The role of CD40 in CD40L- and antibody-mediated platelet activation.
Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.
Langer
Florian
F
Institute of Translational Research, Florida Hospital, Orlando, USA. florian.langer@flhosp.org
Ingersoll
Susan B
SB
Amirkhosravi
Ali
A
Meyer
Todd
T
Siddiqui
Farooq A
FA
Ahmad
Sarfraz
S
Walker
Jamie M
JM
Amaya
Mildred
M
Desai
Hina
H
Francis
John L
JL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000936:Antigen-Antibody Complex / Q000097:blood; D019013:CD40 Antigens / Q000097:blood* / Q000276:immunology; D023201:CD40 Ligand / Q000097:blood* / Q000494:pharmacology; D006487:Hemostasis; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015539:Platelet Activation / Q000187:drug effects / Q000276:immunology*; D017452:Receptors, IgG / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D019013:CD40 Antigens; D017452:Receptors, IgG; D011994:Recombinant Proteins; D023201:CD40 Ligand