Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3947-3950
Characterization of the Murine Platelet 
IIb Gene and Encoded cDNA
By
Michael A. Thornton and
Mortimer Poncz
From the Department of Pediatrics, University of Pennsylvania School
of Medicine, Philadelphia, PA.
 |
ABSTRACT |
The 
IIb/
3 receptor is central to platelet aggregation.
Biological studies of this receptor have been limited by the inability to reproduce IIb/3 function in a cell system. Increasingly, efforts are being directed at studies of this receptor in mice models.
The structure of murine (m) 3 has been reported. We now have
sequenced the mIIb gene and found that it has the same size and
organization as the human gene. The exon/intron borders are reported
here, as are the distances between exons. mIIb protein is 1,033 amino acids (aa), 7 and 5 aa shorter than human (h) and rodent (r)
IIb, respectively, with 79% and 90% homology, respectively. As
part of the comparative analysis of the 3 known IIb chains included
in this report, we found that a particular region of the IIb
N-terminal -propeller is highly conserved and speculate that it
directly participates in ligand binding.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE PLATELET-SPECIFIC integrin
IIb/3 (GPIIb-IIIa, CD41, CD62) binds fibrinogen and other ligands
after platelet activation.1,2 This receptor is densely
packed on the surface of the platelet with 80,000 copies per
platelet3 and also does not bind its ligand with high
affinity until the platelet is activated and the receptors are
"turned on."4 Given the central role played by this
receptor in thrombosis and the successful clinical application of
anti-IIb/3-directed strategies to prevent thrombotic
complications,5 a great deal of effort has been carried out
to establish a functional assay system to study the IIb/3
receptor system ex vivo. These studies have focused on stable
transfections in cell lines such as Chinese hamster ovary (CHO) or in
lymphoid cells.6,7 Both lines suffer from the limited
number of receptors on the surface and the artificial fashion in which
these cells need to be activated. At best, these lines show binding in
the nonactivated state and only modest increases after activation.
Attention has, therefore, focused on setting up more vigorous systems
in in vivo models with an emphasis on murine studies. The m3 cDNA
has been described8 and, subsequently, the m3 knockout
has been produced with a phenotype similar to Glanzmann thrombasthenia.9 Furthermore, studies have been done with
the first m3 knock-in, introducing a mutation into the m3
cytoplasmic tail (Y747F) and showing a mild decrease in
platelet aggregation after agonist activation.10
Additionally, studies have begun introducing human IIb and 3
chains into primary megakaryocytic progenitors. For example, h3
constructs were introduced into m3 knockout marrow cells using a
retroviral system, and the resulting megakaryocytes were rescued with
regard to IIb/3 properties such as clot retraction, presumably
secondary to mIIb/h3 receptor complexes on the surface of the
megakaryocytes.11 Such studies show that cross-species heterodimers readily form and are functional, although there are going
to be important limitations in such studies, as it is known that
IIb/3 receptors appear to have different species related properties such as in their sensitivity to RGD
peptides.12,13
To carry out similar studies for mIIb, we have cloned the gene and
derived the encoded cDNA sequence. The gene organization is remarkably
well preserved. The encoded protein is described and compared with the
known hIIb and rIIb.14,15
| |
MATERIALS AND METHODS |
Isolation of the mIIb 
and bacterial artificial chromosome (BAC)
clones.
Full-length rIIb cDNA was random-primer labeled16 with
32P-dCTP and used to screen a FIX mouse
129SV genomic library (Stratagene, La Jolla, CA) using nitrocellulose
filter lifts of the plated phage. Individual positive colonies were
obtained using repeat rounds of dilutional plating, and these were
grown in large scale and purified using a cesium gradient as previously
described.16
DNA from the genomic clones was characterized by restriction digest and
Southern blotting16 using again the rat IIb cDNA as
probe. A 6-kb BamHI fragment was subcloned from the original positive FIX clone and subcloned into BamHI cut
pBSK+ library (Stratagene).
The sequence in mIIb Intron 12 was used to polymerase chain reaction
(PCR)-screen a BAC mouse 129SV genomic library (Genome Systems, St
Louis, MO), using the primers forward:
5'-ATGGACTTACCCCCATAGAT-3' and reverse:
3'-ACTTCCCCGGGATTCTGCGC-3' to give a 0.5-kb band. The BAC
clone was then used to obtain the sequence through exon 15, and then a
PCR-amplified region for exons 15 and 16 was randomly primer labeled
and used to rescreen the original FIX library to
complete the characterization of the mIIb gene.
mIIb gene characterization.
