Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB–stimulated chemotaxis

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3452-3458 ◽  
Author(s):  
Agneta Siegbahn ◽  
Matilda Johnell ◽  
Charlotte Rorsman ◽  
Mirella Ezban ◽  
Carl-Henrik Heldin ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Tissue Factor ◽  
Active Site ◽  
Factor Viia ◽  
Human Fibroblasts ◽  
Cellular Responses ◽  
Cellular Receptor ◽  
Inositol 1,4,5 Trisphosphate ◽  
Catalytically Active

Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF β-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4,5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.

Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB–stimulated chemotaxis

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3452-3458 ◽  
Author(s):  
Agneta Siegbahn ◽  
Matilda Johnell ◽  
Charlotte Rorsman ◽  
Mirella Ezban ◽  
Carl-Henrik Heldin ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Tissue Factor ◽  
Active Site ◽  
Factor Viia ◽  
Human Fibroblasts ◽  
Cellular Responses ◽  
Cellular Receptor ◽  
Inositol 1,4,5 Trisphosphate ◽  
Catalytically Active

Abstract Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF β-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4,5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Large Enhancement of Functional Activity of Active Site-Inhibited Factor VIIa Due to Protein Dimerization:  Insights into Mechanism of Assembly/Disassembly from Tissue Factor†

Biochemistry ◽  
2005 ◽  
Vol 44 (16) ◽  
pp. 6321-6330 ◽  
Author(s):  
Matthew D. Stone ◽  
Stephen B. Harvey ◽  
Michael B. Martinez ◽  
Ronald R. Bach ◽  
Gary L. Nelsestuen
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Functional Activity ◽  
Factor Viia ◽  
Protein Dimerization ◽  
Large Enhancement

scholarly journals Antibody mapping of tissue factor implicates two different exon-encoded regions in function

1991 ◽  
Vol 278 (3) ◽  
pp. 729-733 ◽  
Author(s):  
W Ruf ◽  
A Rehemtulla ◽  
T S Edgington
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Polyclonal Antibodies ◽  
Factor Vii ◽  
Binary Complex ◽  
Minimum Requirement ◽  
High Affinity ◽  
Transmembrane Glycoprotein ◽  
Catalytically Active ◽  
Exon 4

Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding.

scholarly journals The Location of the Active Site of Blood Coagulation Factor VIIa above the Membrane Surface and Its Reorientation upon Association with Tissue Factor

1996 ◽  
Vol 271 (45) ◽  
pp. 28168-28175 ◽  
Author(s):  
Christine D. McCallum ◽  
Raymond C. Hapak ◽  
Pierre F. Neuenschwander ◽  
James H. Morrissey ◽  
Arthur E. Johnson
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Active Site ◽  
Factor Viia ◽  
Membrane Surface ◽  
Coagulation Factor

scholarly journals Signal Transduction via the Mitogen-activated Protein Kinase Pathway Induced by Binding of Coagulation Factor VIIa to Tissue Factor

1998 ◽  
Vol 273 (11) ◽  
pp. 6228-6232 ◽  
Author(s):  
Lars K. Poulsen ◽  
Nana Jacobsen ◽  
Brit B. Sørensen ◽  
Nils C. H. Bergenhem ◽  
James D. Kelly ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

Characterization Of a New Recombinant Human Factor VIIa (LR769)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3583-3583
Author(s):  
Grandoni Jerry ◽  
Gerald Perret ◽  
Cynthia Forier
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Protein C ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Binding Studies ◽  
Activation Assay

Abstract Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in the replacement therapies. In such cases factor replacement becomes ineffective, and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB/rEVO Biologics was tested. The goal is to provide patients with Hemophilia A and B who develop inhibitors a cost-effective alternative treatment option. Recombinant Human Factor VII was activated during the purification process to yield a highly homogenous rhFVIIa product (LR769). Kinetic enzyme assays and binding studies were used to characterize LR769. Active site titration demonstrated approximately 1 mole of active site per mole of protein. The Km and kcat for activation of FX and FIX were determined using an assay containing recombinant human tissue factor and phospholipid. The Kd for binding to soluble tissue factor was 22.3 ± 1.7 nM as measured using a FX activation assay. The apparent second order rate constant for inactivation by human plasma antithrombin was 5.9 ± 0.4 x103 M-1 sec-1. In all kinetic assays, LR769 behaved as expected for rhFVIIa. Binding studies with isolated human platelets were performed by FACS and indicated that binding was dependent on Ca+2. Activation of the platelets with thrombin and convulxin increased the binding of LR769. Binding of LR769 to Endothelial Protein C Receptor (EPCR) on the surface of cultured HEK 293 cells was determined by FACS analysis and confirmed that LR769 binds to EPCR. This was further demonstrated with a surface plasmon resonance assay using purified soluble EPCR. Binding to EPCR was dependent on Ca+2, and could be stimulated by Mg+2. Specificity of the binding was confirmed by the fact that it was inhibited by excess Protein C. Disclosures: No relevant conflicts of interest to declare.

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