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Wolfram Ruf

Publications and source records attributed to Wolfram Ruf.

31 records · Page 2Linked to original sources
PubMed2004

Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis.

The effects of the pleiotropic serine protease thrombin on tumor cells are commonly thought to be mediated by the thrombin receptor protease-activated receptor 1 (PAR1). We demonstrate here that PAR1 activation has a role in experimental metastasis using the anti-PAR1 antibodies ATAP2 and WEDE15, which block PAR1 cleavage and activation. Thrombin also stimulates chemokinesis of human melanoma cells toward fibroblast conditioned media and soluble matrix proteins. Thrombin-enhanced migration is abolished by anti-PAR1 antibodies, demonstrating that PAR1 cleavage and activation are required. The PAR1-specific agonist peptide TFLLRNPNDK, however, does not stimulate migration, indicating that PAR1 activation is not sufficient. In contrast, a combination of TFLLRNPNDK and the PAR2 agonist peptide SLIGRL mimics the thrombin effect on migration, whereas PAR2 agonist alone has no effect. Agonist peptides for the thrombin receptors PAR3 and PAR4 used alone or with PAR1 agonist also have no effect. Similarly, activation of PAR1 and PAR2 also enhances chemokinesis of prostate cancer cells. Desensitization with PAR2 agonist abolishes thrombin-enhanced cell motility, demonstrating that thrombin acts through PAR2. PAR2 is cleaved by proteases with trypsin-like specificity but not by thrombin. Thrombin enhances migration in the presence of a cleavage-blocking anti-PAR2 antibody, suggesting that thrombin activates PAR2 indirectly and independent of receptor cleavage. Treatment of melanoma cells with trypsin or PAR2 agonist peptide enhances experimental metastasis. Together, these data confirm a role for PAR1 in migration and metastasis and demonstrate an unexpected role for PAR2 in thrombin-dependent tumor cell migration and in metastasis.

Cell Line, Tumor↗
PubMed2003

The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis.

Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease-activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell-signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein-protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma-carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site-directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N-terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible.

Binding Sites↗
PubMed2003

Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry.

Vasculogenic mimicry (VM), the formation of matrix-rich vascular-like networks in three-dimensional culture corresponding with the expression of vascular cell-associated genes, and the lining of matrix-rich networks in situ, has been observed in highly aggressive and malignant melanoma. However, little is known about the molecular underpinnings of this phenomenon. On the basis of gene profiling, protein detection, and immunohistochemistry, aggressive relative to poorly aggressive melanoma showed up-regulation of tissue factor (TF), TF pathway inhibitor 1 (TFPI-1) and 2 (TFPI-2), critical genes that initiate and regulate the coagulation pathways. The procoagulant function of TF on highly aggressive melanoma is shown to be regulated by TFPI-1 but not by TFPI-2. Thus, aggressive melanoma exhibits endothelial cell-like anticoagulant mechanisms that may contribute to the fluid-conducting potential of melanoma cell-lined networks, as studied by correlative in vivo Doppler flow measurements. Antibody inhibition experiments reveal that TFPI-2 is required for VM in vitro, but plasmin is an unlikely target protease of TFPI-2. Blockade of TFPI-2 suppressed matrix metalloproteinase-2 activation, and, therefore, TFPI-2 appears to regulate an essential pathway of VM. TFPI-2 is synthesized by endothelial and tumor cells, which deposit TFPI-2 into extracellular matrices. Culturing poorly aggressive melanoma cells on three-dimensional matrix containing recombinant TFPI-2 produces some of the phenotypic changes associated with aggressive, vasculogenic melanoma cells. Thus, TFPI-2 contributes to VM plasticity, whereas TFPI-1 has anticoagulant functions of relevance for perfusion of VM channels formed by TF-expressing melanoma cells.

Animals↗
PubMed2003

Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation.

