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Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

ABSTRACT: The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis.

SUBMITTER: McEachron TA

PROVIDER: S-EPMC3012597 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

McEachron Troy A TA Pawlinski Rafal R Richards Kristy L KL Church Frank C FC Mackman Nigel N

Blood 20100824 23

The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcino ...[more]

PMID: 20736455

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Project description:BackgroundAstrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway.MethodsPrimary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents.ResultsAstrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor.ConclusionsPlasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

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Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

Publications

Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

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