Preparazioni piastriniche per studi di trascrittomica: caccia al leucocit

E. Tirloni

E. Tremoli

F. Centritto

G. Colombo

L. Facchinetti

M. Brambilla

M. Camera

P. Canzano

AIR Universita degli studi di Milano

1PREPARAZIONI PIASTRINICHE PER STUDI DI TRASCRITTOMICA:CACCIA AL LEUCOCITA M. Brambilla, E. Tirloni, P. Canzano, G. Colombo, F. Centritto, L. Facchinetti, E. Tremoli, M. CameraCentro Cardiologico Monzino IRCCS, Milano Dip. di Scienze Farmacologiche,  Università degli Studi di Milano11° Riunione Gruppo Studio PiastrineGazzada, 3 - 5  Ottobre 2010TISSUE FACTOR and PLATELETS1-Leukocyte contamination in platelet RNA preparations2-IT’S TIME TO TAKE STOCK OF…..BLOOD, 5 AUGUST 2010  VOLUME 116, NUMBER 5To the editor:No evidence for tissue factor on plateletsBeth A. BouchardDepartment of Biochemistry, University of Vermont College of Medicine,Burlington, VTKenneth G. MannDepartment of Biochemistry, University of Vermont College of Medicine,Burlington, VTSaulius ButenasDepartment of Biochemistry, University of Vermont College of Medicine,Burlington, VT…..to resolve this ONGOING CONTROVERSY….…. platelets express active TF after de novo synthesis,2,3 transfer from monocytes,4or release from -granules.5In other studies,6,7 however, the existence of platelet TF could not be demonstrated.2. Panes O et al. Human platelets synthesize and express functional tissue factor. Blood. 20073. Schwertz H, et al. Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets. J Exp Med. 20064. Falati S, Liu Q, Gross P, et al. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand1 and platelet P-selectin. J Exp Med. 20035. Siddiqui FA et al. The presence and release of tissue factor from human platelets. Platelets. 20026. Butenas S, et al. Tissue factor activity in whole blood. Blood. 2005.7. Osterud B et al. What is blood borne tissue factor? Thromb Res. 2009.2Flow cytometric analyses indicated that no TF was expressed by PAR1- and PAR4-activated platelets compared with unactivated platelets  despite maximal -granule release (98% P-selectin–positive platelets)….…no TF antigen was detected by immunoassay after prolonged stimulation of platelets with LPS.No FXa was generated by extrinsic FXase using unstimulated or stimulated platelets as a possible TF source. Similarly, no clot was formed in a plasma-based clotting assay.TF on monocytes or monocyte-derived microparticles is not transferred to platelets.RESULTS:BLOOD, 5 AUGUST 2010  VOLUME 116, NUMBER 5Based on these observations, we conclude that platelets do not express detectable TF antigen or activity. Discrepancies between our data and those published by others may be a result of the assays used to quantify TF antigen and activity in different laboratories. Our assays use specific and highly sensitive anti-TF monoclonal antibodies and physiologically relevant standards and controls, whereas other reported assays use combinations of monoclonal and polyclonal antibodies, which may recognize TF fragments or cross-react with other proteins…..CONCLUSION:BLOOD, 5 AUGUST 2010  VOLUME 116, NUMBER 5? ? ? ?METHODS in figure legend:BLOOD, 5 AUGUST 2010  VOLUME 116, NUMBER 5Figure 1. Search for TF on platelets by flow cytometry. (A) Washed platelets were activated with PAR1 (100µM) and PAR4 (500 µM) agonist peptides (2 hours, 37°C). (B) Platelet-rich plasma was incubated with THP-1 cells in the presence or absence of 250 ng/mL LPS (4 hours, 37°C)……Eureka! ! ! !To the Editor:Tissue factor expression on platelets is a dynamic eventMarina CameraDept. of Pharmacological Sciences, Univ. degli Studi di Milano and Centro Cardiologico Monzino IRCCS, MilanMarta BrambillaCentro Cardiologico Monzino IRCCS, MilanVincenzo ToschiDivision of Haemat and Blood Transfusion and Thrombosis Centre, AO Ospedale San Carlo Borromeo, MilanElena TremoliDept. of Pharmacological Sciences, Univ. degli Studi di Milano and Centro Cardiologico Monzino IRCCS, Milan3RESULTS:Our results confirm that the amount of TF present on the platelet surface, after 2 hours stimulation with PAR-1agonist peptide and TRAP-6, is negligible (..) (Figure 1). After a 15 minutes stimulation, however, PAR-1 andTRAP-6 significantly increase platelet TF expression (..) compared with resting platelets (...)