Peter Kraft

Publications of Peter Kraft

• Antithrombotic Potential of Blockers of Store-Operated Calcium Channels in Platelets.

  Authors: Roger van Kruchten, Attila Braun, Marion A H Feijge, Marijke J E Kuijpers, Ronmy Rivera-Galdos, Peter Kraft, Guido Stoll, Christoph Kleinschnitz, Edouard M Bevers, Bernhard Nieswandt, Johan W M Heemskerk

  Arteriosclerosis, thrombosis, and vascular biology. 05/2012;

  OBJECTIVE: Platelet Orai1 channels mediate store-operated Ca(2+) entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channelsOBJECTIVE: Platelet Orai1 channels mediate store-operated Ca(2+) entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. METHODS AND RESULTS: Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. CONCLUSIONS: Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.

• Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome.

  Authors: Christoph Kleinschnitz, Kinga Blecharz, Timo Kahles, Tobias Schwarz, Peter Kraft, Kerstin Göbel, Sven G Meuth, Malgorzata Burek, Thomas Thum, Guido Stoll, Carola Förster

  Stroke; a journal of cerebral circulation. 02/2011; 42(4):1081-9.

  Glucocorticoids potently stabilize the blood-brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffectiveGlucocorticoids potently stabilize the blood-brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffective in patients with acute ischemic stroke. The reasons for this discrepancy are unresolved. To address the molecular basis for the paradox unresponsiveness of the blood-brain barrier during hypoxia, we used murine brain microvascular endothelial cells exposed to O(2)/glucose deprivation as an in vitro model. In an in vivo approach, mice were subjected to transient middle cerebral artery occlusion to induce brain infarctions. Blood-brain barrier damage and edema formation were chosen as surrogate markers of glucocorticoid sensitivity in the presence or absence of proteasome inhibitors. O(2)/glucose deprivation reduced the expression of tight junction proteins and transendothelial resistance in murine brain microvascular endothelial cells in vitro. Dexamethasone treatment failed to reverse these effects during hypoxia. Proteasome-dependent degradation of the glucocorticoid receptor impaired glucocorticoid receptor transactivation thereby preventing physiological glucocorticoid activity. Inhibition of the proteasome, however, fully restored the blood-brain barrier stabilizing properties of glucocorticoid during O(2)/glucose deprivation. Importantly, mice treated with the proteasome inhibitor Bortezomib in combination with steroids several hours after stroke developed significantly less brain edema and functional deficits, whereas respective monotherapies were ineffective. We for the first time show that inhibition of the proteasome can overcome glucocorticoid resistance at the hypoxic blood-brain barrier. Hence, combined treatment strategies may help to combat stroke-induced brain edema formation in the future and prevent secondary clinical deterioration.

• Sustained reperfusion after blockade of glycoprotein-receptor-Ib in focal cerebral ischemia: an MRI study at 17.6 Tesla.

  Authors: Mirko Pham, Xavier Helluy, Christoph Kleinschnitz, Peter Kraft, Andreas J Bartsch, Peter Jakob, Bernhard Nieswandt, Martin Bendszus, Guido Stoll

  PloS one. 01/2011; 6(4):e18386.

  Inhibition of early platelet adhesion by blockade of glycoprotein-IB (GPIb) protects mice from ischemic stroke. To elucidate underlying mechanisms in-vivo, infarct development was followed byInhibition of early platelet adhesion by blockade of glycoprotein-IB (GPIb) protects mice from ischemic stroke. To elucidate underlying mechanisms in-vivo, infarct development was followed by ultra-high field MRI at 17.6 Tesla. Cerebral infarction was induced by transient-middle-cerebral-artery-occlusion (tMCAO) for 1 hour in C57/BL6 control mice (N = 10) and mice treated with 100 µg Fab-fragments of the GPIb blocking antibody p0p/B 1 h after tMCAO (N = 10). To control for the effect of reperfusion, additional mice underwent permanent occlusion and received anti-GPIb treatment (N = 6; pMCAO) or remained without treatment (N = 3; pMCAO). MRI 2 h and 24 h after MCAO measured cerebral-blood-flow (CBF) by continuous arterial-spin labelling, the apparent-diffusion-coefficient (ADC), quantitative-T2 and T2-weighted imaging. All images were registered to a standard mouse brain MRI atlas and statistically analysed voxel-wise, and by cortico-subcortical ROI analysis. Anti-GPIb treatment led to a relative increase of postischemic CBF vs. controls in the cortical territory of the MCA (2 h: 44.2±6.9 ml/100 g/min versus 24 h: 60.5±8.4; p = 0.0012, F((1,18)) = 14.63) after tMCAO. Subcortical CBF 2 h after tMCAO was higher in anti-GPIb treated animals (45.3±5.9 vs. controls: 33.6±4.3; p = 0.04). In both regions, CBF findings were clearly related to a lower probability of infarction (Cortex/Subcortex of treated group: 35%/65% vs. controls: 95%/100%) and improved quantitative-T2 and ADC. After pMCAO, anti-GPIb treated mice developed similar infarcts preceded by severe irreversible hypoperfusion as controls after tMCAO indicating dependency of stroke protection on reperfusion. Blockade of platelet adhesion by anti-GPIb-Fab-fragments results in substantially improved CBF early during reperfusion. This finding was in exact spatial correspondence with the prevention of cerebral infarction and indicates in-vivo an increased patency of the microcirculation. Thus, progression of infarction during early ischemia and reperfusion can be mitigated by anti-platelet treatment.

• Early detrimental T-cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor thrombus formation.

  Authors: Christoph Kleinschnitz, Nicholas Schwab, Peter Kraft, Ina Hagedorn, Angela Dreykluft, Tobias Schwarz, Madeleine Austinat, Bernhard Nieswandt, Heinz Wiendl, Guido Stoll

  Blood. 03/2010; 115(18):3835-42.

  T cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T-cell receptors (TCRs) and mutations in costimulatory molecules were used to define theT cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T-cell receptors (TCRs) and mutations in costimulatory molecules were used to define the minimal immunologic requirements for T cell-mediated ischemic brain damage. Stroke was induced in recombination activating gene 1-deficient (RAG1(-/-)) mice devoid of T and B cells, RAG1(-/-) mice reconstituted with B cells or T cells, TCR-transgenic mice bearing 1 single CD8(+) (2C/RAG2, OTI/RAG1 mice) or CD4(+) (OTII/RAG1, 2D2/RAG1 mice) TCR, mice lacking accessory molecules of TCR stimulation (CD28(-/-), PD1(-/-), B7-H1(-/-) mice), or mice deficient in nonclassical T cells (natural killer T [NKT] and gammadelta T cells) by transient middle cerebral artery occlusion (tMCAO). Stroke outcome was assessed at day 1. RAG1(-/-) mice and RAG1(-/-) mice reconstituted with B cells developed significantly smaller brain infarctions compared with controls, but thrombus formation after FeCl(3)-induced vessel injury was unimpaired. In contrast, TCR-transgenic mice and mice lacking costimulatory TCR signals were fully susceptible to tMCAO similar to mice lacking NKT and gammadelta T cells. These findings were corroborated by adoptive transfer experiments. Our data demonstrate that T cells critically contribute to cerebral ischemia, but their detrimental effect neither depends on antigen recognition nor TCR costimulation or thrombus formation.

• Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration.

  Authors: Christoph Kleinschnitz, Henrike Grund, Kirstin Wingler, Melanie E Armitage, Emma Jones, Manish Mittal, David Barit, Tobias Schwarz, Christian Geis, Peter Kraft [......] Anja Schrewe, Lore Becker, Valérie Gailus-Durner, Helmut Fuchs, Thomas Klopstock, Martin Hrabé de Angelis, Karin Jandeleit-Dahm, Ajay M Shah, Norbert Weissmann, Harald H H W Schmidt

  PLoS biology. 01/2010; 8(9).

  Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical needIschemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.

• Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice.

  Authors: Peter Kraft, Peter Michael Benz, Madeleine Austinat, Marc Elmar Brede, Kai Schuh, Ulrich Walter, Guido Stoll, Christoph Kleinschnitz

  PloS one. 01/2010; 5(12):e15106.

  Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke areStroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. Focal cerebral ischemia was induced in Vasp(-/-) mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan's Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm(3)±0.5 mm(3) versus 0.8 mm(3)±0.4 mm(3); p<0.0001) were significantly enhanced in Vasp(-/-) mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm(3)±17.3 mm(3) versus 39.3 mm(3)±10.7 mm(3), respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes. Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.

• COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke.

  Authors: Peter Kraft, Tobias Schwarz, Lionel Pochet, Guido Stoll, Christoph Kleinschnitz

  Experimental & translational stroke medicine. 01/2010; 2(1):5.

  Anticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factorAnticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)-deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke functional outcome in mice. C57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Infarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent intracerebral fibrin(ogen) formation. COU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the antithrombotic potential of this novel class of FXII inhibitors can be finally judged.

• Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice are susceptible to intracerebral thrombosis and ischemic stroke.

  Authors: Peter Kraft, Tobias Schwarz, Joost C M Meijers, Guido Stoll, Christoph Kleinschnitz

  PloS one. 01/2010; 5(7):e11658.

  Thrombus formation is a key step in the pathophysiology of acute ischemic stroke and results from the activation of the coagulation cascade. Thrombin plays a central role in this coagulation systemThrombus formation is a key step in the pathophysiology of acute ischemic stroke and results from the activation of the coagulation cascade. Thrombin plays a central role in this coagulation system and contributes to thrombus stability via activation of thrombin-activatable fibrinolysis inhibitor (TAFIa). TAFIa counteracts endogenous fibrinolysis at different stages and elevated TAFI levels are a risk factor for thrombotic events including ischemic stroke. Although substantial in vitro data on the influence of TAFI on the coagulation-fibrinolysis-system exist, investigations on the consequences of TAFI inhibition in animal models of cerebral ischemia are still lacking. In the present study we analyzed stroke development and post stroke functional outcome in TAFI-/- mice. TAFI-/- mice and wild-type controls were subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 hours, functional outcome scores were assessed and infarct volumes were measured from 2,3,5-Triphenyltetrazoliumchloride (TTC)-stained brain slices. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Infarct volumes and functional outcomes did not significantly differ between TAFI-/- mice and controls (p>0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. TAFI deficiency also had no influence on intracerebral fibrin(ogen) formation after tMCAO. Our study shows that TAFI does not play a major role for thrombus formation and neuronal degeneration after ischemic brain challenge.