Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

The use of blood and blood products can be life-saving, but there are also certain risks associated with their administration and use. Packed red blood cells (pRBCs) and platelet concentrates are the most commonly used blood products in transfusion medicine to treat anemia or acute and chronic bleeding disorders, respectively. During the production and storage of blood products, red blood cells and platelets release extracellular vesicles (EVs) as a result of the storage lesion, which may affect product quality. EVs are subcellular structures enclosed by a lipid bilayer and originate from the endosomal system or from the plasma membrane. They play a pivotal role in intercellular communication and are emerging as important regulators of inflammation and coagulation. Their cargo and their functional characteristics depend on the cell type from which they originate, as well as on their microenvironment, influencing their capacity to promote coagulation and inflammatory responses. Hence, the potential involvement of EVs in transfusion-related adverse events is increasingly recognized and studied. Here, we review the knowledge regarding the effect of production and storage conditions of pRBCs and platelet concentrates on the release of EVs. In this context, the mode of processing and anticoagulation, the influence of additive solutions and leukoreduction, as well as the storage duration will be addressed, and we discuss potential implications of EVs for the clinical outcome of transfusion.

Immunothrombosis, an excessive inflammatory response with simultaneous overactivation of the coagulation system, is a central pathomechanism in sepsis and COVID-19. It is associated with cellular activation, vascular damage, and microvascular thrombosis, which can lead to multiple organ failure and death. Here, we characterized factors related to immunothrombosis in plasma samples from 78 sepsis patients. In the course of routine clinical testing, SARS-CoV-2 was detected in 14 of these patients. Viral infection was associated with a higher mortality. Both, COVID-19 negative and COVID-19 positive sepsis patients showed increased levels of effectors of immunothrombosis, including platelet factor 4, D-dimer, nucleosomes, citrullinated histone H3, high mobility group box-1 protein, as well as phosphatidylserine-expressing platelet-derived extracellular vesicles, compared to healthy controls (n = 25). Using a 27-plex cytokine bead array, we found that Interleukin (IL)-1ra, IL-6, IL-8, IL-13, tumor necrosis factor (TNF)-α, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and granulocyte-colony stimulating factor (G-CSF) were elevated in both, COVID-19 negative and COVID-19 positive sepsis patients, as compared to healthy controls. SARS-CoV-2 infection was associated with elevated levels of IP-10, MCP-1, and IL-13, while all other mediators widely overlapped between COVID-19 negative and COVID-19 positive patients.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet–leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP), and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples was associated with CRP, HMGB-1, PF4, and TF as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41⁺ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41⁺CD45⁺) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet-leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP) and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples were associated with CRP, HMGB-1, PF4, and TF, as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41+ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41+CD45+) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.