50 results found
Constantinescu-Bercu A, Wang YA, Woollard KJ, et al., 2022, The GPIbα intracellular tail – role in transducing VWF- and collagen/GPVI-mediated signaling, Haematologica, Vol: 107, Pages: 933-946, ISSN: 0390-6078
<jats:p>The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing
Huang Y, Gu B, Salles II, et al., 2021, Fibrinogen-mimicking, multi-arm nanovesicles for human thrombus-specific delivery of tissue plasminogen activator and targeted thrombolytic therapy, Science Advances, Vol: 7, ISSN: 2375-2548
Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multi-arm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This novel system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.
Salles-Crawley II, 2021, Targeting platelets to improve post-thrombotic syndrome?, Journal of Thrombosis and Haemostasis, Vol: 19, Pages: 355-357, ISSN: 1538-7836
Constantinescu-Bercu A, Wang YA, Woollard K, et al., 2020, The GPIbα intracellular tail - role in transducing VWF- and Collagen/GPVI-mediated signaling, Publisher: Cold Spring Harbor Laboratory
Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GPIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail important for VWF-mediated signaling. GPIbαΔsig/Δsig platelets bound VWF normally under flow but formed fewer filopodia on VWF/botrocetin, demonstrating that the deleted region does not affect ligand binding but appreciably impairs VWF-dependent signaling. Notably, while haemostasis was normal in GPIbαΔsig/Δsig mice, GPIbαΔsig/Δsig platelets exhibited defective responses after collagen-related-peptide stimulation and formed smaller aggregates on collagen-coated microchannels at low and high shears. Flow assays performed with plasma-free blood or in the presence of αIIbβ3-or GPVI-blockers suggested reduced αIIbβ3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.
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
Constantinescu-Bercu A, Grassi L, Frontini M, et 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.
Peghaire C, Dufton N, Lang M, et al., 2019, The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature, Nature Communications, Vol: 10, Pages: 1-17, ISSN: 2041-1723
Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anti-coagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed- specific anti-coagulant pathway in microvasculature exposed to low SS.
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.
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.
Sattler S, Baxan N, Chowdhury R, et al., 2019, Characterization of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by multi-parametric quantitative cardiac MRI, Disease Models & Mechanisms, Vol: 12, Pages: 1-10, ISSN: 1754-8403
Hemorrhagic myocarditis is a potentially fatal complication of excessive levels of systemic inflammation. It has been reported in viral infection, but is also possible in systemic autoimmunity. Epicutaneous treatment of mice with the TLR-7 agonist Resiquimod induces auto-antibodies and systemic tissue damage including in the heart, and is used as an inducible mouse model of Systemic Lupus Erythematosus (SLE).Here, we show that over-activation of the TLR-7 pathway of viral recognition by Resiquimod-treatment of CFN mice induces severe thrombocytopenia and internal bleeding which manifests most prominently as hemorrhagic myocarditis. We optimized a cardiac magnetic resonance (CMR) tissue mapping approach for the in vivo detection of diffuse infiltration, fibrosis and hemorrhages using a combination of T1, T2 and T2* relaxation times, and compared results to ex vivo histopathology of cardiac sections corresponding to CMR tissue maps. This allowed a detailed correlation between in vivo CMR parameters and ex vivo histopathology, and confirmed the need to include T2* measurements to detect tissue iron for accurate interpretation of pathology associated with CMR parameter changes.In summary, we provide detailed histological and in vivo imaging-based characterization of acute hemorrhagic myocarditis as acute cardiac complication in the mouse model of Resiquimod-induced SLE, and a refined CMR protocol to allow non-invasive longitudinal in vivo studies of heart involvement in acute inflammation. We propose that adding T2* mapping to CMR protocols for myocarditis diagnosis will improve interpretation of disease mechanisms and diagnostic sensitivity.
Teraz-Orosz A, Nichola C, Crawley J, et 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
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.
Morren M-A, Jaeken J, Visser G, et al., 2017, PIGO deficiency: palmoplantar keratoderma and novel mutations, Orphanet Journal of Rare Diseases, Vol: 12, ISSN: 1750-1172
BackgroundSeveral genetic defects have been identified in the glycosylphosphatidylinositol (GPI) anchor synthesis, including mutations in PIGO encoding phosphatidylinositol glycan anchor biosynthesis class O protein. These defects constitute a subgroup of the congenital disorders of glycosylation (CDG). Seven patients from five families have been reported carrying variants in PIGO that cause an autosomal recessive syndrome characterised by dysmorphism, psychomotor disability, epilepsy and hyperphosphatasemia.MethodsWhole exome sequencing was performed in a boy with dysmorphism, psychomotor disability, epilepsy, palmoplantar keratoderma, hyperphosphatasemia and platelet dysfunction without a clinical bleeding phenotype.ResultsTwo novel variants in PIGO were detected. The missense variant encoding p. His871Pro was inherited from the boy’s father while the frameshift variant encoding p. Arg604ProfsTer40 was maternally inherited.ConclusionA boy with two novel PIGO variants is reported. The skin phenotype and platelet dysfunction in this patient have not been described in previously reported patients with PIGO deficiency but it is of course uncertain whether these are caused by this disorder. The literature on PIGO deficiency is reviewed.
