Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Characterization of canine coagulation factor VII and its complex formation with tissue factor: canine-human cross-species compatibility.

BACKGROUND: Canine models have been good predictors of efficacy of hemophilia treatments, including recombinant human coagulation factor (F)VIIa (hFVIIa). However, canine FVIIa and tissue factor (TF) have remained incompletely characterized. OBJECTIVE: To explore canine-human cross-species FVIIa-TF compatibility in order to strengthen the predictive value of canine models in research on FVIIa and TF. METHODS: Canine FVIIa (cFVIIa) and canine TF((1-217)) [cTF((1-217))] were produced by recombinant techniques, and canine-human cross-species FVIIa-TF interactions were characterized in vitro. RESULTS: Recombinant cFVIIa and soluble cTF((1-217)) were produced and purified to homogeneity. hFVIIa and cFVIIa bound with comparably high affinities to cTF((1-217)) (K(D)=6.0±0.7 nm and K(D)=6.0±0.3 nm, respectively) and to cell surface-expressed cTF (K(D)=8.4±0.4 nm and K(D)=7.2±1.2 nm, for (125) I-labeled hFVIIa and cFVII, respectively). In contrast, cFVIIa bound to human TF (hTF) with decreased affinity, both in solution and on cell surfaces. The decreased binding resulted in reduced activity of cFVIIa in functional assays with hTF((1-209)) . In direct comparison, cFVIIa was more active than hFVIIa, both in the absence and the presence of cognate TF. CONCLUSION: The present finding that hFVIIa binds to cTF essentially as it does to hTF substantiates the hypothesis that human FVIIa-TF biology can be reliably recapitulated in canine models on administration of hFVIIa to dogs.

Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice.

BACKGROUND: Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. OBJECTIVES: To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. METHODS: We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. RESULTS: ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IkappaBalpha upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-kappaB (NF-kappaB) mediated cell signaling. Levels of nuclear activated NF-kappaB and proteins influenced by the NF-kappaB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). CONCLUSIONS: We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-kappaB mechanism.

Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells.

BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.

Factor VIIa binding and internalization in hepatocytes.

The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization of FVIIa in liver cells using a human hepatoma cell line (HEPG2), and primary rat and human hepatocytes as cell models. 125I-FVIIa bound to HEPG2 cells in a time- and dose-dependent manner. Anti-tissue factor antibodies reduced the binding by about 25%, whereas 50-fold molar excess of unlabeled FVIIa had no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2 cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes. In contrast, annexin V, which binds to phosphatidylserine, blocked the binding and internalization. Consistent with this, binding of gla-domain-deleted FVIIa to hepatocytes was markedly diminished. In summary, the data presented herein reveal differences between HEPG2 cells and primary liver cells in FVIIa binding and internalization, and suggest that the rapid turnover of membrane and not a receptor-mediated endocytosis may be responsible for internalization of FVII/FVIIa in primary hepatocytes.

Active site-inhibited seven: mechanism of action including signal transduction.

Vascular injury brings tissue factor (TF) into contact with blood and its natural ligands, factors VII (FVII) and VIIa (FVIIa). This results in localized FVIIa activity on TF-expressing cells, initiating coagulation, and nonhemostatic activities. Activation of transcription factors, expression of genes for inflammation, tissue remodeling, and wound healing follow, but these mechanisms for maintaining vascular integrity may lead to pathophysiologic states. Recombinant FVIIa is converted into a catalytically inert protein by reactive site residues reacting with Phe-Phe-Arg-chloromethyl ketone. Active site-inhibited FVIIa (ASIS) retains its affinity for TF and competes for FVIIa and FVII binding to TF, blocking FVIIa activity and FVII to FVIIa activation. It therefore acts as an antithrombotic agent and has been shown in previous studies on animal models of sepsis to prevent organ failure associated with fibrin deposition. Mitigation of inflammatory response and prolonged survival were remarkable and additional effects of TF blockage by ASIS not observed with inhibitors of downstream coagulation factors Xa and thrombin. This suggests that FVIIa/TF exerts a noncoagulopathic effect on cellular activities, attenuated by ASIS blocking FVIIa-induced signaling. The precise mechanism remains elusive but blockade of TF/FVIIa activity provides an attractive possibility for pharmaceutical intervention. In vitro measurements of ASIS-TF binding and FVIIa/TF inhibition are described, together with investigation of the FVIIa-induced signaling pathway and gene expression. Additionally, possible implications of ASIS blockage for hemostatic and nonhemostatic aspects of the pathophysiology associated with vascular stress and injury are discussed.

Privacy rules under the Gramm-Leach-Bliley Act and HIPAA.

