The presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin ؊/؊ platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1 IntroductionPlatelets are the body's defense mechanism against excessive bleeding caused by endothelial damage. Activated platelets present at the site of injury provide both a prothrombotic surface and a procoagulant surface. Excessive platelet activation occurs in coronary bypass surgery and may result in thrombotic emboli and neurologic complications. 1 Furthermore, many inflammatory diseases including sepsis, 2,3 psoriasis, 4,5 diabetes, 6-8 and cystic fibrosis 9 are associated with circulating activated platelets. These pathologies are also associated with endothelial inflammation.Platelets induce leukocyte adhesion on preactivated endothelial cells in culture. 10,11 In mouse models of atherosclerosis, the role of activated platelets in exacerbating lesion development has been clearly demonstrated. Activated platelets promote monocyte arrest on atherosclerotic lesions and atherosclerotic lesion growth. 12 Studies in our laboratory have also demonstrated the role of platelet P-selectin, in addition to endothelial P-selectin, in atherosclerotic lesion development and maturation. 13 It is important to note that P-selectin on both platelets and endothelium is expressed on the cell surface only on activation of the cells and granule secretion. 14 Early endothelial inflammation is associated with the rapid release of Weibel-Palade bodies and the consequent surface expression of Pselectin and von Willebrand factor (VWF). These molecules mediate rolling of leukocytes and platelets on endothelial cells. 15 This is followed by transcription and expression of molecules such as E-selectin, vascular-cell adhesion molecule 1 (VCAM-1), and other adhesion receptors. 16 These receptors, in turn, mediate slow rolling and adhesion of leukocytes and have been shown to be up-regulated on treatment with activated platelets in the in vitro studies. [17][18][19][20] This sequence of events leads to leukocyte rolling and extravasation at the site of injury or pathologic conditions.Recently, vascular endothelial growth factor (VEGF) secreted from ␣-granules was sh...

Abstract-Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIb␣ subunit was first described Ͼ25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIb␣ induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIb␣ shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-␣ converting enzyme (TACE ⌬Zn/⌬Zn ) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIb␣ expression in vivo because we observed an Ϸ90% reduction in soluble GPIb␣ (glycocalicin) in plasma of TACE ⌬Zn/⌬Zn chimeras as well as significantly increased levels of GPIb␣ on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE ⌬Zn/⌬Zn platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIb␣. Key Words: platelets Ⅲ TACE Ⅲ GPIb␣, shedding C ellular activation often results in the rapid proteolysis of a wide range of transmembrane proteins such as growth factors/cytokines, growth factor/cytokine receptors, and adhesion molecules. This process, known as ectodomain shedding, regulates many cellular functions and has been implicated in pathologies such as Alzheimer's disease and cancer. 1 The majority of these shedding events are mediated by zinc-dependent proteinases, most importantly members of the matrix metalloproteinase (MMP) 2 and a disintegrin and metalloproteinase (ADAM) 3 family of proteinases. Initially identified as a sheddase for tumor necrosis factor-␣ (TNF-␣), TNF-␣-converting enzyme (TACE; ADAM17) was also shown to mediate the release of many other cell surface transmembrane proteins, including adhesion molecules such as L-selectin and vascular cell adhesion molecule 1 (VCAM-1). 4 -6 Inactivation of the metalloproteinase activity of TACE by targeted deletion of the Zn 2ϩ -binding domain in mice (TACE ⌬Zn/⌬Zn ) results in perinatal lethality, demonstrating the importance of ectodomain shedding in vivo. 4 In platelets, proteolytic cleavage has been identified as a key mechanism to regulate the surface expression of a variety of adhesion receptors, including P-selectin, CD40 ligand, and the glycoprotein (GP) V and GPIb␣ subunits of the von Willebrand factor receptor complex, GPIb-V-IX. 7-9 GPIb-V-IX plays an important role in the adhesion of circulating platelet...

