229 results found
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.
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.
Newnham M, South K, Bleda M, et al., 2019, The ADAMTS13-VWF axis is dysregulated in chronic thromboembolic pulmonary hypertension, European Respiratory Journal, Vol: 53, ISSN: 0903-1936
Chronic thromboembolic pulmonary hypertension (CTEPH) is an important consequence of pulmonary embolism (PE) that is associated with abnormalities in haemostasis. We investigated the ADAMTS13-VWF axis in CTEPH, including its relationship to disease severity, inflammation, ABO groups and ADAMTS13 genetic variants.ADAMTS13 and VWF plasma antigen levels were measured in patients with CTEPH (n=208), chronic thromboembolic disease without pulmonary hypertension (CTED; n=35), resolved PE (n=28), idiopathic pulmonary arterial hypertension (n=30) and healthy controls (n=68). CTEPH genetic ABO associations and protein quantitative trait loci were investigated. ADAMTS-VWF axis abnormalities were assessed in CTEPH and healthy control subsets by measuring ADAMTS13 activity, D-dimers and VWF-multimeric size.CTEPH patients had decreased ADAMTS13 (adjusted β (95% CI)=−23.4 (−30.9– −15.1)%, p<0.001) and increased VWF levels (β=+75.5 (44.8–113)%, p<0.001) compared to healthy controls. ADAMTS13 levels remained low after reversal of pulmonary hypertension by pulmonary endarterectomy surgery and were equally reduced in CTED. We identify a genetic variant near the ADAMTS13 gene associated with ADAMTS13 protein that accounted for ∼8% of the variation in levels.The ADAMTS13-VWF axis is dysregulated in CTEPH. This is unrelated to pulmonary hypertension, disease severity or markers of systemic inflammation and implicates the ADAMTS13-VWF axis in CTEPH pathobiology.
South K, Denorme F, Salles I, et al., 2018, Enhanced activity of ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischaemic stroke, Journal of Thrombosis and Haemostasis, Vol: 16, Pages: 2289-2299, ISSN: 1538-7836
BackgroundADAMTS13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand Factor (VWF) following a substrate‐induced conformational change. A gain of function (GoF) ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally pre‐activated.ObjectivesTo establish how the hyperactivity of GoF ADAMTS13 is manifest in experimental models mimicking the occlusive arterial thrombi present in acute ischaemic stroke.MethodsThe ability of GoF ADAMTS13 to dissolve VWF‐platelet agglutinates was examined using an assay of ristocetin‐induced platelet agglutination and in parallel flow models of arterial thrombosis. A murine model of focal ischaemia was used to assess the thrombolytic potential of GoF ADAMTS13.ResultsWT ADAMTS13 required conformational activation to attain full activity against VWF‐mediated platelet capture under flow. In this assay GoF ADAMTS13 had an EC50 value >5‐fold lower than wild type (WT) (0.73±0.21 nM and 3.81±0.97 nM, respectively). The proteolytic activity of GoF ADAMTS13 against pre‐formed platelet agglutinates under flow was enhanced >4‐fold compared to WT (EC50 values of 2.5±1.1 nM and 10.2±5.6 nM, respectively). In a murine stroke model GoF ADAMTS13 restored cerebral blood flow at a lower dose than WT ADAMTS13 and partially retained the ability to recanalise vessels when administration was delayed by 1 hour.ConclusionThe limited proteolytic activity of WT ADAMTS13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS13 variant translates to a more pronounced protective effect in experimental stroke.
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.
South K, Lane DA, 2017, ADAMTS13 and von Willebrand factor: a Dynamic Duo, Journal of Thrombosis and Haemostasis, Vol: 16, Pages: 6-18, ISSN: 1538-7836
von Willebrand factor (VWF) is a key player in hemostasis, acting as a carrier for factor VIII and capturing platelets at sites of vascular damage. To capture platelets, it must undergo conformational changes, both within its A1 domain and at the macromolecular level through A2 domain unfolding. Its size and this function are regulated by the metalloproteinase ADAMTS-13. Recently, it has been shown that ADAMTS-13 undergoes a conformational change upon interaction with VWF, and that this enhances its activity towards its substrate. This review summarizes recent work on these conformational transitions, describing how they are controlled. It points to their importance in hemostasis, bleeding disorders, and the developing field of therapeutic application of ADAMTS-13 as an antithrombotic agent in obstructive microvascular thrombosis and in cardiovascular disease.
Lane DA, Lynch CJ, Millar C, et al., 2017, A common mechanism by which type 2A von Willebrand Disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand Factor A2 domain FRET construct, PLoS ONE, Vol: 12, ISSN: 1932-6203
Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.
