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HEMATOPOIESIS
From the The Children's Hospital of Philadelphia,
Department of Pediatrics and Pathology and Laboratory Medicine at the
University of Pennsylvania, and the Cardeza Foundation for Hematologic
Research, Departments of Medicine and Pediatrics, Jefferson Medical
College, Philadelphia, Pennsylvania.
The genes for the related human (h) chemokines, PBP (platelet basic
protein) and PF4 (platelet factor 4), are within 5.3 kilobases (kb) of
each other and form a megakaryocyte-specific gene locus. The hypothesis
was considered that the PBP and PF4 genes share a common distal regulatory region(s) that leads to their high-level megakaryocyte-specific expression in vivo. This study examined PBP and
PF4 expression in transgenic mice using 4 distinct human PBP/PF4 gene locus constructs. These studies showed that
within the region studied there was sufficient information to regulate tissue-specific expression of both hPBP and hPF4. Indeed this region
contained sufficient DNA information to lead to expression levels of
PBP and PF4 comparable to the homologous mouse genes in a
position-independent, copy number-dependent fashion. These studies
also indicated that the DNA domains that led to this expression were
distinct for the 2 genes; hPBP expression is regulated by a region that
is 1.5 to 4.4 kb upstream of that gene. Expression of hPF4 is regulated
by a region that is either intergenic between the 2 genes or
immediately downstream of the hPF4 gene. Comparison of the
available human and mouse sequences shows conserved flanking region
domains containing potential megakaryocyte-related transcriptional factor DNA-binding sites. Further analysis of these regulatory regions
may identify enhancer domains involved in megakaryopoiesis that may be
useful in the selective expression of other genes in megakaryocytes and
platelets as a strategy for regulating hemostasis, thrombosis, and inflammation.
(Blood. 2001;98:610-617) Platelet basic protein (PBP) and platelet
factor 4 (PF4) are related, platelet-specific chemokines that are
expressed at high levels during megakaryopoiesis and stored in platelet
Although the biologic roles of these chemokines need further
investigation, their genes offer an opportunity to understand megakaryocyte-specific expression. Like many other genes encoding chemokines, both the PBP and PF4 genes are
encoded by 3 exons, and are preceded by a TATA box in the immediate
5'-flanking region.10,11 Transient transcriptional studies
with the immediate 5'-flanking region defined a PU.1-binding promoter
region upstream of the PBP gene,12 similar to a
thrombopoietin-induced enhancer domain upstream of the
platelet-specific [alpha]IIb
gene.13 Several silencers and promoter domains
have been similarly defined in the immediate 5'-flanking region
of the PF4 gene, including one GATA-1 binding
site.14,15 Reporter gene constructs with 245 bp of the
immediate 5'-flanking region of the hPF4 gene showed that
this region contained sufficient information to drive tissue-specific expression of a LacZ reporter construct16 and that 1.1 kb
of rat PF4 promoter can drive vigorous tissue-specific
expression.17 However, neither of these constructs had
sufficient information to drive position-independent, copy
number-dependent expression.
The genes for PBP and PF4 are closely linked on chromosome 4 with
the PBP gene upstream and both transcripts in the same 5' to
3' orientation.18-20 Because the 2 genes are both
expressed at high levels in developing megakaryocytes, we asked whether the 2 genes are regulated by a common distal regulatory region, similar
to those located flanking the DNA clones studied
The mouse (m) PF4 gene was isolated from mouse 129 SV Establishment of transgenic mice
Positive founder lines were also analyzed by Southern blots as
previously described by us.20 Genomic DNA was digested
with EcoRI and separated on a 0.8% (wt/vol) agarose
gel along with 5 and 10 pg unlabeled probe DNA as controls. Two probes
were radiolabeled using a Random Primers Labeling Kit (Boehringer
Mannheim, Indianapolis, IN). One was a 1.8-kb hPBP genomic fragment to
detect the transgene and the other 1.3 kb fragment was an m Reverse transcription-polymerase chain reaction analysis
