Recombinant Tick Anticoagulant Peptide Research Articles

Abstract 1033: Simultaneous Clot-targeted Factor Xa Inhibition And Selective Blockade Of Activated GPIIb/IIIa On Platelets Results In Delayed But Potent Antithrombotic Effects Without Bleeding Time Prolongation.

Background: We generated phage-display-derived anti-GPIIb/IIIa single-chain antibodies (e.g. scFv SCE5) that specifically bind to the activated GPIIb/IIIa only and thus specifically block activated platelets only. ScFv SCE5 demonstrates strong antithrombotic potency, comparable to the conformation-unspecific blockers tirofiban and eptifibatide. In contrast bleeding times were not prolonged with scFv SCE5. Here we now use the possibility to add effector molecules using molecular biology methods. The highly potent anticoagulant TAP (tick anticoagulant peptide), which is a direct factor Xa (fXa) inhibitor, was used as an effector molecule. Methods and Results: We genetically fused the activation-specific scFv with TAP, expressed the constructs in E.coli and purified the 39 kDa protein via its Histag binding to Nickel beads. Specific binding of the fusion molecules MA2/SCE5-TAP and strong inhibition of fibrinogen binding was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. Prolongation in occlusion time with SCE5-TAP was significantly stronger compared to SCE5 alone, recombinant TAP, non-binding mut-scFv-TAP as well as the clinical used drugs enoxaparine and eptifibatide. In contrast to the other anticoagulants tested, bleeding time was not prolonged by SCE5-TAP. Flow experiments studying platelet adhesion on collagen revealed a possible mechanism for the unique finding of a fully normal bleeding time: LIBS exposure on adhering platelets and as such the anticoagulative targeting potency of SCE5-TAP was delayed until considerable layers of platelets were deposited. Conclusions: The combination of activation-specific GPIIb/IIIa blockade and fXa inhibition in one clot-targeted molecule further improves in-vivo antithrombotic efficiency without causing any bleeding time prolongation. The delay of the observed targeting effect may allow a sealing of injuries with platelet layers but may be in time for the prevention of occlusive platelet aggregates. The described blockers represent a new type of highly selective drugs that warrant further clinical development.

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv(anti-LIBS)) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv(anti-LIBS)-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv(anti-LIBS)-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv(anti-LIBS)-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv(anti-LIBS)-TAP. The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor: BOTH ACTIVE SITE AND EXOSITE INTERACTIONS ARE REQUIRED FOR INHIBITION

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.

The Critical Role of the 185–189-Loop in the Factor Xa Interaction with Na+ and Factor Va in the Prothrombinase Complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism.

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.

Crystallization and preliminary X-ray diffraction data of the complex of recombinant tick anticoagulant peptide (rTAP) and bovine factor Xa.

The complex of bovine factor Xa and recombinant tick anticoagulant peptide (rTAP) was crystallized in two different crystal forms using polyethylene glycol as a precipitant. Form I belongs to space group P42212 with unit-cell dimensions a = b = 133.1, c = 68.8 A. It contains one complex per asymmetric unit and diffracts to 3.0 A resolution. Form II belongs to P41212 (or P43212) with dimensions a = b = 126.5, c = 146.7 A; it contains two complexes per asymmetric unit and diffracts to 2.5 A. The crystals of both forms consist of factor Xa (MW = 45.3 kDa) and rTAP (MW = 6.7 kDa).

Unexpected binding mode of tick anticoagulant peptide complexed to bovine factor Xa

The structure of recombinant tick anticoagulant peptide (rTAP) complexed to bovine factor Xa at 3.0 Å resolution reveals the structural basis for the specificity and the high affinity of rTAP. Three N-terminal residues, Tyr501, Asn502 and Arg503, play a critical role in the complex formation as suggested by earlier mutagenic studies and the ornithodorin-thrombin complex. Unexpectedly, the side-chain of Tyr501 is located in the S1 pocket, although factor Xa favors arginine as a P1 residue. Arg503 is located at the aryl binding pocket and forms a salt-bridge with Glu97 of factor Xa. The autolysis loop, which is disordered in the uninhibited factor Xa structure, is involved in the formation of the complex as a part of the secondary binding site. The C-terminal helix of rTAP interacts with factor Xa as a secondary binding determinant. The N-terminal residues of rTAP reorganize during the formation of the factor Xa-rTAP complex from the conformation found in the solution into an extended conformation. The presence of the secondary binding site confirms the proposed two-step kinetic mechanism based on the results of a mutagenesis study.

