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【摘要】
Objective- 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.
Methods and Results- 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.
Conclusions- 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.
A new strategy of targeting anticoagulants to activated platelets is evaluated. A newly cloned single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa and the potent, direct factor-Xa inhibitor TAP were genetically fused. Anticoagulative efficiency and safety was proven in a mouse model with carotid artery thrombosis and bleeding time measurements.
【关键词】 GPIIb/IIIa anticoagulation singlechain fXa thrombosis
Introduction
Therapeutic anticoagulation is used extensively in many areas of medicine. Despite the overall benefits achieved, the currently used therapeutic anticoagulants are also a major source of mortality and morbidity, caused by limitations in efficacy and even more so by bleeding complications. 1 In an effort to overcome these problems, a plethora of new agents have been developed. 2,3 However, it seems that more efficient therapeutic anticoagulation is inevitably associated with an increase in bleeding complications. Targeting of anticoagulants to the clot may represent a means to break this association. The success of this targeting is dependent on the abundance and specificity of the epitope chosen as the target. We have previously demonstrated that fibrin, which satisfies both requirements, can be used successfully for clot targeting. 4-7 In the present study, we investigated whether activated platelets can be used as an alternative and potentially more efficient clot-target. Platelets are highly abundant in particular in thrombi within the arterial system, as with atherosclerosis-induced thrombi, eg, in myocardial infarction. Activated platelets are highly specific for clots and are not typically found in the circulation. Thus, both requirements for efficient clot-targeting, abundance and specificity, are highly satisfied. Besides these favorable properties, the use of activated platelets as epitopes for clot-targeting may have additional advantages compared with fibrin, because platelet activation may precede fibrin formation. 8
One of the most abundantly expressed molecules on the platelet surface is the glycoprotein (GP) IIb/IIIa (CD41/CD61). This receptor belongs to the adhesion molecule family of integrins and is also termed IIb ß 3. Integrins consist of two noncovalently linked subunits that undergo a conformational change from a low affinity to a high affinity receptor in respect to the binding of the GPIIb/IIIa ligand fibrinogen. 9,10 Besides the exposure of the ligand-binding pocket, this conformational change also induces the exposure of so-called ligand-induced binding sites (LIBS) on GPIIb/IIIa. 10,11 Because these binding sites are specific for the activated and/or ligand-bound GPIIb/IIIa receptor this epitope is uniquely suited for clot targeting.
ScFvs are a new and promising format for the design of recombinant therapeutic agents. They consist of only the variable regions of the antibody?s heavy and light chain fused together via a short linker molecule on a single peptide chain. 12 This small size may be of particular advantage for low immunogenicity and thrombus accessibility/penetration. 12,13 In contrast to chemical coupling, which typically results in a significant loss of both the antibody binding function as well as of the activity of the coupled effector molecules, scFv can be coupled without functional loss using molecular biology techniques. 4,5,14
The anticoagulant to be targeted should have the following properties: It should inhibit a central and important coagulation factor, it should be a highly potent inhibitor, it should be a small molecule, and it should function while fused to an antibody. The soft tick Ornithodoros moubata uses a factor Xa inhibitor, TAP (tick anticoagulant peptide), to suck blood from its prey. 15 This anticoagulant satisfies ideally the above criteria, providing effective anticoagulation because of fXa?s central, up-stream, and rate-determining position in the coagulation cascade. TAP is one of the most potent anticoagulants found in nature and it is a small molecule with only 60 amino acids. 15 Finally, we have previously demonstrated that its anticoagulative function is preserved when it is N-terminally coupled to an antibody. 4
In the present study, we cloned a scFv that allows targeting of anticoagulants to the activated, ligand-bound GPIIb/IIIa receptor. As the targeted agent, we genetically fused the direct fXa inhibitor TAP, which provides strong anticoagulative activity that takes effect early and centrally in the coagulation cascade and at the highly procoagulant surface of clots. 16 We present in vivo data of a mouse model suggesting unique properties of the newly generated anticoagulant with high antithrombotic efficiency but without prolongation of bleeding times.
