Publications by authors named "Abdimajid Osman"

21 Publications

  • Page 1 of 1

Platelet Pathogen Reduction Technologies Alter the MicroRNA Profile of Platelet-Derived Microparticles.

Front Cardiovasc Med 2020 19;7:31. Epub 2020 Mar 19.

Research Center of the CHU de Québec, Quebec, QC, Canada.

Despite improvements in donor screening and increasing efforts to avoid contamination and the spread of pathogens in clinical platelet concentrates (PCs), the risks of transfusion-transmitted infections remain important. Relying on an ultraviolet photo activation system, pathogen reduction technologies (PRTs), such as Intercept and Mirasol, utilize amotosalen, and riboflavin (vitamin B2), respectively, to mediate inactivation of pathogen nucleic acids. Although they are expected to increase the safety and prolong the shelf life of clinical PCs, these PRTs might affect the quality and function of platelets, as recently reported. Upon activation, platelets release microparticles (MPs), which are involved in intercellular communications and regulation of gene expression, thereby mediating critical cellular functions. Here, we have used small RNA sequencing (RNA-Seq) to document the effect of PRT treatment on the microRNA profiles of platelets and derived MPs. PRT treatment did not affect the microRNA profile of platelets. However, we observed a specific loading of certain microRNAs into platelet MPs, which was impaired by treatment with Intercept or its Additive solution (SSP+). Whereas, Intercept had an impact on the microRNA profile of platelet-derived MPs, Mirasol did not impact the microRNA profile of platelets and derived MPs, compared to non-treated control. Considering that platelet MPs are able to transfer their microRNA content to recipient cells, and that this content may exert biological activities, those findings suggest that PRT treatment of clinical PCs may modify the bioactivity of the platelets and MPs to be transfused and argue for further investigations into PRT-induced changes in clinical PC content and function.
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http://dx.doi.org/10.3389/fcvm.2020.00031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096552PMC
March 2020

Genome-wide analyses disclose the distinctive HLA architecture and the pharmacogenetic landscape of the Somali population.

Sci Rep 2020 03 27;10(1):5652. Epub 2020 Mar 27.

Department of Clinical Chemistry, and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.

African populations are underrepresented in medical genomics studies. For the Somali population, there is virtually no information on genomic markers with significance to precision medicine. Here, we analyzed nearly 900,000 genomic markers in samples collected from 95 unrelated individuals in the North Eastern Somalia. ADMIXTURE program for estimation of individual ancestries revealed a homogenous Somali population. Principal component analysis with PLINK software showed approximately 60% East African and 40% West Eurasian genes in the Somali population, with a close relation to the Cushitic and Semitic speaking Ethiopian populations. We report the unique features of human leukocyte antigens (HLA) in the Somali population, which seem to differentiate from all other neighboring regions compared. Current study identified high prevalence of the diabetes type 1 (T1D) predisposing HLA DR-DQ haplotypes in Somalia. This finding may explain the increased T1D risk observed among Somali children. In addition, ethnic Somalis were found to host the highest frequencies observed thus far for several pharmacogenetic variants, including UGT1A4*2. In conclusion, we report that the Somali population displays genetic traits of significance to health and disease. The Somali dataset is publicly available and will add more information to the few genomic datasets available for African populations.
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http://dx.doi.org/10.1038/s41598-020-62645-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101338PMC
March 2020

Assessment of genetic and non-genetic risk factors for venous thromboembolism in glioblastoma - The predictive significance of B blood group.

Thromb Res 2019 Nov 22;183:136-142. Epub 2019 Oct 22.

Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden. Electronic address:

Introduction: Venous thromboembolism (VTE) is a common problem among patients with glioblastoma multiforme (GBM) and with some other cancers. Here, we evaluated genetic and non-genetic potential risk factors for VTE among GBM patients.

Materials And Methods: A cohort of 139 patients treated with concomitant radiotherapy and temozolomide were included in the study. Next generation sequencing and genotyping approaches were applied to assess genetic risk factors in the haemostatic system. Clinical data including surgery, reoperation as well as blood group and patient information such as age and gender were available from patient records. Logistic regression analysis was performed to asses VTE risk.

