Ina Hagedorn

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

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

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

Factor XIIa Inhibitor Recombinant Human Albumin Infestin-4 Abolishes Occlusive Arterial Thrombus Formation Without Affecting Bleeding

Background— Blood coagulation is a tightly regulated process of sequentially activated serine proteases culminating in fibrin formation, which is critical for limiting posttraumatic blood loss but also may contribute to acute thrombotic diseases, most notably myocardial infarction and stroke. Recent studies with factor XII–deficient mice revealed that the factor XII–induced intrinsic coagulation pathway is essential for pathological thrombus formation but dispensable for hemostasis. Consequently, these findings led to the hypothesis that factor XII could be a promising pharmacological target for safe antithrombotic therapy. Methods and Results— The complementary DNA of the previously described factor XIIa inhibitor Infestin-4, cloned from the midgut of Triatoma infestans, was fused to recombinant human albumin (rHA) and analyzed in vitro. The resulting protein rHA-Infestin-4 specifically inhibits factor XIIa and causes prolonged activated partial thromboplastin time in human, mouse, and rat plasma. To assess its inhibitory potency in vivo, mice and rats were injected with rHA-Infestin-4 and challenged in pathological thrombus formation models. In addition, bleeding assays were performed. rHA-Infestin-4 completely abolished occlusive arterial thrombus formation in mice and rats while leaving hemostasis fully intact. Furthermore, rHA-Infestin-4 was highly protective in a murine model of ischemic stroke. Conclusion— These results identify rHA-Infestin-4 as a promising agent to achieve powerful protection from ischemic cardiovascular and cerebrovascular events without affecting hemostasis.

Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

In the absence of PLD1, platelets do not form stable aggregates under high shear conditions. Aggregation Regulation When damage occurs to the endothelium lining a blood vessel and exposes the underlying extracellular matrix, platelets adhere to the site of injury and aggregate to stop blood loss. However, aggregated platelets can cause ischemia if they occlude the vessel, thus creating the need for therapies that can limit platelet aggregation without increasing blood loss. Elvers et al. found that platelets from mice deficient in phospholipase D1 (PLD1) showed reduced activation of αIIbβ3 integrin, a major adhesion receptor, and did not form stable aggregates when experiencing high shear forces (such as those found in small arterioles). PLD1 deficiency conferred protection against thrombosis and cerebral ischemia in vivo, an effect that was seen with Pld1−/− mice and wild-type mice transplanted with bone marrow from Pld1−/− mice. PLD1 deficiency did not, however, increase blood loss after tail wounding. Thus, PLD1 could be a potential therapeutic target to prevent or treat stroke or other ischemic conditions. Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, αIIbβ3 integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate–binding protein or immunoreceptor tyrosine-based activation motif–coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1−/− mice displayed impaired αIIbβ3 integrin activation in response to major agonists and defective glycoprotein Ib–dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction

Myocardial infarction (MI) leads to rapid necrosis of cardiac myocytes. To achieve tissue integrity and function, inflammatory cells are activated, including monocytes/macrophages. However, the effect of monocyte/macrophage recruitment after MI remains poorly defined. After experimental MI, monocytes and macrophages were depleted through serial injections of clodronate‐containing liposomes. Monocyte/macrophage infiltration was reduced in the myocardium after MI by active treatment. Mortality was increased due to thromboembolic events in monocyte‐ and macrophage‐depleted animals (92 vs. 33%; P<0.01). Left ventricular thrombi were detectable as early as 24 h after MI; this was reproduced in a genetic model of monocyte/macrophage ablation. A general prothrombotic state, increased infarct expansion, and deficient neovascularization were not observed. Severely compromised extracellular matrix remodeling (collagen I, placebo liposome vs. clodronate liposome, 2.4±0.2 vs. 0.8±0.2 arbitrary units; P<0.001) and locally lost integrity of the endocardium after MI are potential mechanisms. Patients with a left ventricular thrombus had a relative decrease of CD14+CD16+ monocyte/macrophage subsets in the peripheral blood after MI (no thrombus vs. thrombus, 14.2±0.9 vs. 7.80±0.4%; P<0.05). In summary, monocytes/macrophages are of central importance for healing after MI. Impaired monocyte/macrophage function appears to be an unrecognized new pathophysiological mechanism for left ventricular thrombus development after MI.—Frantz, S., Hofmann, U., Fraccarollo, D., Schäfer, A., Kranepuhl, S., Hagedorn, I., Nieswandt, B., Nahrendorf, M., Wagner, H., Bayer, B., Pachel, C., Schön, M.P., Kneitz, S., Bobinger, T., Weidemann, F., Ertl, G., Bauersachs, J. Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction. FASEB J. 27, 871–881 (2013). www.fasebj.org

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

Combined In Vivo Depletion of Glycoprotein VI and C-Type Lectin-Like Receptor 2 Severely Compromises Hemostasis and Abrogates Arterial Thrombosis in Mice

Objective—Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif–bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. Approach and Results—We investigated whether both (hem)immunoreceptor tyrosine activation motif–bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2–depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. Conclusions—These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti–CLEC-2–based agents in the prevention of thrombotic diseases.

Genetic and antibody-induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl3-induced thrombosis

At sites of vascular injury, platelet adhesion, activation and aggregation occurs on exposed subendothelial collagens and contributes to hemostasis but also to occlusion of diseased vessels leading to a myocardial infarction or ischemic stroke. The activating platelet collagen receptor, glycoprotein (GP) VI [1], is a platelet-specific transmembrane type I receptor that non-covalently associates with the Fc receptor (FcR) c-chain which contains an immunoreceptor tyrosine-based activation motif (ITAM) [2]. Upon ligand-induced GPVI clustering, the ITAM becomes tyrosine phosphorylated and initiates a series of phosphorylation events finally resulting in cellular activation [3]. GPVI has been proposed as an attractive antithrombotic target, particularly because anti-GPVI antibodies have been shown to induce irreversible downregulation of the receptor through ectodomain shedding in circulating platelets in mice, resulting in long-term antithrombotic protection but overall normal tail bleeding times [4–7]. Different mouse models of GPVI-deficiency (FcR c-chain-deficient which also lack GPVI [8], GPVI-immunodepleted or Gp6 mice) have been used to determine the functional relevance of the receptor in thrombus formation in different experimental thrombosis models (laser-, mechanically and chemically induced injuries). AlthoughGPVI is generally regarded as a critical regulator of pathological thrombus formation [9], partially contradictory results have been reported on the relative importance of GPVI in some of these models. For example, GPVI was found to play no [10,11] or only a minor [12,13] role in models of laser-induced vascular injury where thrombus formation is suggested to be largely dependent on thrombin generation, although different results were reported by others [14]. Ferric(III)chloride (FeCl3)-induced vascular injury is a widely used model in thrombosis research because it allows variable levels of injury in different vascular beds and can be monitored by microscopic visualization or blood flow measurements. Previous studies yielded evidence for a role of GPVI in FeCl3-induced thrombus formation in mice [10,15,16], but this was questioned in a very recent study assessing the mechanisms underlying thrombus formation in that model in detail [17]. The authors demonstrated that in their model, FeCl3 diffuses through all vessel wall layers and severely injures the endothelium without damaging the internal elastic lamina (IEL), and only exposes the basement membrane to the vessel lumen. They further report that in their hands GPVI-immunodepleted mice are not protected from vessel occlusion after FeCl3-induced injury of mesenteric arterioles or carotid arteries by measuring thrombus area and time to occlusion. Based on these data, the authors state that FeCl3-induced vascular injury may not be an appropriate model to study the role of subendothelial adhesive proteins and their platelet receptors in thrombosis [17]. This conclusion, if correct, may have major implications for the interpretation of a large number of previous results obtained with this model and also significantly influence its acceptance as a valid experimental system to assess the mechanisms of arterial thrombosis. Therefore, here we assessed the role of GPVI-mediated platelet activation/ adhesion in FeCl3-induced thrombus formation systematically. To do so, we subjected control, Gp6 (details of the knockout generation will be published elsewhere) and GPVIimmunodepleted (100 lg JAQ1 i.p., analysis on day 6 post injection) mice to a model of FeCl3-injury of mesenteric arterioles and carotid arteries, and monitored thrombus formation by intravital fluorescence microscopy or blood flow measurements, respectively. In addition, thrombus formation in these mice was tested in a model of mechanical injury of the abdominal aorta. A drop of 20% FeCl3 was topically applied on small mesenteric arterioles of 4to 5–week-old mice and thrombus formation of fluorescently labeled platelets was monitored for 40 min or until complete occlusion of the vessel had occurred. Kinetics of platelet adhesion (data not shown) and the beginning of first thrombus formation were similar between control, Gp6 and GPVI-immunodepleted mice (Fig. 1A, Correspondence: Bernhard Nieswandt, University Hospital Wurzburg and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Josef-Schneider-Str. 2, 97080 Wurzburg, Germany. Tel.: +49 931 31 80405; fax: +49 931 201 61652. E-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de

Redundant functions of phospholipases D1 and D2 in platelet α-granule release.

Summary.  Background: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD) .