Materials and Methods
The scFvanti-LIBS was cloned from hybridoma cells producing an anti-LIBS IgG. TAP was transferred to a pHOG21 vector, which already included scFvanti-LIBS, thereby creating scFvanti-LIBS-TAP ( Figure 1 ). The fusion protein was produced in Escherichia coli (TG1) and purified via Ni 2+ - Agarose (Qiagen). Human blood was collected by venipuncture with a 21-gauge butterfly needle from healthy volunteers and anticoagulated with citric acid. Mouse blood was collected by intracardial puncture with a 27-gauge needle from C57BL/6 mice and anticoagulated with unfractionated heparin (20 U/mL). Aggregometry was performed on a Biodata PAP-4 aggregometer for 10 minutes at 37°C. Flow cytometry was performed in a FACSCalibur (Becton Dickinson), after staining with a secondary antibody (Penta His Alexa Fluor 488 Conjugat, Qiagen) directed against the Histidin(6)-tag of the scFv. Inhibition of factor Xa (fXa) was determined by the degradation of the chromogenic substrate spectrozyme fXa #222 (American Diagnostica Inc). For the flow experiments, platelets were perfused over a collagen matrix at a shear-rate of 150 s -1 (1.1 dyn/cm 2 ) for 10 min under negative pressure (PHD 2000, Harvard Apparatus), to assess anti-LIBS mAb staining after the initial adhesion step. In vivo functional characterization was evaluated in a mouse ferric chloride thrombosis model. Thrombotic occlusion was measured by a flow meter (T106, Transonic). Mouse bleeding time was measured after tail transection and by template bleeding. Radiolabeling was carried out using the Iodogen method (Pierce) and Na125I (GE Healthcare) following the manufacturer?s instructions. Radioactivity was measured in a -counter (Packard RIASTAR) for 1 min. For immunohistochemistry staining, cryosections (6 µm) were cut and stained with the DAB immunohisto system (Vector Laboratories). Data are presented as mean±standard deviations for the indicated number of mice. The statistical comparisons were made by analysis of variance (ANOVA following a Newmann-Keuls test) and differences were considered to be significant at P <0.05.
Figure 1. Map of pHOG21-scFv anti-LIBS -TAP. R AMP indicates ampicillin resistance gene; ColE1 ORI, origin of replication of E coli; f1 IG, filamentous intergenic region; pelB, leader peptide sequence of pectate lyases pelB; V H /V L, heavy/light chain; TAP, tick anticoagulant peptide; His 6, repeat of 6 histidines.
For expanded Methods, please see the supplemental materials, available online at http://atvb.ahajournals.org.
Results
Cloning of an Anti-LIBS Single-Chain Antibody (scFv) Based on a Hybridoma Cell Line Expressing IgG Anti-LIBS-145
An antibody against a LIBS-epitope was chosen for targeting of anticoagulants to clots. As previously demonstrated, the mAb anti-LIBS-145 (IgG anti-LIBS ) demonstrates ligand-induced binding to GPIIb/IIIa on ADP-activated platelets in the presence of fibrinogen. 11 Thus, this antibody is directed against a target that is highly abundant and specific.
The mAb anti-LIBS-145-expressing hybridoma cells were used as the basis for the cloning of an anti-LIBS single-chain antibody (scFv). mRNA was prepared and reverse transcribed using an oligo-dT primer. The variable regions of the antibody?s heavy and light chain were amplified by polymerase chain reaction (PCR) using primers that anneal to conserved regions at the 5' and 3' ends of the variable regions. The PCR products were cloned into the pHOG21 vector ( Figure 1 ). After transformation of TG1 E coli individual clones were assessed for LIBS-typical binding to GPIIb/IIIa. One clone that revealed a stronger binding compared with the original IgG anti-LIBS-145 mAb in flow cytometry was chosen and sequenced (supplemental Figure I). 12
Construction, Expression, and Purification of the scFv Fusion Protein
Based on our previous results that TAP can be fused without functional loss, 4 we chose to couple this highly potent direct fXa inhibitor to our newly cloned single-chain antibody. TAP was originally synthesized according to published sequence information 15 and was cloned into pHOG21 directly at the C terminus of the variable region of the light chain ( Figure 1 ). pHOG21 contains a pelB-leader sequence for periplasmic localization within the bacteria and a His 6 -tag for Ni 2+ -purification as well as detection. The yield of purified scFv anti-LIBS -TAP was around 0.4 to 0.8 mg from 1 L bacterial culture. After expression and purification, we tested the correct size of our single-chain antibody constructs by Western blot ( Figure 2 ). The molecular weight of the scFv anti-LIBS alone was 32 kDa, of the intact fusion protein scFv anti-LIBS -TAP was 39 kDa, and of the nontargeted mut-scFv-TAP was 42 kDa ( Figure 2 ).
