Ingo Ahrens

Publications of Ingo Ahrens

• New Parenteral Anticoagulants: Focus on Factor Xa and Thrombin Inhibitors.

  Authors: Ingo Ahrens, Christoph Bode

  Current drug discovery technologies. 08/2011;

  For decades, unfractionated heparin (UFH) was the most widely used parenteral anticoagulant in a variety of clinical scenarios requiring rapid and reversible anticoagulation. The shortcomings of UFHFor decades, unfractionated heparin (UFH) was the most widely used parenteral anticoagulant in a variety of clinical scenarios requiring rapid and reversible anticoagulation. The shortcomings of UFH include a high inter-individual variability and the occurrence of heparin induced thrombocytopenia with the potential for serious thromboembolic complications. These shortcomings prompted the successful clinical development of low molecular weight heparins (LMWH) and indirect factor Xa inhibitors such as fondaparinux. LMWH and fondaparinux demonstrated superiority in many clinical scenarios including acute coronary syndromes (ACS). However the long half-life and the lack of a specific antidote still require the usage of UFH in clinical situation where a rapid reversibility of the anticoagulation is required. Especially in ACS patients undergoing PCI, a direct parenteral inhibition of thrombin with the direct thrombin inhibitor (DTI) bivalirudin proved to be an alternative anticoagulant strategy. This has set the stage for the clinical development of other parenteral DTIs. The reduction in clinical ischemic events that were achieved with indirect factor Xa inhibitors in patients with ACS facilitated the clinical development of the first parenteral direct factor Xa inhibitor, otamixaban which is currently investigated in a phase III clinical trial in patients with ACS undergoing PCI. This article discusses novel parenteral factor Xa and thrombin inhibitors currently under clinical investigation including aptamers, a new class of drugs that allows the parallel development of the combination of active drug and reversal agent, which is facilitated by the DNA or RNA structure of which aptamers are composed of.

• Measuring oxidative burden and predicting pharmacological response in coronary artery disease patients with a novel direct activator of haem-free/oxidised sGC.

  Authors: Ingo Ahrens, Jonathon Habersberger, Nadège Baumlin, Hongwei Qian, Belinda K Smith, Johannes-Peter Stasch, Christoph Bode, Harald H H W Schmidt, Karlheinz Peter

  Atherosclerosis. 06/2011; 218(2):431-4.

  The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient'sThe soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy. Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients. We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators.

• Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

  Authors: Ingo Ahrens, Helena Domeij, Denijal Topcic, Izhak Haviv, Ruusu-Maaria Merivirta, Alexander Agrotis, Ephraem Leitner, Jeremy B Jowett, Christoph Bode, Martha Lappas, Karlheinz Peter

  PloS one. 01/2011; 6(8):e23210.

  Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests.Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.

• GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors.

  Authors: Houyuan Hu, Paul C J Armstrong, Elie Khalil, Yung-Chih Chen, Andreas Straub, Min Li, Juliana Soosairajah, Christoph E Hagemeyer, Nicole Bassler, Dexing Huang, Ingo Ahrens, Guy Krippner, Elizabeth Gardiner, Karlheinz Peter

  PloS one. 01/2011; 6(4):e19190.

  Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to plateletStaphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.

• High-mobility group box protein 1 neutralization reduces development of diet-induced atherosclerosis in apolipoprotein e-deficient mice.

  Authors: Peter Kanellakis, Alex Agrotis, Tin Soe Kyaw, Christine Koulis, Ingo Ahrens, Shuji Mori, Hideo K Takahashi, Keyue Liu, Karlheinz Peter, Masahiro Nishibori, Alex Bobik

  Arteriosclerosis, thrombosis, and vascular biology. 11/2010; 31(2):313-9.

  High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. WeHigh-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoE-/- mice. Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE-/- mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-α and interleukin-1β tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers. HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.

• New oral anticoagulant drugs in cardiovascular disease.

  Authors: Ingo Ahrens, Gregory Y H Lip, Karlheinz Peter

  Thrombosis and haemostasis. 07/2010; 104(1):49-60.