Results: In the study 47 patients (34%) were diagnosed for VTE during the course of their disease. When genetic and non-genetic potential risk factors were evaluated, only B blood group was found to be significantly associated with VTE incidence (odds ratio [OR] = 6.91; confidence interval [CI] = 2.19-24.14; P = 0.001). In contrast, A and O blood groups did not correlate with VTE risk. Frontal lobe tumor location also seemed to slightly increase VTE risk compared to other brain sites (OR = 3.14; CI = 1.1-10.7) although the significance level was at borderline (P = 0.05). Current study identified B blood group as the component in non-O blood groups that is responsible for increased VTE risk.

Conclusion: In conclusion, these results suggest for the first time that B blood group is predictive for VTE incidence among patients with glioblastoma, information that may be potentially valuable when selecting GBM patients who are at risk for VTE for anticoagulant prophylaxis.
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http://dx.doi.org/10.1016/j.thromres.2019.10.009DOI Listing
November 2019

Frequency of PAR4 Ala120Thr variant associated with platelet reactivity significantly varies across sub-Saharan African populations.

Blood 2018 11 24;132(19):2103-2106. Epub 2018 Aug 24.

Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

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http://dx.doi.org/10.1182/blood-2018-05-852335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307984PMC
November 2018

Prevalence of common hereditary risk factors for thrombophilia in Somalia and identification of a novel Gln544Arg mutation in coagulation factor V.

J Thromb Thrombolysis 2017 Nov;44(4):536-543

Division of Clinical Chemistry, Region Östergötland, Ingång 64, 581 85, Linköping, Sweden.

Thrombophilia, commonly manifested as venous thromboembolism (VTE), is a worldwide concern but little is known on its genetic epidemiology in many parts of the globe particularly in the developing countries. Here we employed TaqMan genotyping and pyrosequencing to evaluate the prevalence of known common nucleotide polymorphisms associated with thrombophilia in a Somali population in the Puntland region of Somalia. We also employed next generation sequencing (NGS) to investigate other genetic variants in a Somali patient with deep venous thrombosis (DVT). As expected, we found no existence of factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) in the Somali population. The G allele of ABO [261G/delG] polymorphism (rs8176719) was found at a frequency of 29%, similar to that observed in other African populations. We found the lowest so far reported frequency of MTHFR C677T (rs1801133) polymorphism in the Somali population (T allele frequency 1.5%). A novel and deleterious single nucleotide variation in exon 11 of coagulation factor V (c.1631A>G) causing Gln544Arg exchange in factor V was identified in a 29 years old Somali female with DVT. The same patient was heterozygous to VKORC1 Asp36Tyr polymorphism (rs61742245) that predisposes to warfarin resistance. In conclusion, this study shows that common hereditary factors for thromboembolism found in Caucasians are either less frequent or absent in the Somali population-similar to the situation in other Africans. NGS is possibly a better choice to detect genetic risk variants for thrombosis in this ethnic group.
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http://dx.doi.org/10.1007/s11239-017-1543-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658450PMC
November 2017

The platelets' perspective to pathogen reduction technologies.

Platelets 2018 Mar 29;29(2):140-147. Epub 2017 Mar 29.

d CHUQ Research Center/CHUL , 2705 Blvd Laurier, Quebec , QC , Canada.

A wide variety of clinical conditions, associated with low circulating platelet counts, require platelet transfusion in order to normalize hemostatic function. Although single-donor apheresis platelets bear the lowest risk of transfusion-transmitted infections, pathogen reduction technologies (PRT) are being implemented worldwide to reduce this risk further through inactivation of known, emergent and as yet to be discovered nucleic acid-based pathogens. Human blood platelets are now known to harbor a diverse transcriptome, important to their function and comprised of >5000 protein-coding messenger RNAs and different classes of non-coding RNAs, including microRNAs. Our appreciation of the nucleic acid-dependent functions of platelets is likely to increase. On the other hand, the side effects of PRT on platelet function are underappreciated. Recent evidences suggest that PRT may compromise platelets' responsiveness to agonists, and induce platelet activation. For instance, platelets have the propensity to release proinflammatory microparticles (MPs) upon activation, and the possibility that PRT may enhance the production of platelet MPs in platelet concentrates (PCs) appears likely. With this in mind, it would be timely and appropriate to investigate other means to inactivate pathogens more specifically, or to modify the currently available PRT so to better preserve the platelet function and improve the safety of PCs; platelets' perspective to PRT deserves to be considered.
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http://dx.doi.org/10.1080/09537104.2017.1293806DOI Listing
March 2018

