conserved domains and alternative proteolytic ...

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1990 75: 1282-1289

Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing M Poncz and PJ Newman

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Analysis of Rodent Platelet Glycoprotein IIb: Evidence for Evolutionarily Conserved Domains and Alternative Proteolytic Processing By Mortimer Poncz and Peter J. Newman Recently, the full-length primary amino acid sequence for human glycoproteins (GP) Ilb and llla have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPllb/llla receptor with the corresponding rodent receptor. The rodent rGPllb/llla receptor differs from the human receptor, having low affinity for R G D-containing oligopeptides and not binding at all t o the C-terminus of the y chain of human fibrinogen. We describe the structure of rodent platelet GPllb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N-terminal sequence L N L D, agreeing with the consensus derived from other integrin family a heavy chains. All 18 cysteine

residues occur at positions conserved in human GPllb and the vitronectin receptor a subunit VNRa. The putative calcium-binding domains of the GPllbs have a high level of amino acid identity (92%).supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPllb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPllb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPllb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPllb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPllb gene. 0 1990 by The American Society of Hematology.

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tains two disulfide-linked components: a heavy chain of mol wt 125,000 and a light chain of mol wt 23,000.15GPIIIa is a single-chain disulfide rich glycoprotein with an apparent mol wt of 95,000 under nonreducing conditions that increases to 110,000 after disulfide-bond r e d u c t i ~ n . ’Recently, ~ the fulllength primary amino acid sequences for both hGPIIb and hGPIIIa have been derived from cDNA sequence analysis.’”’’ Both hGPIIb and hGPIIIa have N-terminal signal peptides and a single transmembrane region near the Cterminus. The two hGPIIb subchains are derived from a single R N A transcript, with the heavy chain encoded a t the 5’ end. Several lines of evidence suggest that hGPIIb binds calcium.” Based on amino acid sequence similarities with calcium-binding domains in other proteins, there are four potential extracellular calcium-binding domains in the hGPIIb heavy chain. The disulfide-bonded light chain contains the transmembrane domain and has a hydrophilic cytoplasmic domain a t its C-terminus. Against this molecular biology background, recent studies have been directed at defining the structural-functional relationships of the GPIIb/IIIa receptor. One approach using crosslinking Arg Gly Asp peptides has demonstrated an important binding region on GPIIIa corresponding to residues 109 to 171.” An alternative approach, using a comparative analysis of this receptor from a number of different species, is used in this article. Species differences in fibrinogen binding have been previously demonstrated. For example, fibrinogen binding to the activated human GPIIb/ IIIa complex can be competitively inhibited by the Cterminal dodecapeptide of the human y fibrinogen chain, as well as by RGD-containing ~ l i g o p e p t i d e s . ~ *In ~ ~contrast, -’~ rat fibrinogen binding is not as sensitive to competitive inhibition by RGDS oligopeptides.” The human dodecapeptide also does not appear to inhibit fibrinogen binding to the rodent GPIIb/IIIa receptor (J. Hawiger, personal communication, June 1988). The basis for the difference in the two

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H E PLATELET MEMBRANE glycoprotein IIb/IIIa (GPIIb/IIIa) complex is a calcium-dependent heterodimer containing binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin that are exposed by platelet a~tivation.’.~ GPIIb/IIIa is a member of a family of adhesive protein receptors that includes chick fibroblast integrin, the LFA- 1/Mac-1 /pl50,95 complexes of leukocytes, the VLA complexes present on activated T lymphocytes, and the fibronectin and vitronectin receptors found on a variety of c e k 5 These receptors consist of a / @ heterodimers with a subunits analogous to GPIIb and j3 subunits analogous to GPIIIa. Many of these receptors interact with ligands containing the Arg Gly Asp ~ e q u e n c e .The ~ . ~ amino acid sequences for several a and j3 subunits have been determined by analysis of cDNA and have demonstrated homology among the various a! and j3 s u b ~ n i t s . ~ . ‘ ~ Mature human GPIIb (hGPIIb) has an apparent molecular weight (mol wt) of approximately 136,000 on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and con-

