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Article ; Online: The Study of Platelet Receptors Using Artificial Lipid Bilayers.

Dustin, Michael L / Pollitt, Alice Y

Methods in molecular biology (Clifton, N.J.)

2018 Volume 1812, Page(s) 127–137

Abstract: Artificial lipid bilayers are powerful tools that can be used to model the interactions between platelets and membrane-bound ligands. To mimic the interaction of platelets with membrane-bound ligands, biotinylated lipids can be used to couple ... ...

Abstract	Artificial lipid bilayers are powerful tools that can be used to model the interactions between platelets and membrane-bound ligands. To mimic the interaction of platelets with membrane-bound ligands, biotinylated lipids can be used to couple monobiotinylated recombinant ligands to the upper leaflet of an artificial lipid bilayer using streptavidin to bridge the two. Artificial lipid bilayers are generated by preparing liposomes, treating glass coverslips to make them hydrophilic and by assembling the bilayer in a specialized flow chamber. Finally platelets can be added to the flow chamber and the localization of fluorescently labeled molecules followed using microscopy.
MeSH term(s)	Blood Platelets/chemistry ; Lipid Bilayers/chemistry ; Liposomes/chemistry ; Micelles ; Recombinant Proteins/metabolism
Chemical Substances	Lipid Bilayers ; Liposomes ; Micelles ; Recombinant Proteins
Language	English
Publishing date	2018-08-31
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-8585-2_8
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction.

Kenny, Martin / Pollitt, Alice Y / Patil, Smita / Hiebner, Dishon W / Smolenski, Albert / Lakic, Natalija / Fisher, Robert / Alsufyani, Reema / Lickert, Sebastian / Vogel, Viola / Schoen, Ingmar

Research and practice in thrombosis and haemostasis

2024 Volume 8, Issue 1, Page(s) 102322

Abstract: Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in ... ...

Abstract	Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.
Language	English
Publishing date	2024-01-22
Publishing country	United States
Document type	Journal Article
ISSN	2475-0379
ISSN (online)	2475-0379
DOI	10.1016/j.rpth.2024.102322
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Article ; Online: Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.

Clark, Joanne C / Martin, Eleyna M / Morán, Luis A / Di, Ying / Wang, Xueqing / Zuidscherwoude, Malou / Brown, Helena C / Kavanagh, Deirdre M / Hummert, Johan / Eble, Johannes A / Nieswandt, Bernhard / Stegner, David / Pollitt, Alice Y / Herten, Dirk-Peter / Tomlinson, Michael G / García, Angel / Watson, Steve P

Communications biology

2023 Volume 6, Issue 1, Page(s) 376

Abstract: CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked ... ...

Abstract	CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.
MeSH term(s)	Animals ; Humans ; Mice ; Lectins, C-Type/genetics ; Lectins, C-Type/metabolism ; Membrane Glycoproteins/metabolism ; Signal Transduction/physiology ; Single-Domain Antibodies/pharmacology ; Syk Kinase/metabolism
Chemical Substances	CLEC-2 protein, mouse ; Lectins, C-Type ; Membrane Glycoproteins ; Single-Domain Antibodies ; Syk Kinase (EC 2.7.10.2) ; CLEC2B protein, human
Language	English
Publishing date	2023-04-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-023-04766-6
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Article ; Online: Fully automated platelet differential interference contrast image analysis via deep learning.

Kempster, Carly / Butler, George / Kuznecova, Elina / Taylor, Kirk A / Kriek, Neline / Little, Gemma / Sowa, Marcin A / Sage, Tanya / Johnson, Louise J / Gibbins, Jonathan M / Pollitt, Alice Y

Scientific reports

2022 Volume 12, Issue 1, Page(s) 4614

Abstract: Platelets mediate arterial thrombosis, a leading cause of myocardial infarction and stroke. During injury, platelets adhere and spread over exposed subendothelial matrix substrates of the damaged blood vessel wall. The mechanisms which govern platelet ... ...

