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Blood, 12 February 2009, Vol. 113, No. 7, pp. 1535-1542. Prepublished online as a Blood First Edition Paper on November 24, 2008; DOI 10.1182/blood-2008-08-172338.
PLATELETS AND THROMBOPOIESIS Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation1 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON; 2 Division of Hematology/Oncology, Centre Hôspitalier Universitaire Sainte Justine, Montreal, QC; 3 Faculté de Médecine Paris-Ile de France-Ouest, Université de Versailles-St Quentin and Département d'Hématologie, Institut Cochin, Paris, France; and 4 Department of Medicine, McMaster University, Hamilton, ON
Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and  -granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD -granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase II on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of -granule proteins, which was normal. uPA was localized to QPD -granules and it showed extensive colocalization with -granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, -granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with -granule proteins prior to their proteolysis in QPD.
Quebec platelet disorder (QPD) is an unusual inherited bleeding disorder, associated with increased expression and storage of the fibrinolytic enzyme urokinase plasminogen activator (uPA) in platelets and delayed-onset bleeding following trauma or surgery that responds only to fibrinolytic inhibitor therapy.1–3 The genetic cause of QPD has recently been linked to inheritance of a region on chromosome 10 that contains the uPA gene (PLAU).4 The normal uPA in QPD urine5 and plasma (prepared with platelet activation inhibitors),6 and apparent increases in uPA message in platelets (based on Northern blot analysis),2 suggest that the increased uPA in QPD platelets results from increased uPA expression by megakaryocytes.1 However, the expression of uPA by CD34+ progenitors, and by normal and QPD megakaryocytes, at different stages of differentiation, has not been characterized or quantified. Furthermore, it has not been determined whether QPD selectively increases uPA mRNA in platelets or whether it also increases mRNA from VCL and CAMK2G, the flanking genes on chromosome 10 that encode vinculin (a protein normally expressed in platelets)7 and calcium/calmodulin-dependent protein kinase II (CAMK2G), a protein expressed by T lymphocytes8 that has not been studied in platelets.
Normally, blood contains similar molar amounts of uPA and tissue plasminogen activator (tPA) for converting plasminogen to plasmin and only small amounts of uPA in platelets (up to 1.3 ng uPA/109 platelets; reviewed in Diamandis et al1). Unlike normal platelets, QPD platelets contain sufficient uPA (approximately 400-600 ng uPA/109 platelets)2 to trigger extracellular plasmin generation and premature clot lysis when incorporated into forming or preformed clots.6 Within QPD platelets, single-chain (sc) uPA is not evident, as uPA is stored in active forms that include 2-chain uPA (tcuPA) and low-molecular-weight uPA (LMWuPA). In addition, QPD platelets contain uPA complexed with the active forms of platelet plasminogen activator inhibitor 1 (PAI-1), which are consumed in QPD.2 uPA activation within QPD platelets is postulated to result from exposure to plasmin, as QPD platelets, but not plasma, contain elevated levels of plasmin-
To characterize uPA expression during normal and QPD megakaryopoiesis, and investigate uPA storage in QPD
All studies were conducted with approval of the institutional ethics review boards of all participating institutions and in accordance with the Declaration of Helsinki, as last amended in 2004. Sample collection Peripheral blood samples (200 mL/donation) were collected from QPD and healthy control subjects with written informed consent. Samples were collected into sterile acid citrate dextrose anticoagulant (vol/vol = 1:6) containing 1 mM theophylline (Sigma-Aldrich, Oakville, ON), 3 µM prostaglandin E1 (Sigma-Aldrich), and 3 µM aprotinin (Roche Diagnostics, Laval, QC). Isolation of cells from peripheral blood Platelets were harvested from peripheral blood, as previously described (upper two-thirds of platelet-rich plasma; minimal leukocyte contamination was verified by cell counting of selected samples).11 CD34+ hematopoietic stem cells with minimal platelet contamination (< 0.5 platelets per nucleated cell) were isolated from peripheral blood by a modification of the method described for isolating CD34+ cells from cord blood.15 Briefly, after removal of platelet-rich plasma, and density gradient separation using Ficoll,15 mononuclear cells were further purified by an OptiPrep (Axis-Shield, Oslo, Norway; 200g, 12-minute centrifugation) density gradient separation before immunomagnetic isolation of CD34+ cells.15 Mononuclear cells, from Ficoll gradient separations, were also used to obtain control T lymphocytes by immunomagnetic isolation (Human CD3+ selection kit; StemCell Technologies, Vancouver, BC) to quantify CAMK2G mRNA. The final CD34+ cell and T-lymphocyte purities, assessed by flow cytometry,15 were consistently more than 90%. Megakaryocyte cultures