Burdorf L, Riner A, Rybak E, et al., 2016, Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor, XENOTRANSPLANTATION, Vol: 23, Pages: 222-236, ISSN: 0908-665X
Thijs T, Broos K, Soenen SJ, et al., 2015, Artificial MiRNA Knockdown of Platelet Glycoprotein Ib alpha: A Tool for Platelet Gene Silencing, PLOS One, Vol: 10, ISSN: 1932-6203
In recent years, candidate genes and proteins implicated in platelet function have beenidentified by various genomic approaches. To elucidate their exact role, we aimed todevelop a method to apply miRNA interference in platelet progenitor cells by using GPIbαas a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targetingGPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested inCHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introductionof siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown ofGPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAsto miGPIBAs resulted in reduced silencing efficiency, which could however be circumventedby tandem integration of two hairpins targeting different regions of GPIBA mRNAwhere 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBAconstruct displayed a significant decrease in their ability to aggregate characterizedby lower aggregate numbers and size compared to control CHO GPIb-IX cells. Moreimportantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMIcells that exhibited morphological changes associated with actin organization. In conclusion,we here report the successful use of miRNA technology to silence a platelet proteinin megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, webelieve that artificial miRNAs are suitable tools to unravel the role of a protein of interest instem cells, megakaryocytes and platelets, thereby expanding their application to novelfields of basic and translational research.
Gierula M, Salles-Crawley II, Crawley JTB, et 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
South K, Salles-Crawley I, Crawley JT, et 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
Salles-Crawley I, Monkman JH, Lane DA, et 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
Burdorf L, Riner A, Shah A, et al., 2015, Activation and Sequestration of Platelets During Xenogeneic GalTKO. hCD46 Pig Lung Perfusion Is Primarily Mediated By GPIb, GPIIb/IIIa, and VWF, American Transplant Congress, Publisher: WILEY-BLACKWELL, ISSN: 1600-6135
LaMattina J, Burdorf L, Zhang T, et al., 2014, Pig-to-Baboon Liver Xenoperfusion Utilizing GalTKO.hCD46 Pigs and Glycoprotein Ib Blockade., World Transplant Congress, Publisher: LIPPINCOTT WILLIAMS & WILKINS, Pages: 413-413, ISSN: 0041-1337
LaMattina J, Burdorf L, Zhang T, et al., 2014, Pig-to-Baboon Liver Xenoperfusion Utilizing GalTKO.hCD46 Pigs and Glycoprotein Ib Blockade., World Transplant Congress, Publisher: WILEY-BLACKWELL, Pages: 413-413, ISSN: 1600-6135
LaMattina JC, Burdorf L, Zhang T, et al., 2014, Pig-to-baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade, XENOTRANSPLANTATION, Vol: 21, Pages: 274-286, ISSN: 0908-665X
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.
Salles I, Monkman JH, Ahnstroem J, et al., 2013, BMP and activin membrane bound inhibitor (BAMBI): A novel regulator of thrombus formation, Publisher: WILEY-BLACKWELL, Pages: 274-275, ISSN: 1538-7933
Salles II, Crawley JTB, 2012, Blocking VWF platelet binding to treat TTP, BLOOD, Vol: 120, Pages: 3390-3392, ISSN: 0006-4971
Burdorf L, Zhang T, Rybak E, et al., 2012, Combined aGPIb and aGPIIb/IIIa Blockade Prevents Platelet Sequestration in a Pig-to-Human Lung Perfusion Model, 32nd Annual Meeting and Scientific Sessions of the International-Society-for-Heart-and-Lung-Transplantation/Meeting of the ISHLT Academy - Core Competencies in Mechanical Circulatory Support, Publisher: ELSEVIER SCIENCE INC, Pages: S106-S106, ISSN: 1053-2498
Heger M, Salles II, Bezemer R, et al., 2011, Laser-induced primary and secondary hemostasis dynamics and mechanisms in relation to selective photothermolysis of port wine stains, JOURNAL OF DERMATOLOGICAL SCIENCE, Vol: 63, Pages: 139-147, ISSN: 0923-1811
Thijs T, Broos K, Salles II, et al., 2011, Towards platelet-specific RNA interference, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 9, Pages: 710-710, ISSN: 1538-7933
Burdorf L, Barth RN, Zhang T, et al., 2011, Targeting GPIb-vWF Axis by Anti-GPIb Fab Reduces Platelet Sequestration in an Ex Vivo Xenogeneic Pig Liver Perfusion Model, American Transplant Congress, Publisher: WILEY-BLACKWELL, Pages: 108-108, ISSN: 1600-6135
Goodall AH, Burns P, Salles I, et al., 2010, Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function, BLOOD, Vol: 116, Pages: 4646-4656, ISSN: 0006-4971
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