Immobilized Tissue Factor Research Articles

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts.

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy.

Abstract 181: Sub-picomolar Amounts of Coagulation Factor XIa Promote Spatial Clot Growth and Thrombin Generation Inside Propagating Clot

Background: Activated coagulation factor XI (FXIa) was found in the immunoglobulin products that were associated with higher than usual incidence of thrombotic events (TEs). However, TEs after thrombogenic immunoglobulin administration are very rare and are often recorded as late as 48 hours after administration. Objective: This study was aimed to elucidate the mechanisms behind FXIa thrombogenicity. We hypothesized that low amounts of FXIa are not able to activate clotting directly, but can promote on-going coagulation events at a site of vascular lesion. Materials and methods: FXIa activity was characterized by kinetic clotting and thrombin generation (TG) assays. Spatial clot growth and thrombin wave propagation in stagnant plasma were studied using thrombodynamics, a time-lapse videomicroscopy-based approach. To model coagulation events at the site of vascular wall lesion, spatial clot growth was initiated by plastic surfaces covered with immobilized tissue factor (TF). Human plasma deficient in various coagulation factors was used to study pathways of FXI activation. Results: Low plasma FXIa levels of less than 0.3 pM did not induce thrombin generation or clotting, but increased TG in plasma mixed with TF. Higher FXIa levels (>0.3 pM) activated coagulation directly. In stagnant plasma activated by surface with immobilized TF, sub-picomolar amounts of FXIa were also not able to initiate clotting, but was able to enhance TG inside clots. FXIa-induced TG was localized to the propagating edge of the clot and associated with increased clot growth rate. Conclusion: Sub-picomolar amounts of FXIa may promote growth of clot in the vicinity of vascular lesion and have no procoagulant effect in the absence of exposed TF. These in vitro findings suggest that the patient’s condition might contribute to the thrombogenicity of FXIa.

Blood Clot Contraction Dynamics Studied with an Automated Analyzer System

Blood clot contraction plays an important role in hemostasis because it seals a vessel injury, approximates wound edges and restores blood flow past obstructive thrombi. The process is governed by contraction of activated platelets and requires the interaction of platelets with fibrin, however, many aspects of this process are not fully understood. Despite the biological and medical importance, clot contraction has been systematically studied neither in research nor in clinical laboratories. One reason for that is the lack of methodology to follow and quantify clot contraction dynamics. We have developed a novel method to assess blood clot contraction in vitro and successfully applied this technique to investigate the effects of calcium ions, factor XIIIa activity, and different thrombin concentrations and on the rate and degree of clot contraction.The new technique to quantify the kinetics of blood clot contraction is based on the continuous tracking of dynamics of clot size by sensing changes in the light scattering of the clot over time followed by computational processing of the images. The optical platform for this method is the commercially available Thrombodynamics Analyser System (HemaCore, Moscow, Russia) initially designed to monitor the directional growth of a plasma clot triggered on one side of a cuvette with immobilized tissue factor. A standard procedure to analyze clot contraction with this instrument is described next. Plastic cuvettes 12 x 7 x 1 mm are pre-lubricated with a detergent to prevent attachment of fibrin to the walls, which precludes clot contraction. Fresh citrated human blood is incubated with calcium chloride (2 mM final concentration) at 37°C for 3 minutes followed by addition of 1 U/ml of freshly thawed thrombin to initiate blood clotting and platelet activation. Thrombin-activated whole blood sample (80 µl) is quickly transferred to the cuvette, which is placed into the thermostatic chamber (37°C) between a red LED and an optical sensor (CCD camera). The clot contraction is monitored by taking images every 15 seconds over 20 minutes or more. Off-line automated computational analysis of the serial images provides a kinetic curve plotted as the degree of contraction (% retracted clot volume) as a function of time. This curve is characterized by the following parameters: lag period (min), average contraction velocity (Δ clot volume/time), final degree of contraction (%), time to reach 1/4 and 1/2 of the final contraction (min), and area under the curve (a.u.).A general conclusion based on the examination of about 100 kinetic curves is that clot contraction is a non-monotonic process and has at least two phases characterized by distinct rates. Variation of [Ca2+] from zero up to 10 mM revealed that exogenous Ca2+ was not indispensable for clot contraction to occur; however, the clots formed without addition of Ca2+ were less stable and often extruded red blood cells from the clot during contraction (likely due to impaired fibrin cross-linking with factor XIIIa). Recalcification of blood with 2 or 5 mM Ca2+ stabilized the clot, preventing the red blood cell fallout, but had no significant effect on the contraction kinetics. When added at a final concentration of 10 mM, Ca2+ partially inhibited clot contraction as confirmed by significantly reduced average velocity and the degree of retraction compared to the Ca2+-free conditions, perhaps due to suppression of platelet function. These findings indicate that 2 to 5 mM [Ca2+] is optimal for contraction of a whole blood clot in vitro. To see if clot contraction and stability depends on the cross-linking catalyzed by factor XIIIa, we studied clot contraction in the presence of 1 mM iodoacetamide, a potent inhibitor of the transglutaminase activity. Iodoacetamide did not affect blood clot formation but abolished clot shrinkage, confirming that the enzymatic activity of factor XIIIa is essential for clot contraction. As expected, blood clot contraction depended on thrombin concentration within a 0.1-1.0 U/ml range. Thrombin enhanced the rate and degree of clot contraction in a dose-dependent manner.In summary, we developed an accurate and simple assay for blood clot contraction which can be used for research and may be potentially useful for in vitro diagnostics.(Research supported by the Program of Competitive Growth of Kazan Federal University) DisclosuresAtaullakhanov:HemaCore LLC: Employment, Membership on an entity’s Board of Directors or advisory committees.

