Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Integrins are a long family of heterodimeric transmembrane glycoproteins consisting of multiple combinations of noncovalently linked α and β chains, which generate different complex receptors with different expression patterns and ligand binding profiles. The integrins bind to extracellular matrix (ECM) or to cell-surface ligands, regulating numerous downstream pathways (Hynes, 2002). Each integrin binds to only a limited series of ligands, ensuring that cell adhesion and migration are precisely regulated. The α subunit mainly determines the substrate specificity with extracellular matrix molecules (ECM) (Yamada, 1991), while the intracytoplasmic tail of the β chain is predominantly responsible for the integrin interaction with the cell cytoskeleton by binding to vinculin, talin and α-actin (Isenberg, 1991). Thus, this heterodimeric association between α and β subunits allows the integrins to act as bidirectional signaling molecules in the different tissues and cell types in which they are widely distributed, mediating a variety of biological processes so diverse as embryogenesis, haemostasis, tissue repair, migration, cell polarity, immune response and metastatic diffusion of tumor cells (Hynes, 1987, 1992; Hemler et al., 1994). Mammalian integrins have been divided into subfamilies according to their β subunit. The most important β integrin subfamilies are β1, β2 and β3. Within a subfamily, the same β subunit can associate with different α subunits. To date, 18 α and 8 β chains -whose combinations provide up to 24 different integrinshave been described in mammal species (Hynes et al., 2002; Alam et al, 2007).


Introduction
Integrins are a long family of heterodimeric transmembrane glycoproteins consisting of multiple combinations of noncovalently linked α and β chains, which generate different complex receptors with different expression patterns and ligand binding profiles.The integrins bind to extracellular matrix (ECM) or to cell-surface ligands, regulating numerous downstream pathways (Hynes, 2002).Each integrin binds to only a limited series of ligands, ensuring that cell adhesion and migration are precisely regulated.The αsubunit mainly determines the substrate specificity with extracellular matrix molecules (ECM) (Yamada, 1991), while the intracytoplasmic tail of the βchain is predominantly responsible for the integrin interaction with the cell cytoskeleton by binding to vinculin, talin and α-actin (Isenberg, 1991).Thus, this heterodimeric association between αand βsubunits allows the integrins to act as bidirectional signaling molecules in the different tissues and cell types in which they are widely distributed, mediating a variety of biological processes so diverse as embryogenesis, haemostasis, tissue repair, migration, cell polarity, immune response and metastatic diffusion of tumor cells (Hynes, 1987(Hynes, , 1992;;Hemler et al., 1994).Mammalian integrins have been divided into subfamilies according to their βsubunit.The most important β integrin subfamilies are β 1 ,β 2 and β 3 .Within a subfamily, the same βsubunit can associate with different α subunits.To date, 18 α and 8 βchains -whose combinations provide up to 24 different integrins-have been described in mammal species (Hynes et al., 2002;Alam et al, 2007).pET28b (Novagen) following digestion of the PCR product and the vector with BamHI and HindIII.Two different pairs of primers were used: F1rp-EcoRI/R1r-pXhoI and F2rp-BamHI/R2rp-HindIII (Table1).PCR product was ligated into the expression vector pET28b and used to transform Escherichia coli strain BL21 (DE3) (Novagen).Recombinant proteins (rpCD41-F1R1 and rpCD41-F2R2), expression and purification were carried out following previously procedures described by us (Jiménez-Marín et al., 2000).

Antibodies production
A monoclonal antibody, GE2B6, against rpCD41-F2R2 and two polyclonal antibodies, anti-rpCD41-F1R1 and anti-rpCD41-F2R2, were produced using previously described immunization and cells fusion procedures (Arce et al., 2002;Jiménez-Marín et al., 2000).Briefly, female BALB/c mice were immunized with 50 µg of rpCD51.Spleen cells from immune mice were fused with Sp2/0 myeloma cells.Hybridoma clones were selected on the basis of binding secreted antibody to rpCD61 by indirect ELISA.Antibody-producing hybridomas reacting positively were cloned at least twice by limiting dilution.Immunoglobulin classes and subclasses were determined in solid-phase ELISA using rabbit antisera specific for mouse heavy and light chains and a peroxidase-conjugated goat antirabbit immunoglobulin (Sigma).