Peculiarities of studying the effects of pathogen reduction technologies on platelets.

Proteomics Clin Appl 2016 08 17;10(8):805-15. Epub 2016 May 17.

CHUQ Research Center/CHUL, Quebec, QC, Canada.

The transfusion of platelet concentrates (PCs) is mainly used for treatment of thrombocytopenic, trauma or surgery patients. The integrity and safety of these platelet preparations, however, is compromised by the presence of pathogens, such as viruses, bacteria and parasites. The transfer of allogeneic donor leukocytes contaminating PCs can also potentially cause adverse reactions in recipients. These considerations prompted the development and implementation of pathogen reduction technologies (PRT), which are based on chemically induced cross-linking and inactivation of nucleic acids. While the incumbent PRT may provide some protection against transfusion-transmitted infections, they are ineffective against infectious prions and may not inactivate other emerging pathogens. In addition, the safety of PRT concerning platelet viability and function has been questioned in several reports. Recent studies suggest that PRT, such as Intercept, may adversely affect the messenger RNA (mRNA) and microRNA content of platelets, as well as their functional integrity, which may compromise the clinical benefits of PRT. Here, we will discuss about the peculiarities of studying the effects of PRT on platelets, which will need to be taken into account in future studies aimed to characterize further, and polish, the rugged side of this otherwise useful and potentially important approach in transfusion medicine.
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http://dx.doi.org/10.1002/prca.201500124DOI Listing
August 2016

Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems.

PLoS One 2015 14;10(7):e0133070. Epub 2015 Jul 14.

Université Laval CHUQ Research Center / CHUL 2705 Blvd Laurier, Quebec, QC, Canada.

Platelet concentrates (PCs) are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR) systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets' nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE) RNA sequencing (RNA-Seq), we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of >800 mRNAs (P<0.05) compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC) ≥ 2. At the p-value < 0.001, as many as 147 genes were deregulated by ≥ 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA) and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133070PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501785PMC
April 2016

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

Platelets 2015 21;26(2):154-63. Epub 2014 Apr 21.

Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, University of Linköping , Linköping , Sweden .

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.
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http://dx.doi.org/10.3109/09537104.2014.898178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364275PMC
November 2015

Influence of UGT2B7, OPRM1 and ABCB1 gene polymorphisms on postoperative morphine consumption.

Basic Clin Pharmacol Toxicol 2014 Nov 19;115(5):423-31. Epub 2014 May 19.

Unit for Development and Patient Safety, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.

Therapeutic modulation of pain with morphine and other opioids is associated with significant variation in both effects and adverse effects in individual patients. Many factors including gene polymorphisms have been shown to contribute to the interindividual variability in the response to opioids. The aim of this study was to investigate the significance of UGT2B7, OPRM1 and ABCB1 polymorphisms for interindividual variability in morphine-induced analgesia in patients undergoing hysterectomy. The frequency of these polymorphisms was also investigated in forensic autopsies as morphine is also a very commonly abused drug. Blood samples were collected from 40 patients following abdominal hysterectomy, 24 hr after initiation of analgesia through a patient-controlled analgesia (PCA) pump. Samples were genotyped and analysed for morphine and its metabolites. We also genotyped approximately 200 autopsies found positive for morphine in routine forensic analysis. Patients homozygous for UGT2B7 802C needed significantly lower dose of morphine for pain relief. The same trend was observed for patients homozygous for ABCB1 1236T and 3435T, as well as to OPRM1 118A. The dose of morphine in patients included in this study was significantly related to variation in UGT2B7 T802C. Age was significantly related to both dose and concentration of morphine in blood. Regression analysis showed that 30% of differences in variation in morphine dose could be explained by SNPs in these genes. The genotype distribution was similar between the forensic cases and the patients. However, the mean concentration of morphine was higher in forensic cases compared to patients. We conclude that gene polymorphisms contribute significantly to the variation in morphine concentrations observed in individual patients.
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http://dx.doi.org/10.1111/bcpt.12248DOI Listing
November 2014

