Platelet function testing : Current practice among clinical centres in Northern Europe

Introduction Platelet function tests are used to screen and diagnose patients with possible inherited platelet function defects (IPFD). Some acquired platelet dysfunction may be caused by certain drugs or comorbidities, which need to be excluded before testing. Aims To identify current practice among centres performing platelet function tests in Northern Europe. Methods A total of 14 clinical centres from Sweden (six), Finland (two), Denmark (two), Norway (one), Estonia (two) and Iceland (one) completed the survey questionnaire, the population capture area of about 29.5 million. Results Six of the 14 (42.8%) centres providing platelet function assessment represent comprehensive treatment centres (EUHANET status). A Bleeding score (BS) or ISTH bleeding assessment tool (ISTH BAT score) is evaluated in 11/14 (78.6%) centres and family history in all. Five/14 centres (35.7%) use structured preanalytical patient instructions, and 10/14 (71.4%) recorded questionnaire on the preassessment of avoidance of any drugs or natural products affecting platelet functions. Preliminary investigations of screening tests of coagulation are performed in 10/14 (71.4%), while in 4/14 (28.6%), the diagnostic work-up of IPFD and von Willebrand disease (VWD) is performed simultaneously. The work-up of IPFD includes peripheral blood smear in 10/14 (71.4%), platelet aggregometry in all, flow cytometry in 10/14 (71.4%) and Platelet Function Analysis (PFA) in 3/11 (28.6%). Molecular genetic diagnosis is available in 7/14 (50%) centres. Conclusions The considerable variability in the current practice illustrates the need for harmonization between the Northern European centres according to the international registers (i.e. EUHASS) and IPFD guidelines (ISTH, EHA).Peer reviewe

Arachidonic acid increases matrix metalloproteinase 9 secretion and expression in human monocytic MonoMac 6 cells

Background Dietary fatty acids may modulate inflammation in macrophages of the atherosclerotic plaque, affecting its stability. The n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) generally promotes inflammation, while the PUFAs of the n-3 series eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) are considered anti-inflammatory. We determined how these PUFAs influence MMP-9 expression and secretion by the human monocytic cell line (MonoMac 6) at baseline and after 24-hour exposure. MMP-9 protein was measured by zymography and relative levels of MMP-9 mRNA were determined using quantitative real time PCR. Results Supplementation with AA (but not the n-3 fatty acids) increased, in a dose-dependent manner, expression of MMP-9 protein. This stimulation was regulated at the mRNA level. MMP-9 secretion started after 1 h of incubation and could not be prevented by simultaneous presence of n-3 series fatty acids. Finally, the secretion could be attenuated by LY 294002, a specific phosphatidylinositol-3-kinase (PI3K) inhibitor and by SH-5, a selective Akt inhibitor, suggesting that activation of PI3K by AA leads to augmented and sustained MMP-9 production. Conclusion This study shows that of the PUFA studied, AA alone influences the expression of MMP-9, which might have implications in MMP-9 induced plaque rupture.BioMed Central Open acces

Increased ventilatory response to exercise in symptomatic and asymptomatic LMNA mutation carriers : a follow-up study

BackgroundLMNA mutations are an important cause of cardiomyopathy often leading to cardiac arrhythmias, heart failure and even heart transplantation. An increasing number of asymptomatic mutation carriers are identified, as family members of the index patients are screened. Our aim was to study the disease progression in asymptomatic LMNA mutation carriers and in patients with symptomatic cardiolaminopathy by repeated spiroergometric testing in a prospective clinical follow-up study. Methods and ResultsWe studied 26 LMNA mutation carriers once a year during 5years up to 6 times by spiroergometry, clinical assessment, laboratory tests and echocardiography. The 23 control subjects underwent clinical assessment and spiroergometry once. Twelve of the mutation carriers were asymptomatic, and 14 had some clinical manifestations of the mutation ranging from clinically relevant rhythm disturbances to DCM and heart failure. Compared to controls, the symptomatic carriers showed a higher slope of the ventilatory equivalent for CO2 (VE/VCO2 slope) and a lower fraction of end-tidal CO2 (FetCO(2)). The asymptomatic mutation carriers also showed an increased ventilatory response to exercise during the follow-up as indicated by increased VE/VCO2 slope and decreased FetCO(2). ConclusionsThe study suggests that an increased ventilatory response during exercise might reveal a preclinical manifestation of DCM in LMNA mutation carriers.Peer reviewe

Koronainfektion laboratoriodiagnostiikka: Miten laboratorio valitsee menetelmät?