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From the Division of Hematology. The Children’s Hospital of Philadelphia; the Department of Pediatrics. The University of Pennsylvania School of Medicine. Philadelphia, PA; and The Blood Center of Southeastern Wisconsin. Milwaukee, WI. Submitted August 3,1989: accepted November 24,1989. Supported by Grants No. HL40387 (M.P.) and No. HL38166 (P.N.)from the National Institutes of Health, Bethesda, MD; and a basic research grant from the March of Dimes (M.P.). Address reprint requests to Mortimer Poncz. MD, Division of Hematology. The Children’s Hospital of Philadelphia, 34th Si and Civic Center Blvd. Philadelphia, PA 19104. 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 I734 solely to indicate this fact. 6 I990 by The American Society of Hematology. 0006-4971/90/7504-0002$3.00/0 1282

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Blood, Vol75, No 6 (March 15). 1990: pp 1282-1289

From bloodjournal.hematologylibrary.org by guest on July 11, 2011. For personal use only. 1283

RODENT PLATELET GLYCOPROTEIN 118

fibrinogen receptors is unknown, and the determination of the primary amino acid sequences for rodent GPIIb and GPIIIa may provide insights into the structural basis of this functional difference. Cloning of the rodent fibrinogen receptor c D N A s should allow expression studies ex vivo and permit comparative and chimeric studies with the human receptor, localizing the molecular basis for the binding differences between the two species. In this report, we present the primary amino acid sequence of rodent G P I I b (rGPIIb). This sequence represents the first nonhuman G P I I b described. We compare its sequence to the human counterpart as well as other known integrin a subunits and demonstrate regions of unusually high and low amino acid conservation. New information concerning the amino acid sequences near the cleavage sites of the initial GPIIb translational product is also presented. In addition, rGPIIb genomic analysis shows that there are two rGPIIb genes, one of which appears to be a processed pseudogene. EXPERIMENTAL PROCEDURES

Isolation and characterization of cDNA and genomic clones f o r rGPIIb. A 3.3 kilobase (kb) human cDNA GPIIb probet6was used to screen a rodent megakaryocyte cDNA Xgtll library29 (kindly provided by Robert Rosenberg, Massachusetts Institute of Technology, Boston, MA). Plaque lifts with nitrocellulose filters were done as previously de~cribed.~'Prehybridization for 1 hour and overnight hybridization of filter lifts were done at 65OC in 5 x Denharts, 6 x SSC, 50 pg/mL sonicated salmon sperm DNA, and 10 pg/mL poly rA. Approximately 5 x los cpm of a-32Pdeoxycytosinetriphosphate (dCTP) sonicated calf thymus primer random-labeled probe (10' cpm/pg) was used per filter. Washes were as follows: twice in 2 x sodium chloride/sodium citrate solution (SSC), 0.1% SDS, 15 minutes each; and twice at 65OC in 0.5 x SSC, 0.5% SDS, 30 minutes each. Two rodent genomic libraries were screened. A partial Eco RI library in Charon 4A phage (Clontech, Palo Alto, CA) was screened with a 1.3 kb Hind111 subfragment of the rGPIIb cDNA in an identical fashion to the screening of the cDNA library described above. The 5'-most fragment isolated from screening this genomic library was successfully used to screen a second rodent genomic library3' of a partial Sau 3A digest in EMBL3A phage (kindly provided by Richard Hynes, MIT). Isolated positive genomic inserts were rescreened with a 0.3 kb 5' EcoRI-Hind111 rGPIIb cDNA fragment. Positive cDNA and genomic fragments were subcloned into M13mp18 and mp19 phage.32Single-stranded forms of these phages were isolated and sequenced with Sequenase (United States Biochemical Co, Cleveland, OH) using the dideoxy chain-termination t e c h n i q ~ e ' ~and . ~ ~ a universal M13 primer. cDNA clones were sequenced using Bal3 1 nuclease-generated overlapping deletions of the insert.35 Genomic GPIIb sequence was determined using the universal primer and appropriate Sst I subclones. N-terminal sequence determination of the heavy chain of rGPIIb. Rat platelets were isolated by differential centrifugation of citrateanticoagulated whole rat blood, which was purchased from PelFreez Biologicals (Rogers, AK). Rat and human platelet membrane fractions were prepared using essentially the same procedure as previously des~ribed.'~Membrane pellets were solubilized in 2% (wt/vol) SDS, 10 mmol/L N-ethylmaleimide, 50 mmol/L Tris, pH 6.8, to a final concentration of approximately 3 mg/mL, and subjected to the two-dimensional nonreduced/reduced gel electroph~resis'~ to separate GPIIb from other membrane constituents. The discontinuous buffer system of Laemmli was used in both