The intense scrutiny given to the privacy implications of the Gramm-Leach-Bliley Act and the Health Insurance Portability and Accountability Act has led to much confusion regarding which applies to specific entities. The authors attempt to clarify when these Acts would define how confidential medical data are used.

Tissue factor-mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor.

Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface-bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.

More porous fibrin gel structure obtained by interaction with Lys-plasminogen than with Glu-plasminogen.

The effect of Glu1- and Lys78-plasminogen on the assembly and structure of fibrin gels was studied in purified fibrinogen-thrombin system and in plasminogen-free plasma, using turbidity, liquid permeation and three-dimensional (3D) confocal laser microscopy methods. In the purified fibrinogen system using the turbidity method, the final optical density of the fibrin gels increased with increasing concentrations of Lys-plasminogen. The fiber mass/length ratio mu increased with increasing concentrations of both Glu1- and Lys78-plasminogen, the effect of Lys78-plasminogen being much stronger. The permeability coefficient (Ks) analyzed with the permeation method revealed that fibrin gels formed in the presence of Lys78-plasminogen were more permeable (porous) than the control gels. The effect on the gel structure was inhibited by the fibrinolytic inhibitor epsilon-aminocaproic acid. The same results were obtained in plasma milieu for both mu and Ks as in the purified system, i.e. the gels became more porous with increasing concentrations of Lys78-plasminogen. 3D microscopy pictures of the gels verified the findings.

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor.

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.

Exclusion of known protease-activated receptors in factor VIIa-induced signal transduction.

The protease activity is mandatory for intracellular activities induced by coagulation factor VIIa (FVIIa), and in this way it resembles signal transduction induced by thrombin and trypsin caused by specific, proteolytic cleavage of protease activated receptors (PARs). The mechanism for FVIIa-induced signal transduction is, however, not known although a mechanism involving PAR cleavage has been deduced from studies of cytosolic Ca2+ release and p44/p42 mitogen activated protein kinase (MAPK) activation. In the present work we have examined the possibilities that i) FVIIa-induced signal transduction involves the activation of one of the four known PARs, or ii) exposure of cells to FVIIa releases a soluble ligand that is responsible for MAPK activation. For this purpose, we evaluated the effects of FVIIa, thrombin, FXa, trypsin and PAR agonist peptides on the Ca2+ release and MAPK activation in tissue factor-(TF) transfected baby hamster kidney (BHK[+TF]) cells and Madin-Darby canine kidney (MDCK) cells. FVIIa induced a significant MAPK signal in BHK(+TF) cells and in MDCK-I and -II cells whereas no MAPK activation was observed with thrombin, FXa or PAR agonist peptides. Thrombin, trypsin, PAR-1 and PAR-2 agonist peptides induced a prominent Ca2+ response in both cell types. In contrast the cells did not respond with a detectable Ca2+ signal when treated with FVIIa. These results suggest that the intracellular activity induced by FVIIa is distinctly different from that induced by trypsin, thrombin and FXa not involving any of the known PARs. Conditioned medium from BHK(+TF) cells treated with FVIIa failed to induce a MAPK response in untreated BHK(+TF) cells when FVIIa was removed by immunoadsorption from the medium prior to its transfer to the untreated BHK(+TF) cells. Although it is not possible entirely to exclude a transient response close to the cell surface, the data suggest that the intracellular response was not induced by an autocrine release of a soluble mediator to the medium.

Tissue factor-dependent factor VIIa signaling.

Tissue factor (TF) is known primary as a cofactor for factor VIIa-mediated triggering of blood coagulation, which proceeds in a cascade of extracellular reactions. Recent investigations have, however, revealed that intracellular activities can also be induced by the proteolytic activity of factor VIIa bound to cell surface TF. Factor VIIa signal transduction has thus been reported to induce mobilization of intracellular Ca(2+) stores and p44/p42 MAPK phosphorylation and to result in expression of specific genes, which presumably affects a number of cellular functions. The factor VIIa-induced signal transduction is independent of the presence of the TF cytoplasmic domain and it is distinctly different from signaling involving presently known protease-activated receptors (PARs) including receptors for thrombin and factor Xa. This short review summarizes recent advances in our understanding of TF-dependent factor VIIa signaling.

Discordant expression of tissue factor and its activity in polarized epithelial cells. Asymmetry in anionic phospholipid availability as a possible explanation.