Flow-Dependent Functions Of Soluble Or Immobilized Tissue Factor In Mural Thrombus Formation Mediated By Von Willebrand Factor

Mural thrombus formation at sites of damaged vessel wall, essential for both physiologic hemostasis and pathological thrombosis, is established by platelet adhesion/aggregation and blood coagulation mechanisms. Although tissue factor (TF) is up-regulated upon vessel wall damage and plays a pivotal role in the latter process, its functional relevance under physiologic blood flow conditions is poorly understood. Using an in vitro perfusion chamber system, we have therefore studied the relevant role of TF in thrombus formation mediated by von Willebrand factor (VWF), a distinctive flow-dependent thrombogenic surface, under whole blood flow conditions with varying shear rates. Human recombinant TF (Innobin) were co-coated with purified VWF (100 ug/ml) onto a glass plate to prepare ‘surface-immobilized TF/VWF complex'. Surface density of immobilized TF, evaluated by the ELISA-based assay using an anti-TF monoclonal antibody, was increased in a concentration-dependent and saturated manner by soluble TF (1-100 pM) added on a plate. Citrated whole blood, recalcified with 8 mM CaCl2 prior to perfusion, was perfused over a VWF-surface in the presence or absence of surface-immobilized TF. Platelet adhesion and aggregation was evaluated by the surface coverage of generated thrombi in a defined area after 5-min perfusion. Mural thrombi formed on VWF-surface were also double-stained with fluorescently labeled anti-fibrin and anti-fibrinogen antibodies. Fibrin generation was evaluated by confocal laser scanning microscopy as a ratio of fibrin relative to fibrinogen fluorescence within mural thrombi. As a result, surface-immobilized TF significantly augmented flow-dependent fibrin generation as a function of increasing surface density of TF under both low (250 s-1) and high (1500 s-1) shear rate conditions. In this regard, soluble TF, when added to sample blood, similarly increased intra-thrombus fibrin generation in a dose-dependent manner in the absence of immobilized TF. However, coagula formation in sample blood was enormously amplified by soluble TF during perfusion, as judged by the flow-path occlusion time. In addition to the enhancing effects on fibrin generation, immobilized TF significantly up-regulated VWF-dependent platelet adhesion and aggregation under high shear rate conditions, albeit with no appreciable effects under low shear rate conditions. These results suggest a synergistic functional link between immobilized TF and VWF in mural thrombus formation under high shear rate conditions. Our results clearly illustrate the thrombogenic potentials of two distinct forms (soluble or surface-immobilized) of TF, in which surface-immobilized TF plays a concerted role on VWF-dependent thrombus formation with lesser risk of systemic hypercoagulability which may be induced by circulating soluble TF under high shear rate conditions. Disclosures:No relevant conflicts of interest to declare.