dimensions.15To avoid chemically blocking the N-terminus of the proteins, all acrylamide-containing gels were allowed to polymerize for at least 24 hours, and were subsequently pre-electrophoresed at 30 mA for 2 to 4 hours in the presence of 0.1 mmol/L thioglycolate. After pre-electrophoresis, a stacking gel composed of 1.5% Isogel agarose (FMC Corp, Rockland, MA), in place of the normally used 3% acrylamide, was cast.37For the first dimension, 200 to 400 pg of nonreduced solubilized total protein was loaded onto each tube gel, and electrophoresed at 5 mA per tube for 16 hours. The first dimension tube gels were then reduced in the presence of 5% (vol/vol) 2-mercaptoethanol for 60 minutes and placed atop the second dimension slab gels. Isogel agarose was used to form the seal between the tube and slab gel, and also served as the stacking gel for the second dimension. After second dimension electrophoresis, proteins were electrophoretically transferred to Immobilon PVDF membranes (Millipore Corp, Bedford, MA) in a glycine-free buffer composed of 10 mmol/L 3- [cyclohexylamino]- 1-propane-sulfonic acid (CAPS), 10% (vol/vol) methanol, pH 1l.O." Proteins electroblotted onto PVDF membrane were visualized by staining for 5 minutes in 0.1% (wt/vol) Coomassie Blue R-250 dissolved in 50% (vol/vol) methanol, followed by destaining in 50% (vol/vol) methanol. Spots corresponding to rat and human GPIIb were excised with a clean razor blade, placed onto the cartridge block of an Applied Biosystems model 477A pulse-liquid protein sequenator (Foster City, CA), and subjected to 25 cycles of sequential amino acid sequence analysis. Computer analyses of the GPIIb cDNA sequence. Computer analyses, including data storage, hydropathic profiles, and dot matrix comparisons, were done on a Macintosh S E using the DNA Inspector IIe program (Textco, West Lebanon, NH). Rat and human GPIIb amino acid sequences were aligned visually. Alignment with the vitronectin and fibronectin a! subunits (VNRa and FNRa, respectively) were as previously described." The determined rGPIIb cDNA sequence has been submitted to GenBank Genetic Sequence Data Bank. RESULTS

Sequence determination of rCPZZb cDNA. The fulllength sequence of rGPIIb c D N A from the homologous to the polyadenyposition of the proposed hGPIIb c a p site39*40 lation site is 3313 bp. cDNA sequencing was completed on both strands. The length of this sequence is comparable to the 3344 b p of hGPIIb CDNA'~,~' and includes a 26 b p 5'-untranslated region (compared with 3 1 b p in hGPIIb) and a 168 b p 3'-untranslated region (compared with 182 b p for hGPIIb). The coding region is 31 14 b p and has 88% identity with hGPIIb.

Amino acid analysis. T h e initial translation product of rGPIIb is 1037 amino acid residues, compared with 1039 residues of hGPIIb. This is due to four differences in the proteins, including two missing and one additional amino acid in the larger rGPIIb subunit and one less glutamic acid residue at the C terminal in the smaller rGPIIb subunit (Fig 1A). Overall, there is 79% amino acid identity between the two species, as well as 38% and 36% amino acid identity with hFNRa and h V N R a , respectively (Fig 1A); this is very similar to their amino acid identity with hGPIIb." All 18 cysteine residues occur in identical positions, including amino acid residue 602 of the hGPIIb heavy chain, which was misread as a serine in the original description of the hGPIIb cDNA.'~T h e hydropathic profiles of the two proteins are also very similar (Fig 1B) .

