P-selectin in arterial thrombosis

P-selectin is a transmembrane protein present in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells. Following activation, it is rapidly translocated to the cell surface. P-selectin expression in platelets has been shown to be elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells as well as interactions of platelets with leukocytes. Platelet P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes to form platelet-leukocyte aggregates. Furthermore, this interaction of P-selectin with PSGL-1 induces the upregulation of tissue factor, several cytokines in leukocytes and the production of procoagulant microparticles, thereby contributing to a prothrombotic state. P-selectin is also involved in platelet-platelet interactions, i. e. platelet aggregation which is a major factor in arterial thrombosis. P-selectin interacts with platelet sulfatides, thereby stabilizing initial platelet aggregates formed by GPIIb/IIIa-fibrinogen bridges. Inhibtion of the P-selectin-sulfatide interaction leads to a reversal of platelet aggregation. Thus, P-selectin plays a significant role in platelet aggregation and platelet- leukocyte interactions, both important mechanisms in the development of arterial thrombosis.     

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

Thrombocytopenia has been consistently reported following the administration of adenoviral gene transfer vectors. The mechanism underlying this phenomenon is currently unknown. In this study, we have assessed the influence of von Willebrand Factor (VWF) and P-selectin on the clearance of platelets following adenovirus administration. In mice, thrombocytopenia occurs between 5 and 24 hours after adenovirus delivery. The virus activates platelets and induces platelet-leukocyte aggregate formation. There is an associated increase in platelet and leukocyte-derived microparticles. Adenovirus-induced endothelial cell activation was shown by VCAM-1 expression on virus-treated, cultured endothelial cells and by the release of ultra-large molecular weight multimers of VWF within 1 to 2 hours of virus administration with an accompanying elevation of endothelial microparticles. In contrast, VWF knockout (KO) mice did not show significant thrombocytopenia after adenovirus administration. We have also shown that adenovirus interferes with adhesion of platelets to a fibronectin-coated surface and flow cytometry revealed the presence of the Coxsackie adenovirus receptor on the platelet surface. We conclude that VWF and P-selectin are critically involved in a complex platelet-leukocyte-endothelial interplay, resulting in platelet activation and accelerated platelet clearance following adenovirus administration.     

Regulation of Weibel-Palade body exocytosis

Weibel-Palade bodies (WPBs) are endothelial granules that store von Willebrand factor (VWF), P-selectin, and other vascular modulators. Endothelial cells secrete WPBs in response to vascular injury, releasing VWF, which triggers platelet rolling, and externalizing P-selectin, which activates leukocyte trafficking. Endothelial exocytosis is one of the earliest responses to vascular damage and plays a pivotal role in thrombosis and inflammation. This review examines the regulation of WPB exocytosis-the exocytic machinery, activators, and inhibitors of exocytosis-and speculates about the development of novel anti-exocytic drugs.     

The complement factor properdin induces formation of platelet-leukocyte aggregates via leukocyte activation

Both the complement system and platelet-leukocyte aggregates are involved in chronic and acute stages of atherosclerosis. Properdin, a positive regulator of the complement system, is secreted by leukocytes and endothelial cells. In the present study, the role of properdin in the formation of platelet-leukocyte aggregates was investigated. Incubation of human whole blood with properdin (25–200 µg/ml) resulted in a dose-dependent formation of platelet-leukocyte aggregates, with an increase of up to 2.2-fold compared to controls (p < 0.05), as analysed by flow cytometry. In addition, properdin significantly amplified ADP-induced aggregation of platelets with leukocytes by 53% (p < 0.05), while it had no effect on ADP-induced aggregation of platelets alone. Consistent with these results, properdin did not activate platelets as shown by the expression of activated GPIIb/IIIa (PAC-1 epitope) and P-selectin (CD62P) on the platelet surface. However, properdin significantly induced expression of CD11b (MAC-1) on leukocytes by 12-fold (p < 0.05) as a measure of leukocyte activation. In conclusion, the complement system component properdin induces the formation of platelet-leukocyte aggregates via leukocyte activation. The data establish a link between the complement system and platelet-leukocyte aggregates with potential significance in atherosclerotic vascular disease.     

CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin

The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.     

Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin.

P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin.     

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.     

Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation

Platelets are necessary for lung leukocyte recruitment in a murine model of allergic inflammation, and platelet-leukocyte aggregates are formed in circulating blood of patients with asthma after allergen exposure. However, it is unknown how platelets induce pulmonary leukocyte recruitment in asthma. Here, we have investigated the importance of platelet adhesion molecule expression on pulmonary eosinophil and lymphocyte recruitment and on leukocyte CD11b and very late antigen (VLA)-4 expression in mice. Pulmonary leukocyte recruitment in platelet-depleted mice (sensitized and exposed to ovalbumin) transfused with fixed, unstimulated platelets (FUSPs) was abolished, whereas transfusion with platelets stimulated and fixed (FSPs), expressing P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), restored eosinophil and lymphocyte recruitment. Transfusion with platelets from P-selectin-deficient mice, or with FSPs stimulated in the presence of a blocking anti-P-selectin antibody, were unable to restore pulmonary leukocyte recruitment. Flow cytometric analysis revealed increased expression of CD11b and VLA-4 on leukocytes attached to platelets after allergen exposure, and CD11b expression on leukocytes was suppressed in thrombocytopenic mice but was restored with the transfusion of FSPs, but not FUSPs, a phenomenon concurrent with the formation of platelet-leukocyte complexes. P-selectin expression on the surfaces of platelets is a major requirement for pulmonary eosinophil and lymphocyte recruitment, allowing circulating platelets to bind to and stimulate leukocytes for endothelial attachment.     

