,whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly byprotein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins).Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, incombination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantlyimproved neurological scores, reduced cerebral infarct area by ~50% and reducedoedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administrationof tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings,then APC(36-39

Journal article

de Groot R, Lane DA, Crawley JTB, 2015, The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis, Blood, Vol: 125, Pages: 1968-1975, ISSN: 0006-4971

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain–charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

Journal article

South K, Luken BM, Crawley JTB, Phillips R, Thomas M, Collins RF, Deforche L, Vanhoorelbeke K, Lane DAet al., 2014, Conformational activation of ADAMTS13, Proceedings of the National Academy of Sciences of the United States of America, Vol: 111, Pages: 18578-18583, ISSN: 0027-8424

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a “closed” conformation of WT ADAMTS13 and suggested a more “open” conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB–spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.

Journal article

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

Request URL: http://wlsprd.imperial.ac.uk:80/respub/WEB-INF/jsp/search-html.jsp Request URI: /respub/WEB-INF/jsp/search-html.jsp Query String: respub-action=search.html&id=00235428&limit=30&person=true