The role of CD40 in CD40L- and antibody-mediated platelet activation

Abstract

SummaryOur 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 (rhs CD40L), 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 rhs CD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28–5 showed less potential in blocking rhs CD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhs CD40L, G28–5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcγRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhs CD40L induced strong FcγRII-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.Parts of this work have been presented on the 46th Annual Meeting of the American Society of Hematology (San Diego, 2004).

CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia

“…In addition, thrombotic complications have been observed with the expanded use of therapeutic IgG antibodies, such as bevacizumab. [7][8][9] One of the barriers to successful treatment of these thrombotic syndromes is that therapeutic targeting of platelet activation pathways to prevent thrombosis is either not effective or comes with an inherent risk of bleeding complications.…”

“…In addition, thrombotic complications have been observed with the expanded use of therapeutic IgG antibodies, such as bevacizumab. [7][8][9] One of the barriers to successful treatment of these thrombotic syndromes is that therapeutic targeting of platelet activation pathways to prevent thrombosis is either not effective or comes with an inherent risk of bleeding complications.In humans, Fc␥RIIA is expressed on platelets, neutrophils, monocytes, and macrophages and activates these cells following the binding of the Fc region of IgG-coated cells or IgG-containing immune complexes. 10 Mice lack the genetic equivalent of human Fc␥RIIA and, indeed, do not express a platelet Fc receptor.…”

CD40 Ligand and Its Receptors in Atherothrombosis

“…Unfortunately, clinical trials using an anti-CD40L antibody were put on hold due to thromboembolic complications. 97,98 Interactions between CD40 and CD40L-immune complexes at the surface of platelets have been suggested as a possible mechanism by which CD40L therapy induces these complications. 97 Since circulating levels of sCD40L result from platelet activation, indirect targeting of the CD40L system through anti-platelet therapy may represent an alternative approach to suppress this important component.…”

“…97,98 Interactions between CD40 and CD40L-immune complexes at the surface of platelets have been suggested as a possible mechanism by which CD40L therapy induces these complications. 97 Since circulating levels of sCD40L result from platelet activation, indirect targeting of the CD40L system through anti-platelet therapy may represent an alternative approach to suppress this important component. Clopidogrel, a potent inhibitor of the platelet adenosine diphosphate (ADP) receptor, has been reported to block sCD40L release from ADPstimulated platelets.…”

The Thrombogenic Role of Platelets in Valvular Atrial Fibrillation

“…Also, they found that the fraction of peripheral venous platelets expressing P-selection in patients with chronic lone atrial fibrillation did not differ from that of healthy volunteers who were in sinus rhythm and this result provided one possible basic mechanism to clarify the ineffectiveness of aspirin in preventing thromboembolism in patients with non valvular atrial fibrillation according to previous large clinical trials (Petersen et al, 1989;The Stroke Prevention in Atrial Fibrillation Investigatiors,1993). The role of the CD40-CD40L axis in patients with atherothrombotic diseases has generated interest as this molecule may play a pivotal link among platelets (Henn et al, 2001;Langer et al, 2005;Slupsky et al, 1998), inflammation ( Anand et al, 2003;Henn et al, 2001), the endothelium (Slupsky et al, 1998;Zhou et al, 1998), coagulation (Slupsky et al, 1998;Zhou et al, 1998) and, ultimately, thrombosis. The triad of functional activity of CD40L in atherosclerotic models, high content in platelets, and mobilization during platelet thrombosis provide a readily testable hypothesis and places platelet-derived CD40L as a possible important mitigating factor in atrial fibrillation.…”