Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Marjanovic, Jasna; Stojanović, Aleksandra; Brovkovych, Viktor; Skidgel, Randal A.; Du, Xiaoping

doi:10.1074/jbc.m801646200

Cited by 32 publications

(32 citation statements)

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“…This may suggest that the level of NO production in EPCs via iNOS is not sufficient to affect platelet function. Recently, Marjanovic et al 36 have shown that NO synthesis during platelet activation not only involves eNOS but is also mediated by the constitutively expressed platelet iNOS. iNOS-derived NO is important in promoting platelet activation, both ex vivo and in vivo, via a cGMP-dependent mechanism.…”

Section: Discussionmentioning

confidence: 99%

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

et al. 2009

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Background-Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results-Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions-Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis. Key Words: progenitor cells Ⅲ nitric oxide Ⅲ platelets Ⅲ prostaglandins Ⅲ thrombosis E ndothelial progenitor cells (EPCs) are believed to contribute to vascular biology and hemostasis. Although EPCs have been localized in cord blood, bone marrow, and peripheral blood as well as in some regenerative tissues, controversy relative to their isolation, identification, and functionality still exists. Indeed, the term EPC has been employed to describe different populations of multipotent cells that have the capability to differentiate, depending on the environmental cues, into mature endothelial cells (ECs). These cells are extremely rare in peripheral blood, but their number can be markedly increased after treatment with mobilizing cytokines or vascular trauma. 1 Nevertheless, cells with progenitor and endothelial phenotypes have been generated in vitro from peripheral blood mononuclear cells (PBMCs) 2-10 and have shown potential therapeutic applications in vascular tissue engineering and cell-based therapy. 5...

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Section: Discussionmentioning

confidence: 99%

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

et al. 2009

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“…Both eNOS and iNOS are expressed and functional in platelets. [2][3][4][5][6] The major effect of NO is mediated by its cytosolic receptor, soluble guanylyl cyclase (sGC). The roles of the NO/sGC/cGMP pathway in platelet activation have been investigated for Ͼ 3 decades.…”

Section: Introductionmentioning

confidence: 99%

Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation

Zhang

Xiang

Dong

et al. 2011

Blood

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Nitric oxide (NO) stimulates cGMP synthesis by activating its intracellular receptor, soluble guanylyl cyclase (sGC). It is a currently prevailing concept that No and cGMP inhibits platelet function. However, the data supporting the inhibitory role of NO/sGC/cGMP in platelets have been obtained either in vitro or using whole body gene deletion that affects vessel wall function. Here we have generated mice with sGC gene deleted only in megakaryocytes and platelets. Using the megakaryocyte-and platelet-specific sGC-deficient mice, we identify a stimulatory role of sGC in platelet activation and in thrombosis in vivo. Deletion of sGC in platelets abolished cGMP production induced by either NO donors or platelet agonists, caused a marked defect in aggregation and attenuated secretion in response to low doses of collagen or thrombin. Importantly, megakaryocyte-and platelet-specific sGC deficient mice showed prolonged tail-bleeding times and impaired FeCl 3 -induced carotid artery thrombosis in vivo. Interestingly, the inhibitory effect of the NO donor SNP on platelet activation was sGC-dependent only at micromolar concentrations, but sGC-independent at millimolar concentrations. Together, our data demonstrate important roles of sGC in stimulating platelet activation and in vivo thrombosis and hemostasis, and sGC-dependent and -independent inhibition of platelets by NO donors. (Blood. 2011;118(13):3670-3679) IntroductionThe nitric oxide (NO)/cGMP signaling cascade is involved in diverse physiologic and pathophysiologic functions, such as smooth muscle relaxation, vasodilation, neurotransmission, immune responses, and inflammation. 1 NO is a short-lived gaseous molecule, synthesized by a family of enzymes known as nitric oxide synthase (NOS). There are 3 known NOS isoforms, neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). Both eNOS and iNOS are expressed and functional in platelets. [2][3][4][5][6] The major effect of NO is mediated by its cytosolic receptor, soluble guanylyl cyclase (sGC). The roles of the NO/sGC/cGMP pathway in platelet activation have been investigated for Ͼ 3 decades. However, their functions in platelet activation remain controversial. There are 2 major controversies regarding the role of the NO-cGMP pathway in platelets: (1) whether the NO-cGMP pathway plays a stimulatory role, an inhibitory role, or both during platelet activation; and (2) whether the inhibitory effect of NO donors on platelet function is cGMPdependent or not. Early studies in the mid-1970's showed that during platelet activation by agonists, such as ADP and collagen, intracellular cGMP concentrations were enhanced significantly, 7,8 and that exogenous cGMP analogs enhanced platelet aggregation. 9 Therefore, a stimulatory role of cGMP in platelet activation was proposed. This view was soon abandoned because nitric oxide donors inhibited platelet activation, and dramatically increased intraplatelet cGMP concentrations. Since then, it has been generally accepted that cGMP plays an inhibitory...

