Cofactor Tissue Factor Research Articles

EPCR Deficiency Prevents Development of Hemophilic Arthropathy

Recent studies from our laboratory showed that clotting factor VIIa (FVIIa), whose primary function is to initiate the coagulation cascade following its binding to procoagulant cofactor tissue factor (TF), also binds anticoagulant cofactor, endothelial cell protein C receptor (EPCR). EPCR plays a key role in the activated protein C (APC)-mediated anticoagulant pathway by promoting the activation of protein C bound to it. Pharmacological concentrations of rFVIIa could compete with protein C for the EPCR and downregulate APC generation by preventing protein C binding to the EPCR. Our recent studies showed that FVIIa binding to EPCR also induces barrier-protective and anti-inflammatory effects both in vitro and in vivo model systems. Bio-distribution of rFVIIa in mice revealed that a small fraction of rFVIIa administed to mice i.v. accumulates in knee joints and retained there for three to seven days. The present study was conducted to investigate the relevance of FVIIa binding to EPCR in pharmacological FVIIa treatment of joint disease in hemophilia. For this, we first generated hemophilia A mice lacking EPCR or overexpressing EPCR. To generate EPCR deficient FVIII-/- mice, Procrflox/flox and Procr+/floxMeox2+/cre mice were first backcrossed with FVIII-/- mice to generate Procrflox/flox/FVIII-/- and Meox2+/cre/FVIII-/- mice. EPCR deficient FVIII-/- mice (Procr-/-/FVIII-/-) were generated by breeding female Procrflox/flox/FVIII-/- with male Procr+/floxMeox2+/cre /FVIII-/- mice. EPCR- overexpressing FVIII-/- were generated by crossing Tie2-EPCR mice with FVIII-/- mice. We induced joint bleeding in FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice by needle puncture injury. Right knee joint intra articular space was pierced with 30G needle to induce joint bleeding; left knee joint served as an uninjured control. Injured mice were treated with a single dose of rFVIIa (1 mg/kg) at 20 min following the injury or three doses of rFVIIa (1 mg/Kg) at 20 min, 24 h and 72 h following the injury. In controls, saline was administered in place of rFVIIa. The knee injury was evaluated by measuring knee joint diameter and visual bleeding score. At the end of 14 days post injury, mice were euthanized, and the knee joints were excised and fixed for immunohistochemical analysis. Hemophilic arthropathy was evaluated by scoring a set of pathological parameters (e.g., synovial hyperplasia, neoangiogenesis, presence of blood, iron score, villus formation, and cartilage degeneration). A subset of mice was used to collect synovial fluid at 7 days post injury. Assessment of joint bleeding at 5 h following the needle injury, by extracting and measuring hemoglobin levels in joint tissues and monitoring hematocrit, showed an equal amount of bleeding in all three genotypes of mice (FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice). Administration of three doses of FVIIa was effective in preventing hemophilic arthropathy in FVIII-/- and EPCR-overexpressing FVIII-/- mice, whereas a single dose of rFVIIa had a minimal effect in reducing hemophilic arthropathy. Interestingly, a single dose of FVIIa fully corrected the needle injury-induced hemophilic arthropathy in EPCR-deficient FVIII-/- mice. More importantly, EPCR-deficient FVIII-/- mice failed to develop full-blown hemophiic arthropathy even in the absence of any treatment. Measurement of inflammatory cytokines in the synovial fluid showed a robust increase in IL-6 levels in FVIII-/- mice and a single dose of FVIIa substantially reduced IL-6 levels. Interestingly, IL-6 levels in the synovial fluid of injured EPCR-overexpressing FVIII-/- mice were markedly higher compared to injured FVIII-/- mice, three doses of rFVIIa was required to attain a significant reduction in IL-6 levels in these mice. In contrast to these data, we found very little IL-6 levels in the synovial fluids of injured EPCR-deficient FVIII-/- mice. Overall, these data indicate that EPCR levels profoundly influence hemophilic arthorpathy. EPCR-deficiency protects from development hemophilic arthropathy because down-regulation of APC generation in EPCR deficiency could allow sufficient thrombin generation in hemophilia to prevent joint bleeding and bleeding-associated inflammation. Disclosures Rao: Takeda: Research Funding.