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Differential effects of anticoagulants on the activation of platelets ex vivo.

Because activation of platelets and of the coagulation system are interdependent mediators of thrombosis, platelet activation was characterized in whole blood in the presence of anticoagulants used to assess platelet function in vitro or as treatment for patients with occlusive arterial disease. Blood was anticoagulated alone or in combination with citrate, ethylenediaminetetraacetatic acid, corn trypsin inhibitor (CTI, an inhibitor of activated factor XII), heparin, enoxaparin, recombinant tick anticoagulant peptide (rTAP), or recombinant hirudin. Platelet activation in response to adenosine diphosphate (ADP) or collagen was detected by assay of P-selectin on the platelet surface delineated by flow cytometry. Although minimal activation was seen without ADP, the fraction of platelets expressing P-selectin in response to ADP was greatest in blood anticoagulated with citrate compared with CTI and all other anticoagulants. ADP-induced platelet activation was greater in blood anticoagulated with heparin compared with an equipotent anti-Xa concentration of enoxaparin. More variable results were seen with collagen, but platelet activation in the presence of citrate was greater than that with CTI. Interpretation of assays of inhibition of platelet activation by potentially therapeutic agents in vitro requires consideration of the effects of anticoagulants used. In addition, anticoagulants other than standard heparin may potentiate efficacy of antiplatelet drugs.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Transient Interruption of Arterial Thrombosis by Inhibition of Factor Xa Results in Long-term Antithrombotic Effects in Baboons

Recombinant tick anticoagulant peptide (r-TAP) is a potent and specific inhibitor of activated coagulation factor X which effectively interrupts in vivo arterial thrombosis during treatment. It is, however, uncertain if it also affects thrombosis after treatment is stopped. This was tested in a baboon model of arterial thrombosis where platelet deposition onto Dacron vascular graft segments, inserted as extensions into permanent femoral arteriovenous shunts, was measured. The baboons were intravenously treated with 10 micrograms/kg/min (low dose, aPTT = 39 +/- 1 s) and 25 micrograms/kg/min (high dose, aPTT = 58 +/- 2 s) r-TAP for two hours. During treatment the r-TAP inhibited thrombin formation and dose-dependently interrupted platelet deposition onto the graft segment. This effect lasted for up to two hours after treatment with the low dose. Following treatment with the high dose, the graft segments were kept in place for 53 h. After treatment was stopped, platelets again deposited, but at a much lower rate than in control studies. Maximum deposition was approximately 38% lower than in the control studies. Total platelet deposition over 55 h, calculated as the area under the deposition curve, was approximately 40% (p < 0.05) less than in the control studies. A significant shortening in the mean platelet life span and an approximately 15-fold increase in thrombin-antithrombin III complexes during the first 31 h indicated that the thrombus surface remained thrombogenic and that the effect of r-TAP was transient. We have shown that 2 h of treatment with a full antithrombotic dose of r-TAP markedly reduced both the rate of platelet deposition after treatment was stopped and the total number of platelets deposited over 55 h. This was in spite of the finding that the antithrombotic effect of r-TAP was transient.