Electrophoresis and immunoblottings
Platelets (10 8 /sample) were lysated in NP-40 lysis buffer with PMSF 2 mM with vigorous shaking for 1 h at 4ºC, and then centrifuged at 12,000 rpm, 20 min.100 µl supernatant were mixed with 100 µl of sample treatment buffer, and 100 µl were loaded in the gel.Electrophoresis was carried out in 5%-15% gradient polyacrilamide gels.For the 2D electrophoresis, the platelet proteins pellet was resuspended in lyses buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 0,8% ampholytes).Immobilized pH gradient strips (17 cm, 5-8 linear pH gradient, Bio-Rad) were rehydrated with 300 µl (300 µg) of the protein solution for 16 h, and focused in a PROTEAN IEFcell (Bio-Rad).Second dimension was performed on 10% SDS-PAGE.For the immunoblottings, proteins were transferred from gels to PVDF Inmobilon P membranes (Milipore).Membranes were blocked and washed three times in PBS-T, and then incubated with 3 ml antibody or PBS as negative control, overnight at 4ºC in shaking.After three washing ups in PBS-T, the membranes were incubated with rabbit anti-immunoglobulin-peroxidase (Sigma).Afterwards, they were washed up three times in PBS-T, and finally reactions were detected with the ECL TM detection system (Amersham) following the manufactures instructions.

Immunoprecipitation of platelet CD41 proteins
Platelets (10 8 /sample) were incubated with 0.4 mg sulfobiotin (Pierce) with gently shaking for 15 min at 4ºC, and then centrifuged at 3,000 rpm, 15 min.Pellet was washed three times in PBS, and then resuspended in lyses buffer (500 µl/sample) and PMSF 2 mM.After incubation in dark with vigorous shaking for 1 h at 4ºC, it was centrifuged at 13,000 rpm, 20 min, and the supernatant was collected.50 µl of protein G-Sepharose (Pharmacia) were added per ml of supernatant and incubated with shaking overnight at 4ºC, and then centrifuged at 2,000 rpm, 5 min.500 µl lysate were incubated with 1 ml of the anti-porcine CD41 antibody for 2 h at room temperature.At the same time, when monoclonal antibody was going to be used, to increase its binding ability, the G protein is recovered with an anti-mouse immunoglobulin rabbit serum (Pierce) 1/10 in lyses buffer, for 2 h at room temperature.Then, the G protein is washed three times in lyses buffer, and centrifuged at 2,000 rpm, 2 min.This step was not needed when polyclonal antibodies were used.50 µl of the antibody recovered G-Sepharose were added to the lysate containing the anti-CD41 antibody and incubated for 1 h at room temperature with shaking, and then centrifuged at 2,000 rpm, 5 min.The supernatant was collected and washed three times in lyses buffer, the first being in buffer and sucrose.Finally, supernatant was subjected to SDS-PAGE in 5%-15% gels in reducing or not reducing conditions.After electrophoresis, the proteins were transferred to PVDF, Inmobilon P membranes, as described before, and, after be blocked, incubated with a solution of Streptavidin-HRP (Amersham) solution 1/500 in PBS for 1 h in dark.Then, membranes were washed three times in PBS-T and revealed with the ECL TM detection system (Amersham) following the manufactures instructions.

