FXa-Induced Responses in Vascular Wall Cells are PAR-Mediated and Inhibited by ZK-807834

doi:10.1016/S0049-3848(01)00330-9

Thrombosis Research

Volume 103, Issue 4, 15 August 2001, Pages 281-297

https://doi.org/10.1016/S0049-3848(01)00330-9 Get rights and content

Abstract

During thrombosis, vascular wall cells are exposed to clotting factors, including the procoagulant proteases thrombin and factor Xa (FXa), both known to induce cell signaling. FXa shows dose-dependent induction of intracellular Ca²⁺ transients in vascular wall cells that is active-site-dependent, Gla-domain-independent, and enhanced by FXa assembly into the prothrombinase complex. FXa signaling is independent of prothrombin activation as shown by the lack of inhibition by argatroban, hirudin and the sulfated C-terminal peptide of hirudin (Hir^54–65(SO₃⁻)). This peptide binds to both proexosite I in prothrombin and exosite I in thrombin. In contrast, signaling is completely blocked by the FXa inhibitor ZK-807834 (CI-1031). No inhibition is observed by peptides which block interaction of FXa with effector cell protease 1 receptor (EPR-1), indicating that this receptor does not mediate signaling in the cells assayed. Receptor desensitization studies with thrombin or peptide agonists (PAR-1 or PAR-2) and experiments with PAR-1-blocking antibodies indicate that signaling by FXa is mediated by both PAR-1 and PAR-2. Potential pathophysiological responses to FXa include increased cell proliferation, increased production of the proinflammatory cytokine IL-6 and increased production of prothrombotic tissue factor. These cellular responses, which may complicate vascular disease, are inhibited by ZK-807834.

Section snippets

Reagents

FXa was from Enzyme Research Laboratories (South Bond, IN) or Haematological Technologies (Essex Junction, Vermont). FXa-beta, des-Gla FXa, FXa–EGR and α-thrombin were from Haematological Technologies (Essex Junction, Vermont). Recombinant hirudin was from American Diagnostica (Greenwich, CT). Argatroban was a gift from Mitsubishi (Japan). Tick anticoagulant peptide or TAP, peptides and ZK-807834 [33] were made at Berlex Biosciences (Richmond, CA). Cell culture media was from Clonetics (San

FXa Induces Ca²⁺ Transients in Vascular Wall Cells

The ability of human FXa to act as a signaling molecule was assessed in human aortic adventitial fibroblasts (HAOAFs), HASMCs, HAECs and HCASMCs using a fluorescence-based analysis of intracellular Ca²⁺ transients. The Ca²⁺ measurements were made using the FLIPR instrument from Molecular Devices Corporation (Sunnyvale, CA) that measures Ca²⁺ transients on attached cells. FXa induced Ca²⁺ transients dose-dependently in all four cell types (Fig. 1). We observed signaling at concentrations as low

Discussion

The potential effect of FXa exposure to vascular tissue was determined by testing the ability of FXa to induce Ca²⁺ transients in the principle types of vascular wall cells. A dose-dependent induction of Ca²⁺ transients by FXa in the primary vascular wall cell lines HAEC, HAOAF, HASMC and HCASMC was seen. Intracellular Ca²⁺ signals were observed at concentrations as low as 22 nM FXa. In HCASMC and HAEC, the magnitude of the FXa-induced Ca²⁺ response was at least as great as with equimolar

Acknowledgements

We thank J.-H. Lin, R. Pagila and C. Adams for help with TAP production; S. Biancalana for synthesis of peptides; Galina Rumennik for help with prothrombinase; Meina Liang for help using FLIPR; and Dr. William Dole for helpful discussions and support.