Blood was drawn from mice by cardiac puncture and spun at 200g for 10 minutes to obtain platelet-rich plasma (PRP). The platelets were obtained by centrifugation at 800g for 10 minutes. Platelet RNA were isolated using RNA STAT-60 as described above. Reverse transcription-polymerase chain reaction (RT-PCR) was done using the Superscript II Reverse Transcriptase Kit (Life Technologies, Gaithersburg, MD) following the procedures outlined by the manufacturer (which includes no RT-enzyme control samples). RT-PCR used the following sets of sense/antisense primers: hPBP: 5'-ATGAGCCTCAGACTTGATAC-3'/5'-ATCAGCAGATTCATGACCTG-3'10 mPBP: 5'-GCCTGCCCACTTCATAACCTC-3'/5'-GGGTCCAGGCACGTTTTTTG-3' hPF4: 5'-ATCGCACTGAGCACTGAGATC-3'/CTATATAGCAAATGCACACACG-3'11 mPF4: 5'-GTCCAGTGGCACCCTCTTGA-3'/5'-AATTGACATTTAGGCAGCTGA-3' m mHPRT: 5'-CACAGGACTAGAACACCTGCH-3'/5'-GCTGGTGAAAAGGACCTCT-3'23 The use of RT-PCR to obtain quantitative data regarding the expression of the transgene relative to an endogenous gene requires comparison of products at points when both are in the linear range of amplification. For quantitative studies, the antisense primers were 5'-labeled with fluorescent dye Cy-5 (Integrated DNA Technologies, Coralville, IA). Preliminary experiments showed that the human and mouse PCR products were in the linear range of amplification between cycles 14 to 20. Therefore, PCR reaction mixtures were typically divided into several 15-µL aliquots before initiation of the reaction. PCR was performed at 94°C for 2 minutes, followed by cycles at 94°C for 25 seconds, 50°C for 40 seconds, and 72°C for 40 seconds in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA). At selected cycles aliquots from hPF4 and mPF4 or hPBP and mPBP reactions were removed from the thermocycler and placed on ice. The Cy-5-labeled products were then run on a 10% TBE Ready Gel (Bio-Rad Laboratories, Hercules, CA) and analyzed with a Storm imaging system and Imagequant PhosphorImager software (Molecular Dynamics). The log of the signal intensity of each band was plotted against the cycle number to confirm that the amplification was linear. The signal intensity of the human product was normalized with that of the mouse product to compare the transgene expression levels among the different founder lines. Inclusion of RNase free DNase I (1 U/10 µL reaction, Life Technologies) or DNase free RNase A (1 U/10 µL reaction, Sigma, St Louis, MO) were done in the RT step as controls to verify the RNA nature of the amplified material. Total platelet-derived RNA (1 µg) was added to a PCR tube to which 1 µL 10 × reaction buffer (200 mM Tris-HCl, pH 8.4, 20 mM MgCl2; 500 mM KCl) and 1 µL RNase A or DNase I enzyme was added (total volume = 10 µL). Each tube was incubated at 37°C for 30 minutes. The reaction was stopped by adding 1 µL 25 mM EDTA (pH 8.0) and heating at 70°C for 15 minutes. Oligo dT primer was then added and the RT reaction was followed as described above. To control for differences in efficiency between the primer pairs, the mPBP and mPF4 and the hPBP and hPF4 complementary DNAs (cDNAs) were all cloned into a single pBluescript SK+ vector (Stratagene). Following the same procedure as described above, the semiquantitative PCR was done using this multigene construct as template. The differences of intensity of the amplified cDNA run on the gel, hPBP versus mPBP and hPF4 versus mPF4 at the same PCR cycles in the range of exponential growth of PCR reaction, were used to determine the differences of the primer pairs amplification efficiency. This information was then used to normalize the expression values determined in the quantitative RT-PCR studies. Protein detection Human and mouse platelets were isolated by differential centrifugation of whole blood. Collection of blood from healthy human volunteers was approved by the Institutional Human Review Board at the Children's Hospital of Philadelphia. Samples from both humans and mice were collected in acid-citrate-dextrose (ACD) and centrifuged at 200g for 10 minutes at room temperature as previously described.24 PRP was then collected and prostaglandin E1 (Sigma) was added to a final concentration of 1 µM. Platelets were then obtained by centrifugation at 800g for 10 minutes at room temperature. The pellets were washed twice in 134 mM NaCl, 3 mM KCl, 0.3 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES, 5 mM glucose, 0.1% NaHCO3, and 1 mM EGTA, pH 