Immunohistochemistry
Expression of CD41 protein from healthy animals was studied following previously procedures described by us (Jiménez-Marín et al., 2008) using monoclonal antibody GE2B6 supernatant or polyclonal antibodies (1/3000 dilution in PBS) or an irrelevant mAb (as negative control).Briefly, all tissue specimens were fixed in Bouin liquid for 16 hours.Tissues were dehydrated in ascending concentrations of ethanol and xylene and embedded in paraffin.Sections of 5 µm w e r e p l a c e d o n s l i d e s c o a t e d w i t h V e c t a b o u n d ( V e c t o r Laboratiores, Inc.).The tissue slides were kept at 55ºC for 45 min to improve the adherence of sections to glass.The sections were deparaffinized and rehydrated in xylene and descending concentrations of ethanol, respectively.Endogenous peroxidase activity was inhibited with 3% hydrogen peroxidase.The sections were incubated with normal goat serum (1:10 dilution in PBS) (Vector) for 30 min at room temperature.After removing the serum, anti-porcine CD41 antibodies or an irrelevant mAb (as negative control) were added for 18 hours at 4ºC in a wet chamber.The sections were incubated with biotinylated antimouse Ig (Dako) diluted 1/50 in PBS for 30 min at room temperature.Tissue sections were covered with avidin-biotin-peroxidase complex (Sigma) diluted 1/50 with PBS for 1 h in a wet chamber at room temperature, washed and then developed with 3, 3'-diaminobenzidine (Sigma) (5 µg in 10 ml PBS).Sections were counterstained with Mayer hematoxylin and mounted with Eukitt.
2.9 Flow cytometry 100 µl of platelets (10 6 cells/ml) and 100 µl of the antibody (or PBS as a control) were incubated 30 min at 4ºC.After washing with PBS, tubes were centrifuged at 3,000 rpm, 6 min, and the platelets resuspended in 50 µl of a rabbit FITC-anti-immunoglobulin (1/160 in PBS) (Sigma).After incubation at 4ºC 30 min in dark, the platelets were washed three times in PBS and the fixed in 1% PFA/PBS.Samples were analyzed in a FACsort cytometer (Bencton Dickinson) equipped with a CellQuest v 1.2software.

Chromosome localization
The INRA somatic cell hybrid panel (Yerle et al., 1996) was screened with porcine primers (VARP1 and VARP2), which specifically amplify a 212 bp fragment (Table 1).For genotyping of the hybrid panel, 10 ng of DNA from each cell line and control sample (pig, hamster, and mouse) were amplified.PCR products were evaluated on a 1% agarose gel and individual cell lines were evaluated for the presence or absence of a fragment of the correct size.Statistical calculations of the assignment were performed using the software developed by Chevalet et al (1997) (<http://www.inra.toulouse.fr>).The INRA-Minesota porcine radiation hybrid (IMpRH) panel (Yerle et al., 1998;Hawken et al., 1999) was screened with the same porcine specific primers in the same PCR conditions (Table 1).Statistical calculations of the assignment were performed using the IMpRH mapping tools (<http://www.imprh.toulouse.inra.fr>).

Quantitave real time RT-PCR
CD41 cDNA was quantified by real time quantitative PCR (RT-Q-PCR) relative to βactin cDNA reverse transcribed from total RNA from platelets.The PCR reaction was carried out with 0.5 µl of each VARP1/VARP2 andβ−actinF/β-actinR primers (Table 1) (20µM), 12.5 µl of iQ TM SYBR1 Green Supermix (Bio-Rad), and 1.5 µl of the cDNA sample.The PCR conditions included 40 cycles of 30s at 94ºC, 30s at 60ºC and 30s at 72ºC.All experiments were performed three times to confirm accuracy and reproducibility of real-time PCR.The efficiency of the primers (E) was calculated according to the equation ( 1).E = 10 [-1/p]  (1) being p the slope of the standard curve log(fluorescence)/Ct.
The relative abundance of CD41 gene expression was determined by the ratio (R) equation ( 2) (2) being Ct = threshold cycle (cycle at with PCR amplification reaches a significant value).