Exon resequencing of the gene encoding UCma/GRP reveals a common carboxy-terminal 138Thr > Ser polymorphism.

Clin Lab 2013 ;59(11-12):1397-401

Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden.

Background: The upper zone of growth plate and cartilage matrix-associated protein (UCMA), also called G1a-rich protein (GRP), is a novel protein found at sites affected by pathological calcifications.

Methods: We performed a full exon resequencing on DNA samples from 17 chronic kidney disease (CKD) patients (stage 5) and compared the results with 121 healthy controls in a Swedish population.

Results: A novel non-synonymous single nucleotide polymorphism (SNP) causing a carboxy-terminal amino acid exchange was found. This SNP involves an alteration of the last ACC codon for threonine in exon 5 (adjacent to the stop codon) to an AGC serine codon (138Thr > Ser). Six controls and two CKD patients were heterozygous for the 138Thr > Ser polymorphism. Both patients had histories of vascular calcification; however, it is uncertain whether this SNP has any significance for the functional domains of the UCMA protein. In addition, a heterozygous transversion mutation was found in a patient at SNP rs4750328 (A/G) in intron 2, involving an exchange of the ancestral A allele to a T base.

Conclusions: The 138Thr > Ser polymorphism seems to be the only non-synonymous SNP found in the UCMA gene in a Swedish population.
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http://dx.doi.org/10.7754/clin.lab.2013.130123DOI Listing
February 2014

miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells.

Pigment Cell Melanoma Res 2014 May 6;27(3):431-41. Epub 2014 Feb 6.

Division of Microbiology and Molecular Medicine, Faculty of Health Sciences, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

The proteinase-activated receptor 1 (PAR-1) plays a central role in melanoma progression and its expression level is believed to correlate with the degree of cancer invasiveness. Here, we show that PAR-1 is post-transcriptionally regulated by miR-20b microRNA in human melanoma cells. PAR-1 was found to be expressed in metastatic melanoma cells but was barely detectable in primary melanoma. By transducing primary melanoma cells with a lentivirus containing a 3'-UTR construct of PAR-1 mRNA, we could show that endogenous melanoma microRNAs interacted with PAR-1 3'-UTR and silenced a fused luciferase reporter. Transfection of an inhibitor against miR-20b into primary melanoma cells reversed this process. Finally, transfection of miR-20b mimic into metastatic melanoma cells caused downregulation of the luciferase reporter. We conclude that miR-20b regulates expression of melanoma PAR-1 receptor, which may explain the differential expression of PAR-1 observed in human melanoma.
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http://dx.doi.org/10.1111/pcmr.12217DOI Listing
May 2014

Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

PLoS One 2013 11;8(12):e81809. Epub 2013 Dec 11.

Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden.

Background: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA.

Materials And Methods: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts.

Results: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq.

Conclusion: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and tissue types. The in vivo functional significance of the non-mitochondrial transcripts remains to be shown.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081809PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859545PMC
September 2014

MicroRNAs in health and disease--basic science and clinical applications.

Authors:
Abdimajid Osman

Clin Lab 2012 ;58(5-6):393-402

Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, University of Linköping, 581 85 Linköping, Sweden.

