Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII - tissue factor interaction

Wildgoose, Peter; Foster, Donald C.; Schiødt, Jakob; Wiberg, Finn C.; Birktoft, J J; Petersen, Lars C.

doi:10.1021/bi00052a016

Cited by 62 publications

(52 citation statements)

References 41 publications

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“…6 occurs only because Fl-FPR-fVIIa is binding tightly to dcTF, not because Fl-FPR-fVIIa is binding directly to the vesicle surface. Furthermore, the elimination of this energy transfer by EDTA addition shows that fVIIa association with TF at these concentrations is calcium ion-dependent, consistent with the results of previous studies (33)(34)(35).…”

Section: Fluorescent Labeling Of the Active Site Of Fviia-supporting

confidence: 91%

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

McCallum

Hapak

Neuenschwander

et al. 1996

Journal of Biological Chemistry

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The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-Larginyl-fVIIa (Fl-FPR-fVIIa). The location of the activesite probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserinedependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 Å assuming a random orientation of donor and acceptor dyes ( 2 ‫؍‬ 2/3; the orientational uncertainty totals ϳ10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane.When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 Å after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

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Section: Fluorescent Labeling Of the Active Site Of Fviia-supporting

confidence: 91%

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

McCallum

Hapak

Neuenschwander

et al. 1996

Journal of Biological Chemistry

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“…Thus, in the presence of factor VII-depleted plasma, free thrombin generation and prothrombin consumption were negligible for all cell lines over the 25-min time period (data not shown), a result consistent with the requirement of factor VIIa for TF-dependent thrombin generation (62)(63)(64)(65). To specifically examine TF PCA on cell surfaces, wildtype, vector-transfected, or GRP78/BiP-overexpressing cells were grown to confluency and factor Xa generation was measured over 30 min using the chromogenic substrate S-2765.…”

Section: Resultssupporting

confidence: 70%

“…Factor VII Activation Assay-Factor VIIa generation on the surface of T24/83 cells was performed by a one-step assay, which directly measures the conversion of factor VII to factor VIIa, using the chromogenic substrate S-2288 (65). Cell monolayers were washed twice in ABS buffer and incubated for time periods up to 30 min in the absence (blank control) or presence of 108 nM recombinant human factor VII (Enzyme Research Laboratories, South Bend, IN) and 5 mM Ca 2ϩ in ABS buffer.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

Watson

Chan

Berry

et al. 2003

Journal of Biological Chemistry

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Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca 2؉ and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.

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“…The Ca 2ϩ -binding site on the protease domain of PC is located on the 70-80 loop (7). With the exception of prothrombin, the same loop is also known to bind Ca 2ϩ in other vitamin K-dependent coagulation proteases (8,9) and in trypsin (10). The binding of Ca 2ϩ to this loop plays a complex role in PC activation by thrombin: it is inhibitory for activation by thrombin alone but stimulatory for activation by the thrombin-TM complex (11).…”

mentioning

confidence: 99%

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin

Rezaie

Yang

2003

Proc. Natl. Acad. Sci. U.S.A.

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Exosite 1 of thrombin consists of a cluster of basic residues (Arg-35, Lys-36, Arg-67, Lys-70, Arg-73, Arg-75, and Arg-77 in chymotrypsinogen numbering) that play key roles in the function of thrombin. Structural data suggest that the side chain of Arg-35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin (TM). To study the role of these residues in TM-mediated protein C (PC) activation by thrombin, a charge reversal mutagenesis approach was used to replace these residues with a Glu in separate constructs. The catalytic properties of the mutants toward PC were analyzed in both the absence and presence of TM and Ca 2؉ . It was discovered that, with the exception of the Arg-67 and Lys-70 mutants, all other mutants activated PC with similar maximum rate constants in the presence of a saturating concentration of TM and Ca 2؉ , although their affinity for interaction with TM was markedly impaired. The catalytic properties of the Arg-35 mutant were changed so that PC activation by the mutant no longer required Ca 2؉ in the presence of TM, but, instead, it was accelerated by EDTA. Moreover, the activity of this mutant toward PC was improved Ϸ25-fold independent of TM. These results suggest that Arg-35 is responsible for the Ca 2؉ dependence of PC activation by the thrombin-TM complex and that a function for TM in the activation complex is the allosteric alleviation of the inhibitory interaction of Arg-35 with the substrate. P rotein C (PC) is a multidomain vitamin K-dependent plasma serine protease zymogen that, on activation by thrombin in complex with thrombomodulin (TM), down-regulates the coagulation cascade by inactivating factors Va and VIIIa by limited proteolysis (1-3). Similar to the structures of other vitamin K-dependent coagulation proteases, the structure of PC consists of an amino-terminal ␥-carboxyglutamic residue (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and a C-terminal catalytic domain with a trypsin-like primary specificity pocket (4, 5). The Gla domain contains several cooperative low-affinity Ca 2ϩ -binding sites, which mediate the metal ion-dependent interaction of PC with endothelial protein C receptor and the thrombin-TM complex on membrane surfaces (1, 6). In addition to the Gla domain, the amino-terminal EGF domain and the catalytic domain of PC each have a high-affinity Ca 2ϩ -binding site with important roles for the structure and function of the protein (4, 7). The Ca 2ϩ -binding site on the protease domain of PC is located on the 70-80 loop (7). With the exception of prothrombin, the same loop is also known to bind Ca 2ϩ in other vitamin K-dependent coagulation proteases (8, 9) and in trypsin (10). The binding of Ca 2ϩ to this loop plays a complex role in PC activation by thrombin: it is inhibitory for activation by thrombin alone but stimulatory for activation by the thrombin-TM complex (11). The Ca 2ϩ -binding site in the amino-terminal domain does not appear to play a role in PC activation ...

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Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII - tissue factor interaction

Cited by 62 publications

References 41 publications

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

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

Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin

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