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RODENT PLATELET GLYCOPROTEIN Ile

hydrophilic I

Fig 1. Amino acid sequence comparisons between rGPllb, hGPllb, hVNRa, and hFNRa. (A) Amino acid comparison between the deduced rGPllb amino acid sequence (shown in capital letters on the second line) and hGPllb (shown above) and FNRa and VNRa (below rGPllb). All comparisons are with rGPllb. ":" refers t o an amino acid identity with rGPllb. The N-terminus of mature rGPllb determined by amino acid sequence analysis is underlined. Calcium binding domains (19). ;the 12 amino acid mismatch near the cleavage site between rGPllb's heavy and light chains, (B) Hydropathic profile analysis for both GPllb species was done at six amino acid intervals. rGPllb, -; hGPllb, (CI Percent amino acid sequence identity of rGPllb with the other integrin alpha subunits shown in Fig 2A. Analyses were done on consecutive stretches of five amino acid residues. The asterisks shown above the schematic representation of the a chain represent stretches of at least five identical amino acids in all four proteins.

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When comparing hGPIIb and rGPIIb, there are peptide regions that differ greatly from the overall 79% average amino acid identity mentioned above. These regions may demarcate important biologic domains within GPIIb. For example, the previously identifiedcalcium binding dot underlined in Fig 1A have 93% identity between the two species. As shown in Fig 1C as asterisks, there is an identical stretch of at least five amino acids in length between the two GPIIb species and hVNRa and hFNRa in all four calcium binding domains. This high degree of conserved sequence strongly supports the hypothesis that these are biologically important regions. In addition, there are three other regions of unknown biologic function in the midsection of the heavy chain with stretches of at least five identical amino acids. In

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the light chain, there is a high degree of sequence similarity in the transmembrane and intracytoplasmic domains between hGPIIb and rGPIIb. Similar high levels of transmembrane conservation have been previously observed for other membrane protein^.^' The C-terminus of the transmembrane domain and the beginning of the cytoplasmic domains are also very well conserved in all four integrin proteins, with two stretches of at least five identical amino acids (Fig 1B and C). There are also regions of marked amino acid divergence between the two GPIIb species. The N-terminal signal peptides, a functional domain known to show rapid amino acid divergence:' have only 65% identity. More striking is the divergence near the cleavage site between the heavy and light chains of GPIIb. As noted in Fig lB, there is only 56%


1286

PONCZ AND NEWMAN

identity over a stretch of 48 amino acid residues (residues 856 to 903 in rGPllb), including a final stretch of IO amino acids in which there is no identity. This region of GPllb also shows little similarity to the corresponding regions of VNRa and FNRa. GPIIb cleavage sires. The nonreduced/reduced two dimensional gel electrophoresis (Fig 2A) clearly demonstrates that rGPlIb can be well resolved from all other membrane proteins. The mobility of the GPIIb heavy chain well below the diagonal after reduction is characteristic of polypeptides that are disulfide-bonded to another peptide. The N-terminal amino acid sequence of the heavy chain was determined and is aligned in Fig 2B with the sequence derived from the cDNA. There are two discrepancies between the derived amino acid sequence and that predicted by the cDNA, including an N to D conversion at position 15, most likely a result of deamidation during the amino acid

sequencing process. There is also an R versus H discrepancy at position 18, and the human sequence has an E at position 18.16 indicating that this site may be polymorphic with either an arginine or a histidine present. The protein sequence data reveal the presence of the consensus L/F/Y N L/I D sequence at the cleavage site in the signal peptide of the heavy chain of rGPllb. Genomic rCPIlb analysis. Partial DNA sequence was determined for the rGPlIb gene. Two separate clones (designated rGPllb-l and rGPllb-2) were isolated. Both contain a 1.5 kb Ssr I fragment that hybridizes to a 0.3 kb 5' Hind111 rGPllb cDNA fragment (data not shown). Sequence analysis of the 1.5 kb fragments described below reveals that the clone rGPllb-l represents the rGPIlb gene while rGPlIb-2 contains a processed copy of the gene. Figure 3A is a dot matrix comparison between the partial genomic rGPllb-l sequence and the identical region of the

