CONTROLS EARLY THROMBUS FORMATION AND STABILITY BY FACILITATING ΑIIBΒ3 OUTSIDE-IN SIGNALING IN MICE.

with an average area that was approximately twice that of in the treml1 -/- mouse (11.68±0.99 µM µM These results suggested that TLT-1 is responsible for the preliminary stages of clot formation after inflammatory challenge. We also stained for complement factors H (155kD) and I (88kD) to evaluate plasma leakage and found that CXCL2 caused a striking

The relationship between inflammatory and hemostatic systems is dynamic, and although extensively characterized, the complete relationship is not understood (Herter et al., 2014). TREM-Like transcript 1 (TLT-1) is an α-granular platelet receptor that binds fibrinogen and localizes at the platelet surface upon platelet activation (Washington, 2009;Washington et al., 2002;Washington et al., 2004). TLT-1 is abundantly expressed in platelets (Coxon et al., 2017). Based on the works of Simon et. al. (Simon et al., 2014) and those of Rowley et. al (Rowley et al., 2011), TLT-1 is one of the most abundantly expressed proteins in mouse and human platelets (top 3%). These data suggest that it is more abundant than GP1 (top 7%), P-selectin, and integrin 2a (each are in the top 5%). However, TLT-1's role in hemostasis or inflammation remains undefined.
Invocation of the Shwartzman reaction in treml1 -/mice suggests that TLT-1 controls hemorrhage, neutrophil recruitment, and possibly edema during inflammation and points to a role for TLT-1 in the translation of inflammatory response into hemostatic mechanisms (Washington, 2009). The Shwartzman reaction uses lipopolysaccharide and tumor necrosis factor-to induce an immune response (Brozna, 1990;Stetson and Good, 1951;Stetson, 1951b). In the present study, we evaluate the role of TLT-1 in inflammation and determine that TLT-1 mediates early clot formation enabling αIIbβ3 activation by facilitating downstream outside-in signaling. We further demonstrate the relevance of our finding using inflammatory and hemostatic challenges.

Mice
All mice were maintained under specific pathogen-free conditions at the Animal House Facility. Mice were between 8-10 weeks of age and weighed 20-23 g. Animal care was provided in accordance with the NIH Guide for the Care and Use of Laboratory Animals (publication no. 85-23. Revised 1985).

Reverse Arthus Reaction (RAR)
RAR was completed as described in reference (Goerge et al., 2008). For quantification studies of the inflammatory area, edema and hemorrhage were subjected to quantification as described in references (Klopfleisch, 2013;Sylvestre et al., 1996).

Western blotting
Western blotting was completed as in ref (Washington et al., 2002) Whole mount stain of cremaster muscle Anesthetized wild type or treml1 -/mice were intra-scrotally injected with CXCL-2 (2.5 g/mL). After 3.5 hours incubation, the cremaster muscle was prepared for intravital microscopy as previously described (Zhang et al., 2001).

Pulmonary embolism
Thromboplastin induced pulmonary embolism (PE) was achieved through the I.V injection of 5 L thromboplastin [Sigma] and mice were monitored for 30-minutes for breathing cessation after which mortality was recorded and lungs were collected for histology. Collagen/epinephrine PE was induced as in ref (Rowley et al., 2016) using 64 mg/kg of collagen and 5.4 mg/kg of epinephrine and anti-TLT-1 (100ug/100ul) 20 minutes prior to the collagen/epinephrine injection.

Statistics
Paired, two-tailed Student's t test analysis available in Prism, version 7.01 (Graph-Pad Software) was applied to evaluate statistical differences for 2 groups. P < 0.05 was considered statistically significant.

Results and discussion:-
To gain insights into TLT-1 function we injected CXCL2 in to the scrotum of wild type (WT) and treml1 -/mice to visualize the inflammatory changes in the vasculature. CXCL2 attracts neutrophils and leads to clotting in the vasculature. We evaluated the cremaster muscle at 3, 4, and 5 hours after injection and found 3 -4 hours to be the most insightful. WT mice exhibited more and larger clots with an average area that was approximately twice that of those in the treml1 -/mouse (11.68±0.99 µM 2 vs. 5.59±0.80 µM 2 ; figure1a). These results suggested that TLT-1 is responsible for the preliminary stages of clot formation after inflammatory challenge. We also stained for complement factors H (155kD) and I (88kD) to evaluate plasma leakage and found that CXCL2 caused a striking 1145 mobilization of plasma factor H and I from vasculature into the cremaster muscle of treml1 -/mice, which was not observed in WT mice (figure1b). These results support the concept that TLT-1 regulates edema as well.
To determine the basis for the small size of TLT-1 -/thrombi and inhibition of clot retraction, we examined the kinetics of αIIbβ3 surface expression after low dose thrombin activation. Quantification of mean fluorescence intensity demonstrated that, WT platelets exhibited a time dependent increase of αIIbβ3 and TLT-1 surface expression. Although not significant, αIIb seemed to decrease on treml1 -/platelets (figure1c-e). These results suggested that TLT-1 continuously participates in agonist-induced signaling that leads into αIIbβ3 stabilization on the surface. Treml1 -/mice demonstrate reduced clot retraction compared to WT (figure 1f) and collectively our results suggest that TLT-1 affects αIIbβ3 function. We hypothesized that TLT-1 stabilizes clot formation by facilitating outside-in signaling of αIIbβ3. To test this hypothesis, we examined β3 Y773 phosphorylation (pβ3 Y773 ) over time in WT and treml1 -/platelets. pβ3 Y773 is an indicator of early outside-in signaling ( To extend the applicability of TLT-1's mechanism beyond LPS mediated inflammation seen in the Shwarztman reaction, we used the Reverse Passive Arthus reaction which elicits a localized, dermal, immune response using immune complexes (Stetson, 1951a). Evaluations of the skin lesions show that wild type mice exhibited mild petechia surrounding the injection area (figure 2a). Blinded histological grading of the lesions (scale 1 -4) revealed that the treml1 -/mice have a significant increase in bleeding and associated edema when compared with their wild type counterparts (score=2.4±0.23 vs 1.3±0.21). Thus TLT-1 has a broad impact in translating the immune challenge into hemostatic response.
To investigate if TLT-1 responded to hemostatic triggers, we elicited pulmonary embolism (PE) using two different experimental approaches, both of which cause widespread formation of micothrombi in the lungs (Renné et al., 2005). First, we used an I.V. injection of thromboplastin, which mimics tissue factor exposure by activating coagulation through the conversion of pro-thrombin to thrombin (Zacharski and Rosenstein, 1979). Thromboplastin administration caused a significant increase in mortality in treml1 +/heterozygous littermates compared to treml1 -/mice (65% vs 100%; figure 2b). Histological analysis revealed a reduction of clot formation in treml1 -/mice compared to treml1 +/mice. Moreover, quantification of the clots in the lungs WT mice after thromboplastin treatment demonstrated a 40% decrease in clot formation compared to treml1 -/+ mice (p<0.009). Second, we injected I.V. collagen/epinephrine which causes direct platelet activation (Mustonen and Lassila, 1996) , (Renné et al., 2005). Administration of collagen/epinephrine caused a significant increase in mortality in WT compared to treml1 -/mice (p=0.025; figure2c). Accordingly, administration of anti-TLT-1 antibody closely mimics the treml1 -/phenotype (p=0.006; figure 2e). The infusion of Evans' blue after onset of respiratory arrest demonstrated almost complete blockage of perfusion on WT compared to treml1 -/mice (figure 2d, f). To our knowledge, this is the first in vivo demonstration of TLT-1 as a therapeutic target. Based on these results we conclude that TLT-1 contributes to microclot formation after hemostatic challenges as well as immune challenges.
Here, we demonstrate that TLT-1 contributes to αIIbβ3 inside-out signaling and mediates the phosphorylation of the β3 subunit, resulting in enhanced outside-in directed clot stabilization and vessel occlusion. It is possible that TLT-1 may play a receptor scaffolding role, recruiting key signaling molecules to the platelet surface. Like αIIbβ3, TLT-1 is a fibrinogen receptor (Washington, 2009). Where αIIbβ3 must convert to its active form, there is no known TLT-1 activation sequence and consequently TLT-1 should bind fibrinogen upon surface expression. Therefore, TLT-1 could sequestrate fibrinogen on the platelet surface and/or abet fibrin formation. Fibrin(ogen) sequestration would be important in forming a hierarchical thrombus core (Stalker et al., 2013), controlling fibrin deposition, and regulation of clot size (Owaynat et al., 2015).

ISSN: 2320-5407
Int. J. Adv. Res. 6(7), 1143-1149 1146 Author Contributions:-JMO, FR are responsible for the cremaster studies; FR, SO are responsible for the RAR studies; JMO, LR, MKB, MTR are responsible for the PE studies; JMO, BM, NL are responsible for confocal images and analysis; JMO, NL, AVW are responsible for the western blots; and JMO and AVW are responsible for the writing of the manuscript and overall direction of the project (d and f) Mice from d and f were perfused with Evans' blue (500ul) after onset of respiratory arrest or those mice that survived the study; lungs were then excised, the treml1 -/mice showed almost complete perfusion (blue) while in the wild type the perfusion was impaired (pink) compared to wild type (wild type n=4, wild type + antibody-n=4; p=0.006). 1147