CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin alphaIIbbeta3 and to activated platelets in a beta3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an alphaIIbbeta3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.

The plasma concentration of soluble adhesion receptors is increased under pathological circumstances, but their function remains enigmatic. Soluble P-selectin (sP-sel) is shed from activated platelets and endothelial cells. Mice genetically engineered to express P-selectin without the cytoplasmic tail (⌬CT) constitutively show a 3-to 4-fold increase of sP-sel in plasma. We observed that the ⌬CT mice formed fibrin very readily. In an ex vivo perfusion chamber, there was more fibrin deposited at the site of platelet thrombus formation than in wild type (WT), whereas no fibrin deposits were detected using P-selectin-deficient blood during the same interval. Similarly, in vivo, the hemorrhage produced by local Shwartzman reaction was smaller in the ⌬CT mice than in WT. In contrast, we previously showed hemorrhage to be more prominent in P-selectin knock-out mice. Infusion of mouse P-sel-Ig chimera produced the same protective effect in WT mice as seen in the ⌬CT mice, indicating that the effect was due to increased levels of sP-sel. Mice infused with P-sel-Ig showed significantly more fibrin deposited on the luminal face of the injured vessels than control mice. Plasma from ⌬CT mice or mice infused with P-sel-Ig contained higher concentration of pro-coagulant microparticles and clotted one minute faster than WT. This pro-coagulant phenotype of ⌬CT mice could be reversed by a 4-day treatment with PSGL-Ig, a P-selectin inhibitor. We propose that sP-sel should no longer be considered only as a marker of inflammation or platelet activation, but also as a direct inducer of pro-coagulant activity associated with vascular and thrombotic diseases. P-selectin is a member of the selectin family localized in the membranes of ␣-granules of platelets and the Weibel-Palade bodies of endothelial cells (1). A soluble form of P-selectin can be found in the plasma as a circulating protein (2). In vivo, two main physiological roles are attributed to the integral membrane form of P-selectin. First, in inflammation, P-selectin is redistributed onto the surface of activated endothelial cells where it mediates the rolling of leukocytes (3). Second, in thrombosis, P-selectin expressed on activated platelets present in a thrombus supports the recruitment of leukocytes (4). Soluble P-selectin (sP-sel) of healthy individuals has been suggested to originate from the alternatively spliced form found in endothelial cells and platelets (5). Alternatively, elevated levels of sP-sel may reflect platelet activation (6) because P-selectin is proteolytically shed from the plasma membrane in vivo shortly after activation (7,8). Therefore, plasma levels of sP-sel have been considered a useful tool to predict thrombotic consumptive platelet disorders (9-12), but they can also reflect endothelial cell activation (13,14). Although the circulating form of P-selectin is potentially active because only the lectin and epidermal growth factor (EGF) domains are required to bind its receptor, P-selectin glycoprotein ligand-1 (PSGL-1) (15), the biological role of ...

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