Santamaria S, Reglińska-Matveyev N, Gierula M, et al., 2017, Factor V anticoagulant cofactor activity that targets the early phase of coagulation, Journal of Biological Chemistry, Vol: 292, Pages: 9335-9344, ISSN: 0021-9258
Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa (FVIIa). Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV) which has been activated by FXa. Since full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that while FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ~8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa, also enhanced TFPI-mediated inhibition of FXa ~12-fold in the presence of protein S. In contrast, neither activated FV (FVa) nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not FVa) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.
South K, Freitas MO, Lane DA, 2017, A model for the conformational activation of the structurally quiescent metalloprotease ADAMTS13 by Von Willebrand factor, Journal of Biological Chemistry, Vol: 292, Pages: 5760-5769, ISSN: 0021-9258
Blood loss is prevented by the multi-domain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which, in turn, is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KD of 135 ± 10.1 nM and 86.9 ± 9.0 nM, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13∆CUB2, unlike ADAMTS13∆CUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is therefore not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment (KD of 53.7± 2.1 nM and 84.3 ± 2.0 nM, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13∆TSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13-activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.
Lane DA, 2017, Correcting the hemophilic imbalance, Blood, Vol: 129, Pages: 10-11, ISSN: 0006-4971
In this issue of Blood, Polderdijk et al design and evaluate a therapeutic inhibitorof activated protein C.1 They have produced a recombinant variant of a1-antitrypsin (a1AT) incorporating 3 residue changes within the P2-P19 sequence ofits reactive loop. This variant (termed KRK a1AT) exhibits high specificity andinhibitory efficiency toward activated protein C. It is able to restore thrombingeneration in normal and in hemophilia plasmas, when these are supplementedwith soluble thrombomodulin. It is able to restore hemostasis in challengedhemophilia mice. KRK a1AT is therefore a potentially valuable future therapeuticagent for the human hemophilias.
South K, Lane DA, Freitas MO, 2016, Conformational quiescence of ADAMTS13 prevents proteolytic promiscuity, Journal of Thrombosis and Haemostasis, Vol: 14, Pages: 2011-2022, ISSN: 1538-7933
BackgroundRecent work has revealed that ADAMTS13 circulates in a ‘closed’ conformation, only fully interacting with VWF following a conformational change. We hypothesised that this conformational quiescence also maintains the substrate specificity of ADAMTS13 and that the ‘open’ conformation of the protease might facilitate proteolytic promiscuityObjectivesTo identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS13 variant (R568K/F592Y/R660K/Y661F/Y665F)MethodsFibrinogen proteolysis was characterised using SDS PAGE and LC-MS/MS. Fibrin formation was monitored by turbidity measurements and fibrin structure visualised by confocal microscopyResultsADAMTS13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC-MS/MS to be Aα chain Lys225-Met226. Proteolysis of fibrinogen by GoF ADAMTS13 impairs fibrin formation in plasma based assays, alters clot structure and increases clot permeability. While GoF ADAMTS13 does not appear to proteolyse preformed cross-linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to t-PA induced lysis by plasmin and increases the fibrin clearance rate more than 8 fold compared to WT ADAMTS13 (EC50 values of 3.0 ± 1.7 nM and 25.2 ± 9.7 nM, respectively) in in vitro thrombosis modelsConclusionThe ‘closed’ conformation of ADAMTS13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS13 variants are developed as potential therapeutic agents against TTP and other cardiovascular diseases.
Lynch CJ, Lane DA, 2016, N-linked glycan stabilisation of the VWF A2 domain., Blood, Vol: 127, Pages: 1711-1718, ISSN: 0006-4971
Shear forces in the blood trigger a conformational transition in the VWF A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+) binding site and a vicinal cysteine disulphide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilise the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilising intradomain interactions with Y1544 and nearby residues. Substitution of the surface exposed Y1544 to aspartic acid is able to stabilise the domain in the absence of glycosylation and to protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilisation of the VWF A2 domain acts together with the Ca(2+) binding site and vicinal cysteine disulphide bond to control unfolding and ADAMTS13 proteolysis.
