Abstract-To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (Ϫ14%; PϽ0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo. Key Words: transgenic mice Ⅲ adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ vascular inflammation Ⅲ thrombosis C D73/ecto-5Ј-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5Ј-AMP to adenosine. 1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATPdiphosphohydrolase. 3 The product of CD73 is adenosine, a purine nucleoside that has been implicated in many physiological and pathophysiological events. 4 There are four known G-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 , each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution. 5 In human neutrophils, adenosine A 1 and A 2 receptor occupancy mediate opposing roles for adenosine in inflammation: A 1 activation is proinflammatory, whereas the A 2 receptor plays an anti-inflammatory role. 6 A 2 receptor activation inhibits the neutrophil oxidative burst, whereas the A 3 receptor inhibits neutrophil degranulation 7 and may play an important role in inflammation by inhibiting eosinophil migration. 8 Recently, deletion of the A 2A receptor in transgenic mice revealed that this receptor is critical for the limitation a...

Objective-To determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in sickle cell disease (SCD). Methods and Results-Both platelet and neutrophil activation occur in SCD, but the interdependence of these events is unknown. Platelet activation and binding to leukocytes were measured in mice and patients with SCD and in controls.Relative to controls, blood obtained from mice or patients with SCD contained significantly elevated platelet-neutrophil aggregates (PNAs). Both platelets and neutrophils found in sickle PNAs were activated. Multispectral imaging (ImageStream) and conventional flow cytometry revealed a subpopulation of activated neutrophils with multiple adhered platelets that expressed significantly more CD11b and exhibited greater oxidative activity than single neutrophils. On average, wild-type and sickle PNAs contained 1.1 and 2.6 platelets per neutrophil, respectively. Hypoxia/reoxygenation induced a further increase in PNAs in mice with SCD and additional activation of both platelets and neutrophils. The pretreatment of mice with SCD with clopidogrel or P-selectin antibody reduced the formation of PNAs and neutrophil activation and decreased lung vascular permeability. Conclusion-Our findings suggest that platelet binding activates neutrophils and contributes to a chronic inflammatory state and pulmonary dysfunction in SCD. The inhibition of platelet activation may be useful to decrease tissue injury in SCD, particularly during the early stages of vaso-occlusive crises. Key Words: platelet activation Ⅲ sickle cell disease inflammation Ⅲ neutrophil activation Ⅲ oxidative burst Ⅲ clopidogrel Ⅲ P-selectin antiplatelet drugs Ⅲ leukocytes Ⅲ platelets Ⅲ transgenic models S ickle cell disease (SCD) is the most common genetic hematologic disorder in the United States. The vasoocclusive characteristics of SCD have been viewed historically as resulting from deformed red blood cells (RBCs) that mechanically obstruct capillaries to produce tissue hypoxia. 1 Present therapies for SCD are geared toward decreasing the concentration or polymerization rate of sickle hemoglobin. 2 Recently, a modified paradigm has emerged suggesting that the wide spectrum of clinical manifestations of SCD results in part from recurrent episodes of disseminated microvascular ischemia-reperfusion injury. 3,4 Ischemia-reperfusion injury triggers vascular inflammation, characterized by increased adhesion of leukocytes [5][6][7][8] and sickle RBCs 9 to vascular endothelium and activation of coagulation, 10 -12 blood platelets, 13-20 neutrophils, 7 monocytes, 8,21-23 and natural killer T cells. 24 Because blockade of P-selectin-mediated plateletleukocyte aggregation is beneficial in the animal models of vascular injury, 25 we reasoned that platelet-leukocyte aggregation might contribute to the vascular inflammation and tissue injury that occurs in SCD. Although increased formation of platelet-monocyte 21 and platelet-RBC 15 aggregates in SCD is well established, conflicting data exist reg...

Blood platelets play critical roles in hemostasis, providing rapid essential protection against bleeding and catalyzing the important slower formation of stable blood clots via the coagulation cascade. They are also involved in protection from infection by phagocytosis of pathogens and by secreting chemokines that attract leukocytes. Platelet function usually is activated by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas secondary agonists like adrenalin do not induce aggregation on their own but become highly effective in the presence of low levels of primary agonists. Current research has revealed that chemokines represent an important additional class of agonists capable of causing significant activation of platelet function. Early work on platelet alpha-granule proteins suggested that platelet factor 4, now known as CXCL4, modulated aggregation and secretion induced by low agonist levels. Subsequent reports revealed the presence in platelets of messenger RNA for several additional chemokines and chemokine receptors. Three chemokines in particular, CXCL12 (SDF-1), CCL17 (TARC), and CCL22 (MDC), recently have been shown to be strong and rapid activators of platelet aggregation and adhesion after their binding to platelet CXCR4 or CCR4, when acting in combination with low levels of primary agonists. CXCL12 can be secreted by endothelial cells and is present in atherosclerotic plaques, whereas CCL17 and CCL22 are secreted by monocytes and macrophages. Platelet activation leads to the release of alpha-granule chemokines, including CCL3 (MIP-1alpha), CCL5 (RANTES), CCL7 (MCP-3), CCL17, CXCL1 (growth-regulated oncogene-alpha), CXCL5 (ENA-78), and CXCL8 (IL-8), which attract leukocytes and further activate other platelets. These findings help to provide a direct linkage between hemostasis, infection, and inflammation and the development of atherosclerosis.

Platelet activation is normally induced by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas other agonists, such as epinephrine, can play important accessory roles. It is now reported that the macrophage-derived chemokine (MDC), thymus activation-regulated chemokine (TARC), and stromal cell-derived factor one (SDF-1) are highly effective activators of platelet function under a variety of conditions, stimulating platelet shape change, aggregation, and adhesion to collagen or fibrinogen. Chemokine-mediated platelet activation was rapid and maximal (less than 5 seconds) under arterial flow conditions and depended strongly on the presence of low levels of primary agonists such as ADP or thrombin. Concentrations of ADP (0.05-0.25 microM) or thrombin (0.005-0.02 U/mL) that induced minimal aggregation caused major aggregation acting in combination with the chemokines. The ability of apyrase to block chemokine-dependent aggregation or adhesion was consistent with an important role for ADP. Chemokine-stimulated aggregation was also insensitive to indomethacin, suggesting that the activation of cyclo-oxygenase is not involved. TARC, MDC, and SDF-1 increased intracellular calcium concentrations [Ca(2+)](i) when combined with low levels of ADP. The MDC and TARC receptor CCR4 was expressed on platelets, and an anti-CCR4 antibody blocked aggregation induced by TARC or MDC. Treatment of platelets with SDF-1 and MDC rapidly exposed P-selectin (CD62P) on the cell surface but did not induce the secretion of serotonin. These findings suggest that the chemokines MDC, TARC, and SDF-1, which may be produced during inflammatory responses, coupled with low levels of ADP or thrombin, can serve as strong stimuli for activating platelet function.