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Citation Excerpt :
Naturally and synthetic occurring FXa inhibitors exert strong anticoagulant activity and have been shown to be potent antithrombotic agents in animal models of venous and arterial thrombosis (Wong et al., 1996; Perzborn et al., 2007). Independent of the antithrombotic actions, several studies demonstrated that FXa inhibitors exert anti-inflammatory effects (McLean et al., 2001; Hoberg et al., 2004). DX-9065a, the first generation of FXa inhibitors, prevented FXa-induced IL-6 mRNA expression in human gingival fibroblasts and was considered as a useful drug for periodontal disease.
Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, which is activated during atrial fibrillation. Increasing evidence suggests that FXa influences cell signalling in various cell types by activating protease-activated receptors (PARs). It is so far not known if molecular effects of FXa affect atrial signal transduction. To study the effects of FXa, human atrial tissue slices were cultivated with FXa up to 24 h. Additionally, rapid pacing was applied at 4 Hz to resemble atrial fibrillation. The inhibitory impact of FXa antagonist (Rivaroxaban), protease-activated receptor 1 antagonist (SCH79797), and protease-activated receptor 2 antagonist (GB83) were analysed under experimental conditions. The exposure of atrial tissue to FXa resulted in the 1.7 fold upregulation of PAR2-mRNA, activation of MAP kinases (ERK1/2) and NF-κB signalling. Furthermore FXa increased the expression of adhesion molecule ICAM-1 (1.82±0.20), chemokine IL-8 (1.94±0.20), as well as prothrombotic molecule PAI-1 (1.52±0.17). The combination of rapid pacing and FXa caused significant upregulation of PAR1 (2.82±0.22), PAR2 (2.66±0.40), ICAM-1 (2.13±0.25), IL-8 (2.22±0.24), LOX-1 (2.59±0.35), and PAI-1 (2.65±0.52) at the mRNA level. Rivaroxaban and GB83 prevented upregulation of PARs, ICAM-1, LOX-1, IL-8, and activation of MAP kinases. The elevation in the expression of PAI-1 was hindered in the presence of SCH79797, or Rivaroxaban. The present study indicates that FXa mediates inflammatory signalling in atrial tissue. Importantly, FXa and tachyarrhythmia act synergistically to increase expression of protease-activated receptors and inflammatory mediators. Rivaroxaban prevented effectively FXa-induced molecular effects in human atrial tissue particularly during rapid pacing.

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REGULAR ARTICLEFXa-Induced Responses in Vascular Wall Cells are PAR-Mediated and Inhibited by ZK-807834

Abstract

Section snippets

Reagents

FXa Induces Ca2+ Transients in Vascular Wall Cells

Discussion

Acknowledgements

Cell Calcium

Cell

J Biol Chem

Blood

J Biol Chem

Cell Immunol

J Biol Chem

Blood

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Pharmacol

Blood

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Am Heart J

Blood

Blood

Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leukocyte initiation of coagulation

Biochem J

Sustained inhibition of whole-blood clot procoagulant activity by inhibition of thrombus-associated factor Xa

Arterioscler, Thromb, Vasc Biol

Tissue factor mediates prolonged procoagulant activity on the luminal surface of balloon-injured aortas in rabbits

Circulation

Prolonged activation of prothrombin on the vascular wall after arterial injury

Arterioscler, Thromb, Vasc Biol

Pathogenesis of thrombosis in coronary artery disease

Haemostasis

Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes

Circ Res

Atypical protease-activated receptor mediates endothelium-dependent relaxation of human coronary arteries

Circ Res

The role of secondary growth factor production in thrombin-induced proliferation of vascular smooth muscle cells

Semin Thromb Haemostasis

Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism

Thromb Haemostasis

Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons

J Neurobiol

Protease-activated receptors and their role in IL-6 and NF-IL-6 expression in human gingival fibroblasts

J Periodontal Res

Thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation

J Cell Physiol

REGULAR ARTICLE
FXa-Induced Responses in Vascular Wall Cells are PAR-Mediated and Inhibited by ZK-807834

FXa Induces Ca²⁺ Transients in Vascular Wall Cells