The 5'-BamHI pBSK clone, the 5'- and
3'-FIX clones, and the BAC clone described above were end
sequenced with the appropriate primers (eg, T7, SP6, and
T3 primers). Subsequent primers were generated
based on the new data and used to prime the next round of sequencing
reaction. All sequencing used fluorescenated dNTPs, and Sequenase (USB,
Cleveland, OH) and an ABI 373A automated sequencer (PE
Applied Biosystems, Foster City, CA). Sequences were stored and
analyzed using MacDNasis (Hitachi Software, San Diego, CA) and the
BLAST program at the National Center for Biotechnology, which hosts an
internet site at URL: http://www.nlm.nih.gov/.
| |
RESULTS AND DISCUSSION |
Characterization of the mIIb gene.
The only previously characterized IIb cDNAs were those of human and
rat.14,15 Using the rat IIb cDNA, we screened a 129SV murine genomic FIX library. Sequencing of overlapping
IIb+ clones helped to provide the majority of sequence
of the mIIb gene and its surrounding locus. In addition, we
PCR-screened an mBAC 129SV murine genomic library using primers derived
from these sequences. A single IIb+ BAC clone was
obtained and sequenced to fill the remaining sequence. The complete
sequence for the murine IIb gene and cDNA are available through
GenBank at the National Center for Biotechnology (accession nos.
AF170316 and AF169829). Using restriction mapping of the mIIb BAC
clone, we have previously reported that the murine gene is flanked by
the KIAA-O553 gene upstream and the Granulin gene downstream, and that
this organization is conserved in the hIIb locus.17 The
genes themselves are also organized into very similar exons and introns
of virtually identical size. A summation of the exon/intron borders of
the mIIb gene with the distance between the various exons is shown
in Table 1. Since the promoter region,
through the beginning of the coding region of the mIIb gene had been
previously published,18 the data begin with exon 1's
splice donor region. It should be pointed out that the hIIb gene has
a "GC" instead of the canonical "GT" at the splice donor
sequence for exons 5 and 8. This variation is also present for the
mIIb gene (double underlined in Table 1). The 3'-untranslated
region is poorly conserved except in the immediate region around the
purported polyadenylation signal site.
Characterization of the mIIb cDNA.
We extracted the mIIb cDNA from the above sequence and have used
these sequences for reverse transcriptase (RT)-PCR of murine platelet
RNA with amplification of expected band size and have confirmed
portions of the sequence. A comparison with rat and human protein
sequence is shown in Fig 1. mIIb is
1,033 amino acids (aa), 7 and 5 aa shorter than human and rodent
IIb, respectively. There is 79% and 90% homology between hIIb
and rIIb with mIIb, respectively, which is near average for these
cross-species homology.15 As expected, the signal peptides
have lower homology than the remaining mature proteins, but the region
surrounding the start of the mature protein is well conserved (see
arrow in Fig 1). The least-conserved region is near the cleavage site
into the light and heavy chain. Although the cleavage site itself is
conserved (underlined "RR" in Fig 1), homology with hIIb and
rIIb is only 50% and 60%, respectively. In contrast, the
transmembrane and cytoplasmic domains are highly conserved.
View larger version (70K):
[in this window]
[in a new window]
| Fig 1.
Cross-species comparison of mIIb with hIIb and
rIIb. The single-letter amino acid sequence of mIIb is shown at
the top and those of the other 2 species are shown below. A":"
refers to an identical homologous aa. A "-" refers to a missing
aa. The start site for mature IIb is indicated by an arrow. The 3 regions of the N-terminal -propeller regions discussed are grayed in
and labeled. The transmembrane domain is crosshatched grayed in and is
also labeled. The double-arginine cleavage site is underlined.
|
|
Studies of natural-occurring and directed mutations have suggested that
the 3 upper surface loops of the N-terminal -propeller of IIb may
be involved in ligand binding. Our cross-species comparison shows that
there is 100% conservation at the middle of these 3 loops (grayed area
for the "W3 loop" in Fig 1). However, there is poor conservation
in the other 2 loops ("W2/3 loop" and "W3/4 loop" in Fig
1). We would have expected a loop involved in ligand binding to be
highly preserved. One possible explanation for this divergence could be
that these differences in receptor loop structure help to accommodate
for species differences in the primary structure of the fibrinogen
ligand itself. Supporting this, it has been shown that the IIb/3
receptor of different species have different sensitivities to RGD
peptide inhibition of fibrinogen binding so that rodent and murine
platelets are much more resistant to RGD peptide inhibition than human
platelets.12 Whether this difference in species RGD
sensitivity is caused by the rapid evolution of sequences in the W2/3
and W3/4 loops remains to be investigated.
| |
FOOTNOTES |
Submitted June 7, 1999; accepted July 21, 1999.
