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 ...

No abstract

No abstract

We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of ␣IIb␤3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the ␣IIb␤3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a ␣IIb␤3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40 ؊/؊ platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of ␤3. Platelets from the diYF mouse strain, expressing ␤3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to ␣IIb␤3, triggering outside-in signaling by tyrosine phosphorylation of ␤3.

This study demonstrates that GP Ibalpha is a physiologically relevant ligand for alphaMbeta2 and that integrin engagement of GP Ibalpha is critical to leukocyte function and the biological response to vascular injury. These observations establish a molecular target for selectively disrupting leukocyte-platelet complexes that promote inflammation in thrombosis and restenosis.

Summary During the past decade, interrelationships between inflammation and thrombosis have been the subject of extensive works, and it is now commonly recognized that inflammation (notably leucocyte recruitment) directly affects thrombosis, and that thrombosis also constitutes a pro‐inflammatory event. This tight link is partly attributable to P‐selectin, which is functional not only when expressed on the surface of activated platelets and endothelial cells, but also when shed, generating its soluble form, termed sP‐selectin. In this review, we will provide an overview of the relative roles of the different compartments of P‐selectin (platelet, endothelial cell, plasma) in haemostasis and vascular pathologies, and the potential therapeutic benefits achievable in targeting this molecule.

To examine the role of the platelet adhesion molecule von Willebrand factor (vWf) in atherogenesis, vWf-deficient mice (vWf؊/؊) were bred with mice lacking the low-density lipoprotein receptor (LDLR؊/؊) on a C57BL/6J background. LDLR؊/؊vWf؉/؉ and LDLR؊/؊vWf؊/؊ mice were placed on a diet rich in saturated fat and cholesterol for different lengths of time. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules in both genotypes, indicating an increase in P-selectin-mediated adhesion to the endothelium. After 8 weeks on the atherogenic diet, the fatty streaks formed in the aortic sinus of LDLR؊/؊vWf؊/؊ mice of either sex were 40% smaller and contained fewer monocytes than those in LDLR؊/؊vWf؉/؉ mice. After 22 weeks on the atherogenic diet (early fibrous plaque stage), the difference in lesion size in the aortic sinus persisted. Interestingly, the lesion distribution in the aortas of LDLR؊/؊vWf؊/؊ animals was different from that of LDLR؊/؊ vWf؉/؉ animals. In vWf-positive mice, half of all lesions were located at the branch points of the renal and mesenteric arteries, whereas lesions in this area were not as prominent in the vWf-negative mice. These results indicate that the absence of vWf primarily affects the regions of the aorta with disturbed flow that are prone to atherosclerosis. Thus, vWf may recruit platelets/leukocytes to the lesion in a flow-dependent manner or may be part of the mechano-transduction pathway regulating endothelial response to shear stress. Introductionvon Willebrand factor (vWf) is a multimeric glycoprotein essential for thrombus formation at high shear stress. 1 It is found in plasma, platelet ␣-granules, Weibel-Palade bodies of endothelial cells, and the subendothelium. 2 Even though the involvement of vWf in thrombus formation at the site of atherosclerotic plaque rupture is well established, 3 the role of vWf in atherosclerotic lesion development is less clear. Several indications suggest that vWf may directly participate in plaque formation. First, Weibel-Palade bodies are found in great number at the sites of atherosclerotic lesions. 4 Second, oxidized low-density lipoprotein (LDL) and fluid mechanics, 2 factors involved in atherosclerotic lesion development, can induce Weibel-Palade body exocytosis. 5,6 Third, vWf expression is particularly prominent near branch points and bifurcations, areas prone to atherosclerotic lesion development. 7 Fourth, platelets may contribute to the atherosclerotic process by releasing growth factors after their vWf-dependent interaction with a damaged endothelial surface. 8,9 Experimental studies performed in pigs with von Willebrand disease (vWd), though supportive of a role for vWf in atherosclerosis, 3 have not been conclusive because of the genetic heterogeneity existing among the animals. 10 The recent generation of vWf-deficient mice has provided an opportunity to directly test the importance of vWf in atherosclerosis. Because mice are resistant to atherosclerosis, the vWf-deficient mice were first bred with an atherosclerosis...