Figure 2. Western blot analysis of Ni 2+ -purified scFv anti-LIBS, scFv anti-LIBS -TAP, and nontargeted mut-scFv-TAP. MW indicates molecular weight marker (6xHis protein ladder); 1, scFv anti-LIBS; 2, scFv anti-LIBS -TAP; 3, nontargeted scFv-TAP.
In Vitro Functional Evaluation of the Bifunctional Fusion Molecule scFv anti-LIBS -TAP
The function of the scFv-component of the fusion molecule scFv anti-LIBS -TAP was evaluated by flow cytometry. ScFv anti-LIBS - TAP and scFv anti-LIBS demonstrated similar binding properties to activated platelets ( Figure 3 ). Thus, the genetic fusion did not significantly alter the scFv?s binding property. Anti-fXa activity was evaluated by a chromogenic assay. FXa was incubated with a specific chromogenic substrate in the presence of scFv anti-LIBS -TAP, nontargeted mut-scFv-TAP, scFv anti-LIBS, and recombinant TAP ( Figure 4 ). Compared with rTAP, TAP activity was slightly reduced in the fusion constructs but was clearly present. Thus, both functions, antibody binding and fXa inhibition, were retained in the fusion molecule. The scFv anti-LIBS -TAP has no effect on platelet function as shown in flow cytometry by unaffected fibrinogen binding to activated platelets in the presence of different concentrations of scFv as well as normal platelet aggregation after ADP stimulation (supplemental Figure II).
Figure 3. Flow cytometry histograms of specific binding of IgG anti-LIBS, scFv anti-LIBS, and scFv anti-LIBS -TAP to activated but not to nonactivated human platelets in whole blood. Binding to ADP-activated platelets is given by open histograms; binding to nonactivated platelets is given by shaded histograms. Binding of the IgG antibody is detected by a DTAF-conjugated goat anti-mouse antibody, binding of the scFvs is detected by an Alexa Fluor 488 conjugated anti-His-tag antibody.
Figure 4. Inhibition of factor Xa activity by rTAP, scFv anti-LIBS -TAP, and nontargeted mut-scFv-TAP, but not by scFv anti-LIBS. The cleavage of chromogenic substrate (spectrozyme FXa #222) by factor Xa (500 pmol/L) was determined at 405 nm. Bars show optical density (OD) as mean and standard deviation of triplicate measurements of a representative example of 4 experiments.
In Vivo Functional Evaluation of scFv anti-LIBS -TAP
To prove superiority of targeting of anticoagulants to LIBS-epitopes compared with the conventional nontargeted use of anticoagulant, a well-established mouse thrombosis model was chosen. 17-19 However, we first had to ensure that the anti-LIBS antibodies could be used for targeting to fibrinogen-bound activated platelets of mice. We obtained mouse blood and evaluated the binding of the original IgG anti-LIBS, of the scFv anti-LIBS, and of the fusion construct scFv anti-LIBS -TAP to mouse platelets in flow cytometry. Similar to the results in human platelets, we saw a specific binding of the IgG anti-LIBS, but we saw even stronger specific binding of the scFv anti-LIBS antibody alone as well as binding of its fusion protein scFv anti-LIBS -TAP to fibrinogen-bound activated mouse platelets (supplemental Figure III).