Elevated levels of plasma homocysteine (Hcy) correlate with increased risk of cardiovascular and Alzheimer diseases. We studied the effect of elevated Hcy on the blood-brain barrier (BBB) to explore the possibility of a vascular link between the 2 diseases. On a hyperhomocysteinemic diet, cystathionine beta-synthase (Cbs)-heterozygous mice develop hyperhomocysteinemia. Intravital microscopy analysis of the mesenteric venules showed that leukocyte rolling velocity was markedly decreased and numbers of adherent cells were increased in the mutant mice. This was due at least in part to increased expression of P-selectin. BBB permeability was measured by Evans blue dye permeation and was found to be 25% greater in the Cbs(+/-) cortex compared with wild-type controls. Our study suggests an important toxic effect of elevated Hcy on brain microvessels and implicates Hcy in the disruption of the BBB.

Interaction of the platelet receptor GPIb-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbα subunit was first described more than 25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We have recently reported that shedding of GPIbα and platelet clearance induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbα shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-α converting enzyme (TACEΔZn/ΔZn) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of two potent TACE inhibitors, TAPI and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbα expression in vivo, as we observed an ~ 90% reduction in soluble GPIbα (glycocalicin) in plasma of TACEΔZn/ΔZn chimeras as well as significantly increased levels of GPIbα on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Mitochondrial damage in TACEΔZn/ΔZn platelets did not result in reduced posttransfusion recovery or impaired hemostatic function of such platelets in mice, indicating that TACE not only mediates GPIbα shedding but that it may also be crucial for the development of cellular changes leading to platelet clearance. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbα. Inhibition of TACE during platelet storage could be a powerful means to improve the effectiveness of platelet transfusions.

Objective-High-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediated cellular uptake of lipoprotein cholesterol controls HDL structure and plasma HDL and biliary cholesterol levels. In SR-BI knockout (KO) mice, an unusually high plasma unesterified-to-total cholesterol ratio (UC:TC) and abnormally large HDL particles apparently contribute to pathology, including female infertility, susceptibility to atherosclerosis and coronary heart disease, and anemia. Here we examined the influence of SR-BI deficiency on platelets. Methods and Results-The high plasma UC:TC ratio in SR-BI KO mice was correlated with platelet abnormalities, including high cholesterol content, abnormal morphologies, high clearance rates, and thrombocytopenia. One day after platelets from wild-type mice were infused into SR-BI KO mice, they exhibited abnormally high cholesterol content and clearance rates similar to those of endogenous platelets. Platelets from SR-BI KO mice exhibited in vitro a blunted aggregation response to the agonist ADP but a normal response to PAR4. Conclusions-In

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewisx tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.

Objective-Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-␤ play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-␤ signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. Methods and Results-VEGF or TGF-␤ inhibition, accomplished using adenovirus expression of soluble Flt1 or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-␤ resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-␤ was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin ␣IIb␤III, or by leukocyte expression of L-selectin. Soluble vascular cell adhesion molecule (VCAM)-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. Conclusions-VEGF or TGF-␤ neutralization leads to impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-␤ in maintaining the endothelium in a nonactivated state and have implications for therapeutic approaches that neutralize VEGF or TGF-␤. Key Words: VEGF Ⅲ TGF-␤ Ⅲ P-selectin Ⅲ nitric oxide Ⅲ inflammation A key property of the endothelium, which lines the entire vasculature, is maintenance of the nonthrombogenic and noninflammatory vascular surface. During localized infection or injury, the endothelium becomes activated, resulting in expression of adhesion molecules that prevent bleeding and mediate the inflammation that is associated with rolling of immune cells as well as their attachment and extravasation at the site of injury. 1 These events are regulated by interplay between cytokines and adhesion receptors on both the endothelial cells (ECs) and the circulating blood cells. Under nonpathological conditions the endothelium is equipped with several mechanisms to prevent thrombus formation and immune cell adhesion, including production of nitric oxide (NO), 2 which acts as an antithrombotic factor, vasodilator, and modulator of leukocyte adhesion. Acute endothelial dysfunction is often characterized by impaired vascular autoregulation, whereas chronic endothelial dysfunction leads to inflammation and altered expression of cell adhesion molecules. 3 Emerging clinical and experimental observations suggest that vascular endothelial growth factor (VEGF) is required for the maintenance of specialized EC stability and function. 4,5 Selective inhibition of VEGF in the kidney leads to thrombotic glomerular injury. 6 Preeclampsia, a disease mediated in part by high levels of the circulating soluble Flt1 (sFlt1), which binds both VEGF and ...

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