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1 P e p t i d e analysis: L N L D X V K F S V Y T C X I) G X R F

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Fig 2. Two dimensional nonreduced/reduced gel electrophoresis and N-terminal sequence analysis of the rOPllb heavy chain. (A) rGPllb is well resolved from other mambrane proteins. The migration of the heavy chain of rGPllb below the diagonel is characteristic of proteinsthat contain disulfide-linked subunits. ( 6 )The determined peptide sequence for the N-terminus of the rGPllb heavy chain (top line) is compared with that derived from DNA sequence. X, ambiguous residue on peptide sequencing.


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.

Fig 3. Dot matrix comparison of genomic rGPllb and hGPllb. Using eight base matches with two mismatches allowed, the determined partial sequences of genomic clone rGPllb-1 (A) and rGPllb-2 (E) on the ordinate were compared with the homologous l o o 0 bp region of the hGPllb gene DNA sequence on the abscissa. Intron 1 of the hGPllb gene, which is over 2.5 kb, has been truncated as indicated. The coding regions are shown on both axes as thickened lines and numbered on the abscissa for hGPllb.

human genome.40The Sst I fragment of rGPIIb-1 contains a portion of Exon 4 and all of Exon 3 (Fig 1). The splice between Exons 2 and 3 occurs at the cDNA base 344, instead of at the homologous position of base 343 in hGPIIb. The other splice junctions occur at homologous positions in the two species. As can also be seen from this dot matrix, intron 3 and the partial intron 2 shown have obvious DNA sequence homology with their human counterparts. Intron 3 is also of comparable size in the two species. Figure 3B demonstrates that clone rGPIIb-2 is missing introns 1,2, and 3, and contains continuous coding sequence from exons 1, 2, 3, and 4. There is also a double base inversion a t positions cDNA sequence 341 to 342 (CTACGG CTCAGG), not affecting the amino acid sequence. The coding sequence begins at base 191, corresponding to the middle of exon 1 of the hGPIIb gene. Screening of clone rGPIIb-2 with a 0.3 kb 5' Hind111 probe and an oligonucleotide probe to bases 155 to 175 demonstrated that the remaining 5'-coding sequence is not present in the over 2.7 kb of additional 5'-cloned sequence that is available.

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DISCUSSlON

We present cDNA and partial genomic DNA sequence analysis of the rodent GPIIb gene. This study provides the first nonhuman GPIIb sequence and was initiated to allow comparison of the structure and function of the platelet fibrinogen receptor. Both GPIIb and GPIIIa have been previously detected immunologically and biochemically in avian thrombocytes as well as on platelets from diverse mammalian species!' By the criteria of mobility during nonreduced/reduced two dimensional gel electrophoresis, the GPIIb present in all of these species appears to consist of disulfide-linked heavy and light chains. Our analysis extends these earlier observations for rGPIIb and shows that the overall structure and organization of

rGPIIb and hGPIIb appear to be conserved between the two species. The calcium-binding domains present in all of the heavy chains thus far examined are particularly well conserved among the GPIIb species and VNRa and FNRa (Fig 1C). GPIIb/IIIa is a calcium-dependent heteroduplex on platelets, and the binding of calcium may be important in its transformation into a receptor-active form." This function may be ubiquitous to all integrin a subunits and results in highly conserved calcium-binding domains. The biologic function of the other conserved regions is unknown. They may be involved in binding to extracellular ligands or to the integrin p subunit. There are also two well-conserved regions in the light chain of GPIIb. These include the C-terminus of the transmembrane domain and the N-terminus of the intracytoplasmic domain. These regions may be important in either the proper anchoring of the GPIIb protein to the membrane or in rbindingto intracellular ligands such as talin after activation!' It has been previwly suggested that the C-terminus of the heavy chain of hGPIIb may contain the fibrinogen receptor site since its sequence differs greatly from those of hVNRa and hFNRa!6 A comparison of rGPIIb with hGPIIb, however, reveals that this region undergoes rapid species divergence, suggesting that this is a region without a conserved biologic function. The Drosophila integrin receptor's position-specific a subunit PSa has a long 35 Kd additional peptide sequence at the C-terminus of its larger subunit that is variably removed, resulting in a family of 90 to 140 Kd fragments on reducing gels,I3and supporting our hypothesis that the C-terminus of this integrin a heavy chain may not be crucial to the biologic function of this family of receptors. We have demonstrated that the cleavage site between the signal peptide and the heavy rGPIIb subunit is homologous to hGPIIb?' resulting in a mature protein with an Nterminal sequence that is consistent with the integrin a subunit consensus L/F/Y N L/I. D."*13In addition, our