Laboratoriot valitsevat käyttöönsä testit käyttötarkoituksen ja soveltuvuuden perusteella siten, että diagnostiikka on mahdollisimman vaikuttavaa. PCR on luotettavin koronavirustartunnan toteamiseen, mutta etenkin tuoreissa tartunnoissa voidaan käyttää antigeenitestejä.</p

Koronainfektion laboratoriodiagnostiikka : miten laboratorio valitsee menetelmät?

Laboratoriot valitsevat käyttöönsä testit käyttötarkoituksen ja soveltuvuuden perusteella siten, että diagnostiikka on mahdollisimman vaikuttavaa. PCR on luotettavin koronavirustartunnan toteamiseen, mutta etenkin tuoreissa tartunnoissa voidaan käyttää antigeenitestejä.publishedVersio

Koronainfektion laboratoriodiagnostiikka : miten laboratorio valitsee menetelmät?

Laboratoriot valitsevat käyttöönsä testit käyttötarkoituksen ja soveltuvuuden perusteella siten, että diagnostiikka on mahdollisimman vaikuttavaa. PCR on luotettavin koronavirustartunnan toteamiseen, mutta etenkin tuoreissa tartunnoissa voidaan käyttää antigeenitestejä

Lamin A/C mutation affecting primarily the right side of the heart

LMNA mutations are amongst the most important causes of familial dilated cardiomyopathy. The most important cause of arrhythmogenic right ventricular cardiomyopathy (ARVC) is desmosomal pathology. The aim of the study was to elucidate the role of LMNA mutations among Finnish cardiomyopathy patients. We screened 135 unrelated cardiomyopathy patients for LMNA mutations. Because of unusual phenotype, two patients were screened for the known Finnish ARVC-related mutations of desmosomal genes, and their Plakophilin-2b gene was sequenced. Myocardial samples from two patients were examined by immunohistochemical plakoglobin staining and in one case by electron microscopy. We found a new LMNA mutation Phe237Ser in a family of five affected members with a cardiomyopathy affecting primarily the right side of the heart. The phenotype resembles ARVC but does not fulfill the Task Force Criteria. The main clinical manifestations of the mutation were severe tricuspid insufficiency, right ventricular enlargement and failure. Three of the affected patients died of the heart disease, and the two living patients received heart transplants at ages 44 and 47. Electron microscopy showed nuclear blebbing compatible with laminopathy. Immunohisto - chemical analysis did not suggest desmosomal pathology. No desmosomal mutations were found. The Phe237Ser LMNA mutation causes a phenotype different from traditional cardiolaminopathy. Our findings suggest that cardiomyopathy affecting primarily the right side of the heart is not always caused by desmosomal pathology. Our observations highlight the challenges in classifying cardiomyopathies, as there often is significant overlap between the traditional categories.Peer reviewe

