@article{GoeritzerKuentzelBecketal.2023, author = {Goeritzer, Madeleine and Kuentzel, Katharina B. and Beck, Sarah and Korbelius, Melanie and Rainer, Silvia and Bradić, Ivan and Kolb, Dagmar and Mussbacher, Marion and Schrottmaier, Waltraud C. and Assinger, Alice and Schlagenhauf, Axel and Rost, Ren{\´e} and Gottschalk, Benjamin and Eichmann, Thomas O. and Z{\"u}llig, Thomas and Graier, Wolfgang F. and Vujić, Nemanja and Kratky, Dagmar}, title = {Monoglyceride lipase deficiency is associated with altered thrombogenesis in mice}, series = {International Journal of Molecular Sciences}, volume = {24}, journal = {International Journal of Molecular Sciences}, number = {4}, issn = {1422-0067}, doi = {10.3390/ijms24043116}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304052}, year = {2023}, abstract = {Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl\(^{-/-}\)) and platelet-specific Mgl-deficient (platMgl\(^{-/-}\)) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl\(_3\)-induced injury was markedly reduced in Mgl\(^{-/-}\) mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl\(^{-/-}\) mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl\(^{-/-}\) mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.}, language = {en} } @phdthesis{GoebneeKlaus2023, author = {G{\"o}b [n{\´e}e Klaus], Vanessa Aline Domenica}, title = {Pathomechanisms underlying ischemic stroke}, doi = {10.25972/OPUS-28672}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286727}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Every year, stroke affects over 100 million people worldwide and the number of cases continues to grow. Ischemic stroke is the most prevalent form of stroke and rapid restoration of blood flow is the primary therapeutic aim. However, recanalization might fail or reperfusion itself induces detrimental processes leading to infarct progression. Previous studies identified platelets and immune cells as drivers of this so-called ischemia/reperfusion (I/R) injury, establishing the concept of ischemic stroke as thrombo-inflammatory disease. Reduced cerebral blood flow despite recanalization promoted the hypothesis that thrombus formation within the cerebral microcirculation induces further tissue damage. The results presented in this thesis refute this: using complementary methodologies, it was shown that infarct growth precedes the occurrence of thrombi excluding them as I/R injury-underlying cause. Blood brain barrier disruption is one of the hallmarks of ischemic stroke pathology and was confirmed as early event during reperfusion injury in the second part of this study. Abolished platelet α-granule release protects mice from vascular leakage in the early reperfusion phase resulting in smaller infarcts. Using in vitro assays, platelet α-granule-derived PDGF-AB was identified as one factor contributing to blood-brain barrier disruption. In vivo visualization of platelet activation would provide important insights in the spatio-temporal context of platelet activation in stroke pathology. As platelet signaling results in elevated intracellular Ca2+ levels, this is an ideal readout. To overcome the limitations of chemical calcium indicators, a mouse line expressing an endogenous calcium reporter specifically in platelets and megakaryocytes was generated. Presence of the reporter did not interfere with platelet function, consequently these mice were characterized in in vivo and ex vivo models. Upon ischemic stroke, neutrophils are among the first cells that are recruited to the brain. Since for neutrophils both, beneficial and detrimental effects are described, their role was investigated within this thesis. Neither neutrophil depletion nor absence of NADPH-dependent ROS production (Ncf-/- mice) affected stroke outcome. In contrast, abolished NET-formation in Pad4-/- mice resulted in reduced infarct sizes, revealing detrimental effects of NETosis in the context of ischemic stroke, which might become a potential therapeutic target. Cerebral venous (sinus) thrombosis, CV(S)T is a rare type of stroke with mainly idiopathic onset. Whereas for arterial thrombosis a critical contribution of platelets is known and widely accepted, for venous thrombosis this is less clear but considered more and more. In the last part of this thesis, it was shown that fab-fragments of the anti-CLEC-2 antibody INU1 trigger pathological platelet activation in vivo, resulting in foudroyant CVT accompanied by heavy neurological symptoms. Using this novel animal model for CVT, cooperative signaling of the two platelet receptors CLEC-2 and GPIIb/IIIa was revealed as major trigger of CVT and potential target for treatment.}, subject = {Schlaganfall}, language = {en} } @article{NeagoeGardinerStegneretal.2022, author = {Neagoe, Raluca A. I. and Gardiner, Elizabeth E. and Stegner, David and Nieswandt, Bernhard and Watson, Steve P. and Poulter, Natalie S.}, title = {Rac inhibition causes impaired GPVI signalling in human platelets through GPVI shedding and reduction in PLCγ2 phosphorylation}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {7}, issn = {1422-0067}, doi = {10.3390/ijms23073746}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284350}, year = {2022}, abstract = {Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1's mode of action differs between the two species.}, language = {en} } @article{VogelsangEichlerHuntemannetal.2021, author = {Vogelsang, Anna and Eichler, Susann and Huntemann, Niklas and Masanneck, Lars and B{\"o}hnlein, Hannes and Sch{\"u}ngel, Lisa and Willison, Alice and Loser, Karin and Nieswandt, Bernhard and Kehrel, Beate E. and Zarbock, Alexander and G{\"o}bel, Kerstin and Meuth, Sven G.}, title = {Platelet inhibition by low-dose acetylsalicylic acid reduces neuroinflammation in an animal model of multiple sclerosis}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {18}, issn = {1422-0067}, doi = {10.3390/ijms22189915}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284535}, year = {2021}, abstract = {Aside from the established immune-mediated etiology of multiple sclerosis (MS), compelling evidence implicates platelets as important players in disease pathogenesis. Specifically, numerous studies have highlighted that activated platelets promote the central nervous system (CNS)-directed adaptive immune response early in the disease course. Platelets, therefore, present a novel opportunity for modulating the neuroinflammatory process that characterizes MS. We hypothesized that the well-known antiplatelet agent acetylsalicylic acid (ASA) could inhibit neuroinflammation by affecting platelets if applied at low-dose and investigated its effect during experimental autoimmune encephalomyelitis (EAE) as a model to study MS. We found that oral administration of low-dose ASA alleviates symptoms of EAE accompanied by reduced inflammatory infiltrates and less extensive demyelination. Remarkably, the percentage of CNS-infiltrated CD4\(^+\) T cells, the major drivers of neuroinflammation, was decreased to 40.98 ± 3.28\% in ASA-treated mice compared to 56.11 ± 1.46\% in control animals at the disease maximum as revealed by flow cytometry. More interestingly, plasma levels of thromboxane A\(_2\) were decreased, while concentrations of platelet factor 4 and glycoprotein VI were not affected by low-dose ASA treatment. Overall, we demonstrate that low-dose ASA could ameliorate the platelet-dependent neuroinflammatory response in vivo, thus indicating a potential treatment approach for MS.}, language = {en} } @article{MammadovaBachBraun2019, author = {Mammadova-Bach, Elmina and Braun, Attila}, title = {Zinc homeostasis in platelet-related diseases}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {21}, issn = {1422-0067}, doi = {10.3390/ijms20215258}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285554}, year = {2019}, abstract = {Zn\(^{2+}\) deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn\(^{2+}\)-deficient diets, accounting for 1-4\% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn\(^{2+}\) deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn\(^{2+}\) status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn\(^{2+}\) uptake in the gut using different nutritional supplementation of Zn\(^{2+}\) could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn\(^{2+}\) diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn\(^{2+}\) in hemostasis. Storage protein metallothionein maintains or releases Zn\(^{2+}\) in the cytoplasm, and the dynamic change of this cytoplasmic Zn\(^{2+}\) pool is regulated by the redox status of the cell. An increase of labile Zn\(^{2+}\) pool can be toxic for the cells, and therefore cytoplasmic Zn\(^{2+}\) levels are tightly regulated by several Zn\(^{2+}\) transporters located on the cell surface and also on the intracellular membrane of Zn\(^{2+}\) storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn\(^{2+}\) is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn\(^{2+}\) transport and the physiological role of Zn\(^{2+}\) store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn\(^{2+}\) to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn\(^{2+}\) homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.}, language = {en} } @article{UngernSternbergZerneckeSeizer2018, author = {Ungern-Sternberg, Saskia N. I. von and Zernecke, Alma and Seizer, Peter}, title = {Extracellular matrix metalloproteinase inducer EMMPRIN (CD147) in cardiovascular disease}, series = {International Journal of Molecular Sciences}, volume = {19}, journal = {International Journal of Molecular Sciences}, number = {2}, issn = {1422-0067}, doi = {10.3390/ijms19020507}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285014}, year = {2018}, abstract = {The receptor EMMPRIN is involved in the development and progression of cardiovascular diseases and in the pathogenesis of myocardial infarction. There are several binding partners of EMMPRIN mediating the effects of EMMPRIN in cardiovascular diseases. EMMPRIN interaction with most binding partners leads to disease progression by mediating cytokine or chemokine release, the activation of platelets and monocytes, as well as the formation of monocyte-platelet aggregates (MPAs). EMMPRIN is also involved in atherosclerosis by mediating the infiltration of pro-inflammatory cells. There is also evidence that EMMPRIN controls energy metabolism of cells and that EMMPRIN binding partners modulate intracellular glycosylation and trafficking of EMMPRIN towards the cell membrane. In this review, we systematically discuss these multifaceted roles of EMMPRIN and its interaction partners, such as Cyclophilins, in cardiovascular disease.}, language = {en} } @phdthesis{Aigner2023, author = {Aigner, Max}, title = {Establishing successful protocols and imaging pipelines for Expansion Microscopy in murine blood platelets}, doi = {10.25972/OPUS-30900}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-309003}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Platelets play an important role in the body, since they are part of the hemostasis system, preventing and stopping blood loss. Nevertheless, when platelet or coagulation system function are impaired, uncontrolled bleedings but also irreversible vessel occlusion followed by ischemic tissue damage can occur. Therefore, understanding platelet function and activation, mechanisms which are controlled by a variety of platelet membrane receptors and other factors is important to advance out knowledge of hemostasis and platelet malfunction. For a complete picture of platelet function and their modulating behavior it is desired to be able to quantify receptor distributions and interactions of these densely packed molecular ensembles in the membrane. This challenges scientists for several reasons. Most importantly, platelets are microscopically small objects, challenging the spatial resolution of conventional light microscopy. Moreover, platelet receptors are highly abundant on the membrane so even super-resolution microscopy struggles with quantitative receptor imaging on platelets. With Expansion microscopy (ExM), a new super-resolution technique was introduced, allowing resolutions to achieve super-resolution without using a super-resolution microscope, but by combining a conventional confocal microscopy with a highly processed sample that has been expanded physically. In this doctoral thesis, I evaluated the potential of this technique for super-resolution platelet imaging by optimizing the sample preparation process and establishing an imaging and image processing pipeline for dual-color 3D images of different membrane receptors. The analysis of receptor colocalization using ExM demonstrated a clear superiority compared to conventional microscopy. Furthermore, I identified a library of fluorescently labeled antibodies against different platelet receptors compatible with ExM and showed the possibility of staining membrane receptors and parts of the cytoskeleton at the same time.}, subject = {Mikroskopie}, language = {en} } @article{RosaButtHopperetal.2022, author = {Rosa, Annabelle and Butt, Elke and Hopper, Christopher P. and Loroch, Stefan and Bender, Markus and Schulze, Harald and Sickmann, Albert and Vorlova, Sandra and Seizer, Peter and Heinzmann, David and Zernecke, Alma}, title = {Cyclophilin a is not acetylated at lysine-82 and lysine-125 in resting and stimulated platelets}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {3}, issn = {1422-0067}, doi = {10.3390/ijms23031469}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284011}, year = {2022}, abstract = {Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed—neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.}, language = {en} } @phdthesis{Maier2023, author = {Maier, Sophia Edith}, title = {Mapping membrane receptor distribution on resting platelets combining Expansion Microscopy and fluorescence confocal microscopy}, doi = {10.25972/OPUS-30031}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300317}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Stroke and myocardial infarction are the most prominent and severe consequences of pathological thrombus formation. For prevention and/or treatment of thrombotic events there is a variety of anti-coagulation and antiplatelet medication that all have one side effect in common: the increased risk of bleeding. To design drugs that only intervene in the unwanted aggregation process but do not disturb general hemostasis, it is crucial to decipher the exact clotting pathway which has not been fully understood yet. Platelet membrane receptors play a vital role in the clotting pathway and, thus, the aim of this work is to establish a method to elucidate the interactions, clustering, and reorganization of involved membrane receptors such as GPIIb/IIIa and GPIX as part of the GPIb-IX-V complex. The special challenges regarding visualizing membrane receptor interactions on blood platelets are the high abundancy of the first and the small size of the latter (1—3µm of diameter). The resolution limit of conventional fluorescence microscopy and even super-resolution approaches prevents the successful differentiation of densely packed receptors from one another. Here, this issue is approached with the combination of a recently developed technique called Expansion Microscopy (ExM). The image resolution of a conventional fluorescence microscope is enhanced by simply enlarging the sample physically and thus pulling the receptors apart from each other. This method requires a complex sample preparation and holds lots of obstacles such as variable or anisotropic expansion and low images contrast. To increase ExM accuracy and sensitivity for interrogating blood platelets, it needs optimized sample preparation as well as image analysis pipelines which are the main part of this thesis. The colocalization results show that either fourfold or tenfold expanded, resting platelets allow a clear distinction between dependent, clustered, and independent receptor organizations compared to unexpanded platelets.Combining dual-color Expansion and confocal fluorescence microscopy enables to image in the nanometer range identifying GPIIb/IIIa clustering in resting platelets - a pattern that may play a key role in the clotting pathway}, language = {en} } @phdthesis{Gross2022, author = {Gross, Carina}, title = {The role of platelets in hepatic ischemia reperfusion injury in mice}, doi = {10.25972/OPUS-21618}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216180}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Platelets are the second most abundant blood cells and their main function is maintenance of vascular integrity. In addition, platelets are increasingly recognized as cells with immune functions, as they participate in the recruitment of immune cells and modulate the progression and severity of an immune response. So-called lipid mediators, which are - besides other cells - released by activated platelets, influence the immune response. LTB4 is one of these potent lipid mediators and is able to activate neutrophils and induce their infiltration into injured tissue. In order to investigate the involvement of platelets in inflammatory processes, a murine model of hepatic ischemia reperfusion injury as well as confocal intravital microscopy of the liver were established. Both methods were used to analyze the influence of platelets on the inflammation that follows sterile liver inflammation. We found platelet function to be unaltered after three hours of reperfusion and platelet aggregation to be irrelevant for the outcome of hepatic ischemia reperfusion injury. However, a strong impact of the GPIb-vWF axis could be observed, as antibody mediated blockade of GPIb as well as vWF-deficiency significantly reduced liver damage markers and decreased neutrophil infiltration. GPIb-IL-4R mice were used to exclude the possibility that the protective effects of the anti-GPIbα antibody treatment (p0p/B) results from something else than blocking GPIbα. Furthermore, the slope of neutrophil infiltration was decreased in p0p/B-treated mice, leading to overall decreased neutrophil numbers in the liver after three hours of reperfusion. Blockade of the integrin αIIbβ3, however, showed no reduction in neutrophil infiltration into the post-ischemic liver, in line with unaltered liver damage. To study the role of leukotriene B4, conditional and constitutive knockout mice for the LTA4 hydrolase, which catalyzes the last step in LTB4 synthesis, were generated. Lta4h deficiency did not affect general platelet functionality in hemostasis and thrombosis. Interestingly, Lta4h-/- mice were not protected from cellular damage following hepatic ischemia, despite lower neutrophil numbers in the post-ischemic liver. Intravital microscopy of the pancreas was established and revealed increased CD4+ T cell numbers in GPVI-deficient animals compared to WT controls in line with the pre-diabetic phenotype of Gp6-/- mice that was revealed in Grzegorz Sumara's group. Furthermore, platelet 'behavior' in pancreatic islets was observed following glucose injection. We found a high number of platelets adherent to islet sinusoids under basal conditions and no rolling/decelerating of platelets following glucose injection. This was accompanied by temporary sinusoidal constriction and stop of the blood flow. This phenomenon was not observed in control settings (injection of PBS, insulin or L-glucose). In a side project, which was carried out jointly with Tobias Heib, a side by side comparison of the classical syringe-based flushing and the centrifugation-based spinning method to isolate murine bone marrow was conducted. Flow cytometry revealed no differences in the distribution of hematopoietic stem cells and immune cells and functional analysis with primary and cultured megakaryocytes (MKs) showed comparable results in all conducted assays. Thus, our data demonstrated that the faster and more efficient spinning method can be used for the isolation of bone marrow cells.}, language = {en} } @article{EssigBabilonVollmuthetal.2021, author = {Essig, Fabian and Babilon, Lilith and Vollmuth, Christoph and Kollikowski, Alexander M. and Pham, Mirko and Solymosi, L{\´a}szl{\´o} and Haeusler, Karl Georg and Kraft, Peter and Stoll, Guido and Schuhmann, Michael K.}, title = {High mobility group box 1 protein in cerebral thromboemboli}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {20}, issn = {1422-0067}, doi = {10.3390/ijms222011276}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265568}, year = {2021}, abstract = {High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7\% (IQR 0.6-6.2\%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke.}, language = {en} } @phdthesis{Becker2021, author = {Becker, Isabelle Carlotta}, title = {The role of megakaryocytes and platelets in vascular and osteogenic development}, doi = {10.25972/OPUS-21024}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210241}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Platelets, small anucleate cell fragments in the blood stream, derive from large precursor cells, so-called megakaryocytes (MK) residing in the bone marrow (BM). In addition to their role in wound healing, platelets have been shown to play a significant role during inflammatory bleeding. Above all, the immunoreceptor tyrosine-based activation motif (ITAM) receptors GPVI as well as CLEC-2 have been identified as main regulators of vascular integrity. In addition to ITAM-bearing receptors, our group identified GPV as another potent regulator of hemostasis and thrombosis. Surprisingly, concomitant lack of GPV and CLEC-2 deteriorated blood-lymphatic misconnections observed in Clec2-/- mice resulting in severe edema formation and intestinal inflammation. Analysis of lymphatic and vascular development in embryonic mesenteries revealed severely defective blood-lymph-vessel separation, which translated into thrombocytopenia and increased vascular permeability due to reduced tight junction density in mesenteric blood vessels and consequent leakage of blood into the peritoneal cavity. Recently, platelet granule release has been proposed to ameliorate the progression of retinopathy of prematurity (ROP), a fatal disease in newborns leading to retinal degradation. The mechanisms governing platelet activation in this process remained elusive nonetheless, which prompted us to investigate a possible role of ITAM signaling. In the second part of this thesis, granule release during ROP was shown to be GPVI- and partly CLEC-2-triggered since blockade or loss of these receptors markedly deteriorated ROP progression. Proplatelet formation from MKs is highly dependent on a functional microtubule and actin cytoskeleton, the latter of which is regulated by several actin-monomer binding proteins including Cofilin1 and Twinfilin1 that have been associated with actin-severing at pointed ends. In the present study, a redundancy between both proteins especially important for the guided release of proplatelets into the bloodstream was identified, since deficiency in both proteins markedly impaired MK functionality mainly due to altered actin-microtubule crosstalk. Besides ITAM-triggered activation, platelets and MKs are dependent on inhibitory receptors, which prevent overshooting activation. We here identified macrothrombocytopenic mice with a mutation within Mpig6b encoding the ITIM-bearing receptor G6b-B. G6b-B-mutant mice developed a severe myelofibrosis associated with sex-specific bone remodeling defects resulting in osteosclerosis and -porosis in female mice. Moreover, G6b-B was shown to be indispensable for MK maturation as verified by a significant reduction in MK-specific gene expression in G6b-B-mutant MKs due to reduced GATA-1 activity.}, subject = {Megakaryozyt}, language = {en} } @phdthesis{Heck2019, author = {Heck, Johannes}, title = {Role of cyclase-associated protein 2 in platelet function and description of an inherited macrothrombocytopenia}, doi = {10.25972/OPUS-17996}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179968}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Cyclase-associated protein (CAP)2 is an evolutionarily highly conserved actin-binding protein implicated in striated muscle development, carcinogenesis, and wound healing in mammals. To date, the presence as well as the putative role(s) of CAP2 in platelets, however, remain unknown. Therefore, mice constitutively lacking CAP2 (Cap2gt/gt mice) were examined for platelet function. These studies confirmed the presence of both mammalian CAP isoforms, CAP1 and CAP2, in platelets. CAP2-deficient platelets were slightly larger than WT controls and displayed increased GPIIbIIIa activation and P-selectin recruitment in response to the (hem)ITAM-specific agonists collagen-related peptide and rhodocytin. However, spreading of CAP2-deficient platelets on a fibrinogen matrix was unaltered. In conclusion, the functionally redundant CAP1 isoform may compensate for the lack of CAP2 in murine platelets. Moreover, the studies presented in this thesis unveiled a severe macrothrombocytopenia that occurred independently of the targeted Cap2 allele and which was preliminarily termed orphan (orph). Crossing of the respective mice to C57BL/6J wild-type animals revealed an autosomal recessive inheritance. Orph mice were anemic and developed splenomegaly as well as BM fibrosis, suggesting a general hematopoietic defect. Strikingly, BM MKs of orph mice demonstrated an aberrant morphology and appeared to release platelets ectopically into the BM cavity, thus pointing to defective thrombopoiesis as cause for the low platelet counts. Orph platelets exhibited marked activation defects and spread poorly on fibrinogen. The unaltered protein content strongly suggested a defective alpha-granule release to account for the observed hyporesponsiveness. In addition, the cytoskeleton of orph platelets was characterized by disorganized microtubules and accumulations of filamentous actin. However, further experiments are required to elucidate the activation defects and cytoskeletal abnormalities in orph platelets. Above all, the gene mutation responsible for the phenotype of orph mice needs to be determined by next-generation sequencing in order to shed light on the underlying genetic and mechanistic cause.}, subject = {Thrombozyt}, language = {en} } @phdthesis{Semeniak2018, author = {Semeniak, Daniela}, title = {Role of bone marrow extracellular matrix proteins on platelet biogenesis and function}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155857}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis. First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level. Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice. In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation. Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.}, subject = {Knochenmark}, language = {en} } @article{GambaryanSubramanianKehreretal.2016, author = {Gambaryan, Stepan and Subramanian, Hariharan and Kehrer, Linda and Mindukshev, Igor and Sudnitsyna, Julia and Reiss, Cora and Rukoyatkina, Natalia and Friebe, Andreas and Sharina, Iraida and Martin, Emil and Walter, Ulrich}, title = {Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis}, series = {Cell Communication and Signaling}, volume = {14}, journal = {Cell Communication and Signaling}, number = {16}, doi = {10.1186/s12964-016-0139-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161223}, year = {2016}, abstract = {Background Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets. Methods To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni's test and Student's t-test were used as appropriate. Results We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite. Conclusions All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.}, language = {en} } @article{KoesslerSchwarzWeberetal.2017, author = {Koessler, Juergen and Schwarz, Michaela and Weber, Katja and Etzel, Julia and Koessler, Angela and Boeck, Markus and Kobsar, Anna}, title = {The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {11}, doi = {10.1371/journal.pone.0188193}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159879}, pages = {e0188193}, year = {2017}, abstract = {Background: Platelets are important for effective hemostasis and considered to be involved in pathophysiological processes, e.g. in cardiovascular diseases. Platelets provided for research or for therapeutic use are frequently separated from citrated whole blood (WB) stored for different periods of time. Although functionally intact platelets are required, the stability of platelet integrity, e.g. adenosine diphosphate (ADP) mediated responsiveness, has never been thoroughly investigated in citrated WB under ex vivo conditions. Objectives: Platelet integrity was evaluated at different time points in citrated WB units, collected from healthy donors and stored for 5 days at ambient temperature. The analysis included the measurement of activation markers, of induced light transmission aggregometry and of purinergic receptor expression or function. Inhibitory pathways were explored by determination of basal vasodilator-stimulated phosphoprotein (VASP)-phosphorylation, intracellular cyclic nucleotide levels and the content of phosphodiesterase 5A. Fresh peripheral blood (PB) samples served as controls. Results: On day 5 of storage, thrombin receptor activating peptide-6 (TRAP-6) stimulated CD62P expression and fibrinogen binding were comparable to PB samples. ADP induced aggregation continuously decreased during storage. Purinergic receptor expression remained unchanged, whereas the P2Y1 activity progressively declined in contrast to preserved P2Y12 and P2X1 function. Inhibitory pathways were unaffected except for a slight elevation of VASP phosphorylation at Ser\(^{239}\) on day 5. Conclusion: After 5 days of storage in citrated WB, platelet responsiveness to TRAP-6 is sufficiently maintained. However, ADP-mediated platelet integrity is more sensitive to deterioration, especially after storage for more than 2 days. Decreasing ADP-induced aggregation is particularly caused by the impairment of the purinergic receptor P2Y1 activity. These characteristics should be considered in the use of platelets from stored citrated WB for experimental or therapeutic issues.}, language = {en} } @article{KoesslerHermannWeberetal.2016, author = {Koessler, Juergen and Hermann, Stephanie and Weber, Katja and Koessler, Angela and Kuhn, Sabine and Boeck, Markus and Kobsar, Anna}, title = {Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {1}, doi = {10.1371/journal.pone.0147370}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146400}, pages = {e0147370}, year = {2016}, abstract = {Background Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied. Objectives Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation. Results In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets. Conclusion In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.}, language = {en} } @article{WolfBraunHainingetal.2016, author = {Wolf, Karen and Braun, Attila and Haining, Elizabeth J. and Tseng, Yu-Lun and Kraft, Peter and Schuhmann, Michael K. and Gotru, Sanjeev K. and Chen, Wenchun and Hermanns, Heike M. and Stoll, Guido and Lesch, Klaus-Peter and Nieswandt, Bernhard}, title = {Partially Defective Store Operated Calcium Entry and Hem(ITAM) Signaling in Platelets of Serotonin Transporter Deficient Mice}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {1}, doi = {10.1371/journal.pone.0147664}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146399}, pages = {e0147664}, year = {2016}, abstract = {Background Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation. Objective To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke. Results In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice. Conclusion Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.}, language = {en} } @article{KraftDeMeyerKleinschnitz2012, author = {Kraft, Peter and De Meyer, Simon F. and Kleinschnitz, Christoph}, title = {Next-Generation Antithrombotics in Ischemic Stroke: Preclinical Perspective on 'Bleeding-Free Antithrombosis'}, series = {Journal of Cerebral Blood Flow and Metabolism}, volume = {32}, journal = {Journal of Cerebral Blood Flow and Metabolism}, number = {10}, doi = {10.1038/jcbfm.2012.108}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126538}, pages = {1831-1840}, year = {2012}, abstract = {The present antithrombotic drugs used to treat or prevent ischemic stroke have significant limitations: either they show only moderate efficacy (platelet inhibitors), or they significantly increase the risk for hemorrhages (thrombolytics, anticoagulants). Although most strokes are caused by thrombotic or embolic vessel occlusions, the pathophysiological role of platelets and coagulation is largely unclear. The introduction of novel transgenic mouse models and specific coagulation inhibitors facilitated a detailed analysis of molecular pathways mediating thrombus formation in models of acute ischemic stroke. Prevention of early platelet adhesion to the damaged vessel wall by blocking platelet surface receptors glycoprotein Ib alpha (GPIbα) or glycoprotein VI (GPVI) protects from stroke without provoking bleeding complications. In addition, downstream signaling of GPIbα and GPVI has a key role in platelet calcium homeostasis and activation. Finally, the intrinsic coagulation cascade, activated by coagulation factor XII (FXII), has only recently been identified as another important mediator of thrombosis in cerebrovascular disease, thereby disproving established concepts. This review summarizes the latest insights into the pathophysiology of thrombus formation in the ischemic brain. Potential clinical merits of novel platelet inhibitors and anticoagulants as powerful and safe tools to combat ischemic stroke are discussed.}, language = {en} } @article{PoppThielmanNieswandtetal.2015, author = {Popp, Michael and Thielman, Ina and Nieswandt, Bernhard and Stegner, David}, title = {Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5)}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0133429}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125724}, pages = {e0133429}, year = {2015}, abstract = {Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice.}, language = {en} } @article{HofmannBraunPozgajetal.2014, author = {Hofmann, Sebastian and Braun, Attila and Pozgaj, Rastislav and Morowski, Martina and V{\"o}gtle, Timo and Nieswandt, Bernhard}, title = {Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis}, series = {PLoS One}, volume = {9}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0115306}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126477}, pages = {e115306}, year = {2014}, abstract = {Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. \(Cd84^{-/-}\) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of \(Cd84^{-/-}\) mice. In vivo, \(Cd84^{-/-}\) mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.}, language = {en} } @article{NavdaevSubramanianPetuninetal.2014, author = {Navdaev, Alexey and Subramanian, Hariharan and Petunin, Alexey and Clemetson, Kenneth J. and Gambaryan, Stepan and Walter, Ulrich}, title = {Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {4}, issn = {1932-6203}, doi = {10.1371/journal.pone.0093569}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119815}, pages = {e93569}, year = {2014}, abstract = {von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.}, language = {en} } @article{KraftDrechslerGunrebenetal.2014, author = {Kraft, Peter and Drechsler, Christiane and Gunreben, Ignaz and Nieswandt, Bernhard and Stoll, Guido and Heuschmann, Peter Ulrich and Kleinschnitz, Christoph}, title = {Von Willebrand Factor Regulation in Patients with Acute and Chronic Cerebrovascular Disease: A Pilot, Case-Control Study}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {6}, issn = {1932-6203}, doi = {10.1371/journal.pone.0099851}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119588}, pages = {e99851}, year = {2014}, abstract = {Background and Purpose In animal models, von Willebrand factor (VWF) is involved in thrombus formation and propagation of ischemic stroke. However, the pathophysiological relevance of this molecule in humans, and its potential use as a biomarker for the risk and severity of ischemic stroke remains unclear. This study had two aims: to identify predictors of altered VWF levels and to examine whether VWF levels differ between acute cerebrovascular events and chronic cerebrovascular disease (CCD). Methods A case-control study was undertaken between 2010 and 2013 at our University clinic. In total, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HV) were included. Blood was taken at days 0, 1, and 3 in patients with AIS or TIA, and once in CCD patients and HV. VWF serum levels were measured and correlated with demographic and clinical parameters by multivariate linear regression and ANOVA. Results Patients with CCD (158±46\%) had significantly higher VWF levels than HV (113±36\%, P<0.001), but lower levels than AIS/TIA patients (200±95\%, P<0.001). Age, sex, and stroke severity influenced VWF levels (P<0.05). Conclusions VWF levels differed across disease subtypes and patient characteristics. Our study confirms increased VWF levels as a risk factor for cerebrovascular disease and, moreover, suggests that it may represent a potential biomarker for stroke severity, warranting further investigation.}, language = {en} } @phdthesis{Hofmann2013, author = {Hofmann, Sebastian}, title = {Studies on the function and regulation of CD84, GPVI and Orai2 in genetically modified mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87949}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Platelet activation and aggregation at sites of vascular injury are essential processes to limit blood loss but they also contribute to arterial thrombosis, which can lead to myocardial infarction and stroke. Stable thrombus formation requires a series of events involving platelet receptors which contribute to adhesion, activation and aggregation of platelets. Regulation of receptor expression by (metallo-)proteinases has been described for several platelet receptors, but the molecular mechanisms are ill-defined. The signaling lymphocyte activation molecule (SLAM) family member CD84 is expressed in immune cells and platelets, however its role in platelet physiology was unclear. In this thesis, CD84 deficient mice were generated and analyzed. In well established in vitro and in vivo assays testing platelet function and thrombus formation, CD84 deficient mice displayed phenotypes indistinguishable from wild-type controls. It was concluded that CD84 in platelets does not function as modulator of thrombus formation, but rather has other functions. In line with this, in the second part of this thesis, a novel regulation mechanism for platelet CD84 was discovered and elucidated. Upon platelet activation, the N-terminus of CD84 was found to be cleaved exclusively by the a disintegrin and metalloproteinase 10 (ADAM10), whereas the intracellular part was cleaved by calpain. In addition, regulation of the platelet activating collagen receptor glycoprotein VI (GPVI) was studied and it was shown that GPVI is in contrast to CD84 differentially regulated by ADAM10 and ADAM17. A novel role of CD84 under pathophysiological conditions was revealed as CD84 deficient mice were protected from ischemic stroke in the model of transient middle cerebral artery occlusion and this protection was based on the lack of CD84 in T cells. Ca2+ is an essential second messenger that facilitates activation of platelets and diverse functions in different eukaryotic cell types. Store-operated Ca2+ entry (SOCE) represents the major mechanism leading to rise in intracellular Ca2+ concentration in non-excitable cells. The Ca2+ sensor STIM1 (stromal interaction molecule 1) and the SOC channel subunit protein Orai1 are established mediators of SOCE in platelets. STIM2 is the major STIM isoform in neurons, but the role of the SOC channel subunit protein Orai2 in platelets and neurons has remained elusive. In the third part of this thesis, Orai2 deficient mice were generated and analyzed. Orai2 was dispensable for platelet function, however, Orai2 deficient mice were protected from ischemic neurodegeneration and this phenotype was attributed to defective SOCE in neurons.}, subject = {Thrombozyt}, language = {en} } @article{NieswandtMorowskiBrachsetal.2014, author = {Nieswandt, Bernhard and Morowski, Martina and Brachs, Sebastian and Mielenz, Dirk and D{\"u}tting, Sebastian}, title = {The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice}, doi = {10.1371/journal.pone.0107139}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113316}, year = {2014}, abstract = {Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss.}, language = {en} } @phdthesis{Wangorsch2013, author = {Wangorsch, Gaby}, title = {Mathematical modeling of cellular signal transduction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87746}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {A subtly regulated and controlled course of cellular processes is essential for the healthy functioning not only of single cells, but also of organs being constituted thereof. In return, this entails the proper functioning of the whole organism. This implies a complex intra- and inter-cellular communication and signal processing that require equally multi-faceted methods to describe and investigate the underlying processes. Within the scope of this thesis, mathematical modeling of cellular signaling finds its application in the analysis of cellular processes and signaling cascades in different organisms. ...}, subject = {Mathematische Modellierung}, language = {en} } @phdthesis{Mischnik2013, author = {Mischnik, Marcel}, title = {Systembiologische Analyse der ADP- und Prostaglandin-vermittelten Signaltransduktion humaner Thrombozyten}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78807}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Thrombozyten (Blutpl{\"a}ttchen) sind die Vermittler der zellul{\"a}ren H{\"a}mostase. Ihre F{\"a}higkeit zu Aggregieren und sich an das umgebende Gewebe verletzter Blutgef{\"a}sse anzulagern, wird durch ein komplexes intrazellul{\"a}res Signaltransduktionsnetzwerk bestimmt, das sowohl aktivierende, als auch inhibierende Subnetzwerke beinhaltet. Das Verst{\"a}ndnis dieser Prozesse ist von hoher medizinischer Bedeutung. Im Rahmen dieser Arbeit wurde die thrombozyt{\"a}re Signaltransduktion sowohl mittels eines Boole'schen, als auch verschiedener dynamischer Modelle analysiert. Die Boole'sche Modellierung f{\"u}hrte zu interessanten Erkenntnissen {\"u}ber das Zusammenwirken einzelner Subnetzwerke bei der Vermittlung irreversibler Pl{\"a}ttchenaktivierung und zeigte Mechanismen der Interaktion mit dem hemmenden Prostaglandinsystem auf. Das Modell beinhaltet unter Anderem wichtige Systemkomponenten wie Calciumsignalgebung, Aktivierung von Schl{\"u}sselkinasen wie Src und PKC, Integrin-vermitteltes outside-in sowie inside-out Signalgebung und autokrine ADP- und Thromboxan-Produktion. Unter Verwendung dieses Boole'schen Ansatzes wurde weiterhin das System-eigene Schwellenwertverhalten analysiert. Dabei stellte sich eine umgekehrt proportionale Abh{\"a}ngigkeit des relativen aktivierenden Reizes, der notwendig ist um den Schwellenwert zu {\"u}berschreiten, vom absoluten hemmenden Input heraus. Das System adaptiert demnach an h{\"o}here Prostaglandinkonzentrationen durch eine Erh{\"o}hung der Sensitivit{\"a}t f{\"u}r Aktivatoren wie dem van-Willebrandt-Faktor und Kollagen, und erm{\"o}glicht somit auch unter lokal hemmenden Bedingungen eine Pl{\"a}ttchen-vermittelte H{\"a}mostase. Der n{\"a}chste Schritt bestand in der Implementierung eines Differentialgleichungs-basierten Modells der thrombozyt{\"a}ren Prostaglandin-Signaltransduktion, um einen detaillierten {\"U}berblick {\"u}ber die Dynamik des inhibierenden Netzwerkteils zu erhalten. Die kinetischen Parameter dieses Modells wurden teilweise der Literatur entnommen. Der andere Teil wurde anhand einer umfassenden Kombination dosis- und zeitabh{\"a}ngiger cAMP und phospho-VASP Messdaten gesch{\"a}tzt. Der Prozess beinhaltete mehrere Iterationen aus Modellvorhersagen einerseits und experimentellem Design andererseits. Das Modell liefert die quantitativen Effekte der Prostaglandinrezeptoren IP, DP1, EP3 und EP4 und des ADP-Rezeptors P2Y12 auf die zugrunde liegende Signalkaskade. EP4 zeigt den st{\"a}rksten Effekt in der aktivierenden Fraktion, wohingegen EP3 einen st{\"a}rkeren inhibitorischen Effekt aus{\"u}bt, als der durch Clopidogrel hemmbare ADP-Rezeptor P2Y12. Weiterhin wurden die Eigenschaften des negativen feedback-loops der PKA auf den cAMP-Spiegel untersucht, und eine direkte Beeinflussung der Adenylatzyklase durch die PKA festgestellt, in Form einer Reduzierung der maximalen katalytischen Geschwindigkeit. Die Identifizierbarkeit der gesch{\"a}tzten Parameter wurde mittels profile-Likelihood-Sch{\"a}tzung untersucht. In einem dritten Schritt wurde ein sowohl die aktivierenden, als auch die hemmenden Netzwerkteile umfassendes dynamisches Modell implementiert. Die Topologie dieses Modells wurde in Anlehnung an die des Boole'schen Modells auf der Basis von a priori Wissen festgelegt. Die Modellparameter wurden anhand von Western-Blot, Calcium- und Aggregationsmessungen gesch{\"a}tzt. Auch hier wurde die Identifizierbarkeit der Modellparameter durch profile-likelihood-Sch{\"a}tzung {\"u}berpr{\"u}ft. Die bei niedrigen Ligandenkonzentrationen auftretende Reversibilit{\"a}t der Pl{\"a}ttchen-Aggregation konnte mittels dieses Modells reproduziert werden. Jedoch zeigte sich bei mittleren ADP-Konzentrationen ein Fließgleichgewicht in einem teilweise aktivierten Zustand, und damit kein bistabiles Schwellenwertverhalten. Inwiefern dieses Verhalten durch einen Umgebungs-basierteren Mechanismus des Alles-Oder-Nichts-Verhaltens begr{\"u}ndet wird, bei dem der {\"U}bergang von reversibler zu irreversibler Aggregation mehr durch parakrine Effekte des gesammten Thrombus bestimmt wird, als durch spezifische Signaltransduktionseigenschaften der einzelnen Zelle, m{\"u}ssen zuk{\"u}nftige Experimente zeigen. Insgesamt geben die erstellten Modelle interessante Einblicke in die Funktionsweise der Thrombozyten und erm{\"o}glichen die Simulation von pharmakologischen und genetischen Einfl{\"u}ssen, wie Rezeptormodulationen und knock-outs. Sie geben damit Implikationen zur Entstehung und Behandlung pathophysiologischer Zust{\"a}nde, und wertvolle Denkanst{\"o}ße f{\"u}r die weitere Forschung.}, subject = {Thrombozyt}, language = {de} } @phdthesis{Kramer2011, author = {Kramer, Daniel}, title = {Das Phosphatidylinositol-Transfer-Protein PITPnm2 in humanen Thrombozyten}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76638}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Die Analyse des Phosphoproteoms in ruhenden und in aktivierten humanen Pl{\"a}ttchen f{\"u}hrte zur Identifikation des PITPnm2-Proteins. Dieses Protein wird bei einer Stimulation von Thrombozyten mit dem Prostazyklinanalogon Iloprost phosphoryliert. Diese Ergebnisse gaben Anlass zu weiteren Untersuchungen zum Vorkommen und zur Funktion dieses Proteins in Thrombozyten. In der Arbeit wurde gezeigt, dass das PITPnm2-Protein das einzige Protein der PITP-Familie ist, welches in humanen Thrombozyten exprimiert wird. Die membranassoziierten Phosphatidylinositol-Transfer-Proteine PITPnm1 und PITPnm3 sind auf cDNA-Ebene nicht in Thrombozyten nachweisbar. Von den drei zur Zeit der Untersuchung bekannten Splicevarianten des PITPnm2-Proteins konnten zwei Varianten in Thrombozyten mittels RT-PCR identifiziert werden. Diese zwei Varianten unterscheiden sich durch ein unterschiedlich exprimiertes Exon, welches im zentralen Teil des Proteins liegt. Ein weiteres Spliceprodukt, dem die Aminos{\"a}uren 50 bis 328 im vorderen Teil des Proteins fehlen, wird nicht in Thrombozyten exprimiert. PITPNM2 (Splicevariante 1) wurde als Fusionsprotein mit einem sogenannten „Flag-Tag" kloniert und in Eukaryonten exprimiert (pCMV-SC-CF, Stratagene). Mit dem rekombinanten Fusionsprotein wurden gegen PITPnm2 gerichtete Antik{\"o}rper getestet. Der Vergleich mit Flag-Tag-spezifischen Antik{\"o}rpern zeigte, dass der PITPnm2-spezifische Antik{\"o}rper zahlreiche Banden detektiert und f{\"u}r weitere Untersuchungen nicht geeignet ist. Dieser PITPnm2-Klon wird auch in weiterf{\"u}hrenden Arbeiten eingesetzt werden, die sich mit der Identifizierung der Interaktionspartner dieses Proteins, der subzellul{\"a}ren Lokalisierung und der Teilnahme des Proteins an spezifischen Zell-Signalwegen besch{\"a}ftigen werden.}, subject = {Thrombozyt}, language = {de} } @phdthesis{Hubertus2012, author = {Hubertus, Katharina}, title = {Regulation des cAMP Spiegel und der Signaltransduktion in humanen Thrombozyten durch Prostanoid-Rezeptoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71996}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Prostanoide wirken {\"u}ber Prostanoid-Rezeptoren auf die Aktivierung oder Hemmung der Thrombozyten. In dieser Arbeit wurde die Existenz und Funktionsweise der Prostanoid-Rezeptoren anhand synthetischer Agonisten und Antagonisten in humanen Thrombozyten nachgewiesen. Weiter wurde untersucht, {\"u}ber welche Prostanoid-Rezeptoren die Signaltransduktion der nat{\"u}rlichen Agonisten wie PGE2, PGE1 und PGA1 vermittelt wird, sowie das Zusammenspiel der Prostanoid-Rezeptoren auf die Aktivierung oder Hemmung der Thrombozyten gezeigt. Das Vorhandensein der Prostaglandin E2 Synthase 3 wurde nachgewiesen sowie erste Anhaltspunkte f{\"u}r die Existenz eines Komplexes aus Prostaglandin E2 Synthase 3, Hitzeschockprotein-90 sowie Casein Kinase 2 gezeigt.}, subject = {Prostaglandine}, language = {de} } @phdthesis{Neumueller2010, author = {Neum{\"u}ller, Jutta}, title = {Einfluss der Langzeittherapie mit dem Endocannabinoid-Rezeptorblocker Rimonabant auf Thrombozytenaktivierung und proinflammatorische Chemokine bei Diabetes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55045}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Die Volkskrankheit Adipositas zieht eine Reihe von kostentr{\"a}chtigen Komplikationen mit sich wie z. B. Diabetes mellitus Typ 2 und kardiovaskul{\"a}re Erkrankungen. Der Endocannabinoidblocker Rimonabant ist hierbei ein viel versprechendes Medikament, mit dem nicht nur die Adipositas an sich, sondern zus{\"a}tzlich auch ihre weit reichenden Komplikationen im kardiovaskul{\"a}ren Bereich reduziert werden k{\"o}nnen. Im Rahmen der vorliegenden Arbeit konnten an Hand 6 Monate alter diabetischer Ratten, welche f{\"u}r 10 Wochen mit Rimonabant behandelt wurden, aufgezeigt werden, dass Rimonabant auf verschiedenste Weise die Initialphase der Atherogenese positiv beeinflusst. Zum einen konnte die Anzahl der zirkulierenden Monozyten signifikant vermindert und auch die f{\"u}r die initiale Rekrutierung von Thrombozyten und Monozyten wichtigen Chemokine RANTES und MCP-1 reduziert werden. Zum anderen zeigten sich positive Effekte auf das Lipidprofil der Probanden. Ein besonderes Augenmerk lag auf dem Aktivit{\"a}tszustand der Thrombozyten: Mit Rimonabant wurde sowohl die thrombozyt{\"a}re Aktivierung minimiert als auch ein positiver Einfluss auf die Thrombozytenadh{\"a}sion und -aggregation best{\"a}tigt. Folglich reduziert Rimonabant das kardiovaskul{\"a}re Risiko, indem es die pro-inflammatorischen und pro-atherosklerotischen Kaskaden vermindert.}, subject = {Diabetes mellitus}, language = {de} } @phdthesis{Dornieden2009, author = {Dornieden, Catharina}, title = {Aktivierung humaner Thrombozyten durch Streptokokken der Gruppe B: Molekulare Mechanismen und pathophysiologische Bedeutung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-46703}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Infektionen mit Streptokokken der Gruppe B (GBS) sind immer noch die h{\"a}ufigste Ursache f{\"u}r early-onset Erkrankungen des Neugeborenen in den Industriel{\"a}ndern, die zu erh{\"o}hter neonataler Mortalit{\"a}t und Morbidit{\"a}t f{\"u}hren. Bereits bekannt ist, dass neben verschiedenen anderen Bakterien GBS durch die direkte Interaktion mit Thrombozyten eine Aggregation verursachen k{\"o}nnen. In dieser Arbeit wurde das Verhalten von septischen und kolonisierenden GBS-St{\"a}mmen verglichen. Es konnte gezeigt werden, dass alle GBS-St{\"a}mme eine thrombozyt{\"a}re Form{\"a}nderung veranlassen; jedoch f{\"u}hren nur von septischen Patienten isolierte St{\"a}mme zur Thrombozytenaggregation sowie zur P-Selektinexpression. Septische GBS-St{\"a}mme binden Fibrinogen auf ihrer Oberfl{\"a}che und induzieren die thrombozyt{\"a}re Thromboxansynthese und Granulasekretion, w{\"a}hrend kolonisierende GBS-St{\"a}mme dies nicht k{\"o}nnen. p38-mitogen-aktivierte Proteinkinase (p38-MAPK) wurde bevorzugt durch aus septischen Patienten isolierte GBS-St{\"a}mme aktiviert, ebenfalls scheint Proteinkinase C (PKC) durch diese aktiviert zu werden. Alle GBS-St{\"a}mme aktivierten Phospholipase CgammaII (PLCgammaII), Calzium-Calmodulin-abh{\"a}ngige Kinase II (CaMKII) und bewirken Myosinleichtketten (MLC)-Phosphorylierung {\"u}ber Fcgamma-Rezeptor IIA abh{\"a}ngige Signalwege. Es gibt weder einen Unterschied noch einen additiven Effekt zwischen der Aggregation induziert durch GBS und der Aggregation durch GBS und einer geringen Menge Adenosindiphosphat (ADP). Diese Kenntnisse der molekularen Mechanismen von GBS-induzierten Signalwegen in menschlichen Thrombozyten werden zu einem besseren Verst{\"a}ndnis von Bakterieneffekten auf die Thrombozytenaktivierung beitragen und k{\"o}nnen deshalb neue molekulare Ziele f{\"u}r die pharmakologische Behandlung von GBS sein.}, subject = {Thrombozyt}, language = {de} } @phdthesis{Hemberger2009, author = {Hemberger, Melinda}, title = {Verbesserung der vaskul{\"a}ren Dysfunktion bei Diabetes mellitus durch den HMG-CoA-Reduktase-Inhibitor Rosuvastatin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-35304}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Diabetes ist assoziiert mit einer endothelialen Dysfunktion sowie einer vermehrten Aktivierung von Thrombozyten. Beides erh{\"o}ht wahrscheinlich das Risiko eines kardiovaskul{\"a}ren Ereignisses. In der vorliegenden experimentellen Arbeit wurde untersucht, ob der Hydroxy-3-Methyl-Glutaryl (HMG)-CoA-Reduktase-Inhibitor Rosuvastatin zu einer Verbesserung der Endotheldysfunktion und einer Reduktion der Thrombozyten-Aktivierung im diabetischen Tiermodell beitr{\"a}gt. Zu diesem Zweck wurde m{\"a}nnlichen Wistar Ratten einmalig Beta-Zell-toxisches Streptozotocin injiziert und dadurch k{\"u}nstlich ein Diabetes mit persistierender Hyperglyk{\"a}mie erzeugt. Die Behandlung mit Rosuvastatin (20 mg/kg K{\"o}rpergewicht t{\"a}glich) beziehungsweise Placebo wurde zwei Wochen nach Induktion der Hyperglyk{\"a}mie begonnen und {\"u}ber zwei weitere Wochen fortgef{\"u}hrt. Die Gef{\"a}ßfunktion wurde anschließend an isolierten Aortensegmenten im Organbad gemessen, die Bestimmung der Thrombozyten-Aktivierung erfolgte in frischem Vollblut. Die endothelabh{\"a}ngige Relaxation der Gef{\"a}ße, induziert durch den rezeptorabh{\"a}ngigen Agonisten Acetylcholin, war in diabetischen Ratten signifikant vermindert und konnte durch die Rosuvastatin-Therapie verbessert werden. Dies ließ sich haupts{\"a}chlich auf eine deutlich reduzierte Sensitivit{\"a}t der Gef{\"a}ßmuskulatur f{\"u}r Stickstoffmonoxid (NO) zur{\"u}ck f{\"u}hren, welche bei den diabetischen Tieren durch eine gesteigerte Superoxidbildung bedingt war. Rosuvastatin reduzierte signifikant die Bildung der Sauerstoffradikalen und verbesserte dar{\"u}ber hinaus die NO-Sensitivit{\"a}t. Weiterhin konnte durch die HMG-CoA-Reduktase-Inhibition die Bindung von Fibrinogen an aktiviertes GPIIb/IIIa, sowie die P-Selektin-Expression auf der Thrombozytenoberfl{\"a}che als Marker der Thrombozyten-Degranulation reduziert werden, w{\"a}hrend diese beiden Marker der Thrombozyten-Aktivierung in der Placebo-behandelten diabetischen Versuchsgruppe erh{\"o}ht waren. Aus diesen Ergebnissen l{\"a}sst sich schlussfolgern, dass eine Behandlung mit dem HMG-CoA-Reduktase-Inhibitor Rosuvastatin bei diabetischen Ratten die Endotheldysfunktion der Gef{\"a}ße verbessert und die Aktivierung von Thrombozyten durch eine verbesserte Verf{\"u}gbarkeit des endogenen Thrombozyten-Inhibitors NO vermindert. {\"U}bertragen auf das menschliche Gef{\"a}ßsystem k{\"o}nnte dieser Effekt zu einer Verminderung kardiovaskul{\"a}rer Ereignisse durch eine Statin-Therapie bei Patienten mit Diabetes beitragen.}, subject = {Diabetes mellitus}, language = {de} } @phdthesis{Teichmann2007, author = {Teichmann, Lino Lars}, title = {Stromabw{\"a}rts der cGMP-abh{\"a}ngigen Proteinkinase in Thrombozyten - Physiologische und diagnostische Relevanz von VASP und Identifikation neuer Substrate}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-25843}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Clopidogrel hat sich als potentes Medikament zur Verhinderung kardiovaskul{\"a}rer Ereignisse sowohl bei Patienten mit akutem Koronarsyndrom ohne ST-Hebung (non-STE-ACS) als auch bei Patienten mit akutem Myokardinfarkt mit ST-Hebung (STEMI) erwiesen (CURE- und COMMIT-Studie). Insbesondere der Nutzen einer Vorbehandlung mit Clopidogrel bei perkutaner Koronarintervention ist bei Patienten mit non-STE-ACS und STEMI gut belegt (PCI-CURE- und PCI-CLARITY-Studie). Ein betr{\"a}chtlicher Anteil der Patienten zeigt allerdings kein ad{\"a}quates Ansprechverhalten auf Clopidogrel. Wir etablierten deswegen zus{\"a}tzlich zu einem bereits bestehenden FACS-Assay, der den Effekt von Clopidogrel anhand der Phosphorylierung des Proteins VASP quantitativ bestimmt, einen neuartigen auf dem gleichen Prinzip beruhenden enzyme-immuno assay (EIA). In einer Doppelblindstudie mit gesunden Probanden spiegelten im systematischen Vergleich von VASP-EIA, VASP-FACS und anderer verbreiteter Verfahren (Aggregation, p-Selektin Expresssion, PFA100) sowohl die VASP-Assays als auch die Aggregation die Pl{\"a}ttchen-Inhibition deutlich wider. Demgegen{\"u}ber waren weder die p-Selektin Expression noch der PFA100 ein Indikator f{\"u}r den Clopidogrel-Effekt. Die VASP-Assays zeichneten sich im Vergleich zur Aggregation durch bessere Quantifizierbarkeit und eine enge Abh{\"a}ngigkeit von der Zielstruktur des Clopidogrels, dem P2Y12-Rezeptor, aus. So hatte eine Medikation mit Acetylsalicyls{\"a}ure keinen Einfluss auf die VASP-Assays, verminderte allerdings die Aggregation. Weiterhin konnten wir im Rahmen der Probandenstudie durch Verwendung PAR- (protease activated receptor) spezifischer Peptide (SFFLRN, AYPGKF) und dem stabilen Thromboxan A2 Analog U46619 in ex-vivo Versuchen nachweisen, dass die antithrombozyt{\"a}ren Eigenschaften des Clopidogrels zum Teil auf der indirekten Hemmung der Thrombin- und Thromboxan-induzierten Pl{\"a}ttchenaktivierung beruhen. Diese Ergebnisse betonen die zentrale Bedeutung Galpha(i)-vermittelter Signalwege in Thrombozyten. Im zweiten Teil dieser Arbeit strebten wir an, neue Substrate der cGMP-abh{\"a}ngigen Proteinkinase zu identifizieren und das bekannte Substrat VASP weiter zu charakterisieren. Die Pl{\"a}ttchenadh{\"a}sion und -aktivierung an der Zellwand sind initiale Ereignisse der arteriellen Thrombose. Prostacyclin und NO erh{\"o}hen die intrazellul{\"a}re Konzentration zyklischer Nukleotide (cAMP, cGMP). Dies f{\"u}hrt zu einer umfassenden Inhibition der Thrombozyten, haupts{\"a}chlich durch Aktivierung der cAMP- bzw. cGMP-abh{\"a}ngige Proteinkinase (cAK, cGK). Es liegt bisher kein schl{\"u}ssiges Bild davon vor, wie die Substrate der cAK und cGK die Pl{\"a}ttcheninhibition regulieren. Wir identifizierten zun{\"a}chst das adenylylcyclase-associated protein (CAP1) anhand der Analyse des humanen Pl{\"a}ttchen-Phosphoproteoms als neues Substrat der cGK, das innerhalb von Sekunden nach SNP-Stimulation phosphoryliert wird. Die Separation des Phosphoproteoms erfolgte durch 2D-Gelelektrophorese. Wildtyp-CAP1 und Mutanten, bei denen an putativen Phosphorylierungsstellen Serin bzw. Threonin durch Alanin ausgetauscht wurde, wurden anschließend in PtK2-Zellen exprimiert. Bisher konnte die Ko-Transfektion von PtK2-Zellen mit CAP1 und cGK eine Phosphorylierung von CAP1 durch die cGK nicht best{\"a}tigen. Weitere Anstrengungen werden n{\"o}tig sein, CAP1 als cGK-Substrat zu etablieren. Flusskammerversuche zeigten, dass die Hemmung der Thrombusformation durch SNP bei VASP-defizienten M{\"a}usen im Vergleich zu Wildtyp-M{\"a}usen (WT) ineffektiv ist und belegen, dass die Inhibition der Agonisten-induzierten Thrombusformation durch den NO/cGMP-Signalweg in VASP-defizienten Pl{\"a}ttchen gest{\"o}rt ist. VASP hatte in unseren Experimenten keinen signifikanten Einfluss auf die GPIIbIIIa-Aktivierung (gemessen mit dem monoklonalen Antik{\"o}rper JON/A), die Serotonin-Sekretion (delta-Granula) und die p-Selektin-Expression (alpha-Granula). Die Ergebnisse legen nahe, dass die VASP-Deletion zu subtilen St{\"o}rungen von Thrombozytenfunktionen f{\"u}hrt, die erst in Experimenten deutlich zu Tage treten, die ihr Zusammenspiel abbilden.}, subject = {Blutstillung}, language = {de} } @phdthesis{Strehl2006, author = {Strehl, Amrei}, title = {Studies on regulation and signaling of the platelet glycoproteins GPV and GPVI}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22283}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Bei Verletzung einer Gef{\"a}ßwand kommen Blutpl{\"a}ttchen in Kontakt mit den Substanzen des Subendothels; Die Pl{\"a}ttchen werden dadurch aktiviert, sie aggregieren und verschließen die Wunde, wodurch ein hoher Blutverlust verhindert wird. Unter pathologischen Bedingungen, bei Aufbrechen eines artherosklerotischen Plaques an der Gef{\"a}ßwand, k{\"o}nnen sich jedoch große Pl{\"a}ttchenaggregate, die Thromben, formen, die das Gef{\"a}ß verschließen, den Blutfluss stoppen und somit zu Schlaganfall und Herzinfarkt f{\"u}hren k{\"o}nnen. Die kontrollierte Regulation und Signaltransduktion von bzw. durch Pl{\"a}ttchenoberfl{\"a}chenrezeptoren ist wesentlich f{\"u}r das Funktionieren der Zellen und die intakte Balance zwischen physiologischer Pl{\"a}ttchen-Aktivierung und der pathologischen Bildung eines Thrombus. In der vorliegenden Arbeit wird {\"u}ber wichtige Aspekte dieser Signalwege, die in drei Unterprojekten untersucht worden sind, berichtet. In dem ersten Unterprojekt wurde die Regulation von Pl{\"a}ttchenoberfl{\"a}chenrezeptoren, den Glykoproteinen (GP) V und VI, bei M{\"a}usen analysiert. Hier wird beschrieben, dass GPV und GPVI von der Pl{\"a}ttchenoberfl{\"a}che durch Metalloproteinasen geschnitten werden. W{\"a}hrend physiologischer Stress, wie das Entkoppeln der oxidativen Phosphorylierung in den Mitochondrien, das Schneiden von GPVI durch eine unbekannte Proteinase ausl{\"o}st, verursacht die Aktivierung von Pl{\"a}ttchen mit bestimmten Agonisten das Schneiden von GPV. Die daf{\"u}r verantwortliche Metalloproteinase wurde als ADAM17 identifiziert. In dem zweiten Unterprojekt wurde die Rolle der Protein Kinase C (PKC) in der Pl{\"a}ttchenaktivierung einerseits und in der Pl{\"a}ttchen pro-koagulanten Aktivit{\"a}t andereseits untersucht. Die Konformations{\"a}nderung/Aktivierung von alphaIIbeta3-Integrinen und Sekretion von Granula sind charakteristisch f{\"u}r die Pl{\"a}ttchenaktivierung. Calcium-(Ca2+)-abh{\"a}ngige Phosphatidylserin (PS)- Expression auf der Pl{\"a}ttchenoberfl{\"a}che hingegen ist kennzeichnend f{\"u}r die pro-koagulante Aktivit{\"a}t. Der Beitrag von PKC zu den beschriebenen Pl{\"a}ttchenzust{\"a}nden war bisher unklar. In diesem Projekt wurde zum ersten Mal gezeigt, dass PKC eine doppelte Funktion in den Pl{\"a}ttchen besitzt: einerseits f{\"o}rdert PKC die Pl{\"a}ttchen-Aktivierung und -Aggregation, andererseits unterdr{\"u}ckt PKC die pro-koagulant Aktivit{\"a}t. In dem dritten Unterprojekt wurde die Rolle der kleinen GTPase Rac1 in der Pl{\"a}ttchen- Aktivierung und -Aggregation in vitro und in vivo an konditionalen Rac1 M{\"a}usen analysiert. Es wird berichtet, dass Rac1 f{\"u}r die GPVI abh{\"a}ngige Aktivierung von alphaIIbbeta3-Integrinen und dem Freisetzen von Ca2+ in der Zelle, notwendig ist, sowie f{\"u}r GPVI abh{\"a}ngige Pl{\"a}ttchen-Aggregation und Thrombus Bildung. Hiermit wird die GTPase Rac1 zum ersten Mal in den Signalweg unterhalb von GPVI eingeordnet und ihr zudem dort eine essentielle Rolle zugeteilt.}, subject = {Thrombozyt}, language = {en} } @phdthesis{Krohne2005, author = {Krohne, Katharina}, title = {Die Rolle des Proteins VASP f{\"u}r die Proliferation und Differenzierung h{\"a}matopoetischer Stammzellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15639}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {Im Rahmen der Arbeit wurden in mehreren Teilprojekten die Eigenschaften und Funktionen des Vasodilatator stimulierenden Phosphoproteins (VASP) untersucht. Es wurde ein neuer Antik{\"o}rper (5C6) charakterisiert, der f{\"u}r an Serin157 phosphoryliertes VASP spezifisch sein sollte. Es konnte gezeigt werden, dass der 5C6 Antik{\"o}rper spezifisch VASP erkennt, welches an der Stelle Serin157 phosphoryliert ist. Auch konnten mit dem neuen Antik{\"o}rper Ergebnisse best{\"a}tigt werden, die vorher mit anderen Methoden erhoben wurden, n{\"a}mlich, dass Serin157 sowohl cAMP- als auch cGMP-vermittelt phosphoryliert wird. Der Antik{\"o}rper 5C6 stellte sich als ein guter Marker f{\"u}r die Phosphorylierung von VASP an Serin157 durch die PKA dar und erm{\"o}glichte, die Zeitkinetik der VASP-Phosphorylierung zu beschreiben. In einem weiteren Projekt wurde die Rolle des Proteins VASP bei der Proliferation und Differenzierung von Knochenmark-Stammzellen zu Megakaryozyten und Thrombozyten untersucht. Die Stammzellen wurden zus{\"a}tzlich zu Wachstumsfaktoren mit unterschiedlichen Dosen eines cGMP-Analogons stimuliert. Es zeigte sich hierbei, dass 8-pCPT-cGMP einen dualen, konzentrationsabh{\"a}ngigen Effekt auf die Proliferation und die Differenzierung h{\"a}matopoetischer Stammzellen von Wildtypm{\"a}usen hat. Niedrige Dosen hemmten die Proliferation und f{\"o}rderten die Differenzierung, dagegen hatten h{\"o}here Konzentrationen einen proliferationsf{\"o}rdernden und differenzierungshemmenden Effekt auf die Stammzellen. Im Vergleich hierzu ergab eine Stimulation mit 8-pCPT-cGMP bei VASP knock out M{\"a}usen immer einen proliferationsf{\"o}rdernden Effekt, hingegen einen hemmenden Effekt auf die Differenzierung der h{\"a}matopoetischen Stammzellen. Bei den knock out Zellen f{\"u}hrten h{\"o}here Konzentrationen lediglich zu einer st{\"a}rkeren Reaktion als niedrige.}, language = {de} } @phdthesis{Rabie2005, author = {Rabie, Tamer}, title = {Cellular regulation of platelet glycoprotein VI : in vivo and in vitro studies in mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-14267}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {Platelet interaction with the subendothelium is essential to limit blood loss after tissue injury. However, upon rupture of atherosclerotic plaques, this interaction may result in blood vessel occlusion leading to life threatening diseases such as myocardial infarction or stroke. Among the subendothelial matrix proteins, collagen is considered to be the most thrombogenic component as it directly activates platelets. Platelets interact with collagen, either indirectly through glycoprotein (GP) Ib-V-IX receptor complex, or directly through the major collagen receptor on the platelet surface, GPVI. The work presented here focused on studying the cellular regulation of GPVI. In addition, a possible role for GPVI in thrombus formation induced by atherosclerotic plaque material was investigated and it was found that GPVI plays an important role in this process. Using a recently published mitochondrial injury model, it was found that GPVI contains a cleavage site for a platelet-expressed metalloproteinase. Further studies showed that platelet activation by CRP, or thrombin induced down-regulation of GPIb\&\#61537;, but not GPVI. In parallel, cellular regulation of GPV was studied and it was found that GPV is cleaved in vitro by the metalloproteinase ADAM17. In previous studies it was shown that injection of mice with the anti-GPVI mAb, JAQ1, induces GPVI down-regulation, which is associated with a strong, but transient, thrombocytopenia. Using new anti-GPVI mAbs, which bind different epitopes on the receptor, it is shown in this study that GPVI down-regulation occurs in an epitope-independent manner. Further experiments showed that antibody treatment induces a transient, but significant increase in bleeding time. Using different genetically modified mice, it is shown that, upon antibody injection, GPVI is both, shed from the platelet surface and internalized into the platelet. Signaling through the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR\&\#61543; chain is essential for both processes, while LAT and PLC\&\#61543;2 are essential for the shedding process only. Antibody-induced increase in bleeding time and thrombocytopenia were absent in LAT deficient mice, showing that it is possible to uncouple the associated side effects from the down-regulation process. As antibody-induced GPVI internalization still occurs in LAT and PLC\&\#61543;2 deficient mice, this suggests a novel signaling pathway downstream of GPVI that has not been described so far.}, subject = {Maus}, language = {en} } @phdthesis{Brich2004, author = {Brich, Jochen}, title = {Pharmakologie der ADP-Rezeptoren auf humanen Thrombozyten}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9620}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {Die Thienopyridine Ticlopidin und Clopidogrel sind seit mehreren Jahren im klinischen Einsatz als Thrombozytenaggregationshemmer zur Sekund{\"a}rprophylaxe bei arteriosklerotischen Erkrankungen der Herzgef{\"a}ße, zerebralen Gef{\"a}ße sowie der peripheren Arterien. Trotz ihrer nachgewiesenen klinischen Wirksamkeit war der Wirkmechanismus lange Zeit auf die Beschreibung als ADP-Rezeptorantagonist beschr{\"a}nkt. Im Zuge neuerer Erkenntnisse zum Mechanismus der Ausl{\"o}sung einer Aggregation, insbesondere der ADP-vermittelten Aggregation, sollte im Rahmen dieser Dissertation der Wirkmechanismus der Thienopyridine genauer untersucht werden sowie m{\"o}gliche Auswirkungen auf intrazellul{\"a}re Kaskaden, die den aggregationshemmenden Effekt vermitteln. Zu diesem Zweck wurden die Effekte einer Thienopyridin-Einnahme auf die Thrombozytenfunktion gesunder Probanden untersucht. In unseren Versuchen best{\"a}tigte sich die gute Wirksamkeit der Substanzen hinsichtlich der deutlichen Reduktion der ADP-vermittelten Aggregation. Vor dem Hintergrund eines damals neu propagierten Drei-Rezeptoren-Modells f{\"u}r die ADP-vermittelte Aggregation konnten wir erstmals den Wirkmechanismus an humanen Thrombozyten auf eine Inhibierung des P2Yac-Rezeptors eingrenzen. Weiterhin konnten wir unter Thienopyridin-Einnahme eine Aufhebung der ADP-vermittelten Hemmung der PG-E1-vermittelten VASP-(VAsodilator-Stimulated Phosphoprotein) Phosphorylierung feststellen und somit einen wichtigen Beitrag zum Verst{\"a}ndnis der Wirkung der Substanzen beitragen. Zum besseren Verst{\"a}ndnis der zum damaligen Zeitpunkt bereits auf molekularer Ebene bekannten humanen thrombozyt{\"a}ren ADP-Rezeptoren P2Y1 und P2X1 wurden diese aus humanen Thrombozyten kloniert und mit verschiedenen Tags versehen in einer Astrozytoma-Zelllinine transient und stabil exprimiert. Es wurden verschiedene zellbiologische Methoden wie Immunpr{\"a}zipitation, Durchflusszytometrie und Immunfluoreszenz etabliert, die als Grundlage f{\"u}r weitere Untersuchungen der intrazellul{\"a}ren Signalkaskaden der Rezeptoren dienen k{\"o}nnen. Die stabil exprimierenden Zelllinien dienten zur Verifizierung des pharmakologischen Profils der Rezeptoren, weiterhin konnten bereits erste Versuche zu einer m{\"o}glichen Regulation der Rezeptoren durch zyklische Nukleotide durchgef{\"u}hrt werden. Durch die Etablierung dieser Zelllininen wurde insgesamt eine gute Grundlage f{\"u}r eine weitere Charakterisierung dieser ADP-Rezeptoren unter besser standardisierbaren Bedingungen geschaffen.}, language = {de} } @phdthesis{Schwarz2001, author = {Schwarz, Ulrike}, title = {Biochemische und Molekularbiologische Charakterisierung der Wechselwirkungen zwischen Humanen Thrombozyten und Endothelzellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-2030}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {Der Blutkreislauf ist als wichtigstes Transportsystem im menschlichen K{\"o}rper essentiell f{\"u}r die Versorgung der Gewebe und Organe mit Sauerstoff, N{\"a}hrstoffen, Hormonen etc. Zwei Zelltypen, die eine wichtige Rolle bei der Aufrechterhaltung eines funktionell intakten Blutgef{\"a}ßsystems spielen, sind Thrombozyten, die zentralen Mediatoren der Blutgerinnung, und Endothelzellen, welche die luminale Seite der Gef{\"a}ßw{\"a}nde auskleiden. Diese beiden Zellen sind aber auch wesentlich an der Pathologie der Atherosklerose und kardiovaskul{\"a}rer Erkrankungen beteiligt. Durch direkte und indirekte Interaktionen beeinflussen sich diese beiden Zelltypen gegenseitig und regulieren ihre Aktivit{\"a}t. Im Rahmen dieser Arbeit wurde eine Analysenmethode entwickelt, welche den Funktionszustand der Thrombozyten quantitativ erfaßt. Sowohl die Aktivierung als auch die Hemmung humaner Thrombozyten wird durch die Phosphorylierung spezifischer Signalproteine reguliert. Basierend auf der Verwendung phosphorylierungsspezifischer Antik{\"o}rper und der Durchflußzytometrie wurde eine Methode etabliert, welche die Proteinphosphorylierung auf Einzelzellebene erfaßt, schnell quantifizierbare Ergebnisse liefert und f{\"u}r die Analyse im Vollblut geeignet ist. Da die Sekretion von Endothelfaktoren den Phosphorylierungszustand dieser Proteine in den Thrombozyten beeinflußt, kann die Methode auch dazu verwendet werden, indirekt R{\"u}ckschl{\"u}sse auf den Funktionszustand der Endothelzellen zu gewinnen. In einer ersten klinischen Anwendung wurde die Methode eingesetzt, um den Therapieverlauf der antithrombotischen Medikamente Ticlopidin und Clopidogrel, welche gezielt die ADP-induzierte Thrombozytenaktivierung hemmen, zu verfolgen und das Antwortverhalten von Patienten auf diese Medikamente zu messen. Mehrere Personen, bei denen Ticlopidin und Clopidogrel keine Wirkung zeigten, wurden gefunden, ein Hinweis darauf, daß eine Resistenz gegen Thienopyridine vorkommt. Es ist bekannt, daß Endothelfaktoren bestimmte Aspekte der Thrombozytenaktivierung hemmen. In dieser Arbeit wurde gezeigt, daß die Phosphorylierung der p38 und p42 Mitogen-aktivierten Proteinkinasen, die im Verlauf der Thrombozytenaktivierung von zahlreichen Agonisten induziert wird, ebenfalls durch die endothelialen Vasodilatatoren NO (Stickstoffmonoxid) und Prostaglandin gehemmt wurde. Außerdem hemmten diese Substanzen die Translokation der inflammatorischen Molek{\"u}le P-Selektin und CD40 Ligand (CD40L) aus intrazellul{\"a}ren Speicherorganellen auf die Thrombozytenoberfl{\"a}che. P-Selektin und CD40L werden auf aktivierten Thrombozyten exprimiert und sind direkt an der Interaktion von Thrombozyten mit Leukozyten und Endothelzellen beteiligt. Um die Auswirkung von CD40L, P-Selektin und weiteren Faktoren aktivierter Thrombozyten auf humane Endothelzellen zu untersuchen, wurde mit Hilfe von cDNA-Arrays die differentielle Genexpression in Endothelzellen nach Koinkubation mit aktivierten Thrombozyten analysiert. Neben einer bereits bekannten Hochregulierung von Faktoren, die an inflammatorischen Prozessen beteiligt sind, wurde eine verst{\"a}rkte Expression von Transkriptionsfaktoren (c-Jun, Egr1, CREB2), Wachstumsfaktoren (PDGF) sowie von Adh{\"a}sionsrezeptoren f{\"u}r extrazellul{\"a}re Matrixproteine (Integrin av, Integrin b1) gefunden. Diese Faktoren weisen darauf hin, daß aktivierte Thrombozyten die Migration und Proliferation der Endothelzellen anregen und damit die Wundheilung, aber auch pathophysiologische Prozesse wie die Ausbildung atherosklerotischer Plaques induzieren k{\"o}nnten.}, subject = {Thrombozyt}, language = {de} }