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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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Effect of RTL on platelet function and occlusive thrombus formation. The effect of RTL1000 on A) aPTT or B) the clotting time of PPP in the presence of excess molar Ca2+. In selected experiments, plasma was pretreated with vehicle, RTL1000 (10 μg/ml), the anti-FXI mAb 14E11 (20 μg/ml) or tissue factor (TF, 1 pM). C) Washed human platelets (2 × 108/ml) were pretreated with either vehicle or RTL1000 (10 μg/ml) for 2 min prior to stimulation with collagen (2 μg/ml). The extent of platelet aggregation was observed using a Chrono-Log aggregometer. D) Whole human blood in 0.38% sodium citrate was recalcified and perfused through a collagen-coated glass capillary until occlusion. Blood flow was driven by a constant pressure difference. In selected experiments, blood was pre-treated with either 10 or 50 μg/ml RTL1000. Data are reported as mean ± SEM of at least 3 experiments. * P < 0.05 compared to vehicle.
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Figure 7: Effect of RTL on platelet function and occlusive thrombus formation. The effect of RTL1000 on A) aPTT or B) the clotting time of PPP in the presence of excess molar Ca2+. In selected experiments, plasma was pretreated with vehicle, RTL1000 (10 μg/ml), the anti-FXI mAb 14E11 (20 μg/ml) or tissue factor (TF, 1 pM). C) Washed human platelets (2 × 108/ml) were pretreated with either vehicle or RTL1000 (10 μg/ml) for 2 min prior to stimulation with collagen (2 μg/ml). The extent of platelet aggregation was observed using a Chrono-Log aggregometer. D) Whole human blood in 0.38% sodium citrate was recalcified and perfused through a collagen-coated glass capillary until occlusion. Blood flow was driven by a constant pressure difference. In selected experiments, blood was pre-treated with either 10 or 50 μg/ml RTL1000. Data are reported as mean ± SEM of at least 3 experiments. * P < 0.05 compared to vehicle.

Mentions: Next we determined the effect of RTL on the initiation of coagulation. RTL1000 did not affect the activated partial prothrombin time (aPTT) or the clotting time of platelet-poor plasma (PPP) initiated by the addition of excess molar Ca2+ (Figure 7A and 7B, respectively). In contrast, the addition of the anti-FXI mAb, 14E11, prolonged the aPTT (Figure 7A), while the addition of tissue factor (TF, 1 pM) drastically reduced clotting time in recalcified plasma (Figure 7B).


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)

Effect of RTL on platelet function and occlusive thrombus formation. The effect of RTL1000 on A) aPTT or B) the clotting time of PPP in the presence of excess molar Ca2+. In selected experiments, plasma was pretreated with vehicle, RTL1000 (10 μg/ml), the anti-FXI mAb 14E11 (20 μg/ml) or tissue factor (TF, 1 pM). C) Washed human platelets (2 × 108/ml) were pretreated with either vehicle or RTL1000 (10 μg/ml) for 2 min prior to stimulation with collagen (2 μg/ml). The extent of platelet aggregation was observed using a Chrono-Log aggregometer. D) Whole human blood in 0.38% sodium citrate was recalcified and perfused through a collagen-coated glass capillary until occlusion. Blood flow was driven by a constant pressure difference. In selected experiments, blood was pre-treated with either 10 or 50 μg/ml RTL1000. Data are reported as mean ± SEM of at least 3 experiments. * P < 0.05 compared to vehicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effect of RTL on platelet function and occlusive thrombus formation. The effect of RTL1000 on A) aPTT or B) the clotting time of PPP in the presence of excess molar Ca2+. In selected experiments, plasma was pretreated with vehicle, RTL1000 (10 μg/ml), the anti-FXI mAb 14E11 (20 μg/ml) or tissue factor (TF, 1 pM). C) Washed human platelets (2 × 108/ml) were pretreated with either vehicle or RTL1000 (10 μg/ml) for 2 min prior to stimulation with collagen (2 μg/ml). The extent of platelet aggregation was observed using a Chrono-Log aggregometer. D) Whole human blood in 0.38% sodium citrate was recalcified and perfused through a collagen-coated glass capillary until occlusion. Blood flow was driven by a constant pressure difference. In selected experiments, blood was pre-treated with either 10 or 50 μg/ml RTL1000. Data are reported as mean ± SEM of at least 3 experiments. * P < 0.05 compared to vehicle.
Mentions: Next we determined the effect of RTL on the initiation of coagulation. RTL1000 did not affect the activated partial prothrombin time (aPTT) or the clotting time of platelet-poor plasma (PPP) initiated by the addition of excess molar Ca2+ (Figure 7A and 7B, respectively). In contrast, the addition of the anti-FXI mAb, 14E11, prolonged the aPTT (Figure 7A), while the addition of tissue factor (TF, 1 pM) drastically reduced clotting time in recalcified plasma (Figure 7B).

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