Parasite histones are toxic to brain endothelium and link blood barrier breakdown and thrombosis in cerebral malaria

Microvascular thrombosis and blood–brain barrier (BBB) breakdown are key components of cerebral malaria (CM) pathogenesis in African children and are implicated in fatal brain swelling. How Plasmodium falciparum infection causes this endothelial disruption and why this occurs, particularly in the brain, is not fully understood. In this study, we have demonstrated that circulating extracellular histones, equally of host and parasite origin, are significantly elevated in CM patients. Higher histone levels are associated with brain swelling on magnetic resonance imaging. On postmortem brain sections of CM patients, we found that histones are colocalized with P falciparum–infected erythrocytes sequestered inside small blood vessels, suggesting that histones might be expelled locally during parasite schizont rupture. Histone staining on the luminal vascular surface colocalized with thrombosis and leakage, indicating a possible link between endothelial surface accumulation of histones and coagulation activation and BBB breakdown. Supporting this, patient sera or purified P falciparum histones caused disruption of barrier function and were toxic to cultured human brain endothelial cells, which were abrogated with antihistone antibody and nonanticoagulant heparin. Overall, our data support a role for histones of parasite and host origin in thrombosis, BBB breakdown, and brain swelling in CM, processes implicated in the causal pathway to death. Neutralizing histones with agents such as nonanticoagulant heparin warrant exploration to prevent brain swelling in the development or progression of CM and thereby to improve outcomes.

Parasite histones mediate leak and coagulopathy in cerebral malaria

Coagulopathy and leak, specific to the brain vasculature, are central pathogenetic components of cerebral malaria (CM). It is unclear how the parasite, Plasmodium falciparum, triggers these processes. Extracellular histones, released from damaged host cells, bind to cell membranes and cause coagulation activation, platelet aggregation and vascular leak in diverse critical illnesses. In CM patients with P. falciparum, serum histones correlate with fibrin formation, thrombocytopenia, and endothelial activation and predict brain swelling on magnetic resonance imaging and fatal outcome. Post-mortem, histones bind to the luminal vascular surface, co-localizing with P. falciparum-infected erythrocytes (IE), and with thrombosis and leak. Purified P. falciparum histones cause toxicity and barrier disruption in cultured human brain microvascular endothelial cells, as does serum from CM patients, reversed by anti-histone antibodies and non-anticoagulant heparin. These data implicate parasite histones as a key trigger of fatal brain swelling in CM. Neutralizing histones with agents such as non-anticoagulant heparin warrant exploration to prevent brain swelling and improve outcome.

VWF Mediates Platelet Activation and Secretion to Promote Osteosarcoma Metastasis

[Background] As a multimer protein that mediates the adhesion of platelets to vascular surface, VWF has been found to participate in tumor metastasis, and may also be involved in the interaction between platelets and tumor cells. We observed in our clinical studies that the expression of VWF in lung metastatic osteosarcoma tumor is significantly higher than that of in primary osteosarcoma. Based on these observations, we speculate that VWF and platelets may contribute to promoting osteosarcoma metastasis. Thus, this study is to explore the effects of VWF-mediated platelet activation and secretion on the migration and invasion ability of SAOS2 cells with VWF-expressing osteosarcoma cell line SAOS2 as a model, as well as the inhibition of the interaction between VWF and platelets by specific antibodies. [Methods] SAOS2 cells were stimulated with inflammatory factor PMA and resulted in increased expression of vWF. Then the adhesion of labeled platelets to SAOS2 cells was evaluated under static and flow conditions, respectively. The above processes were intervened with SZ-2(anti-GPIb), SZ-123(anti-VWF) and AVW3 (anti-VWF) mAbs which specifically blocked the vWF-GPIbα pathway. Transwell assay was used to explore the effect of vWF-mediated migration and invasion ability of SAOS2 cells. PDGF-BB, TGF-β and VEGF that could be secreted by platelets to promote tumor cells growth in the co-culture system of platelets and SAOS2 cells were detected by ELISA. [Results] Under static and flow conditions, SAOS2 cells treated with PMA exhibited significantly higher platelets-adhesion capacity compared to the cells untreated with PMA, and the adhesion could be partially inhibited by SZ-2 and AVW3 mAbs. Furthermore, platelets significantly promoted the migration and invasion ability of SAOS2 cells, and PDGF-BB and TGF-β secreted by platelets were also increased in these processes. Both of SZ-2 and AVW3 mAbs partially decreased the migration and invasion ability of SAOS2 cells. [Conclusions] vWF were expressed highly in some kind of non-endothelial origin cancer cells, such as SAOS2, which mediates platelet activation and secreting factors such as PDGF-BB and TGF-β to promote tumor cells growth. Activated platelets have a significant promoted effect on the migration and invasion ability of SAOS2 cells and this process can be inhibited by antibodies that specifically block the pathway of VWF-GPIbα. Disclosures No relevant conflicts of interest to declare.