Drug-drug interaction of the anti-TFPI aptamer BAX499 and factor VIII: Studies of spatial dynamics of fibrin clot formation in hemophilia A

In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.

Circulating Procoagulant Microparticles with Coagulation Factors IXa and XIa Cooperatively Induce Spontaneous Clotting in Plasmas of Patients with Hematological Disorders

AbstractAbstract 3402 Background & Objective.Although blood of patients with hematological disorders is known to contain procoagulant material (activated clotting factors, tissue factor, microparticles), its functional significance is poorly understood. Using an in vitro experimental model of immobilized tissue factor-initiated clot growth in platelet-free plasma, we observed formation of activation-surface-independent isolated “spontaneous” clots (SC) throughout the plasma volume in patients with various disorders. The aim of this work was to identify the mechanisms of SC formation. Methods.Platelet-free plasma was prepared from citrated whole blood collected by venipuncture. It was supplemented with corn trypsin inhibitor (CTI), recalcified, placed into a thin flat chamber and brought into contact with a surface covered by immobilized tissue factor. Fibrin clot formation as a function of space and time was determined by imaging light scattering. with a CCD camera1 in a thrombodynamics analyzer device (Hemacore LLC, Moscow, Russia). Flow cytometry was used to monitor annexin V-labeled microparticles and platelets in plasma. Results.SC were reproducibly observed in plasmas of patients with various disoders including: 4 of 32 patients with ischemic cardiomyopathy, 11 of 29 patients with lymphogranulomatosis during chemotherary, 2 of 5 patients with acute leukemia. SC also appeared in 2 of 36 healthy donor plasmas.To determine contribution of intrinsic and extrinsic coagulation activation pathways, we blocked them with corn trypsin inhibitor (CTI) and active site-inhibited recombinant factor VIIa, respectively. Tissue factor-dependent activation was essential in 1 of 10 patients tested in this way. CTI inhibited SC in all samples, but never completely (Fig. 1).To test whether circulating active factors can induce SC, we supplemented normal plasma with factors Va, VIIa, IXa, or XIa known to have long lifetimes (more than 30 min). Addition of factors Va and VIIa led to uniform coagulation thoughout the plasma volume, which did not resemble typical SC patterns observed in the patient plasma (Fig. 2). In contrast, factors IXa and XIa induced isolated clotting centers very similar to those observed in the clinical samples.SC both in patient plasma and in normal plasma supplemented with factors IXa and XIa disappeared after centrifugation for 30 min at 16000 g, indicating also a contribution of microparticles. This was confirmed by inductions of SC in the bottom layers of normal plasma subjected to identical centrifugation. After phospholipid supplementation, SC reappeared in normal plasma supplemented with factors IXa or XIa, as well as in 3 out of 11 patient plasmas. Flow cytometry revealed an increase in procoagulant microparticles in patient plasmas with SC. Conclusions.Here we report observation of a SC phenomenon in vitro in plasma of hematology patients. Although its mechanisms are likely to be different in different illnesses, our data suggest that combination of circulating active factors (specifically, factors IXa and XIa) with circulating procoagulant microparticles is the predominant one. Possible association between the presence of spontaneous clots and clinical manifestations require further study, but prevalence of this phenomenon in the patient plasma indicates possible applicability in diagnostics and. [Display omitted] [Display omitted] Disclosures:Lipets:HemaCore LLC: Employment. Ataullakhanov:HemaCore LLC: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Panteleev:HemaCore LLC: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Thrombin Activity Propagates in Space During Blood Coagulation as an Excitation Wave

