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Besides their central role in haemostasis and thrombosis, platelets are increasingly recognised as versatile effector cells in inflammation, the innate and adaptive immune response, extracellular matrix reorganisation and fibrosis, maintenance of barrier and organ integrity, and host response to pathogens. These platelet functions, referred to as thrombo-inflammation and immunothrombosis, have gained major attention in the COVID-19 pandemic, where patients develop an inflammatory disease state with severe and life-threatening thromboembolic complications. In the CRC/TR 240, a highly interdisciplinary team of basic, translational and clinical scientists explored these emerging roles of platelets with the aim to develop novel treatment concepts for cardiovascular disorders and beyond. We have i) unravelled mechanisms leading to life-threatening thromboembolic complica-tions following vaccination against SARS-CoV-2 with adenoviral vector-based vaccines, ii) identified unrecognised functions of platelet receptors and their regulation, offering new potential targets for pharmacological intervention and iii) developed new methodology to study the biology of megakar-yocytes (MKs), the precursor cells of platelets in the bone marrow, which lay the foundation for the modulation of platelet biogenesis and function. The projects of the CRC/TR 240 built on the unique expertise of our research network and focussed on the following complementary fields: (A) Cell bi-ology of megakaryocytes and platelets and (B) Platelets as regulators and effectors in disease. To achieve this aim, we followed a comprehensive approach starting out from in vitro systems and animal models to clinical research with large prospective patient cohorts and data-/biobanking. Despite the comparably short funding period the CRC/TR 240 discovered basic new mechanisms of platelet biogenesis, signal transduction and effector function and identified potential MK/platelet-specific molecular targets for diagnosis and therapy of thrombotic, haemorrhagic and thrombo-inflammatory disease states.
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
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.
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.
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.
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.
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.
Cyclophilin a is not acetylated at lysine-82 and lysine-125 in resting and stimulated platelets
(2022)
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.
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.
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.