Characterization of human platelet binding of recombinant T cell receptor ligand

Abstract

BackgroundRecombinant 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.MethodsWe 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.ResultsOur 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.ConclusionsPlatelets, 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.

Recombinant T Cell Receptor Ligand Treatment Improves Neurological Outcome in the Presence of Tissue Plasminogen Activator in Experimental Ischemic Stroke

“…Therefore, our data suggest that the anti-inflammatory treatment with RTL1000 is superior to thrombolytic therapy because it provides long-term protection, and because it protects against both vascular injury and downstream parenchymal inflammatory mechanisms of ischemic injury. Interestingly, and in support of vascular mechanism of protection, we recently reported that RTL1000 interacts with human platelets and decreases aggregation [32]. In addition to serving as T cell receptor ligand, RTL is also a platelet ligand.…”

“…In addition to serving as T cell receptor ligand, RTL is also a platelet ligand. Specifically, in our previous study [32], we showed that RTL suppresses collagen-induced platelet aggregation in vitro, and inhibits occlusive thrombus formation on collagen-coated surfaces under physiologically relevant pressure gradients. Therefore, in addition to the anti-inflammatory mechanism of protection, RTL1000 may also prevent platelet re-aggregation after thrombolysis by t-PA.…”

The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

“…For single-platelet calcium analyses, platelets were loaded with 15 M Oregon Green BAPTA1-AM as previously described (22). Loaded platelets were allowed to settle onto fibrinogen-coated coverslips over a period of 30 min at 37°C in the presence or absence of 1 U/ml thrombin.…”

“…Loaded platelets were allowed to settle onto fibrinogen-coated coverslips over a period of 30 min at 37°C in the presence or absence of 1 U/ml thrombin. The fluorescence intensities of single platelets were recorded and analyzed with a customized Matlab application as previously described (22). For comparison purposes, the average max-to-mean ratio Ϯ SE of fluorescence intensities for at least five platelets from three fields of view was calculated for each treatment.…”

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

“…Lysates were denatured in an equal volume of Laemmli sample buffer (Bio-Rad) with 0.5M dithiothreitol (100°C, 5 minutes), separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with indicated antibodies and HRP-conjugated secondary antibodies as previously described. 31 Protein was detected using ECL (Amersham Biosciences). Blot films were digitally scanned and analyzed for band intensity with ImageJ Volume 1.44 software.…”