Cloning and sequence analysis of the porcine CD41 cDNA
The porcine full length CD41 cDNA was obtained by a combination of PCR and RACE (Rapid Amplification of cDNA Extremes).A partial sequence that lacked the 3' cDNA extreme, including part of the coding sequence, was deposited in GenBank (AF170526).So, we first amplified the pig CD41 5' cDNA using forward and reverse primers designed from this sequence.Three pairs of primers were used P3/R4, P1/R2 and F1/R1 (Table1).Three 1352, 870 and 677 bp long overlapping fragments were produced, respectively.Altogether, the three fragments provided a 2701 bp long sequence that belongs to the 5' extreme of the CD41 cDNA (Figure1).To obtain the remaining 3' sequence of the CD41 cDNA we carried out a RACE by using the RACE-out and the RACE-P5 primers shown in Table1.This allowed us to obtain an additional 622 bp long 3' sequence (Figure1).Finally, we obtained the full length CD41 cDNA molecule amplifying RNA from platelets by RT-PCR by using the FcDNA5 and RcDNA3 pair of primers (Table1 and Figure1) and MBLong polimerase.The first 31 amino acid residues of CD41 are predominantly hydrophobic and correspond to the signal peptide sequence.So, the pig mature pre-CD41 molecule consists of 1005 amino acid residues, and, as this amino acid sequence has a proteolytic cleavage site (KR/D) located between amino acids residues 899 and 900 in pre-CD41 (Takada et al., 1989), the mature porcine CD41 polypeptides -lacking the signal peptide-must be composed by two different chains (914 and 91 amino acid residues) linked by disulfide bridges, similar to those reported in homologous CD41 integrins.Other sequences and structural domains contained in other CD41 proteins are also presents in the porcine CD41 chains (Figure2).
The seven FG-GAP tandem repeats are shown as W with arrows marking their initial and final limits.An α helix is shown in red.The long extracellular domain of the porcine CD41 integrin consists of 869 amino acids residues.It contains 8 consensus N-glycosylation sites (Asn-X-Ser/Thr, where X is not Pro) identified by the NetNGlyc 1.0 program (www.expasy.org),and 18 -from 19-cystein residues.As in other α integrins, the extracellular domain of the porcine CD41 contains four Ca ++ binding domains (DX(D/N)XDGXXD) and seven FG-GAP tandem repeats -which are identified by the SABLE 2.0 program (http://sable.cchmc.org/)-eachone containing four helixes similar to those previously described (Springer, 1997;Xiong et al., 2001).The secondary structure of CD41 molecule is shown in Figure3 and the tertiary one, obtained with the Swismodel (www.expasy.org), is shown in Figure4.The stretch sequence of 26 hydrophobic amino acid residues located in the carboxy-terminal portion of the polypeptide must constitute the transmembrane domain.Following it there is a short 20-amino acid sequence that must represent the cytoplasmic domain of the molecule.It contains a GFFKR (1019-1023 in pre-CD41) domain, which is conserved in all human α integrin chains and is involved in the link of both α and β chains of the heterodimeric complex (Rojiani et al., 1991).It also contains a β−like turn (PPLEE) (1026-1030), that in comparison to the α v chain (PPREE) could aid in the ligand interaction of fibronectin and vitronectin with the intact α IIb β 3 heterodimer which is essential for various transductional processes during mammalian organogenesis (Filardo & Cheresh, 1994).

GTCCGGGCAGAGGCCCACCTGGAGCTGAGAGGGAACTCCTTTCCAGCCTCCCTGTTGATGGCGGCAGAAGGCGATTGGGAGAACAGCTCG 2419 V R A E A H L E L R G N S F P A S L L M A A E G D W E N S S 801 GACAATTTGGGCCCCAAAGTGGAGCACACCTATGAGCTCCACAACAATGGCCCTAGTACCGTAAGTGGCCTCCACCTCAGGCTCCACCTG 2509 D N L G P K V E H T Y E L H N N G P S T V S G L H L R L H L 831
Molecular Cloning -Selected Applications in Medicine and Biology 120

Comparative analysis
The deduced protein sequence of the porcine CD41 was compared to their orthologous proteins from six different species: humans, cattle, horses, dogs, rats, rabbits, mice, zebrafish and xenopus (Figure5 and Table2).As shown in Table2, the longest porcine CD41 protein shares a 78% amino acid residue identity with those of humans, cattle and horses, 77% with dogs, 75% with rabbits, 73% with rats, 71% with mice, 42% with Xenopus laevis and 40% with zebrafish.Table2 also shows the percentages of amino acid residue identities of the different regions of the CD41 molecule.
In general, both transmembrane and cytoplasmic domains are more preserved compared to the extracellular one.The phylogenetic tree of CD41 proteins shows that the counterpart closet to porcine CD41 was that of cows (Figure6).

