Platelet CD40L and CD40 enhance tumor cell-induced coagulopathy and experimental metastasis

Abstract number: P0829

Ingersoll S., Amirkhosravi A., Amaya M., Meyer T. V., Desai H., Francis J. L.

Florida Hospital Cancer Institute, USA

The importance of platelets in promoting metastasis is well established. Blood-born tumor cells can activate coagulation, resulting in thrombin generation and platelet activation. CD40 is present resting and activated platelet membranes. Furthermore, upon platelet activation, CD40 ligand (CD40L) is translocated to the platelet surface where it is subsequently cleaved in a CD40 dependent manner, and released in soluble form (sCD40L). Platelet CD40L may stabilize arterial thrombi via interactions with GPIIb/IIIa, identified as an agonist receptor for sCD40L. We studied platelet CD40L and CD40 involvement in tumor cell-induced coagulopathy and experimental metastasis using an injectable model of melanoma metastasis in wild type (wt), Cd40l-deficient (Cd40l^-/-) and Cd40-deficient (Cd40^-/-) C57BL/6 mice. Fifteen minutes after i.v. injection of B16 melanoma cells (10⁶) blood was collected for platelet count, Factor X (FX), and plasma hemoglobin (pHb). Prior to tumor cell injection, there was no significant difference in platelet count, FX levels or pHb levels between wt, Cd40l^-/-, and Cd40^-/- mice. However, following tumor cell injection, platelet counts were reduced by 80 and 65%, respectively, in wt and Cd40^-/- mice (P < 0.001) vs. 18% in Cd40l^-/- mice (P = NS). FX levels were reduced in wt and Cd40^-/- mice by 63 and 57% (P < 0.001), vs. 14% in Cd40l^-/- mice (P = NS). Increases in pHb, indicative of microangiopathic hemolytic anemia resulting from intravascular coagulation, were less pronounced in Cd40l^-/- (16%) mice compared to Cd40^-/- (200%) and wt mice (400%). Also, significantly fewer tumor nodules were later found in the lungs of Cd40l^-/- and Cd40^-/- compared to wt mice (P < 0.01). Interestingly, no difference in platelet aggregation was observed in vitro when blood from untreated wt, Cd40l^-/-, and Cd40^-/- mice was stimulated with collagen/epinephrine using the PFA-100 analyzer, agreeing with previously reported tail vein bleeding times. These results indicate that Cd40l^-/- mice were largely protected from the effects of tumor cell induced coagulopathy as evidenced by less pronounced thrombocytopenia, FX consumption, and hemolytic anemia. This suggests that platelet CD40L is important in promoting experimental metastasis. Interestingly, Cd40^-/- mice were not dramatically different to wt mice in tumor cell induced coagulopathy, but the significant reduction in lung metastasis in Cd40^-/- mice suggests involvement of additional mechanisms.