47 results found
McKinnon TAJ, 2021, Getting under the skin: a new route for factor VIII?, BLOOD, Vol: 137, Pages: 1007-1008, ISSN: 0006-4971
O'Brien HER, Zhang XF, Sanz-Hernandez M, et al., 2021, Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress, Journal of Thrombosis and Haemostasis, Vol: 19, Pages: 358-369, ISSN: 1538-7836
BackgroundVon Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined.MethodsVWF free thiols were blocked with N‐ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF‐C4 domain.ResultsBlockade of VWF free thiols reduced VWF‐mediated platelet capture to collagen in a shear‐dependent manner, with platelet capture virtually abolished above 5000 s−1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen‐bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol‐blockade reduced the lifetime and strength of the VWF‐collagen bond. Pulling simulations of the VWF‐C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free‐thiol exchange.ConclusionsWe conclude that free thiols in the C‐domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.
Prendecki M, Clarke C, McKinnon T, et al., 2021, SARS-CoV-2 antibody point-of-care testing in dialysis and kidney transplant patients with COVID-19., Kidney Medicine, Vol: 3, Pages: 54-59.e1, ISSN: 2590-0595
Rationale & Objective: A number of serologic tests for immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now commercially available, including multiple lateral flow immunoassays (LFIAs), which have the advantage of being inexpensive and easy to use, without the reliance on laboratory facilities. However, data on the development of humoral immunity to SARS-CoV-2 in patients with kidney disease is limited, and the utility of an LFIA to test for antibodies in these patients has not been assessed. Study Design: Observational study. Setting & Participants: 60 patients (40 hemodialysis and 20 kidney transplant recipients) with SARS-CoV-2 infection confirmed by viral reverse transcriptase-polymerase chain reaction (RT-PCR) testing and 88 historic negative-control samples (collected before September 2019). Test: A commercially available LFIA to test for SARS-CoV-2 IgG in patients with infection confirmed by viral RT-PCR testing. Outcomes: Sensitivity and specificity of the LFIA to detect SARS-CoV-2 IgG in dialysis patients and transplant recipients. Results: 56/58 (96.6%) patients (38/39 hemodialysis and 18/19 transplant recipients) tested positive for SARS-CoV-2 IgG. 5/7 (71.4%) patients who were negative on preliminary testing had detectable IgG when retested more than 21 days postdiagnosis. Median times to first and second tests after diagnosis were 17 (interquartile range, 15-20) and 35 (interquartile range, 30-39) days, respectively. Calculation of test characteristics gave sensitivity of 96.6% (95% CI, 88.3%-99.4%) and specificity of 97.7% (95% CI, 92.0-99.6%). Limitations: Possible exposure to other beta-coronaviruses that may cross-react with the antigen used in the LFIA cannot be excluded. Conclusions: Symptomatic dialysis patients and transplant recipients commonly develop an immune response against SARS-CoV-2 infection that can be detected using an LFIA. Used diligently, an LFIA could be used to help scre
Bell RV, McKinnon TAJ, Alton EWFW, et al., 2019, Gene therapy for thrombotic thrombocytopaenic purpura, Annual Conference of the British Society for Gene and Cell Therapy, Publisher: Mary Ann Liebert, Pages: A14-A14, ISSN: 1043-0342
Thrombotic Thrombocytopaenic Purpura (TTP) is a rare (∼1/200,000 people) but life‐threatening disease caused by inherited or acquired deficiencies in ADAMTS13; a metalloprotease responsible for cleavage of large von Willebrand factor (VWF) multimers in the plasma. Reduced cleavage of thrombogenic VWF multimers through deficient ADAMTS13 can lead to spontaneous, wide‐spread accumulation of platelet‐rich thrombi. Without treatment, thrombi accumulation within the microvasculature causes organ failure and death in 90% of acute events. Individuals with TTP receive regular plasma infusions to restore ADAMTS13 levels. Despite current treatments reducing mortality rates, high treatment burden and morbidity associated with donor‐derived plasma warrants the development of a novel therapy for TTP. Gene therapy offers an alternative treatment which could prevent the onset of life‐threatening acute TTP episodes. The UK Cystic Fibrosis Gene Therapy Consortium, has developed a lentivirus pseudotyped with the Sendai virus envelope proteins F and HN for efficient lung gene transfer. Here, we assess whether lungs can be used as ‘factories’ for efficient and persistent ADAMTS13 production. We first cloned ADAMTS13 cDNA into a lentivirus producer plasmid and demonstrated proteolytic activity against VWF following co‐expression in HEK293T cells and subsequent detection of cleaved VWF by SDS‐PAGE. Vector is currently being manufactured using GMP‐compliant production methods. Next, ADAMTS13 knockout mice were characterised to determine suitable biomarkers (e.g. ADAMTS13 plasma levels and VWF cleavage activity) for assessing efficacy of pulmonary gene transfer. Future work will assess the restoration of plasma ADAMTS13 function in knockout mice and protection against TTP‐like symptoms.