Identification and Characterization of Integrin alphaIIbbeta3 Intermediate Affinity State Induced By Gpibalpha Mechanotransduction

During arterial haemostasis, platelets tether to and translocate on disrupted vascular surface via binding of GPIbα to the VWF A1 domain, which triggers activation signals that induce intracellular Ca2+ release and up-regulate integrin αIIbβ3 binding capacity1,2. Inhibition of this signaling pathway or blockade of αIIbβ3 binding has a devastating impact on platelet firm adhesion and aggregate formation1,3,4. We used a newly developed switch biomembrane force probe (BFP) assay to characterize the cooperativity between GPIbα and αIIbβ3 on single-molecular level. VWF-A1 and fibronectin fragment (FNIII7-10) were separately coated on two different probes, enabling controlled presentation of two ligands in separate space and time (Fig. A). Pulling GPIbα by A1 on the first probe with a durable force activated the discoid platelet (Fig. A, upper panel), as revealed by an intracellular calcium flux. Upon switching to the FNIII7-10-bearing bead, the A1-triggered platelet was interrogated for the activity of its surface αIIbβ3 (Fig. A, lower panel). A 25-pN force of >2s duration on a single GPIbα-A1 bond was found to activate αIIbβ3 into an intermediate affinity state, as reflected by a ~30% adhesion frequency with a FNIII7-10 bead, significantly higher than binding to low affinity αIIbβ3 on platelets without A1-triggering but much lower than binding to high affinity αIIbβ3 on platelets pre-incubated with ADP or thrombin (Fig. B), two strong platelet-activating agonists. This submaximal activation agreed with the previous immuno-staining findings2. αIIbβ3 activation and outside-in signaling require the sequential engagement of talin and Gα13 to β3 cytoplasmic tail, respectively5,6. Blocking Gα13-β3 engagement by a synthetic peptide mP6 (gift from Xiaoping Du, UIC)6 had no effect on FNIII7-10 binding of resting and A1-triggered platelets, but significantly suppressed the FNIII7-10 binding of platelets pre-incubated with ADP or thrombin by lowering their adhesion frequencies to a level comparable with A1-triggered platelets (Fig. B). The control peptide mP6Scr showed no effect on the ADP or thrombin stimulated platelets (Fig. B). In addition to binding affinity, bond lifetimes were measured by force-clamp experiment. Platelets with no treatment, A1 triggering and ADP stimulation formed αIIbβ3-FNIII7-10 (Fig. C) and αIIbβ3-fibrinogen (Fig. D) bonds with short, intermediate and long lifetimes, respectively. Interactions with fibrinogen exhibited catch-slip bonds regardless of the αIIbβ3 affinity state. By comparison, only high affinity αIIbβ3 exhibited a catch-slip bond with FNIII7-10 as slip-only bonds were observed for FNIII7-10 interactions with intermediate and low affinity αIIbβ3. These data suggest the existence of three distinct integrin states that correspond to the three affinities and force-dependent bond lifetime patterns, in which the intermediate state was induced by the GPIbα mechanotransduction (Fig. E). Gα13 engagement was not required for the intermediate state, but necessary for the high affinity state (Fig. E). References: 1. Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002). 2. Kasirer-Friede, A. et al. Blood103, 3403-3411, doi:10.1182/blood-2003-10-3664 (2004). 3. Savage, B., Saldivar, E. & Ruggeri, Z. M. Cell84, 289-297 (1996). 4. Warwick, S. N. et al. Nature Medicine15, doi:10.1038/nm.1955 (2009). 5. Gong, H. et al. Science327, 340-343, doi:10.1126/science.1174779 (2010). 6. Shen, B. et al. Nature503, 131-135, doi:10.1038/nature12613 (2013). Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Epidemiology and pathogenesis of thoracic outlet syndrome

The superior thoracic aperture is a place particularly vulnerable to the occurrence of tissue conflict and the development of a number of neurovascular changes carrying a risk of upper limb dysfunction. The triggering factor in this case is the pressure on the nerve vascular elements brought about by too large muscles of the chest and neck, clavicle fracture and dislocation of the upper ribs, anomalies in the form of ribs, in the neck, or by apex of the lung tumors. Each anatomical anomaly may be a cause of a number of lesions and lead to the development of the disease. Due to the nature of the oppressed structures, there are two basic groups: neurogenic and vascular. The most common variant giving clinical symptoms is neurogenic thoracic outlet syndrome. In this, the compression ratio, the brachial plexus, and for this reason, the vascular surface of the upper limb dysfunction is often overlooked. However, the vascular variant, and especially arterial sub-variant, is very dangerous because it can give complications even in the form of aneurysms, and even upper limb ischemia. The aim of the study is to present the most common changes in the thoracic outlet causing functional disorders of the upper limb.