• This indicates that TF expression by platelets in response to certain agonists is an early event, confirming data already published by our group2,3.(REF: Camera et al, ATVB 2003 and ATVB 2008)CONCLUSION:• Our data support the evidence that platelets do express TF as a result of a rapid and dynamic process. Thus its detection may considerably vary if observed at different time points after in vitro cell stimulation.Oggetto: Decision on your Letter to the Editor #BLOOD/2010/307306Title: Tissue factor expression on platelets is a dynamic eventDear Dr. Camera:I am pleased to inform you that your Letter to the Editor has been accepted forpublication in the next available issue of Blood Journal.…………..Sincerely, Cynthia E. Dunbar, M.D.Editor-in-Chief, BloodTISSUE FACTOR and PLATELETS1-Leukocyte contamination in platelet RNA preparations2-IT’S TIME TO TAKE STOCK OF…..4Manuscript Number: TR-D-10-00225R1Gene expression profiling reveals multiple differences in platelets from patients with stable angina or non-ST elevation acute coronary syndromeGualtiero Colombo, Karl Gertow, Giancarlo Marenzi, Marta Brambilla, Monica De Metrio,Elena Tremoli and Marina CameraCentro Cardiologico Monzino IRCCS; Department of PharmacologicalSciences, Università degli Studi di Milano, Milan, ItalyREVISION“The biggest challenge with platelet RNA WGE studies is the contamination with leukocyte RNA. The RNA content of a single leukocyte is estimated to be 3-4 log higher than that of a platelet.  The authors are aware of this drawback and comment on this several times.  They have chosen an arbitrary limit for leukocyte contamination with not more than 5 leucocytes per 105 platelets.  This is a rather liberal limit and the results of Q-PCR tests with myeloid and lymphoid markers have not been shown (bottom of page 8).  There are other means to inspect platelet WGE studies for signals that have been produced by RNA from contaminating leucocytes.Each blood cell type has its own set of "strictly lineage-specific transcripts" and these could have been used to provide reassurance that leukocyte contamination is low enough to not be a confounding factor in the analysis.”Reviewer comment:Methods – Platelet isolation•40 ml blood in ACD/patient•Platelet isolation by sequentialcentrifugation of PRP (top third) withPGE1 and EDTA• Assessment of platelet activation (FACS)• Assessment of leukocyte contamination• RNA extraction and quality controlTopthird10 0 10 1 10 2 10 3 10 4Mouse IgG1 FITCCD45PERCP+CD3FITC norma.006100 101 102 103 104Mouse IgG1 PECD45PERCP+CD20PE.008LINFO BLINFO NON B10 0 10 1 10 2 10 3 10 4Mouse IgG1 PerCPCD14PERCP+CD16PE+ISO FITC.004R2100 101 102 103 104Mouse IgG1 FITCCD45PERCP+CD66FITC.005GRANCD45PerCPCD45PerCPCD45PerCPCD45PerCPCD66b CD20CD3CD14MonocytesGranulocytesLimphocytes TLimphocytes BLeukocyte population markers5GranulocytesMonocytesLimphocytesSet the quadrant Open in G1Only preparations with less than 5 leukocytes/105 platelets were used for RNA isolationAssessment of leukocyte contamination by flow cytometryG11.3 (29%)0.6 (24%)1.3 (35%)0 (71%)0 (76%)0 (65%)NSTE-ACS patients1 (24%)1 (24%)2.4 (30%)0 (76%)0 (76%)0 (70%)Stable angina patientsLymphocytes(CD2+ + CD20+)Monocytes(CD14+)Granulocytes (CD66b+)Mean number of contaminating WBC in 105 platelets, (% of patients)Platelet Preparations used for Microarray, PCR and Western blot experimentsMean number of contaminating WBC in 105 platelets0.7 (100%)2.5 (67%)5.2 (100%)0 (0%)0 (33%)0 (0%)NSTE-ACS patients1.7 (64%)0.8 (38%)5.6 (100%)0 (38%)0 (64%)0 (0%)Stable angina patientsLymphocytes(CD2+ + CD20+)Monocytes(CD14+)Granulocytes (CD66b+)Mean number of contaminating WBC in 105 platelets, (% of patients)Discarded Platelet Preparations1.3 (29%)0.6 (24%)1.3 (35%)0 (71%)0 (76%)0 (65%)NSTE-ACS patients1 (24%)1 (24%)2.4 (30%)0 (76%)0 (76%)0 (70%)Stable angina patientsLymphocytes(CD2+ + CD20+)Monocytes(CD14+)Granulocytes (CD66b+)Mean number of contaminating WBC in 105 platelets, (% of patients)Platelet Preparations used for Microarray, PCR and Western blot experimentsMean number of contaminating WBC in 105 plateletsImageStreamx: multispectral imaging flow cytometer (AMNIS)“By combining the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insight of microscopy, the ImageStreamXovercomes the limitations of both techniques (…)”6Washed platelets Whole blood monocytesCD14 PerCPSSC FITC PE PerCPBFlymphocytesCD3-20 PESSC FITC PE PerCPBFgranulocytesCD66 FITCSSC FITC PE PerCPBFFILTRI Purecell PL (PALL)- Standardization-Time- Platelet recovery- Efficiency of leukocyte removal- Cost- Aggregates removalNI1 hour400 x106<5 leuko /105 plt1 euro/eachNOYES30 min500 x106<5 leuko /105 plt18.40 euro/eachYESTyrode buffer Purecell filterCell sorting NOZZLEPRESSIONEMASCHERA20.000 ev/secGATING STRATEGY- Frequence: 78,1- Drop 1: 260- Gap: 8- Drop Delay: 36,24- Voltage plate: 6000 THRESHOLD70 μm70 PSI0 32 0100 mil ev/ ~2ore7LEUKOCYTE CONTAMINATIONGranulocyte gate (CD45+/CD66+): 0 eventsLimphocyte gate (CD45+/CD20+/CD3+): 0 eventsMonocyte gate (CD45+/CD14+): 0 events- Standardization- Blood volume- Time- Platelet recovery- Efficiency of leukocyte removal- Cost- Specialized technicianNI30 ml1 hour700 x106<5 leuko /105 plt1 euroNOYES5 ml3 hours100 x1060 leuko /105 plt>100 euroYESTyrode buffer SortingCELL SORTING vs WASHING PROCEDUREPAC-10,00,51,0% positive cellsWhole Blood PRP Post SortingCD620,00,51,01,52,0% positive cellsWhole Blood PRP Post SortingNO GP IIbIIIa activationbutdegranulationPOST-SORTING platelet activationPlatelet RNAAssessment of platelet RNA qualityHUVECLADDERVirtual gelPlateletsHUVECElettropherograms18S28S18S28S18S28S8RT-PCRLeukocyte  contamination of the platelet RNA preparations was assessed by amplification of specific markers for T- and B-lymphocytes (CD2 and CD20, respectively), monocytes (CD14), granulocytes (CD66b), and pan-leukocytes (CD45) by RT-PCR.Platelet isolationAssessment of platelet activation (FACS)Assessment of leukocyte contaminationRNA extraction and quality controlMicroarray experimentsMicroarray experimentsNo PBMC signal was detected in PLT preparationsSYMBOL Gene 1 2 3 4 5 6 7 8 9 1 2 PLTavg PBMCavgCD14 CD14 177,37 127,28 173,98 160,45 135,79 246,94 116,53 238,39 294,25 32191,74 30427,67 185,66 31309,71DUSP1 dual specificity phosphatase 1 71,62 86,98 122,31 106,37 102,45 111,64 102,94 171,63 274,06 21102,30 23133,63 127,78 22117,97FCN1 ficolin 135,49 123,21 187,48 151,90 129,41 216,99 126,45 289,58 418,81 25122,01 28195,81 197,70 26658,91HLA‐DRB4 HLA‐DRB4 153,01 116,47 174,56 127,97 115,80 181,80 128,24 178,44 429,92 22961,42 18029,95 178,47 20495,69ITGB2 integrin, beta 2 (antigen CD18 (p95) 189,66 132,15 202,59 155,13 126,41 205,24 125,22 328,13 527,22 27826,67 32995,37 221,31 30411,02LYZ lysozime 361,70 156,90 440,73 154,19 200,61 353,89 256,93 408,17 689,61 51601,36 51003,24 335,86 51302,30Background value=100Positive signal threshold: at least 3 times the background value.PLATELETS PBMCHighest PBMC expressed genes defined as signal intensity > 20000: 227Excuse me!!Have you seena leukocyte?

Preparazioni piastriniche per studi di trascrittomica: caccia al leucocita

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Preparazioni piastriniche per studi di trascrittomica: caccia al leucocita

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