Ishihara J, Ishihara A, Starke RD, et al., 2019, The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing, Blood, Vol: 133, Pages: 2559-2569, ISSN: 0006-4971
During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.
Riddell A, Vinayagam S, Gomez K, et al., 2019, Evaluation of von Willebrand factor concentrates by platelet adhesion to collagen using an in vitro flow assay, RESEARCH AND PRACTICE IN THROMBOSIS AND HAEMOSTASIS, Vol: 3, Pages: 126-135, ISSN: 2475-0379
BackgroundVon Willebrand disease (VWD) results from quantitative or qualitative deficiency of von Willebrand factor (VWF) and is treated using VWF‐containing concentrates. Several studies have compared the function of various VWF containing concentrates however this has not been performed using shear based assays.ObjectivesTo compare the platelet‐capture potential of 10 commercially available, plasma‐derived VWF concentrates under shear conditions.MethodsVWF containing concentrates were assessed for VWF:Ag, VWF:CB, VWF:RCo, factor VIII:C ADAMTS13 content, VWF multimeric profile and glycan content using lectin binding assays. Free‐thiol content of each concentrate was investigated using MPB binding assays. An in vitro flow assay was used to determine the ability of each concentrate to mediate platelet capture to collagen.ResultsVWF multimeric analysis revealed reduction of high molecular weight (HMW) forms in four of the concentrates (Alphante, Octanate and Haemoctin, and 8Y). The high MW multimer distribution of the remaining six concentrates (Optivate, Wilate, Fandhi, Wilfactin, Haemate P, and Voncento) was similar to the plasma control. Lectin analysis demonstrated that 8Y had increased amount of T‐antigen. Although platelet capture after 5 minutes perfusion was similar for all concentrates; Alphante, Octanate, and Haemoctin, demonstrated the lowest levels of platelet capture after 60 seconds of perfusion. Free‐thiol content and ADAMTS13 levels varied widely between the concentrates but was not correlated with function.ConclusionAlphanate, Octanate, and Haemoctin, lacked HMW multimers and had the lowest initial platelet capture levels suggesting that the presence of VWF HMW multimers are required for initial platelet deposition.
Ward SE, O'Sullivan JM, Martinez SA, et al., 2018, Defining the Molecular Mechanisms through Which the Macrophage Galactose Lectin (MGL) Receptor Regulates Von Willebrand Factor Clearance, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Nowak AA, O'Brien HER, Henne P, et al., 2017, ADAMTS-13 glycans and conformation-dependent activity., Journal of Thrombosis and Haemostasis, Vol: 15, Pages: 1155-1166, ISSN: 1538-7933
Background:ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation.Objectives/Methods:We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions.Results:Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain.Conclusions:Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.
Zhang XF, Xu Y, McKinnon TAJ, et al., 2017, Biophysical Mechanisms of Von Willebrand Factor-Collagen Interactions, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 455A-455A, ISSN: 0006-3495
Chion A, O'Sullivan JM, Drakeford C, et al., 2016, N-linked glycan sites within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance, Blood, Vol: 128, Pages: 1959-1968, ISSN: 0006-4971
Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we have investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage-binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and in A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.
Aldabbous L, Abdul-Salam V, McKinnon T, et al., 2016, Neutrophil Extracellular Traps Promote Angiogenesis: Evidence From Vascular Pathology in Pulmonary Hypertension., Arteriosclerosis, Thrombosis, and Vascular Biology, Vol: 36, Pages: 2078-2087, ISSN: 1079-5642
OBJECTIVE: Inflammation and dysregulated angiogenesis are features of endothelial dysfunction in pulmonary hypertension. Neutrophil extracellular traps (NETs), produced by dying neutrophils, contribute to pathogenesis of numerous vascular disorders but their role in pulmonary hypertension has not been studied. We sought evidence of (NETs) formation in pulmonary hypertension and investigated the effect of NETs on endothelial function. APPROACH AND RESULTS: Plasma and lung tissues of patients with pulmonary hypertension were analyzed for NET markers. The effects of NETs on endothelial function were studied in vitro and in vivo. Patients with chronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension showed elevated plasma levels of DNA, neutrophil elastase, and myeloperoxidase. NET-forming neutrophils and extensive areas of NETosis were found in the occlusive plexiform lesions and vascularized intrapulmonary thrombi. NETs induced nuclear factor κB-dependent endothelial angiogenesis in vitro and increased vascularization of matrigel plugs in vivo. Angiogenic responses were associated with increased release of matrix metalloproteinase-9, heparin-binding EGF-like growth factor, latency-associated peptide of the transforming growth factor β1, and urokinase-type plasminogen activator, accompanied by increased endothelial permeability and cell motility. NETs-induced responses depended on myeloperoxidase/H2O2-dependent activation of Toll-like receptor 4/nuclear factor κB signaling. NETs stimulated the release of endothelin-1 in HPAECs and stimulated pulmonary smooth muscle cell proliferation in vitro. CONCLUSIONS: We are the first to implicate NETs in angiogenesis and provide a functional link between NETs and inflammatory angiogenesis in vitro and in vivo. We demonstrate the potential pathological relevance of this in 2 diseases of disordered vascular homeostasis, pulmonary arterial hypertension and chronic thromboembolic pulmonary
