Enhanced Levels of Soluble CD40 Ligand Exacerbate Platelet Aggregation and Thrombus Formation Through a CD40-Dependent Tumor Necrosis Factor Receptor–Associated Factor-2/Rac1/p38 Mitogen-Activated Protein Kinase Signaling Pathway

Abstract

Objective-CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. Methods and Results-sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40 Ϫ/Ϫ mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor-associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40 Ϫ/Ϫ mice. Conclusion-sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases. M ultiple lines of evidence now support a plethora of inflammatory mediators potentially involved in the pathogenesis of vascular disease. Among these, the CD40 ligand (CD40L)/CD40 dyad has been the focus of much attention, and circulating levels of soluble CD40L (sCD40L) are now considered reliable predictors of cardiovascular events. [1][2][3][4] CD40L is a 48-kDa trimeric transmembrane protein belonging to the tumor necrosis factor superfamily originally identified on cells of the immune system. 5,6 Interaction of CD40L with its respective receptor on B cells, CD40, a 39-kDa glycoprotein from the tumor necrosis factor receptor family, is of critical importance for immunoglobulin isotype switching during the immune response. 7 Today, we know that these 2 molecules are also present on cells of the vascular system, including endothelial cells, monocytes/macrophages, smooth muscle cells, and platelets, 8,9 and have important implications in inflammatory reactions, through upregulation of cell adhesion molecules and production of proinflammatory cytokines, chemokines, growth factors, matrix metalloproteinases, and procoagulants. 8,10 -12 The involvement of the CD40L/CD40 dyad in thromboinflammation has been highlighted in all pathogenic phases of atherosclerosis, including endothelial dysfunction, platelet activation, thrombosis, and neointima formation. [13][14][15][16][17][18][19][20] Platelets are highly specialized blood cells of paramount importance in normal hemostasis and thromboinflammatory complications. The pio...

Immunological Considerations for Inducing Skin Graft Tolerance

“…Scientists have since analyzed the cause for the pathophysiological problems identified clinically. Their reports help to identify platelets as a source for soluble CD154 or CD40 ligand and demonstrate a correlation between increased circulating levels of this molecule and increased thrombus formation (Yacoub et al, 2010, Yuan et al, 2010. These studies show further that CD154's concentration is increased in hypertensive patients included non-diabetic and pre-diabetic persons with metabolic syndrome (Unek et al, 2010).…”

Pre-Transplant Therapy in Experimental Heart Transplantation

“…The clinical development of the corresponding biological (humanized monoclonal anti-CD154) antibodies, however was previously thwarted by serious thromboembolic complications (Kawai et al, 2000;Koyama et al, 2004), which are suspected to have been caused by an interference with the stabilizing effect of soluble CD154 on freshly formed thrombi (Andre et al, 2002). In fact, the main source of soluble CD154 are aggregating platelets from which the membrane-bound CD154 is shed upon activation (Kaya et al, 2011;Pamukcu et al, 2011;Silvain et al, 2011;Yacoub et al, 2010). More recently, however, some progress has been made towards the development of monoclonal antibodies with less side effects (Hussein et al, 2010).…”

CD40 Ligand and Its Receptors in Atherothrombosis

“…16 In addition, it has been demonstrated that upon CD40L binding CD40 activation on platelets can enhance platelet function and promote the secretion of inflammatory cytokines involved in plaque formation (this aspect will be discussed in greater detail bellow). [27][28][29] …”