572 Biochemie
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Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances.
One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 % are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered “undruggable” in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments.
Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs.
Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.
Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to “free” inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process.
In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions,
To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases.
The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment.
De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis.
SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis.
SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation.
It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth.
A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation.
In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.
Serum half-life elongation as well as the immobilization of small proteins like cytokines is still one of the key challenges for biologics. This accounts also for cytokines, which often have a molecular weight between 5 and 40 kDa and are therefore prone to elimination by renal filtration and sinusoidal lining cells. To solve this problem biologics are often conjugated to poly(ethylene glycol) (PEG), which is the gold standard for the so called PEGylation. PEG is a synthetic, non-biodegradable polymer for increasing the hydrodynamic radius of the conjugated protein to modulate their pharmacokinetic performance and prolong their therapeutic outcome. Though the benefits of PEGylation are significant, they also come with a prize, which is a loss in bioactivity due to steric hindrance and most often the usage of heterogeneous bioconjugation chemistries. While PEG is a safe excipient in most cases, an increasing number of PEG related side-effects, such as immunological responses like hypersensitivity and accelerated blood clearance upon repetitive exposure occur, which highlights the need for PEG alternative polymers, that can replace PEG in such cases.
Another promising method to significantly prolong the residence time of biologics is to immobilize them at a desired location. To achieve this, the transglutaminase (TG) Factor XIIIa (FXIIIa), which is an important human enzyme during blood coagulation can be used. FXIIIa can recognize specific peptide sequences that contain a lysine as substrates and link them covalently to another peptide sequence, that contains a glutamine, forming an isopeptide bond. This mechanism can be used to link modified proteins, which have a N- or C-terminal incorporated signal peptide by mutation, to the extracellular matrix (ECM) of tissues.
Additionally, both above-described methods can be combined. By artificially introducing a TG recognition sequence, it is possible to attach an azide group containing peptide site-specifically to the TG, recognition sequence. This allows the creation of a site-selective reactive site at the proteins N- or C-terminus, which can then be targeted by cyclooctyne functionalized polymers, just like amber codon functionalized proteins.
This thesis has focused on the two cytokines human Interferon-α2a (IFN-α2a) and human, as well as murine Interleukin-4 (IL-4) as model proteins to investigate the above-described challenges. IFN-α2a has been chosen as a model protein because it is an approved drug since 1986 in systemic applications against some viral infections, as well as several types of cancer. Furthermore, IFN-α2 is also approved in three PEGylated forms, which have different molecular weights and use different conjugation techniques for polymer attachment. This turns it into an ideal candidate to compare new polymers against the gold standard PEG. Interleukin-4 (IL-4) has been chosen as the second model protein due to its similar size and biopotency. This allows to compare found trends from IFN-α2a with another bioconjugate platform and distinguish between IFN-α2a specific, or general trends. Furthermore, IL-4 is a promising candidate for clinical applications as it is a potent anti-inflammatory protein, which polarizes macrophages from the pro-inflammatory M1 state into the anti-inflammatory M2 state.
The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.
Megakaryocytes (MKs) are the largest cells of the hematopoietic system and the precursor cells of platelets. During proplatelet formation (PPF) bone marrow (BM) MKs extent large cytoplasmic protrusions into the lumen of sinusoidal blood vessels. Under homeostatic conditions PPF occurs exclusively in the direction of the sinusoid, while platelet generation into the marrow cavity is prevented. So far, the mechanisms regulating this process in vivo are still not completely understood, especially when PPF is deregulated during disease. This thesis investigated the mechanisms of PPF in native BM and after myeloablation by total body irradiation (TBI).
First, we have identified a specialized type of BM stromal cells, so called CXCL12-abundant reticular (CAR) cells, as novel possible regulators of PPF. By using complementary high-resolution microscopy techniques, we have studied the morphogenetic events at the MK/vessel wall interface in new detail, demonstrating that PPF formation preferentially occurs at CAR cell-free sites at the endothelium.
In the second part of this thesis, we analyzed the processes leading to BM remodeling in response to myeloablation by TBI. We used confocal laser scanning microscopy (CLSM) to study the kinetic of radiation-triggered vasodilation and mapped extracellular matrix (ECM) proteins after TBI. We could demonstrate that collagen type IV and laminin α5 are specifically degraded at BM sinusoids. At the radiation-injured vessel wall we observed ectopic release of platelet-like particles into the marrow cavity concomitantly to aberrant CAR cell morphology, suggesting that the balance of factors regulating PPF is disturbed after TBI. ECM proteolysis is predominantly mediated by the matrix metalloproteinase MMP9, as revealed by gelatin-zymography and by a newly established BM in situ zymography technique. In transgenic mice lacking MMP9 vascular recovery was delayed, hinting towards a role of MMP9 in vessel reconstitution after myeloablation.
In a third series of experiments, we studied the irradiated BM in the context of hematopoietic stem cell transplantation (HSCT). By using mice as BM donors that ubiquitously express the fluorescent reporter protein dsRed we tracked engraftment of donor cells and especially MKs in the recipient BM. We found a distinct engraftment pattern and cluster formation for MKs, which is different from other blood cell lineages.
Finally, we assessed platelet function after TBI and HSCT and were the first to demonstrate that platelets become massively hyporeactive, particularly upon stimulation of the collagen receptor GPVI.
In summary, our findings shed light on the processes of PPF during health and disease which will help to develop treatments for aberrant thrombopoiesis.
While the healthy brain works through balanced synaptic communication between
glutamatergic and GABAergic neurons to coordinate excitation (E) and inhibition (I), disruption
of E/I balance interferes with synaptic communication, information processing, and ultimately
cognition. Multiple line of evidence indicates that E/I imbalance represents the
pathophysiological basis of a wide spectrum of mental disorders. Genetic screening
approaches have identified Cadherin-13 (CDH13). as a risk gene across neurodevelopmental
and mental disorders. CDH13 regulates several cellular and synaptic processes in brain
development and neuronal plasticity in adulthood. In addition to other functions, it is specifically
localized at inhibitory synapses of parvalbumin- and somatostatin-expressing GABAergic
neurons. In support of CDH13’s function in moderating E/I balance, electrophysiological
recordings of hippocampal slices in a CDH13-deficient mouse model revealed an increase in
basal inhibitory but not excitatory synaptic transmission. Moreover, the search for genetic
variants impacting functional expression of the CDH13 gene identified SNP (single nucleotide
polymorphism)) rs2199430 in intron 1 to be associated with differential mRNA concentrations
in human post-mortem brain across the three genotypes CDH13G/G, CDH13A/G and CDH13A/A
.
This work therefore aimed to further validate these findings in a complementary human model
by using induced pluripotent stem cells (iPSCs). The application of human iPSCs in research
has replaced the use of embryonic cells, resolving the ethical conflict of destructive usage of
human embryos. Investigating CDH13’s mode of action in inhibitory synapses was predicted
to facilitate mechanistic insight into the effects of CDH13 gene variants on E/I network activity,
which can then be targeted to reinstate balance.
Genome-wide association studies have identified rare copy number variants (CNVs) resulting
in a deletion (or duplication) of CDH13. To reduce genetic background variance, a set of
isogenic iPSC lines with a gene dose-dependent deficiency of CDH13 (CDH13-/- and CDH13+/-
) was generated by using the Clustered Regulatory Interspaced Short Palindromic
Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. These CRISPRed iPSCs
carrying a single or two allele(s) with CDH13 inactivation facilitate investigation of CDH13
function in cellular processes, at inhibitory synapses and in neuronal network activity. In
addition, iPSCs carrying allelic SNP rs2199430 variants were used to study the effects of
common genetic variation of CDH13. These cell lines were differentiated into pure
glutamatergic and GABAergic neurons and co-cultured to generate neuronal networks allowing
its activity to be measured and correlated with electrophysiological signatures of differential
CDH13 genotypes. The work towards assessment of neuronal network activity of the iPSC
lines was subdivided into three major steps: first, generating rtTA/Ngn2 and rtTA/Ascl1-positive
iPSCs via a lentivirus-mediated approach; second, differentiating pure glutamatergic and
GABAergic neurons from the genetically transduced iPSCs and co-culturing of pure
glutamatergic and GABAergic neurons in a pre-established ratio (65:35) by direct
differentiation upon supplementation with doxycycline and forskolin on a microelectrode array
(MEA) chip; and, finally, recording of neuronal network activity of iPSC lines after 49 days in
vitro, followed by extraction and analyses of multiple MEA parameters.
x
Based on the MEA parameters, it was confirmed that complete CDH13 knockout as well as
heterozygous deficiency influence E/I balance by increasing inhibition. It was further revealed
that common SNP variation alters the signature of neuronal network activity. Specifically,
CDH13 deficiency resulted in a significant reduction in network burst duration (NBD), reduced
number of detected spikes within a network burst and reduction in network burst rate (NBR)
compared to the control (CDH13G/G). CDH13A/G and CDH13A/A showed similarities with the
CRISPRed CDH13-deficient networks by showing a significant reduction in the NBD and a
reduced number of detected spikes within a network compared to CDH13G/G. Strikingly. there
was a significant increase in the NBR of the CDH13A/G and CDH13A/A compared to CDH13G/G
networks. CDH13A/G networks exhibited significant differences in both parameters. At the
cellular level, this indicates that signalling pathways which determine the length and frequency
of network bursts differ among allelic variants of SNP rs2199430, thus confirming functional
relevance of this intronic SNP.
In summary, CDH13-deficient isogenic iPSC lines were generated using CRISPR/Cas9, iPSCs
were genetically transduced via a lentivirus approach, direct differentiation of
glutamatergic/GABAergic neurons derived from transduced iPSCs were used to establish a
scalable co-culture system, and network activity was recorded by MEA using pre-established
parameters to extract and analyze activity information. The results indicate that iPSC-derived
neuronal networks following CRISPR/Cas9-facilitated CDH13 inactivation, as well as networks
with allelic SNP variants of CDH13, moderate E/I balance, thus advancing understanding of
CDH13 function at inhibitory synapses and elucidating the effects of rare and common CDH13
gene variation.
RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease “Neurodevelopmental Disorder with severe Motor Impairment and Absent Language” (NEDMIAL;OMIM#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3’ → 5’ RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome’s mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5’TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.
Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets.
Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin.
In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.
Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research.
Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition.
Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments.
Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation.
As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.