SH2- ja SH3-domeenivuorovaikutukset ja POSH2-proteiinin karakterisointi

Domeenit ovat valkuaisaineiden eli proteiinien itsenäisiä yksiköitä, joilla on tietty rakenne ja toiminta. Domeenit säätelevät soluissa monia proteiinien välisiä vuorovaikutuksia, jotka ovat tärkeitä solunsisäisessä viestinvälityksessä. Toiminnallisen viestivälityskoneistonsa avulla solu pystyy erilaistumaan, kasvamaan ja tarvittaessa myös kuolemaan. Ensimmäiset löydetyt domeenit kuuluivat Src homology-2 (SH2) ja SH3-domeeniperheisiin. Nämä molemmat domeenit ovat kehitysbiologisesti hyvin säilyneitä ja yleisiä solun proteiineissa. SH2-domeeni on noin 100 aminohappoa käsittävä yksikkö, joka sitoo kohdeproteiinissa fosforyloitunutta tyrosiiniaminohappoa ja sitä ympäröivää aluetta. Tyrosiinikinaasit, jotka vastaavat tyrosiiniaminohappojen fosforyloimisesta, luovat toiminnallaan sitoutumispaikkoja SH2-domeeneille. SH3-domeenit ovat puolestaan noin 60 aminohappoa käsittäviä domeeneja, jotka välittävät proteiinivuorovaikutuksia sitoutumalla kohdeproteiinissa olevaan proliinirikkaaseen alueeseen. SH3-domeenin sitoutuminen kohdeproteiiniin on yleisesti ottaen ajateltu olevan heikkoa voimakkuudeltaan (affiniteetti) ja spesifisyydeltään. Tämän tutkimuksen tarkoituksena oli tutkia SH2- ja SH3-domeenien vuorovaikutuksia kohdeproteiineihin. Ensimmäisenä pääkohtana väitöskirjatyössä oli tutkia SH2-domeenien kykyä sitoa tyrosiinifosforyloituja ligandeja sekä solunäytteitä käyttämällä far Western- ja Rosette-menetelmiä. Tulokset vahvistivat SH2-domeenien olevan keskeisiä fosfotyrosiinia sitovia domeeneja ja SH2-domeenien havaittiin tunnistavan spesifisesti tiettyjä solun proteiineja. Tutkimuksessa käytettyjen menetelmien avulla voitiin saada sekä kvantitatiivista että kvalitatiivista tietoa SH2-domeenien sitoutumisesta. Toisena pääkohtana väitöskirjatyössä oli selvittää ihmisgenomissa esiintyvät SH3-domeenit ja identifioida uusia SH3-kohdeproteiineja. Tätä varten kehitettiin SH3-kirjasto, jossa bakteriofaagien pinnalla ilmennettiin SH3-domeeneja. Tällä phage display-menetelmällä pystyttiin seulomaan SH3-domeeneja, jotka sitoutuivat korkealla affiniteetilla käytettyihin kohdeproteiineihin: p21-aktivoituvaan kinaasiin (PAK2), HI-viruksen Nef-proteiiniin ja proteiineja pilkkovaan ADAM15-proteiiniin. Tutkimuksessa kuvattiin uusia, aiemmin tuntemattomia vuorovaikutuksia näiden kolmen kohdeproteiinin ja eri SH3-domeenien välillä. Näistä vuorovaikutuksista esimerkkinä on PAK2:een korkealla affiniteetilla sitoutuva SH3-domeeni, jonka todettiin olevan osa aiemmin kirjallisuudessa kuvaamatonta POSH2-proteiinia. Lisäksi tutkimuksessa havaittiin myös muita SH3-domeeneja, jotka sitoutuivat kohdeproteiiniin spesifisesti ja korkealla affiniteetilla, mikä antaa viitteitä SH3-domeenien oletettua keskeisemmästä roolista solun signaalinvälityksessä. Kolmantena pääkohtana oli karakterisoida uusi POSH-proteiiniperheenjäsen, POSH2, ja tutkia tämän proteiinin toiminnallisuutta. POSH2:n todettiin koostuvan neljästä SH3-domeenista, ubikitiini E3-ligaasiaktiivisuuden sisältämästä RING-domeenista sekä aktiivista GTPaasi-proteiinia, Rac1:tä, sitovasta alueesta. Tutkimuksessa selvitettiin tarkemmin POSH2:n rakenteellisten muutosten vaikutusta Rac1:n sitoutumiseen sekä POSH2:n vaikutusta JNK-kinaasiaktivaatioon. Tutkimustulokset vahvistavat POSH2:n yhtäläisyyttä aiemmin tunnettuihin POSH-proteiineihin ja tuloksien avulla voitiin havaita Rac1:n ja POSH2:n välisen vuorovaikutuksen olevan keskeinen JNK-välitteisessä viestinvälityksessä. Tutkimuksessa havaittu POSH2:n monitoimintainen rooli solussa antaa lähtökohdan proteiinin jatkotutkimukselle. Tulokset antavat uutta tietoa SH2- ja SH3-domeenien vuorovaikutuksista yksittäisiin ligandeihin ja luonteenpiirteistä toimia domeeniperheinä.Modular protein domains mediate protein-protein interactions in various cellular proteins, including adaptors, enzymes and scaffold proteins. Src homology 2 (SH2)domains comprise a relatively large group of domains, which mediate inter- and intramolecular interactions by binding to tyrosine-phosphorylated ligands. Phosphorylation of tyrosine residues in proteins controls many facets of signaling in multicellular organisms, and increased tyrosine phosphorylation is associated with uncontrolled cell growth. Another