The TLT-1 receptor, to date, has only been identified on platelets. The lineage restriction of TLT-1 suggests that TLT-1 plays a specific role in platelet biology. Current work on the TLT-1 receptor shows that antibodies specific to TLT-1 can inhibit platelet aggregation implying that TLT-1 enhances platelet aggregation. Accordingly, the addition of rsTLT-1 significantly enhances platelet aggregation further supporting an important role for TLT-1 during platelet activation and maintenance of vascular integrity. Consistent with this data, studies with platelets from TLT-1 null mice show that these mice are susceptible to bleeding when challenged with lipopolysaccaride. In our recent publication we demonstrated that rsTLT-1 binds to fibrinogen suggesting that interaction with fibrinogen may play a key role to understanding how TLT-1 regulates platelet function. Furthermore, our laboratory has found prominent concentrations of sTLT-1 in patient populations suffering from either sepsis or viral syndrome compared to healthy individuals suggesting a potential role for sTLT-1 in maintenance of vascular hemostasis during inflammatory diseases [16
] (Washington AV, Gibot S, et al;
unpublished observations). Here we provide additional evidence for a role for sTLT-1 in platelet-endothelial cell binding interactions and gain insights to the mechanisms of TLT-1 regulation of platelet function.
We performed a platelet-endothelial adherence assay and demonstrated for the first time that TLT-1 plays a role in platelet adherence to endothelial cells in a static assay. Our results show that the addition of rsTLT-1 to either resting or TxA2 activated BAEC increased platelet adherence to the BAEC over controls. Using activated platelets appeared to cause a reduction of platelet adherence compared to incubations with resting platelets. Activated endothelial cells have been shown to shrink and lose cell to cell contact and detach. The addition of activated platelets may further increase the shrinkage and detachment of the endothelial cells, resulting in a greater variation in the numbers of platelets that adhere under those conditions. Although we consistently found lower numbers of adherent platelets when activated platelets were incubated with activated endothelial cells, the addition of rsTLT-1 increased the total amount of adherent platelets to similar levels seen when resting platelets were used. We have explored the possibility of sTLT-1 increasing adherence to endothelial cells, by investigating the ability of sTLT-1 to directly interact with the extracellular matrix proteins (ECM) vitronectin, collagen, and fibronectin by enzyme linked immunosorbant assay (ELISA). Using the ELISA assay we were, however, unable detect any interactions of sTLT-1 with any of these proteins (data not shown). Therefore we do not believe that the increased adhesion induced by sTLT-1 is mediated through any of these ECM proteins. Our study opens the possibility of a preserving effect of sTLT-1 on endothelial cells and is currently under investigation in our laboratory. This work also suggests that sTLT-1 may play a role in the maintenance of vascular integrity and that addition of rsTLT-1 to sites of vascular injury may accelerate the cession of bleeding.
Another interesting point is that the most significant difference was seen when rsTLT-1 was incubated with resting platelets. Although resting platelets bound activated endothelial cells more effectively than activated platelets, our results indicate that addition of rsTLT-1 resulted in a statistically significant effect on the interaction between resting platelets and endothelial cells that wasn't seen with activated platelets under the same conditions. Based on these results and recent demonstrations of the importance of α-granule components to platelet function[20
] we hypothesize that rsTLT-1 amplifies activation signals from the endothelial cells and/or processing derived signals that may occur during the treatment. A possible mechanism for the increased adherence with resting platelets may lie in the generation of platelet microparticles. It is generally accepted that even with gentle manipulation of platelet samples, platelets will generate microparticles, which have been shown to lower the natural anti-coagulant properties of endothelial cells[21
] as well as contain the procoagulant tissue factor[22
]. The combination of lower endothelial resistance to platelet adherence and low levels of tissue factor in the presence of sTLT-1 may lead to increased platelet degranulation[23
]. We hope to be able to discern some of these possibilities in the future. Our results are consistent with the finding that rsTLT-1 enhances platelet aggregation and may indicate that TLT-1 may enhance both platelet-platelet and platelet-endothelial interactions by a single mechanism.
Because actin polymerization plays a large role in platelet aggregation, adhesion, and functions downstream of the calcium signal needed for fusion of α-granules to the platelet membrane, we investigated the ability of TLT-1 to effect actin polymerization. Recombinant sTLT-1 increased platelet spreading and the amount of platelets that adhered to fibrinogen matrixes on glass slides. On the fibrinogen-only slides you can witness the beginnings of filopodia extension as evidence of increased actin polymerization at 5 min (). However, on the slides with rsTLT-1 there was visually detectable increases in the amount of platelet structures such as filopodia and lamellipodia. These differences were most noticeable on the slides that contained 50 and 100 μg/ml. These results are consistent with the rsTLT-1 mediated adhesion of platelets to the endothelial cells. We attribute the enhanced binding and size of platelets to an increase in the amount of actin structures that were made in the presence of the rsTLT-1 compared to fibrinogen alone. Actin polymerization in platelets is a crucial step in this cascade of events; allowing for a rapid shape change in platelets that leads to the formation of filopodia and lamellipodia on activated platelets. Confocal microscopy revealed that rsTLT-1 promotes platelet cytoskeletal actin polymerization and allows for the formation of filopodia and lamellipodia at the mobile edges of the platelets. Collectively, our studies suggest that TLT-1 may play a visible role in the maintenance of vascular integrity and suggests that the addition of rsTLT-1 to sites of vascular injury may accelerate the cession of bleeding.