The CD40/CD40 Ligand (CD40L) Pathway Plays a Role in Primary Hemostasis and Platelet Function.

Previous studies have suggested a role for platelet CD40L in thrombosis and atherosclerosis. However, there are contradictory reports on the biologic activity of its soluble variant (sCD40L) and the involved receptor signaling pathways (CD40 vs α_IIbβ₃). Furthermore, CD40L mAb-associated thromboembolic complications in recent human and animal studies have raised additional questions about the pro-thrombotic properties of this molecule. This study was conducted to further investigate the function of the CD40/CD40L dyad in primary hemostasis and platelet function. CD40−/− and CD40L−/− mice and mice deficient for both genes (“double knock-out”) showed prolonged tail vein bleeding and platelet function analyzer (PFA-100) closure times as compared to their wild-type littermates, indicating an inherited defect in platelet function. Recombinant human sCD40L (rsCD40L), chemical cross-linking of which yielded a single reaction product compatible with a trimeric structure of the protein in solution, bound to CD40 on resting platelets and induced CD62P (P-selectin) expression in a concentration-dependent manner (0–5 μg/ml) from 1±1 to 23±5% positivity (means±SD, n=4–8; P<0.01). This response was completely abolished by CD40 mAb M3 and not affected by blocking the β₃ integrin (CD61) with abciximab. In contrast, CD40 mAb G28-5 significantly enhanced rsCD40L-induced CD62P expression to 51±5%. This agonistic effect was strongest when G28-5 was added to the platelets after rsCD40L. Pre-incubation of rsCD40L with CD40L mAb M90 also had a potentiating effect, showing an inverted V-shaped dose response curve with maximum levels of CD62P+ platelets (60–95%) at a molar M90/rsCD40L ratio of 1/3. G28-5 and M90 alone had no effect, but their combination in the presence of rsCD40L proved synergistic. Experiments with corresponding F(ab’) fragments and the FcγRII-inhibitory mAb IV.3 demonstrated that G28-5- and M90-mediated additional platelet activation resulted from signaling through FcγRII cross-linked to CD40 and rsCD40L bound to CD40 on the platelet surface, respectively. The CD40L mAb TRAP-1 showed similar characteristics to M90, but its synergistic effect with rsCD40L was less pronounced. Platelet activation by rsCD40L in combination with G28-5 and/or M90 also induced morphological shape changes, fibrinogen binding, dense granule release, microparticle generation, and monocyte-platelet-conjugate formation. Interestingly, consistent with their platelet function-modulating effects, M3 but not G28-5 prolonged PFA-100 closure times of normal human blood. This work provides genetic evidence for a role of CD40 and CD40L in primary hemostasis. Our mechanistic studies further suggest that CD40-mediated platelet activation by CD40L, in its membrane and/or soluble form, may be at least partially involved in this process. The results also offer a potential explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in patients with systemic lupus erythematosus, an inflammatory autoimmune disease characterized by elevated levels of circulating sCD40L. The underlying pathomechanism, in which sCD40L as a platelet-derived, homotrimeric protein causes multimeric clustering of bivalent antibodies on the platelet surface with subsequent FcγRII-mediated platelet activation, resembles other known immune thrombophilias such as the heparin-induced thrombocytopenia (HIT) syndrome.