CD34+ cells were cultured with thrombopoietin (TPO), as described,15 except 50 ng/mL recombinant human TPO (PeproTech, Rocky Hill, NJ) was used. Cell expansion was quantified at different stages of culture, as described,15 with viability determined by trypan blue exclusion. Cells were evaluated by flow cytometry, as described, to quantify the percentage that expressed the mature megakaryocyte marker CD41a (glycoprotein Analyses of mRNA by quantitative reverse-transcription polymerase chain reaction Total cellular RNA was extracted from different cells of the same subjects, including CD34+ cells, cultured megakaryocytes, and platelets, using RNeasy Mini Kits (QIAGEN, Mississauga, ON) as recommended. RNase-Free DNase (QIAGEN) was added to digest any contaminating DNA. Quality and quantity of extracted RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON). Isolated RNA was stored at –80°C until analyzed. For reverse transcription (RT), 72 ng total RNA was incubated at 37°C for 60 minutes in a 20-µL reaction mixture containing recommended Omniscript RT Kit components (QIAGEN), 2.5 µM oligo (deoxythymidine)20 primer (Invitrogen, Burlington, ON), and 20 units RNAse inhibitor RNaseOUT (Invitrogen). Reactions were stopped by a 5-minute incubation at 95°C, followed by rapid cooling on ice.17 RNA from CD34+ cells was used only for quantitative real-time polymerase chain reaction (qPCR) analysis of PLAU transcription, as the quantities harvested precluded other analyses. RNA from day-7 and -13 megakaryocytes and platelets was used for qPCR analysis of PLAU, VWF (control for increased mRNA during megakaryocyte differentiation18), and VCL7 transcription. RNA from platelets was also used to evaluate CAMK2G transcription in QPD. Transcription of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was evaluated simultaneously in all samples as an endogenous control to correct for potential variations in template RNA, RT, and qPCR efficiencies. Supplies and instructions for qPCR were from Applied Biosystems (Foster City, CA). The gene-specific sets of oligonucleotide primers and fluorescent probes for qPCR (from predeveloped TaqMan Gene Expression Assays) were as follows: uPA: Hs00170182_m1; VWF: Hs00169795_m1; vinculin: Hs00243320_m1; CAMK2G: Hs00538454_m1; and GAPDH: 4333764T. All selected amplicons spanned intron-exon boundaries. qPCR singleplex reaction mixtures included: 5 µL cDNA, 12.5 µL TaqMan Gene Expression Master Mix (containing DNA polymerase), 1.25 µL 20x TaqMan Gene Expression Assay reagents, and 6.25 µL RNAse-free water. Assays were done in 96-well TaqMan optical reaction plates in triplicate (or duplicate if there were limiting quantities of template RNA and cDNA) using an ABI PRISM 7900HT real-time thermal cycler (Applied Biosystems), with the following thermal profile: 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. No template controls served to exclude contamination. For each qPCR run, standard curves were obtained by amplifying serially diluted (1:5-1:3125) cDNA that was reverse-transcribed from pooled samples. For uPA, vinculin, and VWF, a pool of total platelet RNA from 5 QPD subjects was used. For CAMK2G, a pool of total T-lymphocyte RNA from 5 control subjects was used. Relative mRNA levels were obtained using the average value for the target gene threshold cycle (Ct), normalized to GAPDH Ct, as described.19 Protein analyses Cell lysates for protein analysis were prepared from CD34+ cells, cultured megakaryocytes, and platelets, by methods described.2,15 Cell-free supernatants were harvested from megakaryocyte cultures on day 7 and day 13, and for some studies daily from days 7 to 13, as described.16 Samples were stored at –80°C until analyzed. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify the following: uPA (modified to include a lower concentration standard of 12.5 pg/mL) and PAI-1 (Oncogene Science, Cambridge, MA); uPA-PAI-1 complexes and platelet factor 4 (PF-4; Hyphen Biomed, Neuville-sur-Oise, France); tPA (lower standards used as described)2; uPA receptor (uPAR; American Diagnostica, Montreal, QC); TSP-111; MMRN111; and VWF.15 Cell lysate and culture supernatant results were expressed in quantities per 106 cells, or per milligram of total cellular protein (comparisons of megakaryocyte and platelet lysates).15 Due to the low numbers of CD34+ cells in peripheral blood, a pooled QPD CD34+ cell lysate from 3 donors was compared with 3 pooled control CD34+ cell lysates (each from 3 donors), to determine whether the protein quantities for QPD samples were within the range for control samples. Western blotting was used to evaluate the mobilities of uPA, TSP-1, VWF, P-selectin, and PAI-1 in platelet lysates (10 µL/lane) and cultured megakaryocyte lysates (45 µL/lane; pooled from 3 individuals to obtain sufficient material) after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), as described.2,9,11,15 Platelet VEGF was also analyzed by Western blotting (primary antibody: 1 µg/mL mouse anti-VEGF [Cedarlane, Burlington, ON]; secondary antibody: 1:25 000 horseradish peroxidase–labeled donkey anti–mouse IgG [Jackson ImmunoResearch Laboratories, West Grove, PA]) after separation (reduced) on a 12% SDS-polyacrylamide gel. Immunofluorescent microscopy
Immunolabeling.