Maturation of coagulation factor IX during Xase formation as deduced using factor VIII-derived peptides.

Blood coagulation involves extrinsic and intrinsic pathways, which merge at the activation step of blood coagulation factor X to factor Xa. This step is catalysed by the extrinsic or intrinsic Xase, which consists of a complex of factor VIIa and its cofactor tissue factor or factor IXa (FIXa) and its cofactor coagulation factor VIIIa (FVIIIa). Upon complex formation with FVIIIa, FIXa is conformationally activated to the Xase complex. However, the mechanistic understanding of this molecular recognition is limited. Here, we examined FVIIIa‐FIXa binding in the context of FIXa's activation status. Given the complexity and the labile nature of FVIIIa, we decided to employ two FVIII‐derived peptides (558‐loop, a2 peptide) to model the cofactor binding of FIX(a) using biosensor chip technology. These two FVIII peptides are known to mediate the key interactions between FVIIIa and FIXa. We found both of these cofactor mimetics as well as full‐length FVIIIa bind more tightly to zymogenic FIX than to proteolytically activated FIXa. Consequently and surprisingly, we observed that the catalytically inactive FIX zymogen can outcompete the activated FIXa from the complex with FVIIIa, resulting in an inactive, zymogenic Xase complex. By contrast, the thrombophilic Padua mutant FIXa‐R170 in complex with the protein–substrate analogue BPTI bound tighter to FVIIIa than to the zymogen form FIX‐R170L, suggesting that the active Xase complex preferentially forms in the Padua variant. Together, these results provide a mechanistic basis for the thrombophilic nature of the FIX‐R170L mutant and suggest the existence of a newly discovered safety measure within the coagulation cascade.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is “locked” into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

4‐Hydroxy‐2‐Nonenal (HNE), a product of Lipid Peroxidation, Induces Tissue Factor Decryption by modulating Thioredoxin system and mitochondrial ROS generation independently

Oxidative stress plays a major role in the pathogenesis of various diseases such as atherosclerosis. Aberrant expression of a coagulation cofactor tissue factor (TF) is thought to contribute to the pathogenesis of atherosclerosis. Recently, we found that 4‐hydroxy‐2‐nonenal (HNE), a major aldehyde produced by oxidation of ω‐6 polyunsaturated fatty acids, increases the procoagulant activity of cell surface tissue factor (TF). These studies revealed that HNE generates ROS and decrypts TF through p38 MAPK‐dependent externalization of phosphatidylserine (PS). The present study is carried out to elucidate the detailed molecular mechanism involved in HNE‐induced TF decryption. Colocalization of ROS generation and mitochondrial staining indicated that mitochondria are the major source of HNE‐induced ROS generation. Inhibitors of the mitochondrial electron transport chain (ETC) complexes III and V significantly inhibited the HNE‐induced ROS generation. These inhibitors also attenuated the HNE‐induced PS externalization and TF activation, indicating that HNE‐induced ROS generation contributes to PS exposure critical for TF decryption. Interestingly, inhibition of ROS by these inhibitors did not block the HNE‐induced p38 MAPK activation. Additional studies showed that HNE inhibits thioredoxin (Trx) and thioredoxin reductase (TrxR) activities in a dose‐ and time‐dependent manner. Antioxidant N‐acetylcysteine (NAC) prevented the HNE‐induced inhibition of TrxR but the inhibitors of mitochondrial ETC that prevented HNE‐induced ROS generation failed to block the HNE‐induced TrxR inhibition. To investigate whether HNE‐induced inhibition of Trx or TrxR is responsible for HNE‐induced TF decryption, we determined whether inhibition of the activity of Trx or TrxR by their pharmacological inhibitors would also activate TF. Treatment of the cells with either curcumin, Auranofin or PMX‐464 (TrxR inhibitors) or PX‐12 (Trx inhibitor) markedly increased TF activity in a PS‐dependent manner. Inhibition of the TrxR/Trx also activated MKK3/6 and p38 MAPK as observed with HNE. Active Trx binds ASK1 and inactivation of Trx disrupts the complex, enabling ASK1 activation that leads to p38 MAPK activation. Therefore, we investigated whether HNE‐induced inactivation of Trx leads to ASK1 activation that could lead to p38 MAPK activation. Our data show HNE neither induces ASK1 activation nor ASK1 inhibitors block HNE‐induced TF decryption, suggesting that p38 MAPK activation is independent of ASK1. In additional experiments, we found that blockade of thiol groups by phenylarsine oxide (PAO) attenuated HNE‐induced PS exposure and TF decryption. In summary, our present data suggest that HNE induces TF decryption by two separate pathways – one is ROS‐dependent but independent of p38 MAPK activation and the second is via Trx‐TrxR‐ and p38 MAPK‐dependent. Both the mechanisms result in the exposure of PS at the cell surface that contributes to TF decryption. Blockade of thiol groups by PAO appears to inhibit HNE‐induced TF decryption by blocking the upstream signaling molecules that regulate HNE‐induced PS exposure.