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PONCZ AND NEWMAN

results show that rGPIIb exists as disulfide-bonded heavy and light chains. However, the site a t which this cleavage occurs is not obvious. Although the light chain sequence of rGPIIb is remarkably well preserved, the 12 amino acid residues immediately upstream (double underlined in Fig 1A) have no interspecies similarity. The dibasic sequence S / K / R - R (Fig l A , arrow 4)that is found at the C-terminus of other integrin a larger subunits and that is thought to be important in defining the cleavage site between the larger and smaller subunits”*’3is not present in rGPIIb. Based on immunologic data, it has been suggested that the initial cleavage site of hGPIIb into heavy and light chains may actually occur within the sequence K . R - D R R that is immediately upstream of the double-underlined area in Fig lA.46 Although the dibasic K - R (Fig lA, arrow 2) is not present in rGPIIb, the dibasic R . R (Fig lA, arrow 3) is present, and occurs immediately upstream of the 12 amino acid divergence noted above. This would suggest that rGPIIb is cleaved after this dibasic R - R (although there is an additional dibasic R - R [Fig lA, arrow 11 eight additional residues further upstream unique to rGPIIb). If cleavage occurs after either R eR dibasic sequence, then the rGPIIb light chain would include the underlined 12 amino acid residues. This comparative analysis also demonstrates that there is only a single common dibasic potential cleavage site in rGPIIb and hGPIIb (arrow 3), supporting previous arguments46that hGPIlb is initially cleaved at this dibasic R R sequence (arrow 3) and that the S R site (arrow 4) may be a secondary cleavage site. Partial genomic analysis demonstrates two rGPIIb genes: the expected normal gene and a processed gene lacking expected introns. Sequence analysis shows that the introns determined for the normal gene are homologous to hGPIIb introns. Additional analysis (to be published in detail else-

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where) demonstrates that the 5’-flanking region of rGPIIb and hGPIIb are homologous. The processed gene lacks three expected introns when compared with hGPIIb, including two that are present in the normal rGPIIb gene. In addition, the 5’ portion of the first exon appears to be missing, with the coding region starting in the middle of the first human exon. Processed genes of this nature are thought to be the result of reverse transcription of mRNA species that are inserted back into the genome.48 As seen here with the processed rGPIIb gene, these processed genes often lack the full 5’ and 3’ sequence. The truncated nature of this processed gene would imply that it is a nonfunctional pseudogene. In contrast, there is only a single copy of the hGPIIb gene per haploid genome with no processed pseudogene.a Further analysis of rGPIIb and rGPIIIa at the molecular level should provide additional understanding of the biologic function of the fibrinogen receptor and the regulation of expression of these proteins in megakaryocytes. Our laboratory and others have recently expressed the human fibrinogen receptor in a transient expression The availability of cDNAs for the rodent counterpart should allow a direct comparison of the receptors, and the construction of interspecies chimeras to attempt to localize the structural basis for species-specific biologic differences. ACKNOWLEDGMENT

The authors thank Drs Elias Schwartz, Saul Surrey (The Children’s Hospital of Philadelphia), and Jack Hawiger (Vanderbilt University, Nashville, TN) for their many useful suggestions and review of the manuscript. We are grateful to Roland Cafaro for technical assistance, and to the Medical College of Wisconsin Protein Sequence Facility for performing amino acid sequence analysis.

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