Recent studies have shown a discrepancy between the level of tissue factor (TF) expression and the level of TF procoagulant activity on the apical and basolateral surface domains of polarized epithelial cells. The present investigation was performed to elucidate possible reasons for the discordant expression of TF and its activity on the surface of polarized epithelial cells using a human intestinal epithelial cell line, Caco-2 and Madin-Darby canine kidney epithelial cells, type II (MDCK-II). Functional activity of coagulation factor VIIa (VIIa) in complex with TF was 6- to 7-fold higher on the apical than the basolateral surface in polarized Caco-2 cells. In contrast, no significant difference was found in the formation of TF/VIIa complexes between the apical and basolateral surface. Confocal microscopy of Caco-2 cells showed TF expression on both the apical and the basolateral surface domains. Studies with MDCK-II cells showed that the specific functional activity of TF expressed on the apical cell surface was 5-fold higher than on the basolateral surface. To test whether differential expression of TF pathway inhibitor (TFPI) on the apical and basolateral surface could account for differences in TF/VIIa functional activity, we measured cell-surface-bound TFPI activity in Caco-2 cells. Small but similar amounts of TFPI were found on both surfaces. Further, addition of inhibitory anti-TFPI antibodies induced a similar enhancement of TF/VIIa activity on both surface domains. Because the availability of anionic phospholipids on the outer leaflet of the cell membrane could regulate TF/VIIa functional activity, we measured the distribution of anionic phospholipids on the apical and basolateral surface by annexin V binding and thrombin generation. The results showed that the anionic phospholipid content on the basolateral surface, compared with the apical surface, was 3- to 4-fold lower. Mild acid treatment of polarized Caco-2 cells, which markedly increased the anionic phospholipid content on the basolateral surface membrane, increased the TF/VIIa activity on the basolateral surface without affecting the number of TF/VIIa complexes formed on the surface. Overall, our data suggest that an uneven expression of TF/VIIa activity between the apical and basolateral surface of polarized epithelial cells is caused by differences in anionic phospholipid content between the two surface domains and not from a polar distribution of TFPI.

Aprotinin attenuates platelet accumulation in ischaemia-reperfusion-injured porcine skeletal muscle.

This purpose of this study was to evaluate the effect of aprotinin, a serine protease inhibitor, in ischaemia- and reperfusion-injured myocutaneous flaps and skin flaps. Flap survival, microcirculatory platelet accumulation, and regional blood flow were investigated in seventeen pigs which had been subjected to 8 h of ischaemia and 18 h of reperfusion. The pigs were randomly assigned to aprotinin treatment (n = 9) or saline (n = 8). In-vitro studies were performed to investigate the influence of aprotinin on the activated partial thromboplastin time. The survival of skeletal muscle correlated positively with the concentration of aprotinin (P = 0.02) and could not be explained by regional changes in blood flow. Platelet accumulation was decreased in aprotinin-treated muscle (P = 0.04). In-vitro (n = 10), 100 kallikrein inactivator units/ml aprotinin prolonged the activated partial thromboplastin time both in plasma (P = 0.001) and in blood (P = 0.002), suggesting an anticoagulant rather than a procoagulant effect. In conclusion, aprotinin at high concentrations may be beneficial for the survival of skeletal muscle and provides protection from platelet accumulation in the microcirculation of skeletal muscle exposed to ischaemia and reperfusion injury.

Factor VIIa-induced p44/42 mitogen-activated protein kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain.

Signal transduction induced by activated factor VII (FVIIa) was studied with baby hamster kidney (BHK) cells transfected with human tissue factor (TF). FVIIa induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in cells expressing TF, BHK(+TF), but not in wild-type BHK(-TF) cells. BHK(+TF) cells responded to FVIIa in a dose-dependent manner, with detectable phosphorylation above 10-20 nM FVIIa. BHK cells transfected with a cytoplasmic domain-deleted version of TF, (des248-263)TF, or a C245S substitution variant of TF also supported FVIIa-induced MAPK activation. Experiments with active site-inhibited FVIIa, thrombin, factor Xa, and hirudin confirmed that the catalytic activity of FVIIa was mandatory for p44/42 MAPK activation. Furthermore, a high concentration of FVIIa in complex with soluble TF induced p44/42 MAPK phosphorylation in BHK(-TF) cells. These data suggest that TF was not directly involved in FVIIa-induced p44/42 MAPK phosphorylation but rather served to localize the action of FVIIa to the cell surface, potentially to cleave a cell surface receptor. Desensitization experiments with sequential addition of proteases suggested that the p44/42 MAPK response induced by FVIIa was distinctly different from the thrombin response, possibly involving a novel member of the protease-activated receptor family.

Inhibitory properties of human recombinant Arg24-->Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2).

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.

Thermal effects on an enzymatically latent conformation of coagulation factor VIIa.

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.

Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation.

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.

13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues.

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.