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“…Bleeding time was measured as described. 10 Submitted March 31, 2010; accepted June 2, 2010. Prepublished online as Blood First Edition paper, June 16, 2010; DOI 10.1182 DOI 10.…”

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confidence: 99%

PTEN regulates collagen-induced platelet activation

Weng

Ding

Zhang

et al. 2010

Blood

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Phosphatidylinositol 3-kinase (PI3K) has been shown to play an important role in collagen-induced platelet activation, but the role(s) of PTEN, a major regulator of the PI3K/Akt signaling pathway, has not been examined in platelets. Here, we report that Pten ؊/؊ mouse blood contains 25% more platelets than Pten ؉/؉ blood and that PTEN deficiency significantly shortened the bleeding time, increased the sensitivity of platelets to collagen-induced activation and aggregation, and enhanced phosphorylation of Akt at Ser473 in response to collagen. Furthermore, we found that PP2, and the combination of apyrase, indomethacin ؉ 1B5, respectively, inhibited collagen-induced aggregation in both PTEN ؉/؉ and PTEN ؊/؊ platelets. In contrast, LY294002 (a PI3K inhibitor) prevented the aggregation of PTEN ؉/؉ , but not PTEN ؊/؊ , platelets. Therefore, PTEN apparently regulates collagen-induced platelet activation through PI3K/Akt-dependent and -independent signaling pathways. IntroductionThe role of collagen/platelet interactions in hemostasis has been studied extensively. Collagen, acting on the glycoprotein VI (GPVI)/Fc receptor ␥ chain (FcR-␥ chain) complex, sequentially activates Src and Syk family tyrosine kinases that propagate potent signaling via phosphatidylinositol 3-kinase (PI3K) that culminates in ␣IIb␤3 activation, and platelet aggregation. [1][2][3] PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a dual-specificity protein phosphatase and an inositol phospholipid phosphatase. The role of PTEN as a tumor suppressor is well established. 4 PTEN dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), thereby negatively regulating oncogenic and nononcogenic PI3K/Akt signaling. 5 PTEN/PI3K/ Akt constitutes an important pathway regulating the signaling of multiple biologic processes, including apoptosis, cell growth, and proliferation. PI3K plays an important role in GPVI-mediated platelet activation, yet the role of PTEN, the major regulator of PI3K/Akt signaling, has not been characterized in GPVI-mediated platelet activation. Here, using inducible and hematopoietic tissuespecific deletion of Pten in mice, we demonstrate that PTEN negatively regulates PI3K-dependent and -independent collageninduced platelet activation. Methods MaterialsCollagen was from Nycomed Arzneimittel. PP2 and LY294002 were from Calbiochem. Apyrase and prostaglandin E 1 were from Sigma-Aldrich. Hamster anti-mouse ␣IIb␤3 antibody (IB5) was a generous gift from Dr Barry Coller (Rockefeller University). Anti-phospho-Akt (Ser473), anti-PTEN, and anti-␤-actin antibodies were from Cell Signaling Technology. Secondary antibodies were from Santa Cruz Biotechnology. AnimalsMx1-Cre ϩ C57BL/6J mice were from Model Animal Research Center of Nanjing University (Nanjing, China). Pten loxp-loxp mice from The Jackson Laboratory were backcrossed onto the C57BL/6J background for 6 generations. The Pten loxp-loxp /Mx1-Cre ؉ mice were produced as described. 6 P...

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Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Cited by 32 publications

References 42 publications

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation

PTEN regulates collagen-induced platelet activation

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