6.5, and resuspended in the same buffer except without EGTA. The platelets were then lysed by freezing and thawing the sample twice. The protein concentration of each lysate was determined by the Pierce BCA Protein Assay kit according to the manufacturer's instructions (Pierce, Rockford, IL). Approximately 20 µg total platelet protein was electrophoresed on a 12% SDS-polyacrylamide gel (SDS-PAGE) and stained with Coomassie blue. A duplicate SDS-PAGE gel was transferred to a polyvinylidene difluoride (PVDF) Immobilon-P Transfer Membrane (Millipore, Bedford, MA). The hPF4 protein was detected using RTO, a mouse anti-hPF4 monoclonal antibody generously provided by Gow Arepally (University of New Mexico, Albuquerque, NM),25 and the hPBP protein was detected using a polyclonal rabbit anti-hNAP-2 antibody (PEPRO Tech, Rocky Hills, NJ) that does not cross-react with mouse PBP. Western blot signals were visualized by enhanced chemiluminescence (NEN Life Science Products, Boston, MA) and measured using Imagequant PhosphorImager Software (Molecular Dynamics).Immunohistochemical staining for hPF4 Tissues from hPF4 transgenic and littermate control mice were immunostained for hPF4 expression using the anti-hPF4 monoclonal antibody RTO. Briefly, formalin-fixed, paraffin-embedded 5-µm sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was quenched with 0.9% peroxide in methanol and unreactive sites blocked with 10% goat serum in 1 × Automation buffer (Biomeda, Foster City, CA) for 20 minutes at 37°C. Slides were then incubated overnight at 4°C with RTO (1 µg/mL), washed in 1 × Automation buffer, and incubated with biotinylated goat-antimouse antibody (Jackson Laboratories, West Grove, PA) diluted 1:200 at 37°C. Slides were washed and incubated with streptavidin-horseradish peroxidase (HRP; Research Genetics, Huntsville, AL) for 30 minutes at 37°C, then were washed and stable DAB chromogen (Research Genetics) was applied for 5 minutes at 20°C. Slides were counterstained with dilute hematoxylin.
Characterization of the PBP/PF4 gene locus The fact that hPBP and hPF4 form a gene locus has been previously described,20 suggesting that these 2 platelet-specific genes may be coordinately regulated during megakaryopoiesis. We have now completed the characterization of the human PBP/PF4 locus and have cloned and characterized the mouse equivalent of the PBP and PF4 genes (GenBank access numbers AF349465 and AF349466). We found that the human genes are 5.3 kb apart. The murine equivalents are also closely linked with both genes oriented in the same 5' 3' orientation, but with only 3.2 kb in the mice
intergenic region. A dot-matrix comparison of the available human and
determined mouse sequences shows that homology exists not only within
the genes and the immediate 5'- and 3'-flanking regions, but also within several more distal regions as well (Figure 1A). The most 5' of
these conserved regions is actually another CXC chemokine, ENA-78 in
human26 and its apparent mouse homologue,
LIX.27 This gene is also oriented in the same 5'3'
orientation as the PBP and PF4 genes. There are
8.9 kb between ENA-78 and hPBP. No other gene was defined up to 40 kb
3' to the PF4 gene by our analysis. Although the human
genome had been shown to contain a nonfunctional duplication of the
PBP/PF4 gene complex,
 PBP/PF4alt,10,20 we could not find such a
duplication of the mouse genes by analysis of genomic Southern blots
(data not shown).
To study the genetic regulation of this region, 4 human transgenic
constructs from the hPBP/PF4 gene locus were studied (Figure 1B). Short-PBP was a 3.2-kb HindIII fragment that
extended 1.4 kb upstream of the hPBP gene's transcriptional
start site and 0.8 kb downstream of its poly A signal site, containing
only the immediately 5'- and 3'-conserved sequence around PBP.
Long-PBP contains an additional 3.0 kb of upstream region,
which includes the conserved region upstream of the PBP gene
(Figure 1A). The Characterization of transgenic mice: genome copy number and tissue-specific expression The number of founder animals obtained for each construct is also shown in Figure 1B. Except for the -PBP/PF4 construct, at
least 4 founders were obtained for each construct. Copy numbers per
haploid genome are also indicated in Figure 1B, and except for the
-PBP/PF4 construct, cover a range of copy numbers.
