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Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
Die vorliegende Arbeit beschreibt die günstigen Auswirkungen der chronischen Aktivierung der löslichen Guanylatcyclase durch HMR 1766 auf die Regulation der Thrombozytenaktivität im experimentellen Diabetes mellitus. Es konnte die zugrunde liegende Hypothese eines aktivierten Zustands von Thrombozyten im Diabetes bestätigt und darüber hinaus die Reversibilität dieser Aktivierung durch medikamentöse, direkte, NO-unabhängige, chronische Stimulation der sGC, dem Schlüsselenzym zur Vermittlung endogener NO-Wirkung, gezeigt werden. Hierzu wurden mit Hilfe durchflusszytometrischer Bestimmungen in frisch entnommenem Vollblut verschiedene thrombozytäre Marker analysiert. Zum einen deuteten die Ergebnisse auf eine verminderte NO-Bioverfügbarkeit beziehungsweise eine verringerte Aktivität des Effektorenzyms im Diabetes und eine gesteigerte Aktivität des Enzyms bei HMR 1766-behandelten Versuchstieren hin, zum anderen konnte ein Rückgang der Expression thrombozytärer Aktivierungsparameter in Richtung des Niveaus gesunder Kontrollen bei Thrombozyten der Tiere gezeigt werden, die den GC-Aktivator erhielten. Darüber hinaus stellte sich eine verminderte Aktivierbarkeit auf externe Stimuli bei in vitro Versuchen in Vollblut als auch in PRP heraus. Bei aggregometrischen Untersuchungen mit PRP zeigte sich eine verminderte Aggregation als Reaktion auf Fractalkine-Inkubation und anschließende direkte Stimulation mit ADP bei dem Material HMR 1766 behandelter Tiere. Zur Untersuchung von Akut-Effekten der Substanz wurden Proben gesunder beziehungsweise diabetischer Tiere teils direkt mit dem Medikament inkubiert, und es wurde einmalig medikamentös behandelten Tieren Blut entnommen. Hier stellte sich eine verminderte ADP-induzierte Aggregation bei behandelten Proben heraus, zudem zeigten sich positive Akut-Effekte hinsichtlich der basalen als auch der mit einem NO-Donor stimulierten VASP-Phosphorylierung. Bei der Betrachtung hämatologischer und metabolischer Parameter fiel bei sonst unauffälligen Ergebnissen das MPV auf, das im Diabetes erhöht, nach Behandlung mit dem GC-Aktivator jedoch wieder auf Kontrollwerte reduziert war. Die vielversprechenden Ergebnisse könnten ein Schritt zur Etablierung dieser Substanz als zukünftiges Medikament zur Prävention beziehungsweise Behandlung kardiovaskulärer Komplikationen bei Diabetes sein.
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.
Effects of cocoa genotypes on coat color, platelets and coagulation parameters in French Bulldogs
(2021)
A nonsense variant in HPS3, c.2420G>A or p.Trp807*, was recently discovered as the cause for a brown coat color termed cocoa in French Bulldogs. Here, we studied the genotype–phenotype correlation regarding coat color in HPS3 mutant dogs that carried various combinations of mutant alleles at other coat color genes. Different combinations of HPS3, MLPH and TYRP1 genotypes resulted in subtly different shades of brown coat colors. As HPS3 variants in humans cause the Hermansky–Pudlak syndrome type 3, which in addition to oculocutaneous albinism is characterized by a storage pool deficiency leading to bleeding tendency, we also investigated the phenotypic consequences of the HPS3 variant in French Bulldogs on hematological parameters. HPS3 mutant dogs had a significantly lowered platelet dense granules abundance. However, no increased bleeding tendencies in daily routine were reported by dog owners. We therefore conclude that in dogs, the phenotypic effect of the HPS3 variant is largely restricted to pigmentation. While an effect on platelet morphology is evident, we did not obtain any indications for major health problems associated with the cocoa coat color in French Bulldogs. Further studies will be necessary to definitely rule out very subtle effects on visual acuity or a clinically relevant bleeding disorder.
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s\(^{−1}\)). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A\(_2\) (TxA\(_2\)), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.
Platelet activation induces cytoskeletal rearrangements involving a change from discoid to spheric shape, secretion, and eventually adhesion and spreading on immobilized ligands. Small GTPases of the Rho family, such as Rac1 and Cdc42, are known to be involved in these processes by facilitating the formation of lamellipodia and filopodia, respectively. This thesis focuses on the role Rac1 and Cdc42 for platelet function and formation from their precursor cells, the megakaryocytes (MKs), using conditional knock-out mice. In the first part of the work, the involvement of Rac1 in the activation of the enzyme phospholipase (PL) C2 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. It was found that Rac1 is essential for PLC2 activation independently of tyrosine phosphorylation of the enzyme, resulting in a specific platelet activation defect downstream of GPVI, whereas signaling of other activating receptors remains unaffected. Since Rac1-deficient mice were protected from arterial thrombosis in two different in vivo models, the GTPase might serve as a potential target for the development of new drugs for the treatment and prophylaxis of cardio- and cerebrovascular diseases. The second part of the thesis deals with the first characterization of MK- and platelet-specific Cdc42 knock-out mice. Cdc42-deficient mice displayed mild thrombo-cytopenia and platelet production from mutant MKs was markedly reduced. Unexpectedly, Cdc42-deficient platelets showed increased granule content and release upon activation, leading to accelerated thrombus formation in vitro and in vivo. Furthermore, Cdc42 was not generally required for filopodia formation upon platelet activation. Thus, these results indicate that Cdc42, unlike Rac1, is involved in multiple signaling pathways essential for proper platelet formation and function. Finally, the outcome of combined deletion of Rac1 and Cdc42 was studied. In contrast to single deficiency of either GTPase, platelet production from double-deficient MKs was virtually abrogated, resulting in dramatic macrothrombocytopenia in the animals. Formed platelets were largely non-functional leading to a severe hemostatic defect and defective thrombus formation in double-deficient mice in vivo. These results demonstrate for the first time a functional redundancy of Rac1 and Cdc42 in the hematopoietic system.
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.
Endothelial–platelet interactions in influenza‐induced pneumonia: A potential therapeutic target
(2020)
Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro‐thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza‐mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza‐induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.
p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase \(A_2\) (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.
Platelet activation and aggregation at sites of vascular injury involves massive cytoskeletal re-organization, which is required for proper platelet function. Moreover, the cytoskeleton plays central roles in megakaryo- and thrombopoiesis. Thus, cytoskeletal protein aberrations can be the underlying reason for many pathological phenotypes. Although intensive research is carried out to identify the key players involved in cytoskeletal reorganization, the signaling cascades orchestrating these complex processes are still poorly understood. This thesis investigates the role of three actin-binding proteins, Coactosin-like (Cotl) 1, Profilin (Pfn) 1 and Thymosin (T) β4, in platelet formation and function using genetically modified mice.
ADF-H-containing proteins such as Twinfilin or Cofilin are well characterized as regulators of thrombopoesis and cytoskeletal reorganization. Although Cotl1 belongs to the ADF-H protein family, lack of Cotl1 did not affect platelet count or cytoskeletal dynamics. However, Cotl1-deficiency resulted in significant protection from arterial thrombus formation and ischemic stroke in vivo. Defective GPIb-vWF interactions and altered second wave mediator release present potential reasons for the beneficial effect of Cotl1-deficiency. These results reveal an unexpected function of Cotl1 as a regulator of thrombosis and hemostasis, establishing it as a potential target for a safe therapeutic therapy to prevent arterial thrombosis or ischemic stroke.
Recent studies showed that the organization of the circumferential actin cytoskeleton modulates calpain-mediated αIIbβ3 integrin closure, thereby also controlling αIIbβ3 integrin localization. The second part of this thesis identified the actin-sequestering protein Pfn1 as a central regulator of platelet integrin function as Pfn1-deficient platelets displayed almost abolished αIIbβ3 integrin signaling. This translated into a profound protection from arterial thrombus formation and prolonged tail bleeding times in vivo which was caused by enhanced calpain-dependent integrin closure. These findings further emphasize the importance of a functional actin cytoskeleton for intact platelet function in vitro and in vivo.
Tβ4 is a moonlighting protein, acting as one of the major actin-sequestering proteins in cells of higher eukaryotes and exerting various paracrine functions including anti-inflammatory, immunomodulatory and pro-angiogenic effects. Although excessively studied, its role for cytoskeletal dynamics, the distinction between endo- and exogenous protein function and its uptake and release mechanisms are still poorly understood. Constitutive Tβ4-deficiency resulted in thrombocytopenia accompanied by a largely diminished G-actin pool in platelets and divergent effects on platelet reactivity. Pre-incubation of platelets with recombinant Tβ4 will help to understand the function of endo- and exogenous protein, which is under current investigation.