The tissue factor (TF)-initiated coagulation pathway plays important roles in hemostasis, inflammation, metastasis, and angiogenesis. Phosphorylation of the TF cytoplasmic domain is functionally relevant in metastasis. How TF cytoplasmic domain phosphorylation downstream of protein kinase C (PKC) activation is regulated in primary vascular cells remains poorly understood. Here, phosphorylation of Ser258, rather than the PKC consensus site Ser253, is identified as the major conformational switch required for recognition by a phosphorylation-specific antibody. With this novel reagent, we demonstrate that the TF cytoplasmic domain is primarily unphosphorylated in confluent endothelial cells. TF cytoplasmic domain phosphorylation can occur in the absence of the autologous TF transmembrane and extracellular domains but requires maturation of TF in the Golgi compartment and cell surface expression. Site-directed mutagenesis and 2-bromopalmitate treatment provide evidence that palmitoylation of the cytoplasmic Cys245 is a negative regulatory mechanism of Ser258 phosphorylation. Profiling with PKC-selective inhibitors identifies PKCalpha as important for TF cytoplasmic domain phosphorylation. Mutagenesis of protein kinase consensus sites are consistent with a model in which PKC-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a Pro-directed kinase. Thus, cell surface location-dependent phosphorylation of the TF cytoplasmic domain is regulated at multiple levels.

Allosteric Regulation↗
PubMed2003

Tissue factor-dependent coagulation protease signaling in acute lung injury.

OBJECTIVES: To review the role of tissue factor-dependent coagulation in acute lung injury. To interpret preclinical and clinical data on therapeutic intervention of the coagulation cascade, focusing on the principles of proteolytic cell signaling of the coagulant and anticoagulant pathways. DATA EXTRACTION AND SYNTHESIS: This review is based on published original research and relevant review articles on cell signaling by coagulation proteases and on experimental models that implicate the tissue factor-initiated coagulation cascade in acute lung injury and systemic inflammation. CONCLUSIONS: The coagulation cascade signals via protease activated receptors in the tissue factor-initiation phase and downstream via the effector protease, thrombin. Bleomycin-induced acute lung injury is an example of thrombin signaling-dependent pathology. Frequently, thrombin signaling is a major contributor to inflammation in the extravascular space but intravascular thrombin signaling is a threshold-regulated event. At low concentrations, intravascular thrombin activates the protein C pathway by converting protein C (bound to endothelial cell protein C receptor) to activated protein C and this generates antiinflammatory signals along the activated protein C-endothelial cell protein C receptor-protease activated receptor 1 pathway on endothelial cells. Direct thrombin signaling only occurs when intravascular thrombin concentrations exceed a coagulant threshold. In systemic bacterial toxin-mediated inflammation, inhibition of thrombin is not sufficient to limit inflammation, whereas tissue factor inhibition interrupts a self-sustaining inflammatory escalation in acute lung injury. Therefore, in the vasculature, inflammatory signaling by the tissue factor initiation complex is favored over thrombin signaling.

Disseminated Intravascular Coagulation↗
PubMed2003

Activated protein C signals through the thrombin receptor PAR1 in endothelial cells.

The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner. Human endothelial cells (HUVECs) express PAR1, PAR2 and EPCR. Stimulation of HUVECs with either APC, or specific receptor activating peptides for PAR1 or PAR2, show that all three agonists induce a very similar set of early response genes as assessed by high density microarray analysis. Only the transcript for monocyte chemo-attractant protein-1 (MCP-1) was selectively induced by APC and the PAR1 agonist, but not by the PAR2 agonist. APC-mediated MAP kinase phosphorylation and gene induction were inhibited by cleavage blocking antibodies to PAR1, demonstrating that APC signals exclusively through PAR1 in endothelial cells. MCP-1 is protective in animal models of endotoxemia, suggesting that APC may prevent lethality in sepsis by inducing MCP-1 expression through EPCR-dependent activation of endothelial cell PAR1. These data demonstrate unexpected protective functions of the major thrombin receptor PAR1 in endothelial cells.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Science review: role of coagulation protease cascades in sepsis.