The RT-PCR analysis of brain, lung, thymus, heart, liver, spleen, small
intestine, adrenal, kidney, testes, skeletal muscle, marrow, and
purified platelets was done using species-specific primers for hPBP,
hPF4, mPBP, mPF4, m
Interestingly, none of the Short-PBP constructs expressed hPBP message well (Figure 2A and solid squares in Figure 2B). Among the 4 founders, only the 8 and 17 copy lines had any detectable hPBP, and this amounted to less than 1% of concurrently expressed mPBP when analyzed over the exponential range of amplification and then corrected for efficiency of amplification of different primer pairs (Figure 2B). Furthermore, 2 other founder lines of the Short-PBP construct with similar or higher copy numbers (7 or 22 copies) did not have any detectable expression. Thus, although the Short-PBP construct may drive tissue-specific expression, it does so inefficiently and not in a copy number-dependent fashion. To see if the inclusion of the conserved region further upstream of the
hPBP gene was involved in the regulated expression of PBP,
the Long-PBP constructs that contained this region were examined (Figure 1A). All of the transgenic lines from this construct expressed high levels of hPBP message (Figure 2A) that on quantitation was approximately equal to endogenous mPBP expression on a per copy
basis (Figure 2B). The 2 The 4 PF4-Only founder lines expressed high levels of hPF4
message, comparable to endogenous mPF4 levels of message (Figure 3A,B).
The level of expression was position independent, copy number dependent
(Figure 3B) with the construct with the highest copy number having the
highest level of expression. The 2 PBP and PF4 protein expression by the transgenic lines Although considerable effort was made to quantitatively analyze mRNA level in the above experiments, we also measured hPBP and hPF4 protein levels as a secondary determination of expression (Figures 4 and 5). Human platelet proteins from 4 volunteers were used as a positive control. Levels of PBP and PF4 expression were done on a per milligram basis rather than on a per platelet basis because mouse platelets are much smaller than human platelets.28 Protein concentrations were determined by optical density measurements and confirmed by Coomassie blue stained SDS-PAGE gels (data not shown).
Figure 4A is an immunoblot of platelet proteins from 4 human volunteers
and from the Long-PBP and Figure 5A is an immunoblot of platelet proteins from the same human
volunteers and from the PF4-Only and It should be noted that levels of hPBP and hPF4 protein found in a number of lines equal that or exceed that of the proteins found in the tested human samples. For hPBP, the maximum level expressed was 1.5 times the level seen in the human controls, and for hPF4, the maximum level seen was 2.5 times the level seen in the 4 controls. None of the lines studied had decreased viability (being transmitted at the expected mendelian frequency) or had abnormalities in their blood counts (data not shown). Immunohistochemical staining for hPF4 Tissues from PF4 transgenic and wild-type control mice were immunostained for hPF4 expression using RTO. Only the bone marrow and spleen showed positive immunostaining, consistent with the RT-PCR studies mentioned above (data not shown). Figure 6 shows that in the spleen, which is a hematopoietic tissue in mice, only mature megakaryocytes show significant staining in the 10-copy PF4-Only and in the 21-copy-PBP/PF4 animals, whereas megakaryocytes were
unstained in the wild-type control. Of interest is that the platelets
in the -PBP/PF4 spleen are so intensely stained that their punctate staining allows one to readily distinguish the red pulp,
which contains circulating platelets, from the white pulp.
Regulation of megakaryocyte-specific genes has been studied by our laboratory and others to gain insights into the mechanisms underlying megakaryocyte differentiation. Given the paucity of megakaryocytes in bone marrow these studies have previously heavily required the use of cell lines with some, but not complete, megakaryocytic features.30-32 Previous studies of megakaryocyte-specific gene expression in transgenic mice have provided additional insights into this process. However, these studies used reporter gene constructs, which in themselves often contain cryptic regulatory components. Further, these studies have not dealt with the important issues of expression level in megakaryocytes, and copy number dependence and position independence. Our studies have focused on the PBP/PF4 gene locus because both genes are highly expressed, megakaryocyte-specific chemokines and are physically linked to one another on human chromosome 4.20 The studies presented show this tight linkage to be conserved for the mPBP and mPF4 genes. Further, our studies are the first to show close physical linkage to an additional related CXC chemokine gene ENA-78/LIX, located 8.9 and 6.9 kb upstream of the transcription start site of hPBP and mPBP genes, respectively. ENA-78 is also expressed in, but not restricted to, megakaryocytes. These data are consistent with our initial supposition that this region could potentially represent a common expression locus for several platelet-specific chemokines. We also proposed that given their proximity, the PBP and PF4 genes might possess common distal regulatory elements. Phylogenetic footprinting data demonstrated in Figure 1A show that not only are PF4, PBP, and ENA-78 highly conserved across mammalian species, but there are a number of regions outside of the coding regions that are highly conserved. Our data show that the PBP and PF4 genes each contain distinct regulatory regions in their flanking regions that allow for their copy number-dependent and position-independent expression. A region upstream to the PBP gene was necessary for these features for PBP gene expression and may also contribute to an approximate 3-fold enhanced expression of the PF4 gene. Despite this apparent expression augmentation, copy number-contingent high levels of PF4 expression can be achieved independent of this element, being regulated by the flanking regions surrounding the PF4 gene. Further studies are needed to define the molecular basis by which these
5'-flanking regions regulate the efficient expression of these 2 genes.