  Oral anticoagulation has been limited to vitamin K antagonists (VKAs) for over 60 years. VKAs are effective and recommended for the prevention of venous and arterial thromboembolism in cardiovascularOral anticoagulation has been limited to vitamin K antagonists (VKAs) for over 60 years. VKAs are effective and recommended for the prevention of venous and arterial thromboembolism in cardiovascular disease, but their pharmacodynamics are difficult to predict and the highly variable interindividual and intraindividual response to treatment accounts for the need of continuous monitoring. This prompted the intensive exploration of numerous substances within the last decade in an attempt to meet the shortcomings of current oral anticoagulation with VKAs. The development and clinical investigation of two novel groups of oral anticoagulants targeting central factors of the coagulation system either factor Xa or thrombin (factor IIa) has now reached the daily clinical practice with the approval of the oral direct thrombin inhibitor dabigatran etexilate and the oral direct factor Xa inhibitor rivaroxaban. Ongoing clinical trials are investigating these substances and other novel oral anticoagulants with similar mechanisms of action in patients with atrial fibrillation and acute coronary syndromes. This review article discusses the clinical evaluation and pharmacological properties of novel oral anticoagulants in late and earlier stages of clinical development, thereby providing a critical analysis and an outlook on the future of oral anticoagulation in cardiovascular disease.

• Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage.

  Authors: Christoph E Hagemeyer, Ingo Ahrens, Nicole Bassler, Natia Dschachutaschwili, Yung C Chen, Steffen U Eisenhardt, Christoph Bode, Karlheinz Peter

  Journal of cellular and molecular medicine. 01/2010; 14(1-2):290-302.

  The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. VascularThe adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin alphaIIbbeta3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of beta3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.

• Novel antiplatelet therapies following percutaneous coronary interventions.

  Authors: Ingo Ahrens, Christoph Bode

  Current opinion in investigational drugs (London, England : 2000). 10/2009; 10(9):902-11.

  Drug-eluting stents effectively reduce the incidence of in-stent restenosis; however, the use of these stents has led to the requirement for more effective antiplatelet therapies than are currentlyDrug-eluting stents effectively reduce the incidence of in-stent restenosis; however, the use of these stents has led to the requirement for more effective antiplatelet therapies than are currently available. This requirement primarily arises from the presence of the antiproliferative drug coating of the stents. These coatings cause the prolonged exposure of platelets to stent struts and denuded endothelium, resulting in an increased potential for prothrombotic clinical sequelae. New antiplatelet therapies are also required because of the inter-individual variability in responses to available antiplatelet drugs. Ideally, new drugs would selectively target distinct mechanisms of platelet activation, avoiding increases in bleeding risk. This review discusses novel antiplatelet therapies under clinical investigation.

• FX-06, a fibrin-derived Bbeta15-42 peptide for the potential treatment of reperfusion injury following myocardial infarction.

  Authors: Ingo Ahrens, Karlheinz Peter

  Current opinion in investigational drugs (London, England : 2000). 10/2009; 10(9):997-1003.

  Several novel interventional and pharmacological therapeutic approaches have been examined in recent years for the prevention of ischemia/reperfusion injury in myocardial infarction; however, mostSeveral novel interventional and pharmacological therapeutic approaches have been examined in recent years for the prevention of ischemia/reperfusion injury in myocardial infarction; however, most approaches have failed to progress to clinical trials, and the underlying pathological mechanisms of ischemia/reperfusion injury remain poorly understood. The fibrin Bbeta chain-derived peptide FX-06, under development by Ikaria Holdings Inc, is a novel candidate in phase II trials for the prevention of ischemia/reperfusion injury. FX-06 competitively binds to vascular endothelial (VE)-cadherin, thereby inhibiting leukocyte transmigration and initiating VE-cadherin-mediated signaling, which tightens the endothelial barrier and reduces capillary leakage. The sequence of FX-06 resembles that of a physiological peptide, and the compound has been well tolerated in clinical trials. In a phase II trial in patients with acute ST-segment elevation myocardial infarction who were undergoing percutaneous coronary intervention, FX-06 significantly reduced the necrotic core zone compared with placebo. Whether FX-06 treatment can have a clinical benefit remains to be established. Nonetheless, the unique mechanism of action and short half-life that distinguish FX-06 from competitive pharmacological agents would allow its use in combination with other drugs, potentially contributing to a successful pharmacological therapy for the prevention of ischemia/reperfusion injury after decades of disappointing results.

• Dissociation of Pentameric to Monomeric C-Reactive Protein on Activated Platelets Localizes Inflammation to Atherosclerotic Plaques.

  Authors: Steffen U Eisenhardt, Jonathon Habersberger, Andrew Murphy, Yung Chih Chen, Kevin J Woollard, Nicole Bassler, Hongwei Qian, Constantin von Zur Muhlen, Christoph E Hagemeyer, Ingo Ahrens, Jaye Chin-Dusting, Alex Bobik, Karlheinz Peter

  Circulation research. 07/2009;

  C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its functionC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis; monocyte activation, as determined by conformational change of integrin Mac-1; generation of reactive oxygen species; and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H2O2-treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects.

• Differences of platelet adhesion and thrombus activation on amorphous silicon carbide, magnesium alloy, stainless steel, and cobalt chromium stent surfaces.

  Authors: Christopher Hansi, Amina Arab, Alexander Rzany, Ingo Ahrens, Christoph Bode, Christoph Hehrlein

  Catheterization and cardiovascular interventions : official journal of the Society for Cardiac Angiography & Interventions. 04/2009; 73(4):488-96.

  Background: Coronary stenting is considered to be the gold standard of percutaneous coronary interventions, because stents are able to reduce early and late elastic recoil (negative remodeling) andBackground: Coronary stenting is considered to be the gold standard of percutaneous coronary interventions, because stents are able to reduce early and late elastic recoil (negative remodeling) and restenosis in comparison with balloon angioplasty alone. Objective: It is known that stent thrombogenicity and neointimal formation are determined by the surface characteristics of the stent platform, electrochemical features of the stent surface, and the degree of degradation after implantation. Metallic stents coated with amorphous silicon carbide and biodegradable stents made of magnesium alloy have been introduced clinically, but there are no data available comparing the biocompatibility of these novel stent materials with conventional stents. Methods: We demonstrate simple and reproducible in vitro methods assessing the rate of platelet adhesion and thrombus activation for biocompatibility tests of different stent surfaces. Results: We show that amorphous silicon carbide and magnesium alloy stent surfaces markedly lower the rate of platelet adhesion and platelet/fibrin activation when compared with uncoated stainless steel or cobalt chromium alloy surfaces. Semiconductor materials on the stent surface reduce platelet and fibrin activation by increasing the critical electron gap to greater than 0.9 eV resulting in a lower electron transfer out of the stent material. Conclusion: Passive stent coatings with specific semiconducting properties such as amorphous silicon carbide or magnesium alloy reduce thrombogenicity and may improve biocompatibility of a stent platform. (c) 2009 Wiley-Liss, Inc.

• Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes.

  Authors: Ingo Ahrens, Christoph Ellwanger, Belinda K Smith, Nicole Bassler, Yung Chih Chen, Irene Neudorfer, Andreas Ludwig, Christoph Bode, Karlheinz Peter

  Journal of leukocyte biology. 07/2008; 83(6):1388-95.

  Selenium therapy in patients with severe sepsis improves clinical outcome and has been associated with increased activity of the selenoprotein glutathione peroxidase. However, the mechanism of theSelenium therapy in patients with severe sepsis improves clinical outcome and has been associated with increased activity of the selenoprotein glutathione peroxidase. However, the mechanism of the observed beneficial effects remains unclear. We determined the effect of selenium treatment on the monocyte adhesion molecule L-selectin and L-selectin-related monocyte functions in vitro and transferred our findings to an in vivo mouse model. Monocytes were purified, cultured, and incubated in the presence or absence of supplemented selenium and metalloproteinase (MP) inhibitors for up to 16 h. Expression of L-selectin was unaffected after 2 and 6 h but decreased after 16 h of incubation in the presence of selenium. Soluble L-selectin (sL-selectin) in the supernatant was determined by ELISA. A 2.3-fold increase as a result of shedding of L-selectin was observed after 16 h of selenium treatment. Addition of the MP inhibitors GM6001, TNF-alpha-converting enzyme inhibitor 2, or GW280264X strongly reduced selenium-induced L-selectin shedding, indicating a MP-dependent mechanism. The functional consequences of L-selectin shedding were examined in a flow chamber model. Selenium-treated monocytes showed significantly decreased rolling and adhesion to the L-selectin ligand Sialyl-Lewis(a) under conditions of venous shear stress (0.5 dyne/cm(2)). Selenium treatment of C57BL6 mice led to increased serum levels of sL-selectin, underscoring the in vivo relevance of our findings. We describe a selenium-induced down-regulation of L-selectin on monocytes as a consequence of MP-dependent shedding of this membrane-anchored adhesion molecule. The impairment of monocyte adhesion by selenium supplementation may represent an important, underlying mechanism for the modulation of inflammatory reactions in patients with severe sepsis.

• Therapeutic integrin inhibition: allosteric and activation-specific inhibition strategies may surpass the initial ligand-mimetic strategies.

  Authors: Ingo Ahrens, Karlheinz Peter

  Thrombosis and haemostasis. 06/2008; 99(5):803-4.

• Humanizing mouse thrombi.

  Authors: Ingo Ahrens, Karlheinz Peter

  Nature biotechnology. 02/2008; 26(1):62-3.

• Direct thrombin inhibition with bivalirudin as an antithrombotic strategy in general and interventional cardiology.

  Authors: Ingo Ahrens, Belinda K Smith, Christoph Bode, Karlheinz Peter

  Expert opinion on drug metabolism & toxicology. 09/2007; 3(4):609-20.

  Antithrombotic therapy is a crucial component of interventional cardiology and currently involves the administration of both anticoagulant and antiplatelet agents. The implementation of standard dualAntithrombotic therapy is a crucial component of interventional cardiology and currently involves the administration of both anticoagulant and antiplatelet agents. The implementation of standard dual or triple antiplatelet therapies has allowed percutaneous coronary intervention (PCI) with stent implantation to become the treatment of choice in most patients with acute coronary syndromes (ACS), particularly in patients with ST-segment elevation myocardial infarction. However, the combined use of antithrombotic agents increases the bleeding risk associated with coronary intervention, which is a concern due to the increasing evidence that bleeding complications are associated with a higher risk of ischaemic events and death. The shortcomings of currently available anticoagulant drugs have promoted the ongoing development of new, powerful anticoagulant agents that have both efficacy in the setting of PCI and a reduced risk of bleeding; one of these classes of agents targets the thrombin molecule, a key factor in the coagulation cascade, and belongs to the class of anticoagulants known as direct thrombin inhibitors (DTIs). Bivalirudin, a synthetic peptide, is a DTI with unique, favourable pharmacological properties that include predictable linear pharmacokinetics. Bivalirudin was approved as an anticoagulant in patients undergoing routine PCI in 2000 by the FDA (in 2004 in Europe and Australia) and more recently in patients with ACS undergoing PCI. The pharmacological properties of bivalirudin, along with current indications for its use, are discussed in this review, with a focus on the major completed and ongoing clinical trials with bivalirudin.

• Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation.

  Authors: Patrick Stoll, Nicole Bassler, Christoph E Hagemeyer, Steffen U Eisenhardt, Yung Chih Chen, Rene Schmidt, Meike Schwarz, Ingo Ahrens, Yasuhiro Katagiri, Benedikt Pannen, Christoph Bode, Karlheinz Peter

  Arteriosclerosis, thrombosis, and vascular biology. 06/2007; 27(5):1206-12.

  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 beOBJECTIVE: 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 mechanistic model for paradoxical platelet activation by ligand-mimetic alphaIIb beta3 (GPIIb/IIIa) antagonists.

  Authors: Nicole Bassler, Christoph Loeffler, Pierre Mangin, Yuping Yuan, Meike Schwarz, Christoph E Hagemeyer, Steffen U Eisenhardt, Ingo Ahrens, Christoph Bode, Shaun P Jackson, Karlheinz Peter

  Arteriosclerosis, thrombosis, and vascular biology. 04/2007; 27(3):e9-15.

  OBJECTIVE: Integrins are attractive therapeutic targets. Inhibition of integrin alphaIIb beta3 effectively blocks platelet aggregation. However, limitations with intravenous alphaIIb beta3OBJECTIVE: Integrins are attractive therapeutic targets. Inhibition of integrin alphaIIb beta3 effectively blocks platelet aggregation. However, limitations with intravenous alphaIIb beta3 antagonists and failure of oral alphaIIb beta3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. METHODS AND RESULTS: Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic alphaIIb beta3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of alphaIIb beta3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic alphaIIb beta3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic alphaIIb beta3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits alphaIIb beta3 antagonist-induced platelet activation. CONCLUSION: These findings can explain limitations of ligand-mimetic anti-alphaIIb beta3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.

• CD40 ligand mediates inflammation independently of CD40 by interaction with Mac-1.

  Authors: Andreas Zirlik, Christoph Maier, Norbert Gerdes, Lindsey MacFarlane, Juliana Soosairajah, Udo Bavendiek, Ingo Ahrens, Sandra Ernst, Nicole Bassler, Anna Missiou, Zsofia Patko, Masanori Aikawa, Uwe Schönbeck, Christoph Bode, Peter Libby, Karlheinz Peter

  Circulation. 04/2007; 115(12):1571-80.

  BACKGROUND: Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classicBACKGROUND: Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. METHODS AND RESULTS: Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1-dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor-deficient mice attenuates lesion development and reduces lesional macrophage accumulation. CONCLUSIONS: These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

• Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.

  Authors: Meike Schwarz, Gerardene Meade, Patrick Stoll, Jari Ylanne, Nicole Bassler, Yung Chih Chen, Christoph E Hagemeyer, Ingo Ahrens, Niamh Moran, Dermot Kenny, Desmond Fitzgerald, Christoph Bode, Karlheinz Peter

  Circulation research. 08/2006; 99(1):25-33.

  Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to designPlatelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

• [Direct Inhibition of Thrombin. A New Strategy for the Treatment of Atrial Fibrillation and Acute Coronary Syndrome?]

  Authors: Ingo Ahrens, Christoph Bode

  Herz. 06/2005; 30(3):197-203.

  Direct inhibition of thrombin is a promising new strategy for the treatment of cardiovascular disease and thrombosis. Three intravenous (lepirudin, bivalirudin and argatroban) and one oral directDirect inhibition of thrombin is a promising new strategy for the treatment of cardiovascular disease and thrombosis. Three intravenous (lepirudin, bivalirudin and argatroban) and one oral direct thrombin inhibitor (ximelagatran) are currently available and approved for some clinical indications in the treatment of cardiovascular disease and venous thrombosis. Further indications are under investigation in large clinical trials. The bivalent direct thrombin inhibitor (DTI) lepirudin is used for anticoagulation of patients with heparin-induced thrombocytopenia (HIT) as is the monovalent DTI argatroban. The bivalent DTI bivalirudin has been studied extensively in the REPLACE 1 and 2 trials. Results from these studies demonstrate that bivalirudin is as effective as heparin for patients undergoing percutaneous coronary intervention (PCI) and leads to less bleeding complications. Efficacy and safety of anticoagulation with bivalirudin in patients with unstable angina or non-ST elevation myocardial infarction (NSTEMI) are currently under investigation in the ACUITY trial. A fixed dose of 36 mg twice daily ximelagatran, an oral prodrug of melagatran, has been shown to be safe and as effective as warfarin for the treatment of patients with nonvalvular atrial fibrillation in the SPORTIF III and V trials. This review focuses on the pharmacology of the DTIs and discusses the results of major clinical trials with DTIs in nonvalvular atrial fibrillation and acute coronary syndromes.