Understanding the Dynamics of Full-Length Tissue Factor (TF1-263) in the Context of Allosteric Modulation of Factor VIIa : A Molecular Dynamics Simulation Approach

Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular injury or endothelial cell rupture, factor VII (FVII) which is present normally in flowing blood as zymogenic form, upon binding with its cofactor TF (present beneath endothelial cell), get converted into its activated form, FVIIa; makes TF-FVIIa protease complex for the conversion of downstream protease members into activated form. Upon successful transformation of FX to FXa, prothrombin to thrombin, soluble fibrinogen get converted into its insoluble form fibrin, which accumulates at the site of vascular injury in the presence of platelets, resulting into the blood clot.Recent studies have shown that TF is solely responsible for the structural modulation of FVIIa. However, due to lack of well-resolved crystal structure of the zymogenic form of FVII and full-length TF-FVIIa binary complex, the actual allosteric mechanism induced by TF and structural changes observed in FVIIa is not fully understood at atomic resolution. Molecular dynamic simulation is the only way to visual the potential interaction between protein-protein and protein-lipid at atomistic resolution. Here we did the simulation of complex and free FVIIa to understand the causative effect of TF in terms of allosteric modulation occurred in FVIIa. The present study was carried out to investigate the domain-wise structural modulation of FVIIa in the presence of full-length TF. We have done the comparative study among full-length (FL) TF1-263-FVIIa, sTF1-219-FVIIa and free FVIIa. Measurements of intrinsic dynamic changes clearly suggest that TF not only made changes in the allosteric modulation of FVIIa but inter as well intradomain communication made between FLTF and FVIIa is also responsible for the structural modulation in the protease domain of FVIIa. Among interdomain communication, two interactions between EGF2 domain and FLTF (Glu94-Ser47 and Asp104-Thr51) guide the potential effect of TF in the structural modulation of FVIIa. Examining the catalytic triad localization reveals that single as well as double interaction regulates the dynamism of protease part in substrate recognition and substrate catalysis, however in the case of no interaction the movement of CT is much higher and in a wide range. In a case of sTF-FVIIa complex, few inter-domain communications ware missing, which causes reduced affinity of sTF towards FVIIa and thus sTF-FVIIa shows less activity compare to FLTF. We proposed the hypothesis that binding of FLTF not only changes the allosteric alteration of SP domain but also induces the structural alteration in each domain of FVIIa, and these alterations in combination contribute to the enhancement of FLTF-FVIIa activity in which two interactions may be the controller for such dynamic changes in FVIIa. In summary, our study provides valuable information to understand the blood coagulation more intrinsically. DisclosuresNo relevant conflicts of interest to declare.

Matriptase activation connects tissue factor–dependent coagulation initiation to epithelial proteolysis and signaling

AbstractThe coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry

Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

Antibody-induced Enhancement of Factor VIIa Activity through Distinct Allosteric Pathways

In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the K(m) and k(cat), the other class mainly affected the K(m). The antibody-induced activity enhancement could be traced to maturation of the S1 substrate binding pocket and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole, and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms.

