Publications by authors named "Simon Stritt"

18 Publications

  • Page 1 of 1

Inherited platelet diseases with normal platelet count: phenotypes, genotypes and diagnostic strategy.

Haematologica 2021 02 1;106(2):337-350. Epub 2021 Feb 1.

Institut Hospitalo-Universitaire LIRYC, Pessac.

Inherited platelet disorders resulting from platelet function defects and a normal platelet count cause a moderate or severe bleeding diathesis. Since the description of Glanzmann thrombasthenia resulting from defects of ITGA2B and ITGB3, new inherited platelet disorders have been discovered, facilitated by the use of high throughput sequencing and genomic analyses. Defects of RASGRP2 and FERMT3 responsible for severe bleeding syndromes and integrin activation have illustrated the critical role of signaling molecules. Important are mutations of P2RY12 encoding the major ADP receptor causal for an inherited platelet disorder with inheritance characteristics that depend on the variant identified. Interestingly, variants of GP6 encoding the major subunit of the collagen receptor GPVI/FcRγ associate only with mild bleeding. The numbers of genes involved in dense granule defects including Hermansky-Pudlak and Chediak Higashi syndromes continue to progress and are updated. The ANO6 gene encoding a Ca2+-activated ion channel required for phospholipid scrambling is responsible for the rare Scott syndrome and decreased procoagulant activity. A novel EPHB2 defect in a familial bleeding syndrome demonstrates a role for this tyrosine kinase receptor independent of the classical model of its interaction with ephrins. Such advances highlight the large diversity of variants affecting platelet function but not their production, despite the difficulties in establishing a clear phenotype when few families are affected. They have provided insights into essential pathways of platelet function and have been at the origin of new and improved therapies for ischemic disease. Nevertheless, many patients remain without a diagnosis and requiring new strategies that are now discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2020.248153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849565PMC
February 2021

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.

Elife 2020 09 8;9. Epub 2020 Sep 8.

Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala, Sweden.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.57732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478896PMC
September 2020

Coactosin-like 1 integrates signaling critical for shear-dependent thrombus formation in mouse platelets.

Haematologica 2020 Jun 3;105(6):1667-1676. Epub 2019 Oct 3.

Institute of Experimental Biomedicine I, University Hospital, University of Würzburg, Würzburg

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage ( to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates , pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of platelets at high shear , indicating a critical role of platelet-derived LT in thrombus formation. , Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.225516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271572PMC
June 2020

Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic vessels.

Development 2018 02 8;145(3). Epub 2018 Feb 8.

Uppsala University, Department of Immunology, Genetics and Pathology, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden

Maintenance of blood vessel integrity is crucial for vascular homeostasis and is mainly controlled at the level of endothelial cell (EC) junctions. Regulation of endothelial integrity has largely been investigated in the mature quiescent vasculature. Less is known about how integrity is maintained during vascular growth and remodeling involving extensive junctional reorganization. Here, we show that embryonic mesenteric blood vascular remodeling is associated with a transient loss of venous integrity and concomitant extravasation of red blood cells (RBCs), followed by their clearance by the developing lymphatic vessels. In wild-type mouse embryos, we observed activated platelets extending filopodia at sites of inter-EC gaps. In contrast, embryos lacking the activatory C-type lectin domain family 1, member b (CLEC1B) showed extravascular platelets and an excessive number of RBCs associated with and engulfed by the first lymphatic EC clusters that subsequently form lumenized blood-filled vessels connecting to the lymphatic system. These results uncover novel functions of platelets in maintaining venous integrity and lymphatic vessels in clearing extravascular RBCs during developmental remodeling of the mesenteric vasculature. They further provide insight into how vascular abnormalities characterized by blood-filled lymphatic vessels arise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.156745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818000PMC
February 2018

TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice.

Arterioscler Thromb Vasc Biol 2018 02 16;38(2):344-352. Epub 2017 Nov 16.

