Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

Miyazaki, Hiroshi; Tamura, Setsuko; Sudo, Tomoko; Suzuki, Takamoto

doi:10.1016/0049-3848(90)90118-v

Cited by 11 publications

(3 citation statements)

References 18 publications

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“…We confirmed tissue-specific expression of many of the constructs by immunomagnetic separation using a murine monoclonal antirat a I l b l P 3 antibody [35]. Using this approach, we demonstrated that all constructs expressing hGH at greater than 10% of the 0.9 kb wild-type, fulllength construct expressed >85% of their activity in a tissue-specific fashion.…”

Section: Expression Studiesmentioning

confidence: 54%

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb

et al. 1996

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The megakaryocyte (MK)-specific integrin, alphaIIb, is the alpha-subunit of the alphaIIb/beta3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alphaIIb gene is specifically expressed in MKs, this gene was studied as a potential model for MK-specific gene expression. Previous studies have defined some of the important regulatory elements in 912 bp of the immediate 5'-flanking region of this gene. These studies defined several important elements including two GATA-binding elements and an Ets-binding element. Using a primary rat marrow expression system, we demonstrated that one of the GATA-binding elements, -454 bp upstream of the transcriptional start site (GATA454), is critical for expression of the alphaIIb gene. A potential negative regulatory element was found between -100 and -200 bp upstream of both the rat and human alphaIIb genes. The biological basis by which this negative regulatory region effects expression is not well understood. Recent studies have focused on the issue of the molecular basis by which this TATA-less gene is properly transcribed. We found that a GA-rich region approximately 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site that interacts with an Ets-consensus site approximately 20 bp further upstream. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alphaIIb gene. In studies looking at more distal regulatory elements, we have found, using the primary rat marrow expression system, that 2.9 kb of 5'-flanking alphaIIb sequence has as high a level of expression as the 912 bp construct. Whether either of these lengths of 5'-flanking region can result in tissue-specific expression in transgenic models is presently being investigated. In addition, while a published report suggests that the two genes alphaIIb and beta3 are physically linked within a 250 kb region of genomic DNA, analysis of yeast artificial chromosome clones and genomic pulsed field gel electrophoresis analysis are consistent with these two genes not being tightly linked and being >1 mb apart, suggesting that these two genes do not form a single, tissue-specific locus.

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Section: Expression Studiesmentioning

confidence: 54%

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb

et al. 1996

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“…We confirmed tissue-specific expression of all our constructs by immunomagnetic separation using a murine monoclonal anti-rodent GPIIb/IIIa antibody kindly provided by Dr. Takamoto Suzuki at the Kirin Brewery in Japan (Fig. 1) [56]. The procedure uses a rat anti-mouse IgG2a coated iron bead as the secondary antibody (Dynal, Great Neck, NY) and a magnetic particle concentrator [57].…”

Section: Regulation Of Rat Gpiib Gene Expressionmentioning

confidence: 57%

Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression

Block

Poncz

1995

STEM CELLS

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Glycoprotein (GP)IIb/IIIa is an integrin complex normally restricted in its expression to platelets and the megakaryocytes from which they are derived. This complex functions as a receptor for fibrinogen and other ligands and is involved in platelet aggregation. The receptor complex is expressed at high levels during final megakaryocyte differentiation. Further, while GPIIIa is expressed in other tissues as part of the vitronectin receptor, GPIIb is only expressed on maturing megakaryocytes and the platelets derived from them. Thus studies of the GPIIb gene may serve as a model of gene regulation during this process. Over the past several years, the genes for both GPIIb and IIIa have been cloned and analyzed. The GPIIb gene contains 30 exons over 18 kilobases (kb). The transcriptional start site has been determined and there does not appear to be a TATA-box immediately upstream of this site. Studies have been done to define regulatory elements upstream of the transcriptional start site. Most of these studies focused on the human promoter and on studies using megakaryocytic cell lines. These studies have defined several important tissue-specific promoter elements including a GATA454 site (454 basepairs upstream of the transcriptional start site that involves a GATA-binding consensus sequence), a GATA54 site and an Ets35 site (that involves an Ets-binding consensus sequence). Expression studies with megakaryocytic cell lines suggest that each of these sites effects expression approximately threefold. Further, an Ets510 site was also described that had a similar effect. While these studies were underway, we pursued studies of the rat 5'-flanking region using a rat primary marrow expression system. Qualitatively, our data support the human data; however, quantitatively, we found significant differences from the human studies done in cell lines. We found that the major tissue-specific promoter element was the GATA454 site. Mutations altering this site result in an approximately fiftyfold drop in expression. In comparison, eliminating the Ets510 site by truncation or point mutation had only a twofold effect on expression. Mutations at the Ets35 site did effect expression at a high level, decreasing expression approximately fifteenfold, while mutations at the GATA54 site effected expression by approximately ninefold. In addition, using 50 bp deletions, we have preliminarily defined two domains from -450 to -351 bp and -150 to -101 bp upstream of the transcriptional start site that effected expression. The former appears to contain a positive regulatory element, while the latter appears to be a silencer element.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…Two days after transfection, cells were transferred to a selection medium containing 500 g/mL G418 (Invitrogen) and 300 g/mL Zeocin (Invitrogen). After 3 weeks of selection, ␣IIb␤3 expression was assessed by flow cytometry using P34, a monoclonal antibody (mAb) recognizing rat, mouse, and human ␣IIb␤3 (a gift from Dr H. Miyazaki, Kirin Brewery, Gunma, Japan), 21 as well as A2A9, a mAb that recognizes the human ␣IIb␤3 heterodimer. 22 Transfected cells were sorted by fluorescenceactivated cell sorting to obtain cell lines expressing high levels of ␣IIb␤3.…”

Section: Stable Expression Of ␣Iib␤3 In Cho Cellsmentioning

confidence: 99%

Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Basani

Zhu

Thornton

et al. 2009

Blood

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Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginineglycine-aspartic acid (RGD) motif. Using chimeric human-rat ␣IIb␤3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the ␣IIb ␤ propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human ␣IIb␤3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the ␣IIb␤3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with ␣IIb in a different manner than with these small molecules. None of these speciesbased substitutions affected the ability of ␣IIb␤3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphatestimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the ␣IIb ␤-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.

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Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

Cited by 11 publications

References 18 publications

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb

Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression

Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

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Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

Cited by 11 publications

References 18 publications

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, &alpha;IIb

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, &alpha;IIb

Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression

Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

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The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb

The Regulated Expression of a TATA&hyphen;Less, Platelet&hyphen;Specific Gene, α_IIb