Allosteric activation of coagulation factor VIIa

Coagulation factor VIIa (FVIIa) is present at subnanomolar concentration and represents a small percentage of the total amount of FVII in the circulation. FVIIa is poised to initiate blood clotting when it encounters its pivotal cofactor tissue factor (TF) which becomes exposed to blood upon vascular rupture. The requirement for complex formation with TF in order for FVIIa to express procoagulant activity ensures thrombin and fibrin generation at the right time and place. Thus TF acts as a guardian of safety of paramount importance to blood coagulation by providing localization to the site of injury and at the same time inducing maturation of zymogen-like free FVIIa to the active cofactor-bound enzyme. This review gives an account of the accumulated knowledge about the structure, function and TF dependence of FVIIa to arrive at a plausible allosteric mechanism by which TF induces maturation of the active conformation of FVIIa.

Cofactor-induced and mutational activity enhancement of coagulation factor VIIa.

Coagulation factor VIIa (FVIIa) is an atypical member of the trypsin family of serine proteases. It fails to attain spontaneously its catalytically competent conformation and requires its protein cofactor tissue factor (TF) to accomplish this. Over a number of years, this unique behaviour of FVIIa has prompted investigations of the TF-induced activation mechanism and the zymogenicity determinants in factor VIIa. Factor VIIa has gained additional interest in the past decade because of its development into a clinically useful haemostatic agent. Here, we present an overview of the current knowledge about the TF-induced allosteric activation of FVIIa and the various molecular approaches to enhance the intrinsic activity and efficacy of FVIIa.

Activity and Regulation of Long-Acting Factor VIIa Analogs.

Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilia patients with inhibitors. However, rFVIIa is cleared from the circulation relatively fast, with circulating half-life of about 2–4 h, requiring repeated administration of rFVIIa for the effective treatment. Therefore, development of FVIIa analogs that could remain in the circulation for a longer period of time would be of a great value for improving the treatment options of rFVIIa. e.g., by prophylaxis. PEGylation of plasma proteins was shown to extend their circulatory half-lives but the PEGylation may also disrupt macromolecular interactions. In the present study we characterized the interaction of two glycoPEGylated analogs of rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, with its cofactor tissue factor (TF), substrate factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Both the PEGylated FVIIa analogs exhibited similar amidolytic activity as of wild-type rFVIIa (wt-rFVIIa) in the absence or presence of relipidated TF. The analogs were as effective as wt-rFVIIa in activating FX in the absence of TF. No significant differences were found between the PEGylated rFVIIa analogs and wt-rFVIIa in TF-dependent FX activation at saturating concentrations of rFVIIa, however, at lower concentrations of rFVIIa (10 to 50 pM), rFVIIa-10K PEG and rFVIIa-40K PEG activated FX at a slightly lower rate, 50% and 75%, respectively, of wt-rFVIIa. Further studies revealed that both AT/heparin and TFPI inhibited the PEGylated rFVIIa-TF complexes effectively but slightly at a lower rate compared to that was noted for wt-rFVIIa-TF. TFPI-Xa inhibited the PEGylated rFVIIa-TF and wt-rFVIIa-TF at a similar rate. On unperturbed HUVEC, wt-FVIIa (10 nM) could activate FX, albeit slowly, (1.7 nM/h) and the PEGylated rFVIIa activated FX even at much lower rates (0.23 nM/h for rFVIIa-10K PEG and 0.15 nM/h for rFVIIa-40K PEG). On stimulated HUVEC expressing TF, the PEGylated rFVIIa variants were slightly less effective at lower concentrations compared to wt-rFVIIa in activating FX, but no significant differences were found among them in activating factor X at saturating concentrations of rFVIIa (80–100 nM/h). The PEGylated rFVIIa analogs bound to cell surface TF were inhibited by TFPI-Xa complex at a similar rate as that was observed for wt-rFVIIa (IC50 in nM: 0.102 ± 0.032 for wt-rFVIIa, 0.111 ± 0.024 for rFVIIa-10K PEG, and 0.096 ± 0.019 for rFVIIa-40K PEG). AT/heparin inhibited rFVIIa-10K PEG bound to endothelial cell TF at a similar rate as it inhibited wt-rFVIIa (IC50 in μg/ml: wt-rFVIIa, 3.42 ± 068; rFVIIa-10K PEG, 3.56 ± 0.073), but the inhibition rate was slightly lower for rFVIIa-40K PEG bound to TF (IC50 5.92 ± 0.44 μg/ml). Overall, our present data suggest that long-acting PEGylated FVIIa analogs retain full enzymatic activity and can interact TF and FX effectively, and are inhibited by AT/heparin and TFPI-Xa as for wt-rFVIIa. Although the pegylated rFVIIa variants exhibited somewhat lower affinity towards TF, this may not critically affect the TF-driven FXa generation. Further work is needed to fully characterize these molecules.

