Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

Background: Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. Objectives: We aimed to explore the contribution of PAD4 to EET formation. Methods: We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. Results: Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. Conclusion: As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents

Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial by inhibiting neutrophil extracellular traps

IntroductionNeutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied.MethodsWe investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis – cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection.ResultsWe observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice.DiscussionThus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis

Towards novel therapeutic strategies in ischemic stroke by targeting von Willebrand factor and promoting fibrinolysis

Ischemic stroke is worldwide the second most common cause of death and the leading cause of permanent disability. In order to improve therapeutic and prophylactic therapy, it is imperative to understand what causes ischemic stroke and how ischemic stroke brain damage progresses. Up until recently, little was known about the thrombi that cause ischemic stroke. Moreover, experimental and clinical data have demonstrated progression of ischemic stroke brain damage, even after removal of the culprit thrombus. This is due to the process of cerebral ischemia/reperfusion injury. The aim of this PhD thesis was to better understand ischemic stroke thrombi in order to improve thrombolytic therapy, and to design strategies that could limit reperfusion injury. The mainstay of current acute ischemic stroke therapy is aimed at achieving early reperfusion via pharmacological thrombolysis. Unfortunately, pharmacological thrombolysis with tissue type plasminogen activator (t-PA) is not always available and it is in many cases not successful. Since the thrombus is the main target of t-PA mediated fibrinolysis, its composition is believed to be an important contributor to t-PA resistance. In chapter 2, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. VWF is a major player in arterial thrombosis, and its thrombogenicity is controlled ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), the VWF cleaving enzyme. We therefore hypothesized that ADAMTS13 can exert a thrombolytic effect on VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. However, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA–resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes. Delayed ADAMTS13 administration, 60 minutes after occlusion, was still effective, but to a lesser extent. These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke. Future studies should focus on the safety and efficacy of combining ADAMTS13 with t-PA, preferentially in larger animal models of stroke sensitive to t-PA mediated fibrinolysis. Besides ADAMTS13, also the mucolytic agent NAC can regulate the activity of VWF by reducing the size of VWF multimers. In chapter 3, we observed that prophylactic NAC treatment delayed thrombus formation and reduced ischemic brain injury. However, unlike ADAMTS13, when NAC was administered after thrombus formation, it did not exert a thrombolytic effect on VWF-rich thrombi and infarct sizes were similar in vehicle and NAC treated animals. In chapter 4, we collected and processed 81 stroke thrombi and aimed to better understand stroke thrombus composition, and correlate histological findings with stroke etiology. The key finding from our study was a link between thrombus age, composition and stroke etiology. Thrombi formed in the heart contained significantly more fibrin, more VWF and less red blood cells. Additionally, cardioembolic thrombi were older compared with thrombi from another known etiology. The distinct features of cardioembolic thrombi support the idea that different thrombi are formed at different places. Future multicenter studies will be needed to confirm these findings and to further evaluate the added value of thrombus histology to stroke etiology determination. In recent years, increasing evidence has shown a clear association between high VWF levels and ischemic stroke, however only a few studies have investigated the correlation between ischemic stroke and ADAMTS13. Therefore, in chapter 5, we measured plasma ADAMTS13 levels in patients with acute ischemic stroke, patients with a chronic cerebrovascular disease and healthy volunteers. We found that ischemic stroke patients had significantly lower ADAMTS13 levels compared with healthy volunteers. Also patients with a chronic cerebrovascular disease had lower ADAMTS13 levels compared with healthy volunteers, however these levels were still higher than the acute stroke patients. Furthermore, when the ratio of VWF/ADAMTS13 was considered, the difference between acute stroke patients, chronic cerebrovascular disease patients and healthy volunteers became even more apparent. These results demonstrate the potential involvement of the VWF/ADAMTS13 axis in acute and chronic cerebrovascular disorders and warrant prospective studies further investigating the causality of this observed association. Interestingly, VWF is not only involved in the acute phase of ischemic stroke, but also plays a major role during cerebral ischemia/reperfusion injury. Indeed, mice without VWF are protected from ischemic stroke. VWF is produced in endothelial cells and megakaryocytes; however, the relative contribution of each storage pool of VWF to ischemia/reperfusion injury was until recently unknown. To be able to dissect the role of the endothelial and platelet pool of VWF, bone marrow transplantations were performed between VWF wild-type and VWF knock-out mice in chapter 7. With these bone marrow transplantations we obtained chimeric mice having only endothelial VWF and chimeric mice having only platelet VWF. These mice were subsequently used to assess the contribution of each VWF storage pool in models of hemostasis, thrombosis and cerebral ischemia/reperfusion injury. As expected, endothelial VWF, which is the main source of circulating VWF, was sufficient to maintain hemostasis, induce arterial thrombosis and aggravate ischemic stroke brain injury. In contrast, both hemostasis and arterial thrombosis were impaired in mice expressing only platelet VWF. However, mice expressing only platelet VWF were not protected from cerebral ischemia/reperfusion injury. Moreover, we could demonstrate that this detrimental effect of platelet VWF was mediated by a platelet GPIb-dependent mechanism. These data suggest that, whereas platelet-derived VWF does not play a crucial role in hemostasis and arterial thrombosis, it aggravates ischemic stroke via a GPIb-dependent mechanism. Besides VWF, also the coagulation cascade is detrimentally involved during ischemia/reperfusion injury. In chapter 8 we assessed the therapeutic potential of inhibiting two inhibitors of fibrinolysis (TAFI and PAI-1) in a mouse model of cerebral ischemia/reperfusion injury. First, we demonstrated that inhibition of either PAI-1 or TAFI with monoclonal antibodies was efficient in reducing cerebral fibrinogen deposition. This was associated with an improved neurological and motor outcome, as well as a reduced ischemic stroke brain damage. Furthermore, when PAI-1 and TAFI were inhibited simultaneously, a cooperative protective effect was observed. Simultaneous inhibition reduced cerebral thrombotic burden, improved cerebral blood flow and reduced