Inhibition of Thrombin Attenuates Stenosis After Arterial Injury in Minipigs

We sought to determine whether brief, profound inhibition of thrombin or prothrombin activation by factor Xa limits neointimal formation and stenosis after arterial injury. Thrombin has been implicated as a mediator of neointimal formation, but adjunctive administration of anticoagulant agents has not proven effective to decrease restenosis in patients undergoing coronary angioplasty. We infused recombinant desulfatohirudin (r-hirudin, bolus of 2 mg/kg body weight followed by 2 mg/kg per h, n = 9), heparin (100 U/kg per h, n = 6) or recombinant tick anticoagulant peptide (rTAP, 1-mg/kg bolus followed by 3 mg/kg per h, n = 5), a specific inhibitor of factor Xa, intravenously, beginning 15 min before and for up to 3 h after repetitive balloon hyperinflations sufficient to disrupt the internal elastic lamina in a carotid artery of minipigs with hypercholesterolemia induced by feeding them an atherogenic diet. Partial thromboplastin time was increased six- to sevenfold over baseline levels at the end of the infusions of the anticoagulant agents. Lumen stenosis measured histologically 4 weeks after balloon-induced carotid injury was 29 +/- 16% (mean +/- SEM) in r-hirudin-treated, 52 +/- 19% in rTAP-treated and 76 +/- 18% in heparin-treated pigs (p < 0.02 for r-hirudin vs. heparin treatment). The marked reduction of stenosis in r-hirudin-treated animals indicates that thrombin plays a major role in neointimal formation after balloon-induced arterial injury. A relatively brief interval of profound, direct inhibition of thrombin may be particularly effective to attenuate restenosis after balloon angioplasty.

Fluorescence study of the conformational properties of recombinant tick anticoagulant peptide (Ornithodorus moubata) using multifrequency phase fluorometry.

Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide (Ornithodorus moubata) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 +/- 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the histidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52, 1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N-bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trp11, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.

Characterization of an Inducible Endothelial Cell Prothrombin Activator

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

ANTITHROMBOTIC ACTIONS OF SELECTIVE INHIBITORS OF BLOOD COAGULATION FACTOR Xa IN RAT MODELS OF THROMBOSIS

The antithrombotic actions of selective factor Xa (FXa) inhibitors, recombinant tick anticoagulant peptide (rTAP) and DX-9065a, were evaluated in experimental thrombosis models in anesthetized rats. In the first model, thrombosis was induced by exposing flowing blood to a silk thread anchored in an arteriovenous (AV) shunt. rTAP, DX-9065a and heparin, given as an iv infusion 1 hr before blood was circulated in the AV shunt, had ID 50s of 0.007, 0.6 μmol/kg/hr and 16 U/kg/hr, respectively. In the model of venous thrombosis which was induced by hypotonic saline (0.225%) followed by 15-min stasis of abdominal vena cava, rTAP and heparin had ID 50s of 0.007 μmol/kg/hr and 3.5 U/kg/hr, respectively. In both models, full inhibition of thrombus formation was achieved with FXa inhibition at doses which only modestly increased ex vivo plasma clotting time APTT (1.26 to 1.82 over the baseline). By contrast, the maximum antithrombotic effect of heparin was associated with high and significant APTT prolongation (> 5 fold over the baseline). Therefore, our study suggests that FXa inhibitors are effective agents in preventing thrombosis in both rat thrombosis models and may have therapeutic antithrombotic potential.

Neointimal thickening after severe coronary artery injury is limited by a short-term administration of a factor Xa inhibitor. Results in a porcine model.

Fibrin- and platelet-rich thrombus formations occur as the initial event after percutaneous transluminal coronary angioplasty. We therefore tested the hypothesis that short-term administration of the recombinant tick anticoagulant peptide (rTAP), a factor Xa inhibitor, would reduce the thickness of neointima at 28 days after injury in a porcine coronary balloon angioplasty model. Continuous intravenous infusion of rTAP (average dose, 194 micrograms . kg-1 . min-1) or placebo (vehicle only) was given to the study pigs for 60 hours. The goal of anticoagulation was to maintain the activated clotting time at 200 seconds. A central venous catheter was inserted 2 days before the procedure. On the day of coronary injury, the animals were administered boluses of rTAP (6.5 mg) and then underwent injury with an oversized metallic coil by standard methods in the right, circumflex, or left anterior descending coronary artery. No significant difference in vascular injury between rTAP and vehicle control was observed after euthanasia at 28 days. Significantly less neointimal thickening occurred in the rTAP-treated animals (thickness, mean +/-SD: 0.30 +/-0.08 mm) compared with the control (0.48 +/- 0.12 mm, P< .001). The specific factor Xa inhibitor rTAP, when given in fully anticoagulant doses for a short duration after coronary artery injury in the porcine model, resulted in a long-term decrease in neointimal thickness. These results implicate thrombin generation in neointimal formation and suggest that administration of a potent antithrombotic for several days immediately after the procedure may influence the long-term outcome of the coronary injury with a decrease in neointimal formation.