Cryptic messages: Is noncoagulant tissue factor reserved for cell signaling?

Tissue factor (TF) is the principal initiator of blood clotting in response to vascular injury and in thrombotic disease (1). TF, an integral membrane protein, is normally excluded from the vascular compartment. Coagulation is initiated when TF is exposed to zymogen coagulation proteases in plasma upon vascular damage. TF first binds factor VII, supporting both its activation to VIIa and activation of factor X (Fig. 1). Factor Xa acts with its cellular cofactor Va to generate thrombin (IIa), which drives both fibrin deposition and platelet aggregation. Thrombin elicits signaling in cells through protease-activated receptors (PARs), a family of G protein-coupled receptors, which are activated proteolytically by unmasking of an N-terminal tethered ligand (Fig. 1) (2). In addition to hemostasis, TF expression and coagulant protease activation have been implicated in inflammation, vascular development, and cancer progression. Signaling by coagulant proteases is an important consequence of TF exposure in these settings. Factors VIIa and Xa have been shown to cleave and activate PAR2, the only PAR not activated by thrombin, and may act in concert with thrombin to elicit cellular responses to coagulation activation (Fig. 1). VIIa requires its cofactor TF to facilitate PAR2 cleavage and activation. The molecular determinants that specify TF function as a cofactor for cellular signaling versus coagulation are not known. In a recent issue of PNAS, Ahamed et al. (3) provide fascinating insight suggesting that coagulant and signaling TF may, in fact, be two different structural entities, that signaling TF may be the long-elusive encrypted TF, and that disulfide isomerization facilitates a dynamic and reversible switch between these two distinct functional TF species.

Disulfide locked variants of factor VIIa with a restricted β‐strand conformation have enhanced enzymatic activity

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Crystal Structures of Uninhibited Factor VIIa Link its Cofactor and Substrate-assisted Activation to Specific Interactions

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69 Å crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder–order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.

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.

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates.

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa-TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa-TF complex, but also in the interaction of FVII with both its activators and cofactor TF.

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa—TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa—TF complex, but also in the interaction of FVII with both its activators and cofactor TF.

Role of residue Phe225 in the cofactor-mediated, allosteric regulation of the serine protease coagulation factor VIIa.

Functional regulation by cofactors is fundamentally important for the highly ordered, consecutive activation of the coagulation cascade. The initiating protease of the coagulation system, factor VIIa (VIIa), retains zymogen-like features after proteolytic cleavage of the activating Arg(15)-Ile(16) peptide bond and requires the binding of the cofactor tissue factor (TF) to stabilize the protease domain in an active enzyme conformation. Structural comparison of TF-bound and free VIIa failed to provide a conclusive mechanism for this catalytic activation. This study provides novel insight into the cofactor-dependent regulation of VIIa by demonstrating that the side chain of Phe(225), an aromatic residue that is common to allosterically regulated serine proteases, is necessary for optimal TF-mediated activation of VIIa's catalytic function. However, mutation of Phe(225) did not abolish the cofactor-induced stabilization of the Ile(16)-Asp(194) salt bridge, previously considered the primary switch mechanism for activating VIIa. Moreover, mutation of other residue side chains in the VIIa protease domain resulted in a reduced level of or no stabilization of the amino-terminal insertion site upon TF binding, with little or no effect on the TF-mediated enhancement of catalysis. This study thus establishes a crucial role for the aromatic Phe(225) residue position in the allosteric network that transmits the activating switch from the cofactor interface to the catalytic cleft, providing insight into the highly specific conformational linkages that regulate serine protease function.