Cultured megakaryocytes and platelets were double labeled as described,16 except resting platelets (2 x 108/mL, prepared as described2) were fixed in suspension (1% paraformaldehyde, room temperature, 10 minutes) and dried on aptex-coated slides overnight, before permeabilization, quenching, and blocking as described.16 Primary antibodies included the following: mouse antibodies to uPA (10 µg/mL; Monosan, Uden, The Netherlands), TSP-1 (CH-1, 3.5 µg/mL),20 MMRN1 (JS-1, 10 µg/mL),20 Image acquisition and processing. Immunolabeled samples were viewed on a DMI6000B wide fluorescent microscope (Leica Microsystems, Richmond Hill, ON) with an Orca ER-AG camera (Hamamatsu, Bridgewater, NJ) and Volocity 4 acquisition software (Improvision, Waltham, MA). Fluorescent crossover between channels was verified to be negligible. Z-series were acquired at 22°C with 100x/1.4NA DIC oil-plan apo objective, using identical microscope settings for samples and negative controls. Images were restored in Volocity 4 using the iterative deconvolution function,21 before importing into MBF-ImageJ22 (McMaster University Biophotonics Facility, http://www.macbiophotonics.ca/imagej/) for linear, uniform brightness and contrast adjustments, background subtraction, and scale bar labeling. Quantitative colocalization analysis. The degree of labeled protein colocalization was assessed using the MBF-ImageJ Intensity Correlation Analysis plugin to determine the Pearson correlation coefficient (rp, range: –1 to 1, with 1 equivalent to complete colocalization)23 and the Manders overlap coefficient (r, range: 0 to 1, with 1 equivalent to complete colocalization).24 A minimum of 10 day-13 cultured megakaryocytes and 30 platelets were evaluated for each protein comparison and data were confirmed using cells from 2 additional subjects. Immunoelectron microscopy To determine the subcellular distribution of uPA, frozen thin sections of platelets were prepared and immunolabeled, as described15 with rabbit anti-uPA (Monosan) and a gold-coupled secondary antibody (British Biocell, Cardiff, United Kingdom). After counterstaining with uranyl acetate, platelets were examined with a Philips CM10 electron microscope (Philips Healthcare, Surenes Cedex, France). Statistical analysis Most quantitative data were expressed as mean plus or minus SEM (range) and compared by 2-tailed unpaired t test. Immunolocalization data (rp and r; expressed as mean ± SD, range) were analyzed by Welch ANOVA with Satterthwaite t post-hoc analysis. Significance was established at P less than .05.
Characteristics of control and QPD-cultured megakaryocytes
QPD and control megakaryocytes showed the typical phenotype and expansion of megakaryocytes grown in culture with TPO from peripheral blood progenitors25 (fold expansion: 8.3 ± 0.8 [range, 5.3-10.0] for 6 QPD cultures; 7.4 ± 2.6 [range, 1.3-16.5] for 5 control cultures; P = .8). Although QPD cultures contained a lower proportion of cells expressing Evaluation of gene expression in CD34+ cells, cultured megakaryocytes, and platelets RT-qPCR analyses indicated that uPA mRNA was not increased in QPD CD34+ cells (Figure 1A); however, it was increased in day-7 (3.7 ± 0.5-fold higher than controls) and day-13 (101 ± 31-fold higher than controls) cultured QPD megakaryocytes (Figure 1A), and in QPD platelets (90.1- ± 18.6-fold higher than control platelets; Figure 1A). Unlike uPA mRNA, VWF and vinculin mRNA were not increased in QPD platelets or megakaryocytes (Figure 1A). In addition, CAMK2G mRNA was not increased in QPD platelets (Figure 1A).