Thrombi were induced in the carotid artery of mice using ferric chloride. The termination of blood flow measured by a nano Doppler-flow probe was used as an indicator of an occlusive thrombus. 19 Sodium chloride solution and the scFv anti-LIBS were used as negative controls, and enoxaparine was used as a positive control representing a current clinical standard. Enoxaparine nearly doubled the occlusion time ( Figure 5 A). Equimolar amounts of recombinant TAP, nontargeted mut-scFv-TAP, and scFv anti-LIBS -TAP caused significant prolongation of the occlusion time that came close to the effects of enoxaparine. A reduction to 1/10 (0.03 µg/g body weight) of the original dose delivered still caused a significant prolongation of the occlusion time ( P =0.002) with the scFv anti-LIBS -TAP, whereas the nontargeted mut-scFv-TAP at the same dose did not cause a prolongation of occlusion time. Thus, the scFv anti-LIBS -TAP delivers a strong anticoagulant effect, even at a dose (1/10 of the original dose) at which the direct control, the nontargeted mut-scFv-TAP does not cause significant anticoagulation. Thus, we could observe an increase in potency of anticoagulation via targeting TAP to LIBS-epitopes.
Figure 5. In vivo evaluation of GPIIb/IIIa-targeted anticoagulation. A, Strong antithrombotic effects of scFv anti-LIBS -TAP at high and low doses in a mouse model with ferric chloride-induced thrombosis in the carotid artery. Thrombus development was evaluated by occlusion time measurements as determined by flow measurement with a nano Doppler-flow probe at the carotid artery. The clot-targeted anticoagulant scFv anti-LIBS -TAP does not cause bleeding time prolongation in contrast to enoxaparine, rTAP, and nontargeted mut-scFv-TAP. Bleeding time in mice was determined by tail transection (B) and incision/template bleeding time measurements (C). rTAP and nontargeted mut-scFv-TAP demonstrated considerable prolongation of bleeding time in contrast to scFv anti-LIBS -TAP. Saline (0.9% NaCl) and the single-chain antibody scFv anti-LIBS were used as negative control. Enoxaparine as a clinically used agent was used as a positive control. rTAP, scFv anti-LIBS -TAP, and nontargeted mut-scFv-TAP were used at a high equimolar dose and scFv anti-LIBS -TAP and nontargeted scFv-TAP were also used at a low equimolar dose. Mean and SD of 8 mice per group are depicted. Probability values are given against control with 0.9% NaCl (* P 0.05, ** P 0.01, *** P 0.001).
To assure that this strong anticoagulative effect is caused by a specific accumulation of scFv anti-LIBS -TAP at the thrombus, we labeled scFvs with I-125. We found a higher amount of radioactivity within the thrombus after the injection of scFv anti-LIBS -TAP in comparison to nontargeted mut-scFv-TAP (supplemental Figure IVA). In addition, using immunohistochemistry we could demonstrate the binding of scFv anti-LIBS -TAP at the side of injury in contrast to the control experiment (supplemental Figure IVB). To rule out that this specific accumulation is caused by a different plasma clearance of scFv anti-LIBS -TAP in contrast to nontargeted mut-scFv-TAP, we determined the blood elimination rate of the two constructs and found equal results within the experimental time frame (supplemental Figure IVC).
Because we believe that a major benefit of clot-targeted anticoagulation will be the reduction of bleeding complications, the evaluation of bleeding times is a key element of our study. We determined two types of bleeding times: (1) The standardized surgical tail transaction, and (2) an incision method, which resembles the template bleeding times normally measured in humans. As expected, saline and scFv anti-LIBS did not cause bleeding time prolongations, whereas enoxaparine and in particular recombinant TAP caused a considerable prolongation. At the dose of 0.3 µg/g body weight at which both nontargeted mut-scFv-TAP and scFv anti-LIBS -TAP demonstrated a strong anticoagulant effect ( Figure 5 A), only the nontargeted mut-scFv-TAP caused a significant prolongation in bleeding times ( P <0.01, Figure 5B and 5 C). In clear contrast, the clot-targeted scFv anti-LIBS -TAP did not cause prolongation in bleeding times at all. Also the lower dose of scFv anti-LIBS -TAP, which still demonstrated a clear anticoagulant effect, did not cause bleeding time prolongation.
To investigate the mechanism behind the unique situation of a strong anticoagulative effect without prolongation of bleeding times, we evaluated the time course of LIBS-expression on platelets adhering on collagen under flow conditions. Initially, adhering platelets do not expose LIBS-epitopes ( Figure 6 A), only platelets adhering for 5 minutes or more and in particular larger aggregates bind the anti-LIBS antibody. In contrast, as a positive control the anti-CD41 signal is detectable on platelets right from the beginning of platelet adhesion ( Figure 6 B). Thus, the delayed exposure of LIBS-epitopes on adhering platelets may allow a "sealing" of vessel injuries but may prevent the formation of larger thrombi.
