Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.     

An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates.

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8-11D12 and TF9-9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9-9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8-11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8-11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8-11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.     

Novel anticoagulant activity of polybrene: inhibition of monocytic tissue factor hypercoagulation following bacterial endotoxin induction.

The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear. Nor is the effective anticoagulation well established. The search for arresting hypercoagulation is of antithrombotic relevance. The ability of polybrene (PB) to inhibit tissue factor (TF)-initiated extrinsic blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma samples. In a single-stage clotting assay, PB dose-dependently offset bacterial endotoxin (lipopolysaccharide)-induced monocytic TF (mTF) hypercoagulation and inhibited rabbit brain thromboplastin (rbTF) procoagulation. Consistent with these findings, the significantly prolonged prothrombin time indicated the depressed extrinsic coagulation by PB. However, PB showed no effect on thrombin time. We dissected the extrinsic pathway to further determine the inhibitory site(s) of PB. A two-stage chromogenic assay monitoring S-2288 hydrolysis showed that PB readily blocked mTF-dependent or rbTF-dependent FVII activation, which was verified by the diminished activated factor VII (FVIIa) formation derived from the proteolytic cleavage of its zymogen factor VII on Western blotting analyses. PB had no effect on FVIIa and activated factor X amidolytic activity. Nor was the dissected TF/FVIIa-catalyzed factor X activation affected. In conclusion, the preferential downregulation of factor VII activation was responsible for the depressed extrinsic coagulation. PB could present a novel anticoagulant antagonizing the extrinsic hypercoagulation for the prevention of thrombotic complication following sepsis and inflammations.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex

Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.     

New insights into how blood clots: implications for the use of APTT and PT as coagulation screening tests and in monitoring of anticoagulant therapy.

Blood coagulation occurs efficiently on cell surfaces such as activated platelets and monocytes, and fibroblasts. It is initiated by limited amounts of tissue factor (TF) exposed at the sites of vascular injury that complexes with trace amounts of circulating factor VIIa (FVIIa). Additional FVIIa-TF complexes are formed from FVII-TF involving positive feedback loops, including FVIIa-TF as well as factors Xa and IXa as they are formed in subsequent steps. For sustained normal coagulation to proceed, effective in vivo activation of factor X requires the participation of factor IXa generated via the FVIIa-TF complex. This may, in part, be due to effective inhibition of factor Xa and FVIIa-TF complex by tissue factor pathway inhibitor that results in blockage of direct activation of factor X by the FVIIa-TF complex. Additional generation of factor Xa at injury sites may then proceed via the FIXa-VIIIa pathway. Thrombin generated from prothrombin via complex formation of prothrombin with FXa and FVa on phospholipid surfaces (prothrombinase complex) powerfully accelerates coagulation by activation of FVIII and FV, and sustains coagulation through activation of FXI. Thus, in light of our current understanding of how blood clots in vivo, it is clear that both prothrombin time (PT) and activated partial thromboplastin time (APTT) are highly artificial in vitro systems with major limitations. Nevertheless, these tests are quite useful as global screening tests for abnormalities in the intrinsic or extrinsic, as well as common, pathways of coagulation and for monitoring of anticoagulant therapy.     

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TF-induced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.     

Relevance of tissue factor in cardiovascular disease

Overexpression and exposition of tissue factor (TF) in atherosclerotic plaques and/or arterial thrombi are critical events in atherothrombosis. TF, the receptor for factor VII (FVII) and activated factor VII (FVIIa), is the principal initiator of blood coagulation and induces thrombin generation leading to fibrin formation and platelet activation. TF also plays a major role in cell migration and angiogenesis. TF activity is downregulated by Tissue Factor Pathway Inhibitor (TFPI), a Kunitz-type inhibitor, which forms a neutralizing complex with TF, FVIIa and activated factor X. In physiological conditions, TF is absent from vascular cells which come into contact with flowing blood and is present as an inactive pool in fibroblasts and smooth muscle cells (SMC). In contrast, TF is widely expressed in atherosclerotic plaques and is found in macrophages, SMCs, and foam-cells and also in extracellular matrix and acellular lipid-rich core. TF expression is up-regulated by inflammatory cytokines and oxidized lipids. Plaque thrombogenicity is directly correlated to their TF content. After fibrous cap disruption, TF is exposed on plaque surface and triggers thrombus formation leading to arterial lumen occlusion and/or downstream embolization. In coronary and carotid plaques, TF content was found to be higher in plaques from symptomatic than asymptomatic patients. Soluble forms of TF and microparticles of monocyte and platelet origin, and bearing TF, constitute "blood-born TF". The contribution of this TF pool to arterial thrombosis is still under discussion. TF pathway is a target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TFPI and antibodies against TF or peptides interfering with TF-FVIIa complex activity.     

Characterization of the inhibition of tissue factor in serum

Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K- dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half- maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa- Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.     

Reduction of salivary tissue factor (thromboplastin) activity by warfarin therapy

The coagulant of normal human saliva has been identified as tissue factor (thromboplastin, TF) by virtue of its ability to cause rapid coagulation in plasmas deficient in first-stage coagulation factors and to activate factor x in the presence of factor VII and by virtue of the fact that its activity is expressed only in the presence of factor VII and is inhibited by an antibody to TF. The TF is related to cells and cell fragments in saliva. Salivary TF activity has been found to be significantly reduced in patients taking warfarin. The decline in TF activity during induction of warfarin anticoagulation occurs during the warfarin-induced decline in vitamin-K-dependent clotting factor activity, as judged by the prothrombin time. The decrease in TF activity is not related to a reduction in salivary cell count or total protein content or to a direct effect of warfarin on the assay. It is hypothesized that the mechanism by which warfarin inhibits TF activity may be related to the mechanism by which it inhibits expression of the activity of the vitamin-K-dependent clotting factors. Inhibition of the TF activity may be involved in the antithrombotic effect of warfarin.     

Studies of the activation of factor VII bound to tissue factor [see comments]

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.     

Initiation of the tissue factor pathway of coagulation in the presence of heparin: control by antithrombin III and tissue factor pathway inhibitor

Activation of factor X by both the unactivated tissue factor:factor VII complex (TF:VII) and the activated tissue factor:factor VIIa complex (TF:VIIa) has been studied in the presence of tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and heparin. At near-plasma concentrations of TFPI, ATIII, and factor X, factor X activation that occurs in response to TF:VII is essentially abolished in the presence of heparin (0.5 micromol/L). This effect requires both inhibitors, acting on different targets: (1) ATIII, which in the presence of heparin blocks the activation of TF:VII, and (2) TFPI, which inhibits the TF:VIIa that is generated. In the absence of ATIII, TFPI alone with heparin reduces but does not abolish factor X activation. Conversely, in the absence of TFPI, ATIII + heparin reduces but does not abolish TF:VIIa generation and allows continuing activation of factor X. These results indicated that when the unactivated TF:VII complex is the initiating stimulus, heparin-dependent reduction in the rate and extent of factor X activation requires both ATIII and TFPI. In contrast, if TF:VIIa is used to initiate activation, only TFPI is involved in its regulation.     

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

We report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild-type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (K(d) from 4.4 nmol/l for FVIIaWT to 17.3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (k(cat)/K(m) from 2.3 x 10(7)/mol/l s for FVIIaWT to 8.7 x 10(5)/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen-like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF-catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the K(m) value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.     

Altered regulation of in-vivo coagulation in orthopedic patients prior to knee or hip replacement surgery.

Up to 20% of patients develop venographically proven deep-vein thrombosis after elective orthopedic surgery even under the cover of heparin or low molecular weight heparin. The extent to which the chronic inflammation of osteoarthritis requiring elective orthopedic surgery alters in-vivo coagulation and whether any specific alteration influences the development of postoperative thrombosis are unknown. This study compared the concentrations of activated factor VII (FVIIa), tissue factor pathway inhibitor (TFPI), activated factor X (FXa)-TFPI, thrombin-antithrombin, and prothrombin fragment 1+2 (F1+2) in plasmas of 535 healthy individuals (ages 17-76) with those in the preoperative plasmas of 306 arthritis patients (ages 30-92) scheduled for elective knee or hip replacement surgery. C-reactive protein was also measured in the plasmas of approximately 15% of the participants. Age-adjusted concentrations of FVIIa, F1+2, and C-reactive protein were higher in patients than controls, while the concentrations of thrombin-antithrombin, TFPI and FXa-TFPI were similar. Chronic inflammation in the patients was thus associated with increased coagulation in vivo. Without compensatory increases in the concentrations of TFPI (natural inhibitor of prothrombinase), the elevated concentrations of FVIIa in the preoperative plasmas and the trauma associated with surgery may enhance the risk for developing postoperative deep-vein thrombosis.     

Tissue factor: (patho)physiology and cellular biology.

The transmembrane glycoprotein tissue factor (TF) is the initiator of the coagulation cascade in vivo. When TF is exposed to blood, it forms a high-affinity complex with the coagulation factors factor VII/activated factor VIIa (FVII/VIIa), activating factor IX and factor X, and ultimately leading to the formation of an insoluble fibrin clot. TF plays an essential role in hemostasis by restraining hemorrhage after vessel wall injury. An overview of biological and physiological aspects of TF, covering aspects consequential for thrombosis and hemostasis such as TF cell biology and biochemistry, blood-borne (circulating) TF, TF associated with microparticles, TF encryption-decryption, and regulation of TF activity and expression is presented. However, the emerging role of TF in the pathogenesis of diseases such as sepsis, atherosclerosis, certain cancers and diseases characterized by pathological fibrin deposition such as disseminated intravascular coagulation and thrombosis, has directed attention to the development of novel inhibitors of tissue factor for use as antithrombotic drugs. The main advantage of inhibitors of the TF*FVIIa pathway is that such inhibitors have the potential of inhibiting the coagulation cascade at its earliest stage. Thus, such therapeutics exert minimal disturbance of systemic hemostasis since they act locally at the site of vascular injury.     

Activation of human factor VII in the initiation of tissue factor- dependent coagulation

We have used activation peptide release assays to compare factor VII and activated factor VII (VIIa) activation of factor X, normal factor IX (IXN), and a variant factor IX (IXBmLE), which, after activation, is unable to back-activate factor VII. In purified systems, factor VII and VIIa each rapidly activated factor X, but after a one minute lag for factor VII. VIIa also readily activated both IXN and IXBmLE. Factor VII initially failed to activate substantial amounts of either IXN or IXBmLE; on further incubation factor VII activated IXN but not IXBmLE. Activation of IXN began when approximately 10% of factor VII had been converted to VIIa, as measured by 125I-factor VII radioactivity profiles. Adding factor VII to VIIa slowed its activation of IXBmLE. However, in the presence of factor X, factor VII alone rapidly activated IXBmLE. Unlike purified systems, 1 nmol/L VIIa added to factor VII-deficient plasma failed to activate factor IX. Increasing factor VII to 10 nmol/L (plasma concentration) either as native VII or VIIa yielded similar activation curves for factor IX and similar activation curves for factor X. Adding 5% VIIa to factor X-deficient plasma and to factor XII-deficient plasma substantially shortened the dilute tissue factor clotting time of only the former. These data support the hypothesis that factor VII/tissue factor complex initiates tissue factor-dependent clotting through a minimal generation of Xa. This Xa then rapidly back-activates a small amount of factor VII, following which the rates of activation of both factors IX and X increase dramatically.     

Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells

Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.     

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H- labeled factor X to the plasma resulted, after a short lag, in burst- like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.     

Variants of recombinant factor VIIa with increased intrinsic activity

Following vascular damage, blood clotting is triggered when factor VIIa (FVIIa) forms a complex with tissue factor (TF). In hemophilia A and B, the propagation phase of blood coagulation is disrupted due to the lack of factors VIII (FVIII) and IX (FIX), leading to excessive bleeding. However, high doses of recombinant FVIIa (rFVIIa) can bypass the FVIII/FIX deficiency and ameliorate bleeding problems. Although the precise mechanism of action of rFVIIa at pharmacological doses remains a matter of debate, rFVIIa-catalyzed (TF-independent) activation of factor X (FX) on the surface of the activated platelet appears to be important. Variants of rFVIIa with increased intrinsic (TF-independent) activity have been developed, which may offer improved treatment of bleeding episodes, for example, in hemophiliacs with inhibitory antibodies to FVIII; they can also help us to understand how FVIIa works at the molecular level. This article reviews the properties of these molecules.