Cell and tissue expression of porcine CD41 transcripts
To investigate the pattern of the porcine CD41 mRNA expression, RT-PCR analysis was conducted with a variety of pig adult tissues and cell types using VARP1/VARP2 genespecific primers (Table1).The highest level of CD41 transcripts was detected in platelets, although a moderate level was detected in bone marrow and a low level in ganglions and lungs.No CD41 transcripts were detected in the rest of tissues and cells analyzed (Figure9).

Cell and tissue expression of porcine CD41 proteins
The precise localization of the CD41 protein was studied by immunohistochemistry and by flow cytometry with antibodies developed against two different porcine CD41 recombinant proteins.

Expression and purification of porcine recombinant CD41 proteins
Two different cDNA fragments belonging to the functional region of the porcine CD41 protein were amplified and subcloned in the pET-28b expression vector.One, 996 bp long and amplified with primers CD41-F2 and CD41-R2 (Table1), contained the coding sequence for amino acids 32 to 363, and the other one, 677 bp long and amplified with primers CD41-F1 and CD41-R1, contained the coding sequence for amino acids 684 to 909, a highly antigenic region selected by the Jameson-Wolf method (Jameson & Wolf, 1988).The recombinant constructions, named respectively pET-F2R2 and pET-F1R1, were transfected and expressed in E. coli (DE3).Two different recombinant CD41 proteins were purified: rCD41-F1R1 (about 26 kDa) and rCD41-F2R2 (about 46 kDa) (Figure10).

Production of antibodies against porcine rCD41 proteins
An anti-rCD41-F2R2 monoclonal antibody (GE2B6), and two anti-rCD41-F2R2 and anti-rCD41-F1R1 polyclonal antibodies were produced, and their specific reactivity against the rCD41 proteins tested in immunoblottings.Before being used in immunohistochemical assays, their ability to specifically recognize the platelet CD41 molecules were carried out through immuno precipitations of platelets lysates in non-reduced conditions.An

Immunohistochemical detection of CD41 proteins
The reactivity of the anti-CD41 monoclonal and polyclonal antibodies was tested by immnohistochemestry on a variety of porcine tissues and cells types.Results are shown in Figures 13 and 14.Immunoreactivity was only detected in the membranes of megakaryocytes from bone marrow.No reactivity was detected in any of the tissues checked, including ganglion, in which a weak CD51 transcription was detected by RT-PCR.

Detection of CD41 proteins by flow cytometry
In order to identify the possible PBL cells that express CD41 proteins we carried out a flow cytometry analysis by using both anti-CD41 polyclonal antibodies.Figure15 shows the results in platelets, lymphocytes, granulocytes and erythrocytes with anti-CD41-F2R2.CD41 proteins were only detected in platelets by both polyclonal antibodies.Furthermore, to test the platelet porcine specificity of the antibodies produced in this study we test their reactivity with platelets from pigs, humans, dogs, horses, goats, chats, sheep and cows by flow cytometry.Both anti-CD41-F2R2 and anti-CD41-F1R1only reacted with porcine platelets (Figure 16), confirming the porcine CD41 specificity of both antibodies.

Effect of the platelet activation on the expression of porcine CD41
Previous results obtained in our lab using a two dimension differential in gel electrophoresis (2D-DIGE) technique had shown that the proteome of thrombin activated porcine platelets showed a reduced number of proteins affected in their expression level, among which CD41 was not found.Although CD41 is strongly expressed in platelets, the membrane proteins are usually poorly represented in the gels as a consequence of their high hydrophobicity.As we had produced specific anti-CD41 polyclonal antibodies, we used the anti-rCD41-F2R2 to check, using immunoblotting, if CD41 was or not present in a similar gel than that used in our previous study.Results are shown in Figure 17 in which CD41 integrin was clearly detected.
In order to test if the CD41 transcripts level was or not modified in the platelets after activation by thrombin, we carried out a real time quantitative PCR (rt-q-PCR) with RNAs from unstimulated and stimulated platelets.Results are shown in Figure18 in which a higher but not significant change in the CD41 transcripts level was detected after the activation by thrombin (the significant value is 1.5).Three replicates were assayed with very similar results.

