Genetic Evidence That CD154 (CD40 Ligand) Plays a Pathophysiologic Role in the Systemic Coagulopathy Associated with Murine Models of Experimental Metastasis and Endotoxemia.
Authors: Ingersoll Susan B. , Amirkhosravi Ali , Langer Florian , Walker Jamie , Meyer Todd , Amaya Mildred , Francis John L. 
Published: April 2020

Abstract
Platelet-derived CD154 has been shown to play an important role in platelet function and arterial thrombus formation in vivo. These properties of CD154 may be mediated by signaling via the CD40 receptor and/or the α IIβ 3 integrin on the platelet surface. CD154 expressed on activated platelets can also induce an inflammatory response, including the production of tissue factor (TF) in endothelial cells and monocytes, in a CD40-dependent manner. TF-driven disseminated intravascular coagulation (DIC) with activation and subsequent sequestration of platelets are common features in murine models of hematogenous metastasis and endotoxemia. In both models, platelet- and fibrin-rich thrombi are formed in the microvasculature, and in the model of metastasis, these microthrombi are believed to enhance tumor cell lodgement, extravasation and colony formation. It is therefore reasonable to hypothesize that the CD154 pathway also plays a critical role in the processes of hematogenous tumor cell dissemination and LPS-induced consumptive coagulopathy. We have used a gene knockout model to test these hypotheses. To assess tumor cell-induced coagulopathy, wild-type (WT) mice and mice deficient for CD154 (Cd154−/−), CD40 (Cd40−/−) or both (Dbl−/−), (n=5–12 per group) were injected intravenously with B16 melanoma cells (1X106). Fifteen minutes later, blood was collected by cardiac puncture to measure platelet counts, factor X (FX) activity and plasma hemoglobin (pHb), a sensitive marker of microangiopathic hemolytic anemia following microvascular thrombosis. To assess metastasis, mice (n=10–20 per group) were injected via the tail vein with B16 tumor cells (2x105) and surface tumor nodules were counted macroscopically 18 days later. To assess endotoxin-induced coagulopathy, mice (n=10 per group) were injected intraperitoneally with LPS (50 mg/kg) and platelet count, FX and fibrinogen levels were measured after 8 hours. All groups of animals had similar baseline parameters. However, following tumor cell injection, platelet counts were reduced by 73% and 72%, in WT and Cd40−/− mice respectively, compared to 50% in Cd154−/− and 54% in Dbl−/− mice (P&lt;0.01 for both Cd154−/− and Dbl−/− compared to WT). FX levels were reduced in WT (37%) and Cd40−/− (29%) mice, compared to 20% (P&lt;0.05) and 0% (P&lt;0.01) in Cd154−/− and Dbl−/− mice, respectively. Increases in pHb levels were less pronounced in Cd154−/− (2.5-fold), Cd40−/− (1.5-fold), and Dbl−/− (2-fold) as compared to WT (&gt;4-fold) mice (P&lt;0.05 for all groups). In addition, significantly fewer tumor nodules were found in the lungs of Cd154−/− (44% reduction), Cd40−/− (52%) and Dbl−/− (82%) than in WT mice (P&lt;0.01). Consistent with our observations in the model of tumor cell-induced coagulopathy, Cd154−/− mice injected with LPS were partially protected against DIC, as evidenced by a less pronounced fall in platelet counts and reduced consumption of plasma FX and fibrinogen levels. In contrast, WT and Cd40−/− mice showed similar responses to endotoxin challenge. These results provide genetic evidence for a pathophysiologic role of the CD154 pathway in two murine models of TF-driven intravascular coagulation and platelet activation. The prothrombotic function of CD154 in these models appears to be independent of CD40. The role(s) of CD40 in hematogenous metastasis warrants further investigation.