Cellular signaling by proteases of the blood coagulation cascade through members of the protease-activated receptor (PAR) family can profoundly impact on the inflammatory balance in sepsis. The coagulation initiation reaction on tissue factor expressing cells signals through PAR1 and PAR2, leading to enhanced inflammation. The anticoagulant protein C pathway has potent anti-inflammatory effects, and activated protein C signals through PAR1 upon binding to the endothelial protein C receptor. Activation of the coagulation cascade and the downstream endothelial cell localized anticoagulant pathway thus have opposing effects on systemic inflammation. This dichotomy is of relevance for the interpretation of preclinical and clinical data that document nonuniform responses to anticoagulant strategies in sepsis therapy.

Anticoagulants↗
PubMed2002

Role of zymogenicity-determining residues of coagulation factor VII/VIIa in cofactor interaction and macromolecular substrate recognition.

Factor VIIa (VIIa) remains in a zymogen-like state following proteolytic activation and depends on interactions with the cofactor tissue factor (TF) for function. Val(21), Glu(154), and Met(156) are residues that are spatially close in available zymogen and enzyme structures, despite major conformational differences in the corresponding loop segments. This residue triad displays unusual side chain properties in comparison to the properties of other coagulation serine proteases. By mutagenesis, we demonstrate that these residues cooperate to stabilize the enzyme conformation and to enhance the affinity for TF. In zymogen VII, however, substitution of the triad did not change the cofactor affinity, further emphasizing the crucial role of the activation pocket in specifically stabilizing the active enzyme conformation. In comparison to VIIa(Q156), the triple mutant VIIa(N21I154Q156) had a stabilized amino-terminal Ile(16)-Asp(194) salt bridge and enhanced catalytic function. However, proteolytic and amidolytic activities of free VIIa variants were not concordantly increased. Rather, a negatively charged Asp at position 21 was the critical factor that determined whether an amidolytically more active VIIa variant also more efficiently activated the macromolecular substrate. These data thus demonstrate an unexpected complexity by which the zymogenicity-determining triad in the activation pocket of VIIa controls the active enzyme conformation and contributes to exosite interactions with the macromolecular substrate.

Amino Acid Sequence↗
PubMed2002

Plasmin-induced migration of endothelial cells. A potential target for the anti-angiogenic action of angiostatin.

Angiostatin, a plasminogen fragment containing 3-4 N-terminal kringle domains, is a potent inhibitor of tumor-induced angiogenesis, but its mechanism of action is unclear. Angiostatin is a ligand for integrin alphavbeta(3) but does not induce stress fiber formation upon integrin binding, suggesting that angiostatin is a potential integrin antagonist. Plasmin, the parent molecule of angiostatin and a major extracellular protease, induces platelet aggregation, migration of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types. In the current study, we found that plasmin specifically bound to alphavbeta(3) through the kringle domains and induced migration of endothelial cells. In contrast, angiostatin did not induce cell migration. Notably, angiostatin, anti-alphavbeta(3) antibodies, RGD-peptide, and a serine protease inhibitor effectively blocked plasmin-induced cell migration. These results suggest that plasmin-induced migration of endothelial cells requires alphavbeta(3) and the catalytic activity of plasmin and that this process is a potential target for the inhibitory activity of angiostatin.

Angiogenesis Inhibitors↗
PubMed2002

Activation of endothelial cell protease activated receptor 1 by the protein C pathway.

The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.

Animals↗
PubMed2002

Orchestration of coagulation protease signaling by tissue factor.

Protease-activated receptors (PARs) are vascular sensors for signaling of the trypsinlike coagulation serine proteases that play key roles in cardiovascular medicine. In the initiation phase of coagulation, tissue factor (TF) orchestrates the assembly of VIIa with substrate X, forming a ternary complex in which product Xa is generated. The resulting TF-VIIa-Xa complex is an efficient activator of PAR1 and PAR2. TF initiation of the coagulation cascade is thus intimately linked to inflammatory cell signaling. Inflammation is an increasingly appreciated component of the vulnerable atherosclerotic plaque. Targeting inflammatory cell signaling events of the coagulation system may become an important aspect of efforts to improve antithrombotic therapy.

Animals↗