MicroRNAs (miRs) are small RNAs that fine-tune gene expression at posttranscriptional level. They are involved in virtually all physiological processes, such as development and differentiation, heart function, metabolism, haemostasis, and apoptosis. Anucleated human platelets were revealed to contain comparatively many different miR species relative to nucleated cells, suggesting a functional impact of miRs on these small cells. In recent years, numerous studies have established the significance of miRs in different physiological states. Sufficient evidence exists to suggest that miRs are involved in a range of diseases, including cancer and cardiovascular disorders. Aberrant expression levels of miRs are found in tissues and in sera from patients with different forms of malignant tumours. Utilising these miRs as biomarkers for disease is a valuable diagnostic strategy. In addition, a novel class of synthetic oligonucleotides, called antagomirs, has recently been introduced as efficient silencers of overexpressed miRs in cancer. Overall, miRs represent an important class of small RNAs with a huge impact on health and disease. Future studies will further illuminate the potential value of miRs in diagnostics and therapeutics.
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July 2012

The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets.

Thromb Res 2012 Apr 26;129(4):e51-8. Epub 2012 Jan 26.

Department of Clinical and Experimental Medicine, Linköping University, Sweden.

Introduction: Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet α-granule proteins such as VEGF and endostatin.

Materials And Methods: Human isolated platelets were incubated with thrombin (0.5 U/ml), PAR1-activating peptide (AP) (0.4-30 μM) or PAR4-AP (1.5-300 μM) for up to 24 hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin.

Results: Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24 hours incubation of platelets.

Conclusions: PAI-1 and VEGF, but not endostatin, might be stored in the same α-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24 hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.
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http://dx.doi.org/10.1016/j.thromres.2011.12.021DOI Listing
April 2012

Characterization of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes.

Platelets 2011 25;22(6):433-41. Epub 2011 Mar 25.

Linköping University, Clinical and Experimental Medicine, Department of Clinical Chemistry, SE-581 85 Linköping, Sweden.

Platelets are anucleate blood cells that play a crucial role in thrombosis and hemostasis. Despite their lack of nuclear DNA, platelets contain significant amounts of microRNA (miRNA) that may have vital functions in post-transcriptional gene regulation. Here, we combined comprehensive miRNA expression profiling by quantitative PCR with target prediction analysis for the most abundant miRNAs in human platelets. A network composed of predicted platelet miRNA target genes was then constructed, using annotations available in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In addition, we evaluated possible differences in miRNA levels between resting and thrombin-stimulated platelets. We identified 281 transcripts, including 228 mature miRNAs and 53 minor miRNAs (or miR*), of which six miRNAs (miR-15 a, miR-339-3 p, miR-365, miR-495, miR-98, and miR-361-3 p) were up- or down-regulated in activated human platelets (P ≤ 0.001). A redundancy-reduced network was established that encompassed 246 genes in five statistically significant functional clusters representing platelet miRNA regulating pathways. Comparison of the 246 network genes with the platelet mRNA expression data available at ArrayExpress database confirmed that most of these genes (89%) are expressed in human platelets. In conclusion, this work affirms a recent microarray study reporting a wide-spread existence of miRNAs in human platelets. Further, we observed that thrombin stimulation was associated with altered levels of some miRNAs in platelets. The proposed functional network, combining computational prediction analysis with annotations from experimental observations, may in addition provide some information about probable miRNA target pathways in human platelets.
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http://dx.doi.org/10.3109/09537104.2011.560305DOI Listing
December 2011

A genotyping method for VKORC1 1173C > T by Pyrosequencing technology.

Scand J Clin Lab Invest 2008 ;68(5):427-30

Department of Clinical Chemistry, Laboratory Medicine, University Hospital, Linköping, Sweden.

Vitamin K epoxide reductase complex subunit 1 (VKORC1) is the site of inhibition by warfarin and other antivitamin K drugs during oral anticoagulant therapy. The SNP rs9934438 in intron 1 of VKORC1 (c.173+1000C > T or 1173C > T) discriminating the VKORC1*2 haplotype is associated with low warfarin dose requirement and unstable prothrombin time - international normalized ratio. To genotype this SNP, we have developed a rapid method using Pyrosequencing technology. The proposed method takes a post-PCR sample preparation of less than 1 h and a DNA sequencing time of less than 15 min to genotype 96 samples. The current method was compared with a dHPLC method that we reported previously. Genotype frequencies at VKORC1 1173C>T for our Swedish population were 38% wild-type, 40% heterozygote and 22% homozygote. The frequency of the T-allele was 0.42, which exactly matches the frequency previously reported for Germans. The current method can be used to determine whether patients initiating warfarin therapy are carriers of SNP 1173 C>T that is strongly associated with low warfarin dose requirement.
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http://dx.doi.org/10.1080/00365510701810621DOI Listing
February 2009

A possible ethanol-catalyzed rearrangement of vitamin K(1) detected by gas chromatography/mass spectrometry.