Figure 6. LIBS-epitope expression on platelets adhering under flow conditions. A, Phase contrast and fluorescence pictures of platelets stained with activation-specific anti-LIBS are shown after 3, 5, 10, and 15 minutes as well as 10 µmol/L ADP activated platelets after 10 minutes (positive control). Complete movies are available in the online supplemental materials (3, 5, 15 minutes LIBS.avi). Initially, adhering platelet are negative for LIBS-epitopes during the first 5 minutes. During further platelet adhesion and thrombus formation the signal intensity increases. B, Phase contrast and fluorescence pictures of platelets stained with nonactivation specific anti-CD41 IgG are shown after 3 and 5 minutes. Platelets are positive for CD41 right from the beginning of the flow. The complete movie is available in the online supplemental materials (3 minutes CD41.avi). Platelets stained with secondary antibody alone showed no specific signal (not shown).
Discussion
Platelets are obvious targets for clot-targeting of anticoagulants. The anionic membrane surface of activated platelets provides the prerequisite to assemble and catalyze the intrinsic tenase (VIIIa/IXa), prothrombinase (Va/Xa), and XIa complexes, leading to explosive thrombin generation, fibrin formation, and consolidation of fibrin-platelet clots. 8 This cell-based model of coagulation 8,20 explains the important role of platelets in coagulation and underlines the hypothesis that activated platelets are an ideal epitope for clot-targeted anticoagulation, considering both their early and their spatial involvement in coagulation. GPIIb/IIIa LIBS-epitopes are specific and highly abundant on activated platelets and for this reason have been used to detect platelet activation in clinical settings. 10,21 Furthermore, the exclusive expression of GPIIb/IIIa on platelets is already the basis for one of the most successful pharmacological strategies, which is antiplatelet therapy with GPIIb/IIIa blockers. 9,10,22 In addition, a long-lasting localized anticoagulative effect may be achievable by a stable fixation of anti-fXa activity at the clot via anchoring of TAP to activated platelets by the targeting antibody. And finally, targeting TAP to the membrane of activated platelet is attractive, because fXa inhibition takes place where it is most needed. The membrane surface of activated platelets is a major constituent of the prothrombinase complex potentiating fXa activity of up to 300 000-fold compared with free fXa. 8
Clot-targeting is an attractive therapeutic concept that has been addressed by us and others. 4-7,14,23 Fibrin as a relative clot-specific component has been used to increase the potency of anticoagulants such as hirudin, 6,7 tick anticoagulant peptide (TAP), 4 as well as fibrinolytics. 14,24 However, the development of this fibrin-targeting approach is hampered by the species selectivity of the targeting antibodies. Up to now, only a few in vivo experiments have been reported in baboons. 24,25 An alternative epitope for clot-targeting has been recently described: Phosphatidyl- L -serine/phosphatidyl-ethanolamine, which is exposed on activated platelets and microparticles. 26 A 3- to 10-fold increase in anticoagulant potency could be achieved by this targeting strategy. 26 Another recent approach used P-selectin, which is expressed on activated platelets as well as on activated endothelial cells, as an epitope to target Desmodus-rotundus salivary plasminogen activator alpha1. 27 In this only partially selective targeting approach, no functional gain or even a functional loss was seen. 27 In addition to these reports, which focused on potential gain in potency, we could provide unique data that targeting also reduces the clinically highly relevant bleeding effect.
Another promising approach based on antibody-targeting is prophylactic thrombolysis. Targeting fibrinolytics to erythrocytes or endothelial cells has been shown to inhibit clot development in injured arteries and to promote lysis of emboli, eg, in pulmonary arteries. 28,29 This strategy is not based on enrichment of fibrinolytics at the clot, but it provides a constant prophylactic level of fibrinolytics either circulating on erythrocytes or localized in pulmonary vessels. The use of platelets that ectopically express urokinase-type plasminogen activator as transgene resulted in resistance against occlusive artery thrombi and in rapid resolution of pulmonary emboli in mice. 30 This intriguing report further underlines the attractiveness of platelets as targets for antibody targeting of anticoagulants and/or fibrinolytics.