From the Institute of Experimental Biomedicine, University Hospital of Würzburg (S.K.G., W.C., I.C.B., K.W., S.S., B.N., A.B.), Rudolf Virchow Center (S.K.G., W.C., I.C.B., K.W., S.S., B.N., A.B.), and Institute of Clinical Epidemiology and Biometry, Comprehensive Heart Failure Center (P.K.), University of Würzburg, Germany; Department of Hepatology (H.M.H.) and Department of Neurology (P.K., G.S.), University Hospital of Würzburg, Germany; Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig Maximilian University of Munich, Germany (S.Z., V.C., T.G.); Department of Immunology and Microbiology, University of Colorado, Denver (D.R., A.-L.P., C.S.); National Jewish Health, Denver, CO (D.R., A.-L.P., C.S.); Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan (M.M.); Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany (R.P.Z.); School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, United Kingdom (P.J.N., M.G.T.); and Department of Bioinformatics, Functional Genomics and Systems Biology Group, University of Würzburg Biocenter, Germany (T.D.).

Objective: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg and Ca permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown.

Approach And Results: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( ) causes a marked signaling defect in platelets. platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo.

Conclusions: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca entry in platelets. The protection of mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.117.310391DOI Listing
February 2018

Twinfilin 2a regulates platelet reactivity and turnover in mice.

Blood 2017 10 25;130(15):1746-1756. Epub 2017 Jul 25.

Institute of Experimental Biomedicine I, University Hospital and.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice () display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2017-02-770768DOI Listing
October 2017

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis.

Nat Commun 2017 06 15;8:15838. Epub 2017 Jun 15.

Institute of Experimental Biomedicine, University Hospital and University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms15838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481742PMC
June 2017

A novel two-nucleotide deletion in HPS6 affects mepacrine uptake and platelet dense granule secretion in a family with Hermansky-Pudlak syndrome.

Pediatr Blood Cancer 2017 05 4;64(5). Epub 2016 Dec 4.

Institute of Experimental Biomedicine I, University Hospital Würzburg, Germany.

Background: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by oculocutaneous albinism and platelet dysfunction. We report on a novel HPS6 homozygous frameshift variant (c.1919_1920delTC; p.Val640Glyfs*29) in a nonconsanguineous Caucasian family with two affected siblings (index patients) who presented with oculocutaneous albinism at birth and a mild bleeding phenotype during childhood and adolescence.

Procedure: Genetic analysis was conducted by panel-based next-generation sequencing (NGS) and Sanger sequencing. Platelets of the index patients, their parents, and the unaffected sister were then comprehensively evaluated by luminoaggregometry, whole blood flow cytometry, immunoblotting, immunofluorescence, and transmission electron microscopy.

Results: The homozygous frameshift variant in HPS6 gene detected by panel-based NGS and its segregation in the family was confirmed by Sanger sequencing. Flow cytometric analysis of the patients' platelets revealed a substantially decreased mepacrine uptake and release upon activation with a thrombin receptor agonist. Electron microscopy of resting platelets confirmed diminished dense granule content and enhanced vacuolization. Reduced release of adenosine triphosphate and CD63 neoexposition upon activation indicated not only a lack of dense granule content, but even an impairment of dense granule release.

Conclusions: Our results demonstrate that the novel loss-of-function variant in the HPS6 subunit of biogenesis of lysosome-related organelles complex 2 is pathologic and leads to a reduced platelet dense granules and their release. The findings are compatible with an impaired platelet function and hence an enhanced bleeding risk. In future, a valid genotype-phenotype correlation may translate into best supportive care, especially regarding elective surgery or trauma management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pbc.26320DOI Listing
May 2017

Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg(2+) homeostasis and cytoskeletal architecture.

Nat Commun 2016 Mar 29;7:11097. Epub 2016 Mar 29.

Chair of Experimental Biomedicine, University Hospital, University of Würzburg, Josef-Schneider-Strasse 2, 97078 Würzburg, Germany.