Production of uPA and other proteins during QPD megakaryopoiesis Pooled QPD CD34+ cells contained approximately 60 pg uPA/106 cells, which was within the range observed for pooled control samples (n = 3; 40 ± 20 pg/106 [range, 10-70 pg/106]). Although day-7 and day-13 QPD megakaryocyte cultures contained normal amounts of PAI-1, PF-4, TSP-1, VWF, and MMRN1, they contained increased amounts of uPA (Table 1). Some day-13 QPD megakaryocyte cultures also contained small amounts of uPA–PAI-1 complexes (Table 1). The increased uPA production by QPD megakaryocytes coincided with the increased production of PF-4, TSP-1, and VWF in QPD and control cultures (Figure 1B). By day 13, QPD megakaryocytes contained 19% to 27% of the QPD platelet uPA/mg cellular protein (paired analyses of samples from 4 QPD individuals). tPA was undetectable (< 1.6 ng/mL) in all CD34+ cells (n = 3 QPD and 3 control lysates) and megakaryocyte cultures (n = 4 QPD and 6 control day-7 and day-13 lysates and supernatants), indicating that uPA was the only plasminogen activator produced during megakaryopoiesis.
Plasminogen was undetectable in all megakaryocyte cultures (< 0.8 ng/mL in n = 3 QPD and 3 control day-13 lysates and supernatants) unless the cells were cultured in media containing plasma (ng/106 cells, in lysate from cultures with plasma, n = 3: QPD: 73 ± 13 [range, 46-90], control: 27 ± 8 [range, 11-36]; P = .06). The amounts of uPAR in cultured megakaryocytes were not quantified as there were similar, low amounts of uPAR antigen in control and QPD platelets (ng/109 platelets, n = 4: QPD: 0.7 ± 0.1 [range, 0.6-0.9]; control: 0.4 ± 0.1 [range, 0.2-0.9]; P = .1), which were undetectable by immunostaining of platelets and megakaryocytes (not shown). Distribution of uPA in cultured QPD megakaryocytes and platelets
Immunofluorescent microscopy (Figure 2) indicated that there was strong uPA labeling of all differentiated QPD cells that showed strong labeling for
Immunoelectron microscopy of platelets confirmed that uPA was stored in QPD -granules, without significant labeling of other platelet structures (Figure 3A) or control platelets (not shown). Further analyses by immunofluorescent microscopy indicated that there was extensive colocalization of QPD platelet uPA with TSP-1 (Figure 3B; Table 2) and plasminogen (Figure 4Q plt; Table 2), and extensive but less complete colocalization of QPD platelet uPA with VWF, VEGF (Figure 3B; Table 2), osteonectin, fibrinogen, and factor V (Table 2). Colocalization of QPD platelet uPA with MMRN1 could not be quantified, as unlike QPD megakaryocytes (Figure 2), QPD platelets showed only very faint immunolabeling for MMRN1 (not shown). Western blot analyses indicated that like other -granule proteins, QPD platelet VEGF was abnormally degraded as QPD platelets contained decreased amounts of VEGF (Figure 3C) that also had an abnormally lower mobility (bands indicated by arrow in Figure 3C that were evident in all QPD samples on longer exposures not shown).
In day-13 QPD megakaryocytes cultured with plasma, plasminogen was found predominantly in peripheral structures and not in uPA-containing granules (Figure 4Q MK). Quantitative analyses confirmed that there was less colocalization of uPA and plasminogen in day-13 QPD megakaryocytes (rp: 0.39 ± 0.08 [range, 0.30-0.53]; r: 0.67 ± 0.07 [range, 0.55-0.74]) compared with QPD platelets (Table 2; P < .001). These data indicated that there was incomplete trafficking of plasminogen into uPA-containing structures in cultured QPD megakaryocytes compared with QPD platelets.
Forms of uPA and In contrast to QPD platelets, which contained mostly tcuPA, day-13 QPD cultured megakaryocytes contained mainly scuPA (Figure 5A), even when cultured with plasma (not shown). Like QPD platelets, QPD megakaryocytes contained some high-molecular-weight uPA complexes (Figure 5A, bands indicated by * and **) that included forms (*) recognized by PAI-1 antibodies (data not shown), as suggested by uPA–PAI-1 complex ELISA (Table 1). Control megakaryocytes contained undetectable uPA by Western blotting (not shown).