Nowak AA, Laffan M, McKinnon TAJ, 2015, The N-linked glycans of ADAMTS13 are critical determinants of conformation and proteolytic activity, 57th Annual Meeting of the American Society of Hematology, Publisher: American Society of Hematology, ISSN: 0006-4971
Smith K, Starke RD, Mckinnon TAJ, et al., 2015, RECIPROCAL REGULATION OF ANGIOPOIETIN 2 & VON WILLEBRAND FACTOR EXPRESSION IN ENDOTHELIAL CELLS, Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 51-52, ISSN: 1018-1172
Chion A, O'Sullivan J, Bergsson G, et al., 2014, N-Linked Glycans within the A1A2A3 Domains of VWF Play a Critical Role in Modulating Macrophage-Mediated Clearance, BLOOD, Vol: 124, ISSN: 0006-4971
Shiltagh N, Kirkpatrick J, Cabrita LD, et al., 2014, Solution structure of the major factor VIII binding region on von Willebrand factor, Blood, Vol: 123, Pages: 4143-4151, ISSN: 0006-4971
Although much of the function of von Willebrand factor (VWF) has been revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D′ region of VWF, which constitutes the major FVIII binding site. D′ consists of 2 domains, trypsin-inhibitor–like (TIL′) and E′, of which the TIL′ domain lacks extensive secondary structure, is strikingly dynamic and harbors a cluster of pathological mutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbone malleability of TIL′ is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL′, which is supported by the rigid scaffold of the TIL′ and E′ domain β sheets. Furthermore, surface-charge mapping of the TIL′E′ structure reveals a potential mechanism for the electrostatically guided, high-affinity VWF⋅FVIII interaction. Our findings provide novel insights into VWF⋅FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD.
Shapiro SE, Nowak AA, Wooding C, et al., 2014, The von Willebrand factor predicted unpaired cysteines are essential for secretion, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 12, Pages: 246-254, ISSN: 1538-7933
Nowak AA, McKinnon TAJ, Hughes JM, et al., 2014, The O-linked glycans of human von Willebrand factor modulate its interaction with ADAMTS-13, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 12, Pages: 54-61, ISSN: 1538-7933
Fallah MA, Huck V, Niemeyer V, et al., 2013, Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions, BIOMICROFLUIDICS, Vol: 7, ISSN: 1932-1058
Starke RD, Paschalaki KE, Dyer CEF, et al., 2013, Defective angiopoietin-2 release from von Willebrand disease patients' blood outgrowth endothelial cells, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 175-175, ISSN: 1538-7933
Shapiro SE, Laffan MA, McKinnon TAJ, 2013, Potential thiol isomerase activity of conserved CXXC motifs in D3, D4 and C1 domains of von Willebrand factor, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 551-552, ISSN: 1538-7933
Starke RD, Paschalaki KE, Dyer CE, et al., 2013, Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells, Blood, Vol: 121, Pages: 2773-2784
Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease
Starke RD, Paschalaki KE, Dyer CEF, et al., 2013, Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells, BLOOD, Vol: 121, Pages: 2773-2784, 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
Nowak AA, Laffan M, McKinnon TAJ, 2012, The Role of Sialic Acid in Von Willebrand Function Under Shear Stress, 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Mckinnon TAJ, Shaperio S, Nowak AA, et al., 2012, The Effect of Unpaired Cysteine Residues and the C Domains On the Expression of Von Willebrand Factor, 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Canis K, McKinnon TAJ, Nowak A, et al., 2012, Mapping the N-glycome of human von Willebrand factor, BIOCHEMICAL JOURNAL, Vol: 447, Pages: 217-228, ISSN: 0264-6021
Nowak AA, Canis K, Riddell A, et al., 2012, O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions, BLOOD, Vol: 120, Pages: 214-222, ISSN: 0006-4971
McKinnon TAJ, Nowak AA, Cutler J, et al., 2012, Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: correlation of clinical phenotype with flow-based platelet adhesion, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 10, Pages: 1409-1416, ISSN: 1538-7933
Starke RD, Paschalaki KE, Ferraro F, et al., 2012, Endothelial Von Willebrand factor regulates angiogenesis, 6th European Meeting on Vascular Biology and Medicine (EMVBM), Publisher: ELSEVIER SCIENCE INC, Pages: 318-319, ISSN: 1537-1891
Shapiro SE, Laffan MA, McKinnon TAJ, 2012, Nine predicted unpaired cysteine residues in von Willebrand factor are essential for VWF secretion, 52nd Annual Scientific Meeting of the British-Society-for-Haematology, Publisher: WILEY-BLACKWELL, Pages: 9-9, ISSN: 0007-1048
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