Discussion
In general, the study of genes expressed in platelets is difficult since platelets are enucleated cells that show a reduced level of protein synthesis, and megakaryocytes, the platelets precursors, represent only 0.1% of bone marrow cells (Bray et al., 1987).Nevertheless, in the present study we describe for first time the cloning and characterization of the full-length cDNA for the porcine CD41 (α IIb ) integrin chain.The porcine CD41 proteins share common structural elements, including cytoplasmic, transmembrane and extracellular domains and the position of the proteolytic cleavage sites with the CD41 protein of other species.The porcine CD41 integrin showed an average of 75% amino acid identity with their mammal orthologous molecules, being the conservation in the transmembrane and cytoplasmic regions higher than in the extracellular one in all the The cytoplasmic region of the porcine α IIb integrin is also highly conserved (80-89% of identity when compared to their homologous mammalians).A short GFFKR motif, which was involved in the activation of the integrin receptors, is present in the cytoplasmic region of all the human αintegrins.In humans, mutations in the GFFKR motif of the α IIb β 2 integrin receptor induce a permanent activation of the integrins.As expected, in all the species compared in this study, the porcine α IIb integrin contains this motif near to the transmembrane region.The porcine α IIb integrin also contains in the cytoplasmic region the PPLEE motif, present in all the mammalian α IIb integrin compared in this study, whose modifications determine changes that interfere with the specific recognizing of the ligands (Filardo & Cheresh, 1994).
Once the porcine α IIb integrin was characterized, we used a porcine radiation hybrid panel and a somatic cell hybrid panel to map the pig CD41 (α IIb ) gene into swine chromosome 12 (Sscr 12), region p11(2/3)-p13.This chromosomal localization is in total concordance with heterologous painting data that demonstrate the correspondence between the swine Sscr 12 and the human Hsap 17 chromosomes (Rettenberger et al., 1995), where CD41 (α IIb ) gene maps in the human Hsap 17 q21 region (Bray et al., 1987), homologous to the porcine Sscr 12 p11-p13 one (Figure 19).It is interesting to note that the swine CD41 (α IIb ) and CD61 (β 3 ) genes are closely locatedwhich confirms our previous results (Morera et al., 2002)-, the same as in humans, where both genes map together in chromosome 17, q21 region (Thornton & Poncz, 1999).This is exceptional for genes coding for α and β integrins belonging to the same receptor, and it must have a functional significance, as both genes are simultaneously expressed in human megakaryocytes (Bennett et al, 1983).Therefore, the chromosomal assignment of pig CD41(α IIb ) gene provides additional evidence of the conserved linkage homology in these chromosome regions among pigs and humans.
We also checked in this study the porcine α IIb expression profile in different cells and t i s s u e s .W h e n w e u s e d R T -P C R t o d e t e c t t h e α IIb transcripts level, we observed, as expected, a strong expression in platelets and in bone marrow.However, we also detected a lower expression in lymphatic ganglion and lung, which we explain by the probable presence of platelets or blood cells in them.When we used immnunohistochemisty and flow cytometry to locate accurately the CD41 (α IIb ) protein expression with specific antibodies produced by us, we confirmed that the presence of CD41 proteins was restricted to platelets and megakaryocytic membranes.The same restricted expression pattern of α IIb proteins have been detected in other species, like humans and mice, although some studies suggest that α IIb could be a differentiation marker expressed in early stages of the cellular hematopoietic differentiation (Mitjavila- García et al., 2002) or to be over expressed in tumor cells (Raso et al., 2004).In fact, the α IIb /β 3 integrin expression in tumor cells has been controversial as α ΙΙβ andα v integrin have similar structures, and although the role of the CD51 integrin in tumor metastasis and angiogenesis is well documented (Chen, 1992(Chen, , 1997;;Mitjans et al., 2000), these studies have been carried out using antibodies that could cross react with the α v /β 3 receptor (Chen et al., 1992;Chen 2006).However, some studies have revealed that the α ΙΙβ /β 2 receptor mediates interactions between platelets and tumor cells, detecting an over expression in the filopodia emitted by the platelets in the focal adhesion plates, with the filopodia being the first contact sites between tumor cells and platelets (Chopra et al., 1992).
In this sense, it is worthy to note that in our study we have produced the two first specific anti-porcine α IIb antibodies, whose specificities we have demonstrated by flow cytometry in cross reactions against platelets from humans, dogs, horses, goats, cats, sheep and cows.Finally, as α IIb /β 3 is involved in adhesion and aggregation of platelets after their activation, we checked if the platelet activation was or not associated with changes in the α IIb transcripts level.Changes in the proteome of platelet activated by thrombin, the strongest platelet activator, was previously studied in our laboratory, detecting some differential modification in only a small number of proteins, among which the CD41integrin was not included, even though a very sensitive two dimension differential in gel electrophoresis (2D-DIGE) technique was used (Esteso et al., 2008).As CD41 is strongly represented in platelets and it plays an essential role in their activation, we took advantage of the specific anti-porcine α IIb antibodies produced for the studies presented in this chapter to check if CD41 integrin was or not present in the gels used to carry out those studies.
Immunoblotting results clearly showed that CD41protein was detected in the platelet proteome, which confirmed our previous results that showed that platelets did not modify their CD41 protein level after thrombin activation.Moreover, although platelets are enucleated cells that lack their nucleus during the megakaryocytic cells cytoplasm fragmentation, it is well established that they conserve ribosomes, mRNAs, as well as the post-translationally modifying protein mechanisms (Dittrich et al., 2005).For this, we used a real time PCR to check if some change was produced in the α IIb transcripts level as a consequence of the platelet activation by thrombin.Results showed that although a small increase was detected, this was not statistically significant.It is well established that most changes produced after platelets activation involve post-translational modifications that affect the interactions between transmembrane and cytoplasmic domains of α andβ chains (Russ & Engelman, 1999).So, our results support that the changes produced after thrombin platelet activation, which seems to disrupt the helical interface between the integrin and subunit transmembrane domains, favoring homomericα IIb (and β 3 ) transmembrane domain interactions in the α IIb β 3 receptor (Luo et al., 2004;Li et al., 2005;Partridge et al., 2005;Yin et al., 2006), must be produced by post-translational regulation, without affecting neither the transcript nor the protein level in the α IIb .