The use of TAP as the targeted anticoagulant has major advantages. Thrombus-associated fXa, but not thrombin, is primarily responsible for the procoagulant activity on the surface of thrombi. 31 In addition, the direct inhibition of fXa by TAP has been proposed as advantageous compared with the indirect, antithrombin-III-mediated inhibition, eg, as mediated by heparins. Clot-bound as well as prothrombinase-associated fXa are resistant to antithrombin-III-mediated inhibition but are well inhibited by direct fXa inhibitors. 32 During recanalization of clotted vessels by mechanical angioplasty or by therapeutic fibrinolysis highly thrombogenic material, especially fXa, is released from the lysed clot, often resulting in reocclusion of the initially recanalized vessel, and fXa inhibition, in particular by TAP, may provide more benefits than inhibition of thrombin. 33-35
The chosen format of recombinant single-chain antibodies offers several advantages. Compared with the production of antibodies in hybridoma cells, scFvs can be produced in bacteria at low cost and are easily purified via attached tags and are amenable to large scale production. Furthermore, scFvs can be directed against complex epitopes that include function-specific conformations of targeted molecules using scFv display techniques on phages, yeast, bacteria, or ribosomes. 36-38 The newly generated anti-LIBS scFv provides interesting perspectives as diagnostic marker and imaging tool. Preliminary results demonstrate that anti-LIBS scFv can be used to diagnose platelet activation in clinical settings such as acute coronary syndromes and to detect thrombi/emboli in MRI (von zur Muhlen C, unpublished data, 2007). ScFvs can be tailored in size or generated as alternative formats, eg, Fab-fragments. 12,39 The Fab-fragment abciximab, which is a blocker of GPIIb/IIIa, is the prototype for the successful use of antibodies as therapeutic agents. 22 Overall, the unique properties of the single-chain antibody technology causes major interest in drug discovery programs in particular in cancer therapy. 40-42
Our studies on LIBS-epitope expression on platelets adhering under flow conditions provide a mechanistic model that may explain the differential effects of scFv anti-LIBS -TAP on thrombus formation and bleeding time. The delayed expression of LIBS-epitopes may allow the formation of a "sealing" platelet layer at the site of vascular injury. However, once activated platelets start to form aggregates/clots, the scFv?s targeted anti-fXa function may then prevent thrombus formation.
In summary, we generated a single-chain antibody that specifically binds to a LIBS-epitope on platelet GPIIb/IIIa. This antibody allows targeting of effector molecules to activated platelets in clots. With the goal to enrich an anticoagulant at the clot, we genetically fused the direct fXa inhibitor TAP to this single-chain antibody. This molecule can be easily modified by molecular biology methods and provides a highly flexible, cost-effective, and up-scalable production method. In vitro assays prove that the antibody fragment as well as the fXa inhibitor retain their individual function in the fusion molecule. In vivo evaluation demonstrates effective anticoagulation without prolongation of bleeding times. The results obtained in a mouse model have to be interpreted with caution and may not be fully transferable to the human situation. However, our data warrant further evaluation of the described novel pharmacological approach that promises effective anticoagulation with reduced bleeding risks.
Acknowledgments
We are grateful to Dr S. Krishnaswamy for the generous gift of recombinant TAP and to Dr B. Kingwell for the critical reading of the manuscript.
Sources of Funding
N.B., C.E.H. and K.P. are supported by the National Health and Medical Research Council of Australia. K.P. is supported by the Heart Foundation of Australia. C.E.H. is supported by the Deutsche Forschungsgemeinschaft (DFG Ha 5297/1-1).
Disclosures
A patent explanation has been filed to protect the intellectual property of the single-chain antibody and its derivatives.
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作者单位：Centre for Thrombosis & Myocardial Infarction (P.S., N.B., C.E.H., S.U.E., Y.C.C., K.P.), Baker Heart Research Institute, Melbourne, Australia; the Departments of Cardiology (P.S., M.S., I.A., C.B.) and Anaesthesiology (R.S.), University of Freiburg, Germany; the National Institutes of Health (Y