Abstract	Platelets mediate arterial thrombosis, a leading cause of myocardial infarction and stroke. During injury, platelets adhere and spread over exposed subendothelial matrix substrates of the damaged blood vessel wall. The mechanisms which govern platelet activation and their interaction with a range of substrates are therefore regularly investigated using platelet spreading assays. These assays often use differential interference contrast (DIC) microscopy to assess platelet morphology and analysis performed using manual annotation. Here, a convolutional neural network (CNN) allowed fully automated analysis of platelet spreading assays captured by DIC microscopy. The CNN was trained using 120 generalised training images. Increasing the number of training images increases the mean average precision of the CNN. The CNN performance was compared to six manual annotators. Significant variation was observed between annotators, highlighting bias when manual analysis is performed. The CNN effectively analysed platelet morphology when platelets spread over a range of substrates (CRP-XL, vWF and fibrinogen), in the presence and absence of inhibitors (dasatinib, ibrutinib and PRT-060318) and agonist (thrombin), with results consistent in quantifying spread platelet area which is comparable to published literature. The application of a CNN enables, for the first time, automated analysis of platelet spreading assays captured by DIC microscopy.
MeSH term(s)	Blood Platelets ; Deep Learning ; Image Processing, Computer-Assisted ; Platelet Activation
Language	English
Publishing date	2022-03-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-08613-2
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Article: Super-Resolution Fluorescence Microscopy Reveals Clustering Behaviour of

Danson, Amy E / McStea, Alex / Wang, Lin / Pollitt, Alice Y / Martin-Fernandez, Marisa L / Moraes, Isabel / Walsh, Martin A / MacIntyre, Sheila / Watson, Kimberly A

Biology

2020 Volume 9, Issue 10

Abstract: Chlamydia ... ...

Abstract	Chlamydia pneumoniae
Language	English
Publishing date	2020-10-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2661517-4
ISSN	2079-7737
ISSN	2079-7737
DOI	10.3390/biology9100344
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Article ; Online: Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

Morán, Luis A / Di, Ying / Sowa, Marcin A / Hermida-Nogueira, Lidia / Barrachina, María N / Martin, Eleyna / Clark, Joanne C / Mize, Todd H / Eble, Johannes A / Moreira, David / Pollitt, Alice Y / Loza, María I / Domínguez, Eduardo / Watson, Steve P / García, Ángel

Thrombosis and haemostasis

2022 Volume 122, Issue 8, Page(s) 1361–1368

Abstract: Background: CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, ... ...

Abstract	Background: CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives: To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. Methods: ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin-CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and conclusion: We developed a CLEC-2-podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)'2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.
MeSH term(s)	Blood Platelets/metabolism ; Humans ; Inflammation/metabolism ; Lectins, C-Type/metabolism ; Ligands ; Membrane Glycoproteins/metabolism ; Platelet Activation ; Thrombosis/metabolism
Chemical Substances	Lectins, C-Type ; Ligands ; Membrane Glycoproteins
Language	English
Publishing date	2022-02-15
Publishing country	Germany
Document type	Journal Article
ZDB-ID	518294-3
ISSN	2567-689X ; 0340-6245
ISSN (online)	2567-689X
ISSN	0340-6245
DOI	10.1055/a-1772-1069
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Article ; Online: Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

Joanne C. Clark / Eleyna M. Martin / Luis A. Morán / Ying Di / Xueqing Wang / Malou Zuidscherwoude / Helena C. Brown / Deirdre M. Kavanagh / Johan Hummert / Johannes A. Eble / Bernhard Nieswandt / David Stegner / Alice Y. Pollitt / Dirk-Peter Herten / Michael G. Tomlinson / Angel García / Steve P. Watson

Communications Biology, Vol 6, Iss 1, Pp 1-

2023 Volume 17

Abstract: Fluorescence correlation spectroscopy on crosslinked nanobodies tested on C-type lectin-like receptor 2 (CLEC-2) show different outcomes for divalent and tetravalent nanobody ligands on CLEC-2 clustering, also dependent on CLEC-2 expression levels. ...

Abstract	Fluorescence correlation spectroscopy on crosslinked nanobodies tested on C-type lectin-like receptor 2 (CLEC-2) show different outcomes for divalent and tetravalent nanobody ligands on CLEC-2 clustering, also dependent on CLEC-2 expression levels.
Keywords	Biology (General) ; QH301-705.5
Language	English
Publishing date	2023-04-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
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Article: WASP and SCAR/WAVE proteins: the drivers of actin assembly.