Rapid Commun Mass Spectrom 2008 Dec;22(23):3861-6

Department of Clinical Chemistry, University Hospital, SE-581 85 Linköping, Sweden.

We studied vitamin K(1)(20), vitamin K(1)(25), and vitamin K(1) epoxide in n-hexane and ethanol solutions by gas chromatography/mass spectrometry (GC/MS) utilizing a DB-5 MS fused-silica capillary column. In ethanol solutions of K(1), we observed an extra peak eluting from the GC column with somewhat longer retention time than K(1)(20). A similar peak following K(1)(25) was also found. These peaks were not found in n-hexane solutions of K(1). A close examination of the mass spectra of these peaks indicated that they were vitamin K(1) variants containing a base peak at m/z 225 characteristic of the methylnaphthoquinone ring with a four-carbon side chain. In addition, they contained the molecular ions of K(1)(20) and K(1)(25), respectively. We conclude that K(1)(20) and K(1)(25), but not K(1) epoxide, might undergo rearrangements in ethanol involving an intramolecular proton transfer and a shift of the beta,gamma-double bond on the phytyl side chain toward the ring. The conjugation of the phytyl double bond with the quinone ring is probably the driving force of the rearrangement. We emphasize, however, that our conclusion is based only on mass spectral analysis and would require further investigation by other spectroscopic methods.
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http://dx.doi.org/10.1002/rcm.3807DOI Listing
December 2008

Plasma S/R ratio of warfarin co-varies with VKORC1 haplotype.

Blood Coagul Fibrinolysis 2007 Apr;18(3):293-6

Department of Clinical Chemistry, Laboratory Medicine, University Hospital, Linköping, Sweden.

We recently reported that the low-dose VKORC1*2 haplotype is an important genetic determinant for warfarin dose requirement and is associated with difficulties to attain stable therapeutic prothrombin time--International Normalized Ratio in patients undergoing anticoagulation therapy. The aim of this study was to investigate whether patients with VKORC1*2 compared with patients carrying high-dose haplotypes VKORC1*3 or VKORC1*4 had different warfarin S/R ratios in their plasma, and whether that was related to CYP2C9 variants CYP2C9*2 and CYP2C9*3 or other factors. Samples from patients previously haplotyped for VKORC1 and measured for plasma warfarin concentration were genotyped for the CYP2C9 variants CYP2C9*2 and CYP2C9*3. Nonparametric statistical analysis was performed to elucidate whether there was any significant difference in the warfarin S/R ratio between the two patient groups. Our result shows that there is a significant difference (P<0.01) in warfarin S/R ratios between VKORC1*2 and VKORC1*3 or VKORC1*4 patients. This difference did not originate from CYP2C9 variants CYP2C9*2 and CYP2C9*3. We speculate that VKORC1 haplotypes possibly are linked to some unidentified factors involved in the metabolic clearance of warfarin enantiomers. Dose-dependent variations in (S)-warfarin and (R)-warfarin clearance in these patients can also be a probable explanation for the difference in warfarin S/R ratios.
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http://dx.doi.org/10.1097/MBC.0b013e3280444bfdDOI Listing
April 2007

A new high-performance liquid chromatographic method for determination of warfarin enantiomers.

J Chromatogr B Analyt Technol Biomed Life Sci 2005 Nov 6;826(1-2):75-80. Epub 2005 Sep 6.

Department of Clinical Chemistry, Laboratory Medicine, University Hospital, S-58185 Linköping, Sweden.

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.
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http://dx.doi.org/10.1016/j.jchromb.2005.08.011DOI Listing
November 2005
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