Such important regulatory elements are likely to be conserved, and
indeed, phylogenetic footprinting data analyses for other genes have
been useful in localizing important distal regulatory
elements,33,34 and may apply to our data as well. For
example, our expression data show that the Short-PBP and
Long-PBP constructs differ only in that the
Long-PBP includes the region 1.4 to 4.4 kb upstream of the
hPBP gene. Thus, this region is necessary for high-level
tissue-specific and copy number-dependent expression of the
hPBP gene. Within this region is a highly conserved approximate 700-bp domain, showing about 60% nucleotide conservation (Figures 1A and 7A). We analyzed this
region in the mouse and human sequences for transcription factor
consensus binding sequences implicated in the control of megakaryocytic
and hematopoietic gene regulation, focusing on GATA-1, NF-E2, Ets,
PU.1, and AML-1 binding sites.35-47 Of note is a
homologously conserved NF-E2 site beginning 3696 (2151) bp upstream of
the PBP start site (with the mouse distance in parenthesis), and a
GATA-1 site 3140 (1644) bp upstream (Figure 7A). Whether either site is
biologically relevant will now need to be tested. Additionally, several
regions contain other GATA-1 and Ets binding sites that are present
within about 60 bp of each other in the mouse and human sequences but
that may nevertheless be functionally significant. Studies of enhancers have classically demonstrated independence for physical attributes such
as distance and orientation. It is possible that there is no
evolutionary constraint in this context to keep these binding sites
precisely aligned, but rather generally present within the boundaries
of the enhancer domain.
Expression of hPF4 from all animals, including the 4 PF4-Only founder lines and the 2 It is intriguing that the Our study clearly shows we have defined important domains for high
level, tissue-specific expression within the flanking regions of the
PBP and PF4 genes. With the identification of
ENA-78 gene immediately upstream of the PBP/PF4
gene locus, the question of whether there is a larger
megakaryocyte-specific gene locus involved in the regulation of all 3 genes is raised. Given that all known CXC chemokines localize to a
single locus on human chromosome 420,48,49 and that our
studies show that ENA-78, PBP, and PF4 are clustered and transcribed in
the same 5' For over 40 kb downstream of the PF4 gene, we found no other
CXC chemokine genes. Whether this means that the
ENA-78/PBP/PF4 genes are at one end of the CXC chemokine
locus is not clear yet. Furthermore, we have yet to determine the
precise relative location of the inactive, duplicated
The protein levels of hPBP and hPF4 in the founder lines are consistent
with the semiquantitative RT-PCR data. The 2
We wish to thank Dr Gowthami Arepally at the University of New Mexico, Albuquerque, NM, for providing the mouse anti-hPF4 monoclonal antibody RTO, and Dr Katya Ravid, Boston University, Boston, MA, for sharing unpublished sequences with us. We would also like to thank Zheng Cui, Diana Cassel, and Saul Surrey at the Thomas Jefferson University as well as Jean Richa at the Transgenic Mouse Facility of the University of Pennsylvania for their technical help.
Submitted October 24, 2000; accepted March 15, 2001.
Supported in part by grants from the American Society of Hematology (to C.Z.), the National Institutes of Health HL40387 (to M.P.), HL40387 (to S.E.M.), and HL61865 (to S.E.M. and M.P.R.), and the Nemours Foundation (to S.E.M. and M.P.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Chunyan Zhang, The Children's Hospital of Philadelphia, Abramson Research Center, Rm 314, 34th St and Civic Center Blvd, Philadelphia, PA 19104; e-mail: zcy118{at}yahoo.com.