Comparison of Sustained Antithrombotic Effects of Inhibitors of Thrombin and Factor Xa in Experimental Thrombosis

In the pathogenesis of (recurrent) thrombosis, clot-associated thrombin appears to play an important role. Antithrombin III-independent thrombin inhibitors have been shown to neutralize clot-bound thrombin effectively. We compared the sustained antithrombotic effects and the effects on endogenous fibrinolysis of several of these agents with recombinant tick anticoagulant peptide (rTAP), a selective factor Xa inhibitor, and low-molecular-weight heparin (LMWH) in an experimental venous thrombosis model. Rabbits received either recombinant hirudin (rHir), Hirulog-1, CVS#995 (a novel direct inhibitor of thrombin), rTAP, LMWH, or saline. The effect on thrombus growth was assessed by measuring the accretion of 125I-labeled fibrinogen onto preformed nonradioactive thrombi, and the effect on endogenous fibrinolysis was assessed by measuring the decline in radioactivity of preformed 125I-labeled thrombi in rabbit jugular veins. All direct thrombin inhibitors induced a sustained antithrombotic effect compared with either LMWH and rTAP. In addition, CVS#995 also further decreased thrombus size after stopping its infusion, which was due to a significant enhancement of endogenous fibrinolysis. Direct thrombin inhibition by rHir, Hirulog-1, or CVS #995 induces a sustained antithrombotic effect compared with rTAP and LMWH, which is most likely due to inhibition of clot-bound thrombin. CVS#995 was shown to also enhance the extent of endogenous fibrinolysis to a greater degree compared with rHir and might therefore be an interesting new antithrombotic agent for the treatment of venous and arterial thrombosis.

Effect of selective factor Xa inhibition on arterial thrombus formation triggered by tissue factor/factor VIIa or collagen in an ex vivo model of shear-dependent human thrombogenesis.

Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of factor Xa. TAP has shown good antithrombotic efficacy in experimental animal models of disseminated intravascular coagulation and venous and arterial thrombogenesis. In the present study we evaluated the effect of recombinant TAP (rTAP) on acute thrombus formation in human nonanticoagulated blood triggered either by tissue factor (TF) or by collagen at arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation by use of an optimal inhibitory concentration of rTAP. Blood was drawn directly from an antecubital vein by a pump over the respective thrombogenic surfaces, which were positioned in a parallel-plate perfusion chamber. rTAP was mixed homogeneously into the flowing blood by a heparin-coated device positioned proximal to the perfusion chamber. The passage of blood through this device caused minor activation of coagulation but little activation of platelets. Fibrinopeptide A and beta-thromboglobulin levels after 5 minutes of blood perfusion were, on average, 14 ng/mL and 45 IU/mL, respectively. rTAP at a plasma concentration of 0.90 mumol/L completely inhibited TF/factor VIIa-dependent thrombus formation at wall shear rates of 650 and 2600 s-1. These shear conditions are comparable to those in medium-sized arteries and in moderately stenosed small arteries, respectively. In contrast to the TF-coated surface, rTAP was less efficient in reducing collagen-induced thrombus formation. While a significant reduction of 53% was observed at 650 s-1, thrombus formation at 2600 s-1 was not affected by rTAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Blockade at Specific Levels of the Coagulation Cascade on Restenosis in a Rabbit Atherosclerotic Femoral Artery Injury Model