Injury-induced bleeding is stopped by a hemostatic plug formation that is controlled by a complex nonlinear and spatially heterogeneous biochemical network of proteolytic enzymes called blood coagulation. We studied spatial dynamics of thrombin, the central enzyme of this network, by developing a fluorogenic substrate-based method for time- and space-resolved imaging of thrombin enzymatic activity. Clotting stimulation by immobilized tissue factor induced localized thrombin activity impulse that propagated in space and possessed all characteristic traits of a traveling excitation wave: constant spatial velocity, constant amplitude, and insensitivity to the initial stimulation once it exceeded activation threshold. The parameters of this traveling wave were controlled by the availability of phospholipids or platelets, and the wave did not form in plasmas from hemophilia A or C patients who lack factors VIII and XI, which are mediators of the two principal positive feedbacks of coagulation. Stimulation of the negative feedback of the protein C pathway with thrombomodulin produced nonstationary patterns of wave formation followed by deceleration and annihilation. This indicates that blood can function as an excitable medium that conducts traveling waves of coagulation.

Predicting prothrombotic tendencies in sepsis using spatial clot growth dynamics

Inflammation in sepsis is associated with hypercoagulation that may lead to thrombosis and disseminated intravascular coagulation. Conventional diagnostic assays are poorly sensitive to procoagulant changes in sepsis. Objectives of the article is to study changes in hemostatic state of septic patients using spatial clot growth assay (currently being developed under the trademark of thrombodynamics) and to compare the sensitivity of this method with the sensitivity of conventional methods. Sixteen patients with hematological malignancies and sepsis were enrolled in the study. All patients had been surveyed for a month following the infection onset. Spatial clot growth assay monitors fibrin clot development in a nonstirred thin layer of platelet-free plasma activated by immobilized tissue factor. Clotting time tests, thromboelastography, D-dimer assays were also performed. Spatial clot growth revealed hypercoagulation in six patients. D-dimer levels increase (with vein thrombosis in one case) was subsequently observed in five of them. D-dimer levels did not increase when spatial clot growth was normal. At the next time point, after spatial clot growth assay showed hypercoagulation, the mean D-dimer concentration was significantly higher than after a normal analysis (457 versus 234 μg/l; P < 0.05); there was no such correlation for other assays. The remaining 10 patients had elevated D-dimer levels on the first day; this either decreased gradually or remained elevated. Spatial clot growth showed normalization in survivors and growing hypocoagulation in nonsurvivors. Measuring spatial clot growth dynamics has potential diagnostic utility for the evaluation of thrombotic risk.

Improvement of spatial fibrin formation by the anti‐TFPI aptamer BAX499: changing clot size by targeting extrinsic pathway initiation

Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m(-2) by shortening lag time and increasing clot size by up to ~2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m(-2) , both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates in vitro blood coagulation similarly to thromboplastin on fibroblasts

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.

Computational Modeling of Factor Xa Inhibition by Immobilized Tissue Factor Pathway Inhibitor

Coating surfaces of implanted devices with anticoagulants can reduce thrombosis and studies using a recombinant form of endogenous tissue factor pathway inhibitor (rTFPI) are promising. The anticoagulant function of immobilized rTFPI is thought to occur primarily by its inhibition of plasma clotting factor Xa (FXa); however the kinetics of this reaction at a surface are as yet unknown. To better understand the surface inhibition reaction under flow conditions, a theoretical model was developed delineating the roles of mass transport and reaction kinetics for an in vitro parallel plate device used in prior experimental studies [Hall et al., J. Biomech. Eng. 120:484-490, 1998]. As a first approximation, the kinetics of inhibition of FXa by rTFPI reported for static, homogeneous systems was considered. The unsteady convection-diffusion equation was solved for different wall-shear rates and inlet concentrations of FXa using the computational fluid dynamics software CFD-ACE (ESI Software Group). The results show that the heterogeneous inhibition reaction is diffusion controlled prior to saturation of the rTFPI. The experimental results compare favorably with the model at the lower shear rates (100-400 s(-1)). At higher shear rates (>400 s(-1)) the theoretical results follow the same trend as the experimental results but show a greater inhibition of FXa, implying an effect of flow or shear on the inhibition reaction.

Selection and characterization of a new class of peptide exosite inhibitors of coagulation factor VIIa.

A new series of peptide inhibitors of human Factor VIIa (FVIIa) has been identified and affinity matured from naive and partially randomized peptide phage libraries selected against the immobilized tissue factor x Factor VIIa (TF x FVIIa) complex. These "A-series" peptides contain a single disulfide bond and a 13-residue minimal core required for maximal affinity. They are exemplified by peptide A-183 (EEWEVLCWTWETCER), which binds at a newly identified exosite on the FVIIa protease domain, described in the accompanying report [Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531]. A-183 was obtained from a trypsin digest of A-100-Z, a recombinant protein comprising A-183 and the Z domain of protein A. Surprisingly, A-183 was a very potent inhibitor of TF x FVIIa, inhibiting activation of Factor X (FX) and Factor IX and amidolytic activity of Chromozym t-PA with IC50 values of 1.6 +/- 1.2, 3.5 +/- 0.3, and 8.5 +/- 3.5 nM, respectively. Kinetic analysis revealed that A-183 was a partial (hyperbolic) mixed-type inhibitor of FX activation having a Ki of 200 pM as well as a partial competitive inhibitor of amidolytic activity. The A-series peptides were also specific and potent inhibitors of TF-dependent clotting as measured in a prothrombin time (PT) clotting assay and had no effect on the TF-independent activated partial thromboplastin time. At saturating concentrations of peptide, the maximal extent by which A-183 and A-100-Z inhibited the rate of FX activation was 78 +/- 3 and 89 +/- 6%, respectively. The degree of inhibition of the rate of FX activation correlated with a maximum fold prolongation in the PT assay of 1.8-fold for A- 183 and 3.3-fold for A-100-Z. The A-series peptides represent a new class of peptide exosite inhibitors that are capable of attenuating, rather than completely inhibiting, the activity of TF x FVIIa, potentially leading to anticoagulants with an increased therapeutic window.

Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood.

Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.

Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display.

Potent active-site inhibitors of human tissue factor-Factor VIIa (TF.FVIIa) have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries displayed on phage. Eight randomized positions on the extended primary binding loop (P5 through P4') and positions 34 and 39 were examined in three separate libraries. Libraries contained from 3.2 x 10(5) to 3.2 x 10(6) potential variants resulting from replacing up to 5 positions with all 20 amino acids. Following 4 rounds of selection against FVIIa associated with immobilized tissue factor (TF), 12 clones from each library were sequenced. Variants were purified by trypsin affinity chromatography and reverse-phase high performance liquid chromatography, and characterized for their ability to inhibit TF.FVIIa chromogenic activity. Measured apparent equilibrium dissociation constants (Ki*) ranged from about 10 to 500 nM. From sequence and activity data, an overall consensus sequence, TF7I-C, was constructed by site-directed mutagenesis. TF7I-C differed from APPI at 4 key residues, T11P, M17L, S19L, and G39Y, and inhibited TF.FVIIa with a Ki* = 1.9 +/- 0.4 nM, which represented an increase in binding affinity of more than 150-fold compared to APPI. At 40 microM, TF7I-C prolonged the clotting times 3.5-fold in a prothrombin time assay and > 10-fold at 7 microM in an activated partial thromboplastin time assay. Prolongation of the activated partial thromboplastin time correlates with potent inhibition of FXIa (Ki* = 0.8 nM) and plasma kallikrein (Ki* = 1.2 nM). TF7I-C also inhibited plasmin (Ki* = 40 nM) and FXa (Ki* = 55 nM), but not activated protein C, thrombin, or FXIIa (Ki* > 10 microM each).

The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor

The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VII to factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51–88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the β-galactosidase fusion proteins containing amino acid residues 51–88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.