Maino A, Siegerink B, Lotta LA, et al., 2015, Plasma ADAMTS-13 levels and the risk of myocardial infarction: an individual patient data meta-analysis, Journal of Thrombosis and Haemostasis, Vol: 13, Pages: 1396-1404, ISSN: 1538-7933
BackgroundLow ADAMTS-13 levels have been repeatedly associated with an increased risk of ischemic stroke, but results concerning the risk of myocardial infarction are inconclusive.ObjectivesTo perform an individual patient data meta-analysis from observational studies investigating the association between ADAMTS-13 levels and myocardial infarction.MethodsA one-step meta-analytic approach with random treatment effects was used to estimate pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for confounding. Analyses were based on dichotomous exposures, with the 5th and 1st percentiles of ADAMTS-13 antigen levels as cut-off values. Quartile analyses, with the highest quartile as a reference category, were used to assess a graded association between levels and risk (‘dose’ relationship). Additionally, we assessed the risk of the combined presence of low ADAMTS-13 and high von Willebrand factor (VWF) levels.ResultsFive studies were included, yielding individual data on 1501 cases and 2258 controls (mean age of 49 years). Low ADAMTS-13 levels were associated with myocardial infarction risk, with an OR of 1.89 (95% CI 1.15–3.12) for values below the 5th percentile versus above, and an OR of 4.21 (95% CI 1.73–10.21) for values below the 1st percentile versus above. Risk appeared to be restricted to these extreme levels, as there was no graded association between ADAMTS-13 levels and myocardial infarction risk over quartiles. Finally, there was only a minor synergistic effect for the combination of low ADAMTS-13 and high VWF levels.ConclusionsLow ADAMTS-13 levels are associated with an increased risk of myocardial infarction.
Andreou AP, Efthymiou M, Yu Y, et 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
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.
South K, Luken BM, Crawley JTB, et 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.
Reglinska-Matveyev N, Andersson HM, Rezende SM, et al., 2014, TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain, Blood, Vol: 123, Pages: 3979-3987, ISSN: 0006-4971
Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest–specific 6 chimeras, with either the whole sex hormone–binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest–specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane.
Salles-Crawley II, Monkman JH, Ahnstroem J, et al., 2014, Vessel wall BAMBI contributes to hemostasis and thrombus stability., Blood, Vol: 123, Pages: 2873-2881, ISSN: 0006-4971
Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the transforming growth factor-β superfamily, and is highly expressed in platelets and endothelial cells. We previously demonstrated its positive role in thrombus formation using a zebrafish thrombosis model. In the present study, we used Bambi-deficient mice and radiation chimeras to evaluate the function of this receptor in the regulation of both hemostasis and thrombosis. We show that Bambi−/− and Bambi+/− mice exhibit mildly prolonged bleeding times compared with Bambi+/+ littermates. In addition, using 2 in vivo thrombosis models in mesenterium or cremaster muscle arterioles, we demonstrate that Bambi-deficient mice form unstable thrombi compared with Bambi+/+ mice. No defects in thrombin generation in Bambi−/− mouse plasma could be detected ex vivo. Moreover, the absence of BAMBI had no effect on platelet counts, platelet activation, aggregation, or platelet procoagulant function. Similar to Bambi−/− mice, Bambi−/− transplanted with Bambi+/+ bone marrow formed unstable thrombi in the laser-induced thrombosis model that receded more rapidly than thrombi that formed in Bambi+/+ mice receiving Bambi−/− bone marrow transplants. Taken together, these results provide strong evidence for an important role of endothelium rather than platelet BAMBI as a positive regulator of both thrombus formation and stability.
Lynch CJ, Lane DA, Luken BM, 2014, Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF, BLOOD, Vol: 123, Pages: 2585-2592, ISSN: 0006-4971
Millar CM, Lane DA, 2013, Blocking direct inhibitor bleeding, BLOOD, Vol: 121, Pages: 3543-3544, ISSN: 0006-4971
Ahnstroem J, Andersson HM, Hockey V, et al., 2012, Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S, BLOOD, Vol: 120, Pages: 5059-5062, ISSN: 0006-4971
Greaves M, Lane DA, 2012, Final message from the current Editors in Chief, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 10, Pages: 2417-2417, ISSN: 1538-7933
Andersson HM, Siegerink B, Luken BM, et al., 2012, High VWF, low ADAMTS13, and oral contraceptives increase the risk of ischemic stroke and myocardial infarction in young women, BLOOD, Vol: 119, Pages: 1555-1560, ISSN: 0006-4971
Crawley JTB, de Groot R, Xiang Y, et al., 2011, Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor, BLOOD, Vol: 118, Pages: 3212-3221, ISSN: 0006-4971
Xiang Y, de Groot R, Crawley JTB, et al., 2011, Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 11602-11607, ISSN: 0027-8424
Ahnstroem J, Andersson HM, Canis K, et al., 2011, Activated protein C cofactor function of protein S: a novel role for a gamma-carboxyglutamic acid residue, BLOOD, Vol: 117, Pages: 6685-6693, ISSN: 0006-4971
de Groot R, Lane DA, Crawley JTB, 2010, The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity, BLOOD, Vol: 116, Pages: 3064-3072, ISSN: 0006-4971
Greaves M, Lane DA, 2010, Aspiring for quality and service, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 8, Pages: 1885-1885, ISSN: 1538-7933
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.