Supported in part by National Institutes of Health Grant No. HL40387.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mortimer Poncz, MD, The Children's
Hospital of Philadelphia, 34th St and Civic Center Blvd, Philadelphia,
PA 19104; e-mail: poncz{at}emailchop.edu.
| |
REFERENCES |
1.
Phillips DR, Charo IF, Parise LV, Fitzgerald LA:
The platelet membrane glycoprotein IIb-IIIa complex.
Blood
71:831, 1988[Free Full Text]
2.
Ginsberg MH, Xiaoping D, O'Toole TE, Loftus JC, Plow EF:
Platelet integrins.
Thromb Haemost
70:87, 1993[Medline]
[Order article via Infotrieve]
3.
Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE:
Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets.
Blood
88:907, 1996[Abstract/Free Full Text]
4.
Shattil SJ, Ginsberg MH:
Integrin signaling in vascular biology.
J Clin Invest
100:S91, 1997 (11 suppl)
5.
Coller BS:
Platelet GPIIb/IIIa antagonists: The first anti-integrin receptor therapeutics.
J Clin Invest
100:S57, 1997 (11 suppl)
6.
Tozer EC, Baker EK, Ginsberg MH, Loftus JC:
A mutation in the alpha subunit of the platelet integrin alphaIIbbeta3 identifies a novel region important for ligand binding.
Blood
93:918, 1999[Abstract/Free Full Text]
7.
Loh E, Qi W, Vilaire G, Bennett JS:
Effect of cytoplasmic domain mutations on the agonist-stimulated ligand binding activity of the platelet integrin alphaIIbbeta3.
J Biol Chem
271:30233, 1996[Abstract/Free Full Text]
8.
Cieutat AM, Rosa JP, Letourneur F, Poncz M, Rifat S:
A comparative analysis of cDNA-derived sequences for rat and mouse beta3 integrins (GPIIIa) with their human counterpart.
Biochem Biophys Res Commun
193:771, 1993[Medline]
[Order article via Infotrieve]
9.
Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M, Ross FP, Coller BS, Teitelbaum S, Hynes RO:
Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.
J Clin Invest
103:229, 1999[Medline]
[Order article via Infotrieve]
10.
Law DA, DeGuzman F, Ministri K, Phillips DR:
Demonstration of a role for the 3 cytoplasmic tyrosine residues in platelet function using mice expressing a mutant (Y747F) 3.
Blood
92:701a, 1998 (abstr, suppl 1)
11.
Wilcox DA, Hodivala-Dilke KM, Hynes RO, White GC II:
Of mice and men: Detection of a functional murine IIb-human 3 heterodimer complex on the surface of megakaryocytes derived from retrovirus transduced bone marrow cells from 3-knockout mice.
Blood
92:702a, 1998 (abstr, suppl 1)
12.
Cox D, Motoyama Y, Seki J, Aoki T, Dohi M, Yoshida K:
Pentamidine: A non-peptide GPIIb/IIIa antagonist
In vitro studies on platelets from humans and other species.
Thromb Haemost
68:731, 1992[Medline]
[Order article via Infotrieve]
13.
Jennings LK, White MM, Mandrell TD:
Interspecies comparison of platelet aggregation, LIBS expression and clot retraction: Observed differences in GPIIb-IIIa functional activity.
Throm Haemost
74:1551, 1995[Medline]
[Order article via Infotrieve]
14.
Poncz M, Eisman R, Heidenreich R, Silver SM, Vilaire G, Surrey S, Schwartz E, Bennett JS:
Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors.
J Biol Chem
262:8476, 1987[Abstract/Free Full Text]
15.
Poncz M:
Newman PJ. Analysis of rodent platelet glycoprotein IIb: Evidence for evolutionarily conserved domains and alternative proteolytic processing.
Blood
75:1282, 1990[Abstract/Free Full Text]
16.
Sambrook J,
Fritsch EF,
Maniatis T
(eds):
Molecular Cloning: A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989
17.
Thornton MA, Poncz M, Korostishevsky M, Yakobson E, Usher S, Seligsohn U, Peretz H:
The human platelet IIb gene is not closely linked to its integrin partner 3.
Blood
94:2039, 1999[Abstract/Free Full Text]
18.
Denarier E, Martin F, Martineau S, Marguerie G:
PCR cloning and sequence of the murine GPIIb gene promoter.
Biochem Biophys Res Commun
195:1360, 1993[Medline]
[Order article via Infotrieve]
TOP
ABSTRACT
INTRODUCTION