The relation among the coagulation cascade, its individual proteins, and the response to vascular injury is largely undefined. We have evaluated the effect of four probes that block specific levels of coagulation cascade on neointimal hyperplasia in the atherosclerotic rabbit arterial injury model. Focal femoral atherosclerosis was induced by air-desiccation injury and hypercholesterolemic diet in 48 New Zealand White rabbits, followed by balloon angioplasty. Active-site inactivated factor VIIa (DEGR-VIIa), which blocks the binding of factor VIIa to tissue factor, was administered (n = 12 arteries) by intravenous bolus (1 mg/kg) at the time of balloon angioplasty and followed by infusion of 50 micrograms.kg-1.h-1 for 3 days; for the control (n = 13 arteries), 150 U heparin was injected as bolus and followed by infusion of saline at 50 microL.kg-1.min-1. Recombinant tissue factor pathway inhibitor (TFPI), which binds factor Xa and inhibits the tissue factor-factor VIIa complex and factor Xa, was given as a 1 mg/kg bolus followed by 15 micrograms.kg-1.min-1 infusion for 3 days (n = 17 arteries). Recombinant tick anticoagulant peptide (TAP; n = 15 arteries) and hirudin (n = 14 arteries), which block factor Xa and thrombin, respectively, were administered as a 1 mg/kg bolus followed by 5 micrograms.kg-1.min-1 infusion for 3 days. These three groups had their own controls (n = 14 arteries). There were no differences among treatment groups in preangioplasty and postangioplasty minimal luminal diameter (MLD) by angiography. The mean MLD 21 days after balloon angioplasty was significantly different between control and DEGR-VIIa-treated groups (0.74 +/- 0.25 and 1.24 +/- 0.27 mm, respectively; P = .0001) and between the TFPI-treated group and others (0.88 +/- 0.21 mm for control, 0.97 +/- 0.22 mm for hirudin-treated, 0.98 +/- 0.14 mm for TAP-treated, and 1.32 +/- 0.21 mm for TFPI-treated arteries; P = .0001 by ANOVA). By quantitative histological analysis, the ratio of neointimal cross-sectional area compared with the area of internal elastic lamina in the DEGR-VIIa-treated group was significantly less than control (0.48 +/- 0.12 versus 0.67 +/- 0.12, P = .0001), and the ratio of neointimal cross-sectional area to the area demarcated by the internal elastic lamina of the TFPI-treated group was significantly reduced compared with the other groups (0.46 +/- 0.20 for TFPI-treated, 0.67 +/- 0.15 for hirudin-treated, 0.61 +/- 0.15 for TAP-treated, and 0.64 +/- 0.13 for control groups; P = .003). Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.

New developments in antiplatelet and antithrombotic therapy.

Several agents which inhibit platelet aggregation (aspirin, ticlopidine, dipyridamole), and anticoagulants (vitamin K antagonists, unfractionated heparin, low molecular weight heparins and heparinoids) are in clinical use. The search for more effective antiaggregating agents has resulted in the development of clopidogrel, a chemical analogue of ticlopidine with minimal bone-marrow suppressing effects, thromboxane synthase inhibitors and receptor blockers, and antagonists of platelet receptor glycoproteins Ib and IIb/IIIa. In addition there is increasing therapeutic experience with chimeric monoclonal antibodies against the platelet receptors, glycoprotein IIb/IIIa, and, to a minor extent, with synthetic peptides or non-peptide inhibitors against the same receptors. Although new anticoagulants have become available, their efficacy has only been tested in animal models of thrombosis: tissue factor pathway inhibitor, factor Xa inhibitors (recombinant tick anticoagulant peptide, antistasin, natural pentasaccharide and DX-9065), recombinant thrombomodulin and recombinant protein C have been tested in this manner. Considerable clinical progress has been made with direct thrombin inhibitors, such as recombinant hirudin and hirulog which appear to be effective antithrombotic agents in patients. There is also clinical experience with argatroban, an arginine derivative which is a competitive antagonist to thrombin. However, PPACK, a tripeptide synthetic compound which irreversibly blocks the active catalytic site of thrombin, has not been investigated in the clinical setting.