Ongoing projects in coagulation
TF expression and procoagulant activity
Individual variations of TF expression and activity in monocytes have been established, but still little is known of cellular and genetic factors regulating the magnitudes of TF expression and activity. We identified the novel 5466 A>G SNP in the TF gene, coding for increased TF expression and activity in monocytes. This SNP was subsequently shown to be associated with myocardial infarct and cardiovascular death in acute coronary syndrome. Very recently, thrombin formation following vascular injury and thrombin-lowering effect of statins in patients with CAD were found to be genetically determined by the TF 5466A>G polymorphism. We are continuing our studies how the tissue factor gene is regulated on the molecular level. During 2014 we have started a collaboration with Professor Johann Wojtas research group in Vienna concerning different subsets of monocytes and found that a subset of monocytes, CD14+ and CD16+, express higher levels of TF induced by LPS and the cytokine IL-33. The ultimate goal being to identify novel mechanisms, genetic, epigenetic and microRNAs, governing tissue factor gene regulation.
A cocktail of cytokines was shown to express TF in pancreatic islets. TF/FVIIa signalling was also demonstrated to augment beta-cell death in response to cytokines.
Microparticles; methods and biological functions
Upon activation platelets, leukocytes and endothelial cells form MPs. Circulating platelet MPs have been found in inflammatory diseases and are related to the severity of disease. We have during the year developed a new flow cytometry method to calculate the amount of MPs with different cellular origin in whole blood. The new method is superior to earlier used methods, and is now implemented in a number of new clinical studies in patients with CAD and pulmonary arterial hypertension. Characterization of the biological effects induced by purified platelet MPs upon interaction with a number of human cells and whether new antiplatelet/antithrombotic drugs can interfere with this interaction are a subject of our ongoing experimental studies.
TF non-coagulant, signalling and biological functions
The TF-induced signalling events eventually changes cell fate and behaviour, rendering cells and tissues pro-migratory, resistant to apoptosis and proliferative. This experimental work is paralleled by clinical observations of increased TF expression in conditions such as metastatic cancers and the atherosclerotic plaque, where cell survival, migration and proliferation are paramount to the pathological process. We reported for the first time that TF/FVIIa induces the transactivation of receptor tyrosine kinases, i.e the PDGFRβ, and thereby identified a new signalling pathway involved in cell migration.
We have continued our work on TF/FVIIa-induced signaling and biological consequences. We showed that IGF-1R is a key player in TF/FVIIa-induced cell survival. TF/FVIIa induces transactivation of the IGF-1 receptor, which then translocates to the nucleus, and binds to chromatin and induces generegulation.
We have also shown that Eph RTKs are novel proteolytical targets of TF/FVIIa and cleaved in their ectodomains by TF/FVIIa. We have identified the exact cleavage site in the receptors. The cleavage controls EphB2-mediated cell segregation. Cleavage of EpHA2 by TF/FVIIa complex leads to potentiation of EphA2-ligand induced cytoskeleton reorganization. Moreover, we have demontsrated that TF/FVIIa phosporylates serine 897 in the cytoplasmic domain of EphA2. EphA2/ephrinA1 pathway is a novel proinflammatory mediator and one regulator of atherosclerotic plaque development.
MicroRNA: TF regulation and arrays for clinical studies
Not much is known about the molecular regulation of the human TF gene. We have recorded 211 differentially expressed microRNAs during TF down-regulation. One of these, was identified to regulate the transcription of the human TF gene by directly binding to its target sequence in the 3’UTR. In a patient cohort with ACS, we found that expression of this microRNA is reduced one year after the acute event, and this reduction correlates with an increase in TF on the surface of platelets and circulating platelet microparticles.
A novel high-throughput and cost effective qPCR-method for measuring relative microRNA expression levels is currently being established using the BioMark HD at the Clinical Biomarkers facility, SciLifelab (headed by me). So far 34 miRNAs have been successfully amplified using this system. This method will be used for screening microRNAs in our large studies on ACS and AF.
Identification of biomarkers in atherothromboembolic diseases
The purpose is to identify new biomarkers and establish new tools with higher sensitivity to be used in the understanding of pathophysiologic mechanisms, diagnosis and for estimation of prognosis and treatment efficacy in these diseases.
Plasma samples from our well-characterized patients with CAD have been analysed with a new plasma proteomic multiplex assay, the proximity extension assay, where 90 samples and 92 biomarkers in each sample are analysed simultaneously. A case control study of 400 patients with MI included in the PLATO study has been analyzed with the multiplex PEA. Using this assay and also conventional assays a number of the new biomarkers of importance for new events have been identified, among others the stem cell factor, SCF. GDF-15 has been demonstrated to be an excellent prognostic biomarker for bleeding in patients with AF and NOAC treatment.
We have been actively involved in the design of the substudy programs of biomarkers, genome wide association studies and the analyses of the plasma samples and to translate candidate genes and proteins into functional studies. During the last year we have in close collaboration with the Cardiology research group at IMV been very actively involved in establishing new clinical tools for improving the identification of risk of stroke, MI and bleeding during anticoagulant treatment. These tools are based on age, biomarkers and previous cardiovascular events, and therefore called ABC-risk scores. The first version of three different scores, based on biomarker results analysed in our large trials in ACS and AF, have recently been presented.