Pollitt, Alice Y / Insall, Robert H

Journal of cell science

2009 Volume 122, Issue Pt 15, Page(s) 2575–2578

MeSH term(s)	Actins/metabolism ; Humans ; Wiskott-Aldrich Syndrome Protein/physiology ; Wiskott-Aldrich Syndrome Protein Family/physiology
Chemical Substances	Actins ; WAS protein, human ; WASF1 protein, human ; Wiskott-Aldrich Syndrome Protein ; Wiskott-Aldrich Syndrome Protein Family
Language	English
Publishing date	2009-07-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2993-2
ISSN	1477-9137 ; 0021-9533
ISSN (online)	1477-9137
ISSN	0021-9533
DOI	10.1242/jcs.023879
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Article ; Online: Loss of Dictyostelium HSPC300 causes a scar-like phenotype and loss of SCAR protein.

Pollitt, Alice Y / Insall, Robert H

BMC cell biology

2009 Volume 10, Page(s) 13

Abstract: Background: SCAR/WAVE proteins couple signalling to actin polymerization, and are thus fundamental to the formation of pseudopods and lamellipods. They are controlled as part of a five-membered complex that includes the tiny HSPC300 protein. It is not ... ...

Abstract	Background: SCAR/WAVE proteins couple signalling to actin polymerization, and are thus fundamental to the formation of pseudopods and lamellipods. They are controlled as part of a five-membered complex that includes the tiny HSPC300 protein. It is not known why SCAR/WAVE is found in such a large assembly, but in Dictyostelium the four larger subunits have different, clearly delineated functions. Results: We have generated Dictyostelium mutants in which the HSPC300 gene is disrupted. As has been seen in other regulatory complex mutants, SCAR is lost in these cells, apparently by a post-translational mechanism, though PIR121 levels do not change. HSPC300 knockouts resemble scar mutants in slow migration, roundness, and lack of large pseudopods. However hspc300-colonies on bacteria are larger and more similar to wild type, suggesting that some SCAR function can survive without HSPC300. We find no evidence for functions of HSPC300 outside the SCAR complex. Conclusion: HSPC300 is essential for most SCAR complex functions. The phenotype of HSPC300 knockouts is most similar to mutants in scar, not the other members of the SCAR complex, suggesting that HSPC300 acts most directly on SCAR itself.
MeSH term(s)	Amino Acid Sequence ; Animals ; Dictyostelium/genetics ; Dictyostelium/growth & development ; Dictyostelium/metabolism ; Genes, Protozoan ; Molecular Sequence Data ; Movement ; Multiprotein Complexes ; Mutation ; Phenotype ; Protozoan Proteins/chemistry ; Protozoan Proteins/genetics ; Protozoan Proteins/metabolism ; Sequence Homology, Amino Acid ; Signal Transduction
Chemical Substances	Multiprotein Complexes ; Protozoan Proteins ; SCAR protein, Dictyostelium
Language	English
Publishing date	2009-02-19
Publishing country	England
Document type	Journal Article
ISSN	1471-2121
ISSN (online)	1471-2121
DOI	10.1186/1471-2121-10-13
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Article ; Online: Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis.

Lombard, Stephanie E / Pollitt, Alice Y / Hughes, Craig E / Di, Ying / Mckinnon, Tom / O'callaghan, Chris A / Watson, Steve P

Platelets

2017 Volume 29, Issue 7, Page(s) 716–722

Abstract: The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In ...

Abstract	The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A
MeSH term(s)	Animals ; Biomarkers ; Biomechanical Phenomena ; Blood Platelets/physiology ; Endothelial Cells/metabolism ; Gene Expression ; Hemostasis ; Humans ; Lectins, C-Type/genetics ; Lectins, C-Type/metabolism ; Membrane Glycoproteins/genetics ; Membrane Glycoproteins/metabolism ; Mice ; Platelet Activation ; Platelet Adhesiveness ; Platelet Aggregation ; Protein Binding ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism
Chemical Substances	Biomarkers ; CLEC-2 protein, mouse ; Gp38 protein, mouse ; Lectins, C-Type ; Membrane Glycoproteins ; Recombinant Fusion Proteins
Language	English
Publishing date	2017-11-01
Publishing country	England
Document type	Journal Article
ZDB-ID	1034283-7
ISSN	1369-1635 ; 0953-7104
ISSN (online)	1369-1635
ISSN	0953-7104
DOI	10.1080/09537104.2017.1356919
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