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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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Characterization of platelet adhesion and spreading on RTL1000. Platelets were pipetted onto glass surfaces coated with RTL1000 or fibrinogen (FG) for 45 min at 37°C and imaged using DIC microscopy. In selected experiments, platelets were pretreated with vehicle, the ADP scavenger apyrase (2 U/ml) and cyclooxygenase inhibitor indomethacin (10 μM), the integrin αIIbβ3 antagonist eptifibatide (20 μg/ml), inhibitors to Src family kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM).
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Figure 5: Characterization of platelet adhesion and spreading on RTL1000. Platelets were pipetted onto glass surfaces coated with RTL1000 or fibrinogen (FG) for 45 min at 37°C and imaged using DIC microscopy. In selected experiments, platelets were pretreated with vehicle, the ADP scavenger apyrase (2 U/ml) and cyclooxygenase inhibitor indomethacin (10 μM), the integrin αIIbβ3 antagonist eptifibatide (20 μg/ml), inhibitors to Src family kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM).

Mentions: We next aimed to determine the intracellular mechanisms that mediate human platelet spreading on RTL1000. Addition of the ADP-scavenger apyrase in combination with the cyclooxygenase inhibitor indomethacin had no effect on platelet spreading on RTL1000 (Table 1). In contrast, treatment of platelets with inhibitors to Src kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM) abrogated platelet lamellipodia formation on RTL1000-coated surfaces. Platelet lamellipodia formation on RTL1000 was also inhibited in the presence of the αIIbβ3 receptor antagonist, eptifibatide, whereas adhesion to fibrinogen was eliminated in the presence of eptifibatide (Figure 5 and Table 1)


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)

Characterization of platelet adhesion and spreading on RTL1000. Platelets were pipetted onto glass surfaces coated with RTL1000 or fibrinogen (FG) for 45 min at 37°C and imaged using DIC microscopy. In selected experiments, platelets were pretreated with vehicle, the ADP scavenger apyrase (2 U/ml) and cyclooxygenase inhibitor indomethacin (10 μM), the integrin αIIbβ3 antagonist eptifibatide (20 μg/ml), inhibitors to Src family kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Characterization of platelet adhesion and spreading on RTL1000. Platelets were pipetted onto glass surfaces coated with RTL1000 or fibrinogen (FG) for 45 min at 37°C and imaged using DIC microscopy. In selected experiments, platelets were pretreated with vehicle, the ADP scavenger apyrase (2 U/ml) and cyclooxygenase inhibitor indomethacin (10 μM), the integrin αIIbβ3 antagonist eptifibatide (20 μg/ml), inhibitors to Src family kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM).
Mentions: We next aimed to determine the intracellular mechanisms that mediate human platelet spreading on RTL1000. Addition of the ADP-scavenger apyrase in combination with the cyclooxygenase inhibitor indomethacin had no effect on platelet spreading on RTL1000 (Table 1). In contrast, treatment of platelets with inhibitors to Src kinases (PP2, 20 μM), Syk kinase (BAY 61-3606, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM) or an intracellular Ca2+ chelator (BAPTA-AM, 10 μM) abrogated platelet lamellipodia formation on RTL1000-coated surfaces. Platelet lamellipodia formation on RTL1000 was also inhibited in the presence of the αIIbβ3 receptor antagonist, eptifibatide, whereas adhesion to fibrinogen was eliminated in the presence of eptifibatide (Figure 5 and Table 1)

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