Unlike the degraded -granule proteins in QPD platelets, the TSP-1, VWF, and P-selectin in day-13 QPD cultured megakaryocytes, grown with or without added plasma, had the mobility of the normal forms in control platelets (Figure 5B) and megakaryocytes (not shown). QPD megakaryocytes also contained normal quantities of MMRN1, even when cultured in media with plasma (Table 1 and data not shown). Analyses of cultures with plasma, by Western blotting, indicated that there was no detectable conversion of plasminogen to plasmin in QPD megakaryocytes (not shown), unlike QPD platelets.
The magnitude and timing of increased uPA transcription and storage during QPD megakaryopoiesis has been uncertain. Our study indicates the differentiation of QPD CD34+ progenitors into megakaryocytes results in differentiation-dependent, log-fold increases in uPA message and protein, without increasing VWF, vinculin, or CAMK2G expression (Table 1; Figure 1). Interestingly, the temporal increased production of uPA, during QPD megakaryopoiesis, mirrored the increased production of -granule proteins, which were made in normal quantities (Table 1; Figure 1B) and costored with uPA within QPD -granules (Figures 3,4; Table 2). We found that unlike QPD platelets, QPD megakaryocytes contained undegraded -granule proteins and scuPA (Figure 5), which has low catalytic activity.26 These data, and the different distributions of plasminogen in cultured QPD megakaryocytes compared with platelets (Figure 4), suggest that the activation of uPA and proteolysis of -granule proteins occur late, after plasminogen traffics into QPD -granules and is converted to plasmin. Importantly, our study provides new evidence that profibrinolytic abnormalities of QPD platelets reflect increased expression of the uPA gene as hematopoietic progenitors differentiate into megakaryocytes, without altering expression of the flanking genes on chromosome 10 that encode vinculin and CAMK2G.
The expression of genes during hematopoietic stem cell differentiation into megakaryocytes is highly regulated and coordinated by transcription factors synthesized during megakaryopoiesis (for reviews, see Battinelli et al,27 Pang et al,28 and Chang et al29). This likely contributes to the sustained low levels of uPA expression, and increased expression of PAI-1 and other
Heterogeneity has been noted in the contents of normal platelet
The exposure of plasminogen to scuPA is known to generate plasmin.26 Our current study provides indirect evidence that the uptake of plasminogen for costorage with uPA is required to generate sufficient plasmin to trigger
Our current study establishes that overexpression of uPA in QPD emerges as QPD hematopoietic progenitors differentiate into megakaryocytes without increasing expression of the flanking genes or the production of other
Contribution: D.K.V. recruited subjects, designed and performed experiments, interpreted results, and wrote the paper; G.E.R. recruited subjects and participated in writing of the paper; M.D. and J.B. did experimental work and participated in writing of the paper; E.M.C.-B. designed experiments, interpreted results, and participated in writing of the paper; and C.P.M.H. supervised the project, designed experiments, interpreted results, and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Catherine P. M. Hayward, McMaster University Health Sciences Centre, Room 2N30, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada; e-mail: haywrdc{at}mcmaster.ca.
The authors thank Ms Francine Derome for sample collections, Mr Jean-Marc Massé for electron microscopy, and Dr Kalathil Suresh for help with planning RT-qPCR experiments. This work was supported by grant T5888 from Heart and Stroke Foundation of Ontario, Toronto, ON (C.P.M.H.) and Bayer Canada, Toronto, ON (G.E.R.). D.K.V. is the recipient of a Canadian Institute of Health Research/Heart and Stroke Foundation of Canada Focus on Stroke Doctoral Research Award, Ottawa, ON. M.D. is the recipient of an Ontario Graduate Student Scholarship, Thunder Bay, ON. C.P.M.H. is the recipient of a Career Investigator Award from Heart and Stroke Foundation of Ontario, Toronto, ON, and a Canada Research Chair in Molecular Hemostasis from the Government of Canada, Ottawa, ON.
Submitted August 21, 2008; accepted October 27, 2008.
Prepublished online as Blood First Edition Paper, November 24, 2008
DOI: 10.1182/blood-2008-08-172338
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Abstract
Introduction
B activation. Mol Cell Biol. 2006;26:5497–5508.