group of domains, Src homology 3 (SH3) domains, mediates protein interactions by binding to ligands containing a polyproline type II (PPII) helix. SH3 domains mediate generally relatively low specificity and affinity interactions, although this might be due to the use of short ligands lacking the regions flanking the binding interface of SH3 domains. These outside regions are known to contribute to the ligand binding of SH3 domains. POSH proteins, which have been implicated as scaffold proteins in the JNK-mediated apoptosis signaling cascade, contain SH3 domains. POSH proteins also participate in protein trafficking and the regulation of protein degradation via ubiquitination. The importance of tyrosine phosphorylation for many biological processes has led to an interest in profiling global tyrosine phosphorylation in cells. However, many of the methods utilized are either not suitable for comprehensive phosphotyrosine profiling or have technical limitations regarding their applicability for high-throughput analysis. In our study, we exploited the fact that the tyrosine phosphorylation of proteins creates binding sites for SH2 domains. A far-Western approach and a newly developed reversephase Rosette assay were used to profile the tyrosine phosphorylation of selected ligands (platelet endothelial cell adhesion molecule-1; PECAM-1 and p21-activated kinase 2; PAK2) as well to assess global tyrosine-phosphorylation levels from cell lysates. SH2 domains exhibited different protein binding preferences,which reflected in their ability to recognize a specific subset of cellular proteins. Our methods elucidated SH2 interactions at the protein level and demonstrated increased binding of SH2 domains to adherent cells in addition to adhesion-specific SH2/ligand interactions. The human genome was found to contain 296 different SH3 domains, and these domains were used in a phage display approach to determine which SH3 domains bound most strongly to our target proteins of interest: Nef, PAK2 and a disintegrin and metalloprotease 15 (ADAM15). An unbiased system for simultaneously assaying the complete human SH3 proteome was used to determine the preferred SH3/ligand interactions; this approach provided valuable information without the limitations caused by short peptide ligands or the skewing of variables caused by more indirect methods. Our approach identified both previously reported and novel SH3 domains that were capable of binding to the target proteins with nanomolar affinities. In addition to providing information regarding the SH3-mediated binding kinetics, this method also identified novel signaling proteins, such as the PAK2-binding scaffold protein plenty of SH3 domains 2 (POSH2). POSH2 was found to be a highly homologous new member of the previously identified POSH family of proteins. POSH2 was shown to contain four SH3 domains and a RING domain, which provides ubiquitin E3 ligase activity to the protein. Activated Rac1, a GTPase, was shown to interact with POSH2, and this interaction was mediated by the partial Cdc42/Rac1 interactive binding (CRIB) domain. Moreover, the interaction of POSH2 and Rac1 suggests that POSH2 acts downstream of Rac1 in JNK-mediated apoptosis. In conclusion, our results from the SH2 domain study provided information regarding likely in vivo interactions and changes in the concentration of SH2 binding sites under various conditions. Deciphering the global phosphotyrosine pattern and identifying activated signaling pathways are also important for understanding aberrant cellular functions. Thus, global phosphotyrosine profiling and quantification by SH2 domains could be used as a diagnostic tool in clinical applications. SH3 domains were suggested to have a more prominent role in mediating cellular interactions, and these domains bind with higher specificity than was previously anticipated. Moreover, the SH3 phage library system proved to be a valuable tool for deciphering the wiring of SH3-dependent signaling networks within the cell. POSH2 contains an SH3 domain and is the third member of the POSH protein family. These POSH proteins contain a highly homologous domain structure consisting of a RING finger domain, multiple SH3 domains and a partial CRIB domain, which is crucial for binding to Rac1