Our previous work has suggested that the TLT-1 extracellular domain is shed from platelets during activation (9
). The potential biological significance of the sTLT-1 fragment is reinforced by the existence of 2 splice variants with limited or absent intracellular domains. The first is the most abundant TLT-1 mRNA species and possesses an extracellular domain identical to that of full-length TLT-1 but only a 16-aa cytoplasmic domain (18
). The second form was recently identified in our laboratory and encodes only the TLT-1 extracellular domain (A.V. Washington, unpublished observations). From an evolutionary perspective, the presence of these truncated and soluble species in both mice and humans argues for an important role for the extracellular domain in physiology. Therefore, here we addressed the possibility that sTLT-1 might be released under pathological conditions, resulting in changes in platelet function during those disease states. Based on this paradigm, we hypothesized that during rife, unfocused platelet activation associated with sepsis (11
), we might detect abnormally high levels of sTLT-1.
Data from 2 independent cohorts confirmed the presence of increased levels of sTLT-1 in sepsis patients. Our initial study incorporated patients in the very early stages of sepsis, as indicated by their relatively low SOFA scores, as compared with our in-depth study. Our original patients also had much higher levels of sTLT than the cohort, with higher SOFA scores suggesting that sTLT-1 may spike early in the septic response. However, clinical characteristics of some patients in our initial study, including HIV infection, limit these interpretations. Our finding that patients who died during sepsis had increasing levels of sTLT-1 in the 2 days following admission to the ICU, whereas those who survived showed declines in sTLT-1 during this same period, suggest that monitoring of sTLT-1 levels could be an important prognostic indicator. Similarly, the correlation between sTLT-1 and D-dimers indicates association of sTLT-1 with the clinical manifestations of DIC. In support of this conclusion, ROC curve analysis showed that at 50 μg/ml, sTLT-1 has good sensitivity and specificity as an indicator for DIC. In fact, a recent report found that 3 criteria included in the current ISTH classification of DIC (platelet count, AUC 0.67; prothrombin time, AUC 0.74; and fibrinogen AUC, 0.70) all had AUC values below what we find for sTLT-1 as an indicator of DIC (19
). Taken together, these data clearly demonstrate the involvement of TLT-1 in the host response to sepsis and indicate that sTLT-1 may provide a significant clinical tool for the diagnosis of DIC associated with sepsis, perhaps becoming readily detectable well before other manifestations of DIC.
The ability of anti–TLT-1 scFv to block aggregation of washed platelets suggested that TLT-1 facilitates thrombosis by interacting with a ligand or ligands on or in activated platelets (10
). Our finding here that clinically relevant levels of sTLT-1 directly promote platelet aggregation in vitro suggests that TLT-1 may be a novel, platelet-specific, secondary activation factor, poised to promote aggregation in situations where only low levels of agonist are present yet vascular integrity must be maintained. This conclusion is bolstered by our demonstration of significant platelet aggregation defects and extended bleeding times in Treml1–/–
mice. Surprisingly, we even detected aggregation defects in Treml1–/–
platelets stimulated with ADP, an agonist not normally associated with the release of α-granules. However, our analysis of TLT-1 expression in whole blood isolated from human donors or mice confirmed the ability of ADP to induce TLT-1 expression on platelets, this despite the recent confirmation of TLT-1’s location within α-granules via ultrastructural analysis (20
). Oddly enough, our flow cytometrical analysis of sTLT-1–mediated amplification of platelet aggregation failed to demonstrate any direct binding of sTLT-1 to resting or activated platelets (A.V. Washington, unpublished observations). Instead, we found that TLT-1 binds fibrinogen. These data are consistent with a model in which, during platelet activation, stored fibrinogen is secreted and cross-linked by both sTLT-1 and cell surface TLT-1. The inability of TLT-1 to interact with vitronectin or fibronectin suggests that unlike GPIIb/IIIa, TLT-1 likely does not interact with RGD-type sequences found in fibrinogen, a conclusion not unexpected given the distinct structural properties of integrins and TREM (9
). Rather, the use of unique binding sites suggests that TLT-1 may work in concert with GPIIb/IIIa to facilitate fibrinogen/platelet interactions and/or higher-order platelet aggregation. Future detailed biochemical analysis of the TLT-1/fibrinogen interaction will clarify these possibilities.
Our biochemical analysis also showed that the TLT-1 cytoplasmic tail interacts strongly with the ERM protein moesin. Thus, TLT-1 becomes the second ITIM-containing receptor in platelets shown to interact with the ERMs, PECAM being the other (21
). Although others have suggested that moesin is the only ERM in mouse platelets, moesin-null mice don’t show an aggregation defect as Treml1–/–
mice do (15
). We could readily detect ezrin and radixin in purified mouse platelets and found that both these proteins also interact with TLT-1, leading us to conclude that in the absence of moesin, TLT-1 couples to other members of the ERM family. The ERMs are implicated in the formation of filopodia and lamellipodia in various cell types, including platelets. Moesin signaling is regulated downstream of Rho by phosphorylation at Thr558 in a process controlled by myosin phosphatase and Rho kinase, both of which play a role in platelet activation during shape change (15
). Thus, the interaction of TLT-1 with moesin we report here is consistent with our previous scFv studies that suggested that TLT-1 functions after shape change (10
). Moreover, when we assessed CD62 expression by flow cytometry, we found no differences between Treml1–/–
and WT platelets, suggesting that initial agonist signaling is not affected by the lack of TLT-1 (A.V. Washington, unpublished observations). Collectively, these findings support a model in which platelet activation causes TLT-1 to be brought to the platelet surface, facilitating the release of sTLT-1. After shape change and degranulation, TLT-1 binds fibrinogen and guides rapid pseudopodia formation in platelets though interaction with moesin and other ERM proteins, resulting in enhanced higher-order platelet aggregation. This model places TLT-1 in an emerging class of platelet regulatory molecules, including CD40L (25
), Gas6 (26
), CD36 (27
), and the eph kinases (28
), that assist thrombin, fibrinogen, and collagen with control of the more subtle aspects of platelet aggregation, providing a critical mechanism allowing for hemostasis without thrombosis.
Our establishment of a direct role for TLT-1 in the regulation of platelet aggregation together with the elevated sTLT-1 levels in septic patients and its association with DIC raised the possibility that the release of sTLT-1 during sepsis may be beneficial in maximizing platelet aggregation at sites of vascular damage during the inflammatory response. Alternatively, by virtue of their ability to reduce the aggregation threshold in the periphery, the high systemic levels of sTLT-1 detected in patients might contribute to aberrant platelet aggregation at distal sites, potentiating the development of DIC and the subsequent depletion of coagulation factors that contribute to morbidity (1
). When we assessed TLT-1 during endotoxemia in mice, we found detectable levels of sTLT-1 within 2 hours of LPS administration. The levels of sTLT-1 were in strong inverse correlation with platelet counts. Moreover, in these experiments, we did not detect an increase in cell surface TLT-1 on platelets remaining in the circulation, and when stimulated ex vivo, these platelets expressed normal levels of TLT-1 (D.W. McVicar and A.V. Washington, unpublished observations). Therefore, sTLT-1 is most likely released only by platelets as they leave the circulation during endotoxemia, not from the remaining circulating pool.
In normal mice, LPS causes a spike in TNF levels 2 hours after injection; these levels fall as sTLT-1 levels increase. This relationship opens the possibility of a direct feedback mechanism involving sTLT-1 and TNF-producing cells. Indeed, activated platelets have been reported to express a TREM-1 ligand, interact with neutrophils and monocytes, and enhance neutrophil response to LPS via TREM-1 (29
). Although these initial reports suggested TLT-1 is not itself the TREM-1 ligand on activated platelets, we cannot currently rule out inhibition of these platelet leukocyte interactions as the mechanism whereby TLT-1 tempers inflammation (29
Our challenge of Treml1–/–
mice with LPS confirmed a role for TLT-1 in both the inflammatory and consumptive phases of sepsis. Although LPS-induced leukocytopenia was largely unaffected in Treml1–/–
mice, these mice had higher serum levels of TNF and higher levels of D-dimers following LPS than did WT mice. However, these changes translated into only a limited survival benefit for the Treml1–/–
mice, likely because DIC does not play a significant role in the mortality associated with endotoxemia in mice (30
). Thus, we propose that TLT-1 primarily supports platelet aggregation at sites of inflammatory vascular injury, thereby controlling vascular integrity during the septic response. This interpretation is strongly supported by the results of our analysis of localized Shwartzman reactions in Treml1–/–
mice. Although not all models of inflammation-induced hemorrhage are dependent on platelet adhesion, the Shwartzman reaction is, as indicated by exacerbated hemorrhage in P selectin–null mice (2
). Accordingly, we found that removal of TLT-1 slightly increased neutrophil influx at the site of injection and that Shwartzman lesions were demonstrably more hemorrhagic in Treml1–/–
mice. Thus, our data suggest that TLT-1 may minimize vessel damage by regulating the production of TNF and augment platelet aggregation at the site of vessel injury, preventing hemorrhage. Therefore, we suggest that the high levels of sTLT-1 detected in patients who die from sepsis likely indicate the hemostatic system’s aggressive attempts at maintaining vascular integrity as well as efforts by the host to contain the inflammatory response.
In summary, our data establish TLT-1 as a molecule capable of fine-tuning platelet aggregation and inflammation for the control of vascular integrity. As such, its characterization has revealed a unique opportunity for the therapeutic separation of the benefits of hemostasis from the detriment of thrombosis. Based on our findings, we predict that specific intervention with TLT-1 or TLT-1–mediated signals might have potential in the therapy of a variety of hypercoagulatory states, including those associated with sepsis.