Mg(2+) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg(2+)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7(fl/fl-Pf4Cre)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7(fl/fl-Pf4Cre) MKs, which is rescued by Mg(2+) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820538PMC
March 2016

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss.

Blood 2016 06 24;127(23):2903-14. Epub 2016 Feb 24.

School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2015-10-675629DOI Listing
June 2016

Megakaryocyte rupture for acute platelet needs.

J Cell Biol 2015 May;209(3):327-8

Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, 97070 Würzburg, Germany.

Circulating platelets were thought to arise solely from the protrusion and fragmentation of megakaryocyte cytoplasm. Now, Nishimura et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410052) show that platelet release from megakaryocytes can be induced by interleukin-1α (IL-1α) via a new rupture mechanism, which yields higher platelet numbers, occurs independently of the key regulator of megakaryopoiesis thrombopoietin, and may occur during situations of acute platelet need.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201504026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427795PMC
May 2015

Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization.

Blood 2015 Jun 20;125(26):4069-77. Epub 2015 Mar 20.

Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;

Platelet aggregation at sites of vascular injury is not only essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemi-immunoreceptor tyrosine-based activation motif-containing C-type lectinlike receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and spleen tyrosine kinase (Syk)-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 not only contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis, and prevention of inflammatory bleeding, making it a potential pharmacologic target to modulate these processes. We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immunodepletion occurs through Src-family kinase-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with platelet-specific Syk deficiency, INU1-induced CLEC-2 internalization/degradation was fully preserved whereas the associated thrombocytopenia was largely prevented. These results show for the first time that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2014-11-611905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598192PMC
June 2015

In vitro platelets in sight.

Blood 2014 Sep;124(12):1849-50

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2014-08-592493DOI Listing
September 2014

Rap1-GTP-interacting adaptor molecule (RIAM) is dispensable for platelet integrin activation and function in mice.

Blood 2015 Jan 21;125(2):219-22. Epub 2014 Oct 21.

Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany.

Platelet aggregation at sites of vascular injury is essential for hemostasis but also thrombosis. Platelet adhesiveness is critically dependent on agonist-induced inside-out activation of heterodimeric integrin receptors by a mechanism involving the recruitment of talin-1 to the cytoplasmic integrin tail. Experiments in heterologous cells have suggested a critical role of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM) for talin-1 recruitment and thus integrin activation, but direct in vivo evidence to support this has been missing. We generated RIAM-null mice and found that they are viable, fertile, and apparently healthy. Unexpectedly, platelets from these mice show unaltered β3- and β1-integrin activation and consequently normal adhesion and aggregation responses under static and flow conditions. Similarly, hemostasis and arterial thrombus formation were indistinguishable between wild-type and RIAM-null mice. These results reveal that RIAM is dispensable for integrin activation and function in mouse platelets, strongly suggesting the existence of alternative mechanisms of talin-1 recruitment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2014-08-597542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347307PMC
January 2015

Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott-Aldrich syndrome-like platelet defect.

Nat Commun 2014 Sep 4;5:4746. Epub 2014 Sep 4.

1] Department of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg 97080, Germany [2] Rudolf Virchow Centre, University of Würzburg, Würzburg 97080, Germany.

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the WAS gene and is characterized by immunodeficiency, eczema and microthrombocytopenia. The molecular link between WAS mutations and microthrombocytopenia is unknown. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins, including the WAS protein (WASp)/WASp-interacting protein (WIP) complex. Here we report that mice with a megakaryocyte/platelet-specific Pfn1 deficiency display microthrombocytopenia due to accelerated turnover of platelets and premature platelet release into the bone marrow. Both Pfn1-null mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyperstable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms5746DOI Listing
September 2014

Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

Chem Biol 2014 Mar 13;21(3):357-68. Epub 2014 Feb 13.

Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany. Electronic address:

Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2013.12.018DOI Listing
March 2014