Interactions of platelets with subendothelium and endothelium

In this review, the authors summarize how platelets interact with subendothelium when the vessel wall is damaged or with intact endothelium in the inflammatory state. When subendothelium is exposed to rapidly flowing blood upon vessel damage, platelets adhere rapidly to the exposed surface, decelerate, and aggregate to arrest bleeding. Under high shear stress, such as is found in the microcirculation, the interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor, glycoprotein (GP) Ib-IX-V, is required to slow down platelets and allow the platelet collagen receptors aloha2beta1 and GP VI to bind to collagen. GP VI and alpha2beta1 play important roles to activate platelets in the early stage and work with GP Ib-IX-V to fully activate platelets to form thrombi. GP Ib-IX-V and GP VI employ similar signaling pathways for platelet activation and the signals from both receptors are down-modulated by PECAM-1 (platelet-endothelial-cell adhesion molecule 1) to prevent unnecessary platelet activation under high shear. During inflammatory states, intact endothelial cells release VWF and P-selectin from their Weibel-Palade bodies. Both molecules are ligands for GP Ib-IX-V. The newly released VWF is larger and stickier than the form normally found in plasma and binds platelets spontaneously. Normally, VWF is processed by proteolysis by the plasma metalloprotease ADAMTS-13. Failure of this processing results in the microvascular thrombotic disorder thrombotic thrombocytopenic purpura. In this review, the authors also use available crystal structures of platelet receptors and ligands to explain the details of their interactions.     

Role of p-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

BACKGROUND: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. METHODS: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. RESULTS: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. CONCLUSION: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction.     

Dimerization of P-selectin in platelets and endothelial cells

P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor-activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.     

A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.     

C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression

The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS(-/-)) or under acute inflammatory conditions (induced by interleukin-1beta or histamine). CNP suppressed basal leukocyte rolling in eNOS(-/-) mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF(4-23). CNP also suppressed leukocyte rolling induced by IL-1beta or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders.     

Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules

Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P- selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P- selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.     

The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.     

Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates

OBJECTIVES: We investigated the role of P-selectin in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates. BACKGROUND: Plaque rupture followed by thrombus formation is a fundamental pathophysiology of acute coronary syndromes. Although the adhesive interaction between platelets and leukocytes via P-selectin is known to mediate platelet-rich thrombi, the true function of P-selectin in thrombus formation in vivo is unknown. METHODS: In wild-type (P(+/+)) and P-selectin-deficient (P(-/-)) mice with ferric chloride (FeCl(3))-induced carotid arterial thrombosis model, we measured in vivo platelet P-selectin expression and adenosine diphosphate (ADP)-induced ex vivo platelet aggregation. We also measured ex vivo ADP-induced whole blood aggregations and their size distribution by flow cytometry. RESULTS: Time to thrombotic occlusion was longer in P(-/-) mice than in P(+/+) mice. Spontaneous reflow after total thrombotic occlusion was observed in 8 of 10 P(-/-) mice but not in any P(+/+) mice. ADP-induced ex vivo platelet aggregation was not different between the two groups. However, ADP-induced ex vivo whole blood aggregation was inhibited in P(-/-) mice compared to P(+/+) mice. FeCl(3) application increased in vivo expressions of platelet P-selectin in P(+/+) mice but not in P(-/-) mice. The number of leukocytes within thrombi was less in P(-/-) mice than in P(+/+) mice. In flow cytometric analysis of size distribution of ADP-induced whole blood aggregates, the number of large aggregates was less in P(-/-) mice than in P(+/+) mice. Using platelet and leukocyte fluorescence makers, the large aggregates were confirmed as platelet-leukocyte aggregates. CONCLUSIONS: Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates.     

Molecular and cellular biology of von Willebrand factor

von Willebrand factor (VWF) is a plasma protein that performs 2 main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries and protects coagulation factor VIII. VWF is synthesized through a multistep process in endothelial cells and megakaryocytes as a very large polymer composed of identical disulfide-linked 250-kd subunits. In endothelial cells, VWF not only directs the formation of its own storage granules, the Weibel-Palade bodies, but it also acts as a chaperone molecule to direct other proteins, such as P-selectin, into these granules. Upon stimulation of the endothelium, the Weibel-Palade bodies will be translocated to the plasma membrane, and their contents will be secreted into the plasma milieu. The expression of VWF can be regulated at different levels by a number of genetic and environmental factors, resulting in control of its activity. New roles for VWF, especially in inflammatory processes, have recently been suggested, indicating that some aspects of this well-studied protein remain to be investigated.     

Plasma P-selectin is increased in thrombotic consumptive platelet disorders

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P- selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P- selectin may be a useful new marker for thrombotic diseases.     

Ceramide triggers Weibel-Palade body exocytosis

The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing von Willebrand factor (vWF) and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. We added ceramide to human aortic endothelial cells and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF released into the media. Exogenous ceramide induces vWF release from endothelial cells in a dose-dependent manner. Activators of endogenous ceramide production, neutral sphingomyelinase, or tumor necrosis factor-alpha also induce Weibel-Palade body exocytosis. We next studied NO effects on ceramide-induced Weibel-Palade body exocytosis because NO can inhibit vascular inflammation. The NO donor S-nitroso-N-acetylpenicillamine decreases ceramide-induced vWF release in a dose-dependent manner, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester increases ceramide-induced vWF release. In summary, our findings show that endogenous ceramide triggers Weibel-Palade body exocytosis, and that endogenous NO inhibits ceramide-induced exocytosis. These data suggest a novel mechanism by which ceramide induces vascular inflammation and thrombosis.