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Characterization of human platelet binding of recombinant T cell receptor ligand.

Itakura A, Aslan JE, Sinha S, White-Adams TC, Patel IA, Meza-Romero R, Vandenbark AA, Burrows GG, Offner H, McCarty OJ - J Neuroinflammation (2010)

Bottom Line: The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor.The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, USA.

ABSTRACT

Background: Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.

Methods: We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function.

Results: Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.

Conclusions: Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

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Real-time imaging of intracellular Ca2+ in platelets. Platelets were loaded with the calcium reporter dye Oregon Green BAPTA 1-AM and imaged upon contact with RTL1000 or fibrinogen-coated surfaces. Graphs show the observed fluorescent intensity (arbitrary units) of a single platelet over the period of 6 minutes. The arrows indicate the platelets' arrival at the region of interest.
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Figure 4: Real-time imaging of intracellular Ca2+ in platelets. Platelets were loaded with the calcium reporter dye Oregon Green BAPTA 1-AM and imaged upon contact with RTL1000 or fibrinogen-coated surfaces. Graphs show the observed fluorescent intensity (arbitrary units) of a single platelet over the period of 6 minutes. The arrows indicate the platelets' arrival at the region of interest.

Mentions: Upon activation, platelets rapidly mobilize intracellular stores of calcium and assemble actin-rich structures such as filopodia, lamellipodia and stress fibers. Fluorescent labeling of the actin cytoskeleton showed the formation of stress fibers in the human platelets on immobilized RTL1000 (Figure 3). Real-time imaging of platelets loaded with the calcium-reporter dye, Oregon Green BAPTA 1-AM, revealed that platelets generated a rapid burst of oscillating intracellular Ca2+ following binding to RTL1000, which subsequently declined over a period of 3-10 min (Figure 4). In contrast, a rhythmic series of Ca2+ spikes were observed on fibrinogen (Figure 4).


Characterization of human platelet binding of recombinant T cell receptor ligand.

Itakura A, Aslan JE, Sinha S, White-Adams TC, Patel IA, Meza-Romero R, Vandenbark AA, Burrows GG, Offner H, McCarty OJ - J Neuroinflammation (2010)

Real-time imaging of intracellular Ca2+ in platelets. Platelets were loaded with the calcium reporter dye Oregon Green BAPTA 1-AM and imaged upon contact with RTL1000 or fibrinogen-coated surfaces. Graphs show the observed fluorescent intensity (arbitrary units) of a single platelet over the period of 6 minutes. The arrows indicate the platelets' arrival at the region of interest.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992052&req=5

Figure 4: Real-time imaging of intracellular Ca2+ in platelets. Platelets were loaded with the calcium reporter dye Oregon Green BAPTA 1-AM and imaged upon contact with RTL1000 or fibrinogen-coated surfaces. Graphs show the observed fluorescent intensity (arbitrary units) of a single platelet over the period of 6 minutes. The arrows indicate the platelets' arrival at the region of interest.
Mentions: Upon activation, platelets rapidly mobilize intracellular stores of calcium and assemble actin-rich structures such as filopodia, lamellipodia and stress fibers. Fluorescent labeling of the actin cytoskeleton showed the formation of stress fibers in the human platelets on immobilized RTL1000 (Figure 3). Real-time imaging of platelets loaded with the calcium-reporter dye, Oregon Green BAPTA 1-AM, revealed that platelets generated a rapid burst of oscillating intracellular Ca2+ following binding to RTL1000, which subsequently declined over a period of 3-10 min (Figure 4). In contrast, a rhythmic series of Ca2+ spikes were observed on fibrinogen (Figure 4).

Bottom Line: The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor.The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, USA.

ABSTRACT

Background: Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.

Methods: We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function.

Results: Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.

Conclusions: Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

Show MeSH
Related in: MedlinePlus