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  D. Cervi, T.-T. Yip, N. Bhattacharya, V. N. Podust, J. Peterson, A. Abou-Slaybi, G. N. Naumov, E. Bender, N. Almog, J. E. Italiano Jr, et al. Platelet-associated PF-4 as a biomarker of early tumor growth Blood, February 1, 2008; 111(3): 1201 - 1207. [Abstract] [Full Text] [PDF]
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M. A. Kowalska, S. A. Mahmud, M. P. Lambert, M. Poncz, and A. Slungaard Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge Blood, September 15, 2007; 110(6): 1903 - 1905. [Abstract] [Full Text] [PDF]
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M. P. Lambert, L. Rauova, M. Bailey, M. C. Sola-Visner, M. A. Kowalska, and M. Poncz Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications Blood, August 15, 2007; 110(4): 1153 - 1160. [Abstract] [Full Text] [PDF]
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L. Rauova, L. Zhai, M. A. Kowalska, G. M. Arepally, D. B. Cines, and M. Poncz Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications Blood, March 15, 2006; 107(6): 2346 - 2353. [Abstract] [Full Text] [PDF]
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D. E. Eslin, C. Zhang, K. J. Samuels, L. Rauova, L. Zhai, S. Niewiarowski, D. B. Cines, M. Poncz, and M. A. Kowalska Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin Blood, November 15, 2004; 104(10): 3173 - 3180. [Abstract] [Full Text] [PDF]
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A. Schaffner, C. C. King, D. Schaer, and D. G. Guiney Induction and antimicrobial activity of platelet basic protein derivatives in human monocytes J. Leukoc. Biol., November 1, 2004; 76(5): 1010 - 1018. [Abstract] [Full Text] [PDF]
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Y. Okada, E. Matsuura, Z. Tozuka, R. Nagai, A. Watanabe, K. Matsumoto, K. Yasui, R. W. Jackman, T. Nakano, and T. Doi Upstream stimulatory factors stimulate transcription through E-box motifs in the PF4 gene in megakaryocytes Blood, October 1, 2004; 104(7): 2027 - 2034. [Abstract] [Full Text] [PDF]
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A. El-Gedaily, G. Schoedon, M. Schneemann, and A. Schaffner Constitutive and regulated expression of platelet basic protein in human monocytes J. Leukoc. Biol., March 1, 2004; 75(3): 495 - 503. [Abstract] [Full Text] [PDF]
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H. V. Yarovoi, D. Kufrin, D. E. Eslin, M. A. Thornton, S. L. Haberichter, Q. Shi, H. Zhu, R. Camire, S. S. Fakharzadeh, M. A. Kowalska, et al. Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment Blood, December 1, 2003; 102(12): 4006 - 4013. [Abstract] [Full Text] [PDF]
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D. Kufrin, D. E. Eslin, K. Bdeir, J.-C. Murciano, A. Kuo, M. A. Kowalska, J. L. Degen, B. S. Sachais, D. B. Cines, and M. Poncz Antithrombotic thrombocytes: ectopic expression of urokinase-type plasminogen activator in platelets Blood, August 1, 2003; 102(3): 926 - 933. [Abstract] [Full Text] [PDF]
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M. A. Thornton, C. Zhang, M. A. Kowalska, and M. Poncz Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent, high-level megakaryocyte expression Blood, November 15, 2002; 100(10): 3588 - 3596. [Abstract] [Full Text] [PDF]
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M. P. Reilly, S. M. Taylor, N. K. Hartman, G. M. Arepally, B. S. Sachais, D. B. Cines, M. Poncz, and S. E. McKenzie Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through Fc{gamma}RIIA Blood, October 15, 2001; 98(8): 2442 - 2447. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-granules.
-thromboglobulin (
X HaeIII marker.
(B) Relationship between the copy number for the PBP-expression
transgenic lines Short-PBP, Long-PBP, and
, Short-PBP; 
,
Long-PBP; 
, 
, PF4-Only; 
245 bp of 5'-flanking region from the human platelet factor 4 gene is sufficient to drive megakaryocyte-specific expression in vivo.
Blood.
1998;91:2326-2333
RIIA. Blood. In
press.