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Conclusion
Integrins are a family of heterodimeric transmembrane glycoproteins consiting of varying combinations of noncovalently bound α and β chains that generate several receptors with different expression patterns and ligand binding profiles.α IIb β 3 (CD41/CD61) integrin is the most abundant platelet receptor being responsible for the platelet aggregation.Most of the studies with α IIb β 3 integrin have been carried out in humans and mice but little is known about the expression ofα IIb β 3 integrin in porcine tissues, although pig is generally accepted as an optimal experimental model for different areas, as cardiovascular diseases, because of its similarity to humans.We have previously cloned and characterized the porcine gene coding for the β 3 (CD61) chain of theα IIb β 3 integrin; however, the one coding for α IIb (CD41) chain -the only α subunit for the β 3 one-remained to be characterized.We describe in this chapter the molecular cloning, the structural and comparative analysis, and the expression patterns of the porcine gene coding for the α IIb integrin chain.Additionally, we also describe the chromosomal localization of the gene.
We used a combined strategy of PCR and RACE reactions to obtain a full porcine α IIb cDNA sequence from platelet RNA.The pig α IIb cDNA was 3336-pb long and contained an ORF 3111 b long that encodes a pre-α IIb protein composed by 1036 amino acid residues, from which, 961, 26 and 10 belong to the NH2-extracellular, the transmembrane and the cytoplasmic-COOH domains, respectively.The porcine α IIb shares with α IIb from other species: identical structure, a high % amino acid identity, common domains (α−Ι,Ca ++ binding, MIDAS), N-glycosylation sites, and the seven FG-GAP tandem repeats.However, in relation to other mammalian αchains, the porcine α IIb shares lower identities with those homologous in mammals (78% with humans, horses and cows, 78% with dogs, 75% with rabbits, 73% with mice and 71% with rats).A phylogenetic tree identifies cows CD41 as the closest to pigs.By using both somatic cell hybrid and irradiated cell hybrid panels, we localized the gene coding for the porcine α IIb integrin in chromosome Sscr 12 region p11-(2/3 p13), in the same region where we previously localized the porcine β 3 integrin gene, region that corresponds to the human homologous Hsap 17(q21) in chromosome 17.
As expected, the porcine α IIb mRNAs were predominantly detected in platelets, but they were also detected in bone marrow and ganglion, in which platelets or megakaryocytes -the platelets precursors-were probably presents.To locate accurately the pattern expression of the α IIb protein, immunohistochemical, immunocytochemical and flow cytometry analysis were carried out.For this, monoclonal and polyclonal antibodies against porcine recombinant α IIb integrins (rα IIb ) were previously produced.Citometry flow analysis determined the antibodies specificity for porcine platelets, being the first antibodies described with this characteristic.Immunohistochemical assays confirmed that the α IIb expression is restricted to the membranes of megacariocytes present in bone morrow.Flow cytometry analysis of PBC confirmed the α IIb expression in platelet but not in lymphocytes, erythrocytes or granulocytes.Finally, we checked by RT-Q-PCR if any change was produced in the level of αIIb transcripts in thrombin activated platelets, no detecting significant ones.This result, together to previous ones obtained by us, support that no change were produced in neither the transcript nor the protein level of αIIb, supporting α IIb post-translational changes in the α IIb β 3 platelet receptor after thrombin activation.

Fig. 2 .
Fig. 2. The nucleotide and deduced amino acid sequences of pig CD41 cDNA.The predicted signal peptide is remarked in light green, the transmembrane domain in dark green, and the cytoplasmic region, containing the GFFKR sequence, in purple.The putative polyadenylation sequence is remarked in a black box.Potential N-glycosylation sites are indicated in red.Cysteine residues are marked as C in yellow.Putative cleavage sites are shown as ▲.Ca++ binding domains are remarked in pink.Primers used for cloning are marked with arrowheads.
Fig. 3. Secondary structure of the porcine CD41 molecule.Sequences in β antiparallel sheets are shown in green.

Fig. 4 .
Fig. 4. Three-dimensional structure of the porcine CD41 molecule.The seven FG-GAP tandem repeats are shown as W, each one composed by four β antiparallel chains.

Fig. 5 .
Fig. 5. Comparison of the porcine CD41 amino acid sequence to other homologous molecules.The sequences were derived from GenBank entries with accession numbers shown in materials and methods.Signal peptide is in green.Heavy and light chains are shown by orange and black lines, respectively.Ca ++ binding domains are remarked in pink boxes.Potential N-glycosylation sites (N) and cysteine residues (C) are respectively marked in red and yellow in the respective sequences.Amino acids residues conserved in all the sequences are shown in light grey.

Fig. 7 .
Fig. 7. Diagram showing results for the presence/absence of the CD41 gene in the INRA somatic hybrid cell panel.

Fig. 9 .
Fig. 9. RT-PCR expression patterns of CD51 transcripts in different pig cells and tissues.RU: Relative units.18S RNA amplification was used as control.

Fig. 17
Fig. 17.A: Two dimension gel electrophoresis showing the platelet proteome stained with Comassie blue.B: Immunoblotting of platelet proteome with anti-rCD41-F2R2.Red circle shows detection of CD41 protein.

Table 1 .
Primers used in PCRs and RACE.

2701 bp 622 bp CD41 cDNA 3336 bp/1036 aa FcDNA53 RcDNA3
Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor 117 www.intechopen.com Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor