Alice Assinger

Platelets and Infection – An Emerging Role of Platelets in Viral Infection

Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen–antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses.

Platelets Mediate Oxidized Low-Density Lipoprotein–Induced Monocyte Extravasation and Foam Cell Formation

Objective— A growing body of evidence indicates that platelets contribute to the onset and progression of atherosclerosis by modulating immune responses. We aimed to elucidate the effects of oxidized low-density lipoprotein (OxLDL) on platelet–monocyte interactions and the consequences of these interactions on platelet phagocytosis, chemokine release, monocyte extravasation, and foam cell formation. Approach and Results— Confocal microscopy and flow cytometric analysis revealed that in vitro and in vivo stimulation with OxLDL resulted in rapid formation of platelet–monocyte aggregates, with a preference for CD16+ monocyte subsets. This platelet–monocyte interaction facilitated OxLDL uptake by monocytes, in a process that involved platelet CD36–OxLDL interaction, release of chemokines, such as CXC motif ligand 4, direct platelet–monocyte interaction, and phagocytosis of platelets. Inhibition of cyclooxygenase with acetylsalicylic acid and antagonists of ADP receptors, P2Y1 and P2Y12, partly abrogated OxLDL-induced platelet–monocyte aggregates and platelet-mediated lipid uptake in monocytes. Platelets also enhanced OxLDL-induced monocyte transmigration across an endothelial monolayer via direct interaction with monocytes in a transwell assay. Importantly, in LDLR−/− mice, platelet depletion resulted in a significant decrease of peritoneal macrophage recruitment and foam cell formation in a thioglycollate-elicited peritonitis model. In platelet-depleted wild-type mice, transfusion of ex vivo OxLDL-stimulated platelets induced monocyte extravasation to a higher extent when compared with resting platelets. Conclusions— Our results on OxLDL-mediated platelet–monocyte aggregate formation, which promoted phenotypic changes in monocytes, monocyte extravasation and enhanced foam cell formation in vitro and in vivo, provide a novel mechanism for how platelets potentiate key steps of atherosclerotic plaque development and plaque destabilization.

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2

Summary.  Background: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram‐negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. Objectives: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. Methods: Phagocytosis, cell–cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet‐neutrophil‐bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. Results: In the presence of plasma neutrophil‐mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet–neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet–neutrophil aggregate formation but is required for efficient phagocytosis. Conclusions: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.

Evidence for serotonin as a relevant inducer of liver regeneration after liver resection in humans

Liver regeneration (LR) involves a complex interplay of growth factors and antagonists. In this context, platelet‐derived serotonin (5‐HT) has been identified as a critical inducer of LR in mice. Clinical evidence for a role of 5‐HT in LR in humans is lacking. Accordingly, serum and plasma 5‐HT was monitored perioperatively in 60 patients undergoing liver resection, of which 35 served as exploration and 25 as validation sets. Intraplatelet (IP) levels of 5‐HT were calculated by subtraction of plasma 5‐HT from serum values. Serum markers of liver function were used to evaluate LR and liver dysfunction (LD). In the exploration setting, IP 5‐HT levels significantly decreased after liver resection (P < 0.001) and gradually recovered during the first week. IP 5‐HT measured before surgery specifically predicted LD in the subsequent 7 days (area under the curve: 0.721; P = 0.029). Patients suffering from postoperative LD and morbidity were found to have reduced IP 5‐HT levels during the entire perioperative period. Furthermore, we validated that reduced preoperative IP 5‐HT (<73 ng/mL) was associated with an increased incidence of postoperative LD and morbidity (P =0.045 and P = 0.021) and were able to demonstrate that IP 5‐HT levels were an independent predictor of poor clinical outcome. Conclusions: These findings provide evidence that IP 5‐HT correlates with LR in humans: Patients with low IP 5‐HT before liver resection suffered from delayed hepatic regeneration. Therefore, IP 5‐HT levels may prove a helpful clinical marker to predict postoperative LD and clinical outcome before hepatic resection and initiate suitable interventions. (Hepatology 2014;60:257‐266)

Platelet Interaction with Innate Immune Cells.

Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis.

Smoking alters circulating plasma microvesicle pattern and microRNA signatures

Circulating plasma microvesicles (PMVs) and their microRNA content are involved in the development of atherosclerosis and could serve as biomarkers for cardiovascular disease (CVD) progression. However, little is known on how smoking influences the levels of PMVs and microRNA signatures in vivo. Therefore, we aimed to investigate the effects of smoking on circulating PMV levels and CVD-related PMV-derived microRNAs in young, healthy smokers. Twenty young (10 female, 10 male; 25 ± 4 years) healthy smokers (16 ± 6 cigarettes per day for 8 ± 4 years) and age- and sex-matched controls were included in this study. While complete blood count revealed no differences between both groups, smoking significantly enhanced intracellular reactive oxygen species in platelets and leukocytes as well as platelet-leukocyte aggregate formation. Total circulating PMV counts were significantly reduced in smokers, which could be attributed to decreased platelet-derived PMVs. While the number of endothelial PMVs remained unaffected, smoking propagated circulating leukocyte-derived PMVs. Despite reduced total PMVs, PMV-derived microRNA-profiling of six smoker/control pairs revealed a decrease of only a single microRNA, the major platelet-derived microRNA miR-223. Conversely, miR-29b, a microRNA associated with aortic aneurysm and fibrosis, and RNU6-2, a commonly used reference-RNA, were significantly up-regulated. Smoking leads to alterations in the circulating PMV profile and changes in the PMV-derived microRNA signature already in young, healthy adults. These changes may contribute to the development of smoking-related cardiovascular pathologies. Moreover, these smoking-related changes have to be considered when microRNA or PMV profiles are used as disease-specific biomarkers.

VWF excess and ADAMTS13 deficiency: a unifying pathomechanism linking inflammation to thrombosis in DIC, malaria, and TTP

Absent or severely diminished activity of ADAMTS13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motif, member 13) resulting in the intravascular persistence and accumulation of highly thrombogenic ultra large von Willebrand factor (UL-VWF) multimers is the pathophysiological mechanism underlying thrombotic thrombocytopenic purpura. Reduced VWF-cleaving protease levels, however, are not uniquely restricted to primary thrombotic microangiopathy (TMA), e. g. thrombotic thrombocytopenic purpura, but also occur in other life-threatening thrombocytopenic conditions: severely decreased ADAMTS13 activity is seen in severe sepsis, disseminated intravascular coagulation (DIC) and complicated malarial infection. The clinical relevance of these secondary thrombotic microangiopathies is increasingly recognised, but its therapeutic implications have not yet been determined. The presence of a secondary TMA in certain diseases may define patient groups which possibly could benefit from ADAMTS13 replacement or a VWF-targeting therapy. This short-review focuses on the role of UL-VWF multimers in secondary TMA and discusses the potential of investigational therapies as candidates for the treatment of TTP. In conclusion, prospective clinical trials on the effectiveness of protease replacementin vivo seem reasonable. Carefully selected patients with secondary TMA may benefit from therapies primarily intended for the use in patients with TTP.

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

Summary.  Background:  Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis.

Oxidation by hypochlorite converts protective HDL into a potent platelet agonist

Addition of hypochlorite‐oxidized HDL to human platelets results in an immediate and transient raise in intracellular calcium, surface expression of P‐selectin and platelet aggregation. The observed effects are dose dependent and can be blocked by an antibody directed against the lipoprotein‐binding domain of platelet thrombospondin‐ and scavenger receptor CD36.

Human Cytomegalovirus–Platelet Interaction Triggers Toll-Like Receptor 2–Dependent Proinflammatory and Proangiogenic Responses

Objective—Human cytomegalovirus (HCMV) is a widespread pathogen that correlates with various clinical complications, including atherosclerosis. HCMV is released into the circulation during primary infection and periodic viral reactivation, allowing virus–platelet interactions. Platelets are important in the onset and development of atherosclerosis, but the consequences of platelet–HCMV interactions are unclear. Approach and Results—We studied the effects of HCMV–platelet interactions in blood from healthy donors using the purified clinical HCMV isolate VR1814. We demonstrated that HCMV bound to a Toll-like receptor (TLR) 2–positive platelet subpopulation, which resulted in signal transduction, degranulation, and release of proinflammatory CD40L and interleukin-1&bgr; and proangiogenic vascular endothelial–derived growth factor. In mice, murine CMV activated wild-type but not TLR2-deficient platelets. However, supernatant from murine CMV–stimulated wild-type platelets also activated TLR2-deficient platelets, indicating that activated platelets generated soluble mediators that triggered further platelet activation, independent of TLR2 expression. Inhibitor studies, using ADP receptor antagonists and apyrase, revealed that ADP release is important to trigger secondary platelet activation in response to HCMV. HCMV-activated platelets rapidly bound to and activated neutrophils, supporting their adhesion and transmigration through endothelial monolayers. In an in vivo model, murine CMV induced systemic upregulation of platelet–leukocyte aggregates and plasma vascular endothelial–derived growth factor in mice and showed a tendency to enhance neutrophil extravasation in a TLR2-dependent fashion. Conclusions—HCMV is a well-adapted pathogen that does not induce immediate thrombotic events. However, HCMV–platelet interactions lead to proinflammatory and proangiogenic responses, which exacerbate tissue damage and contribute to atherogenesis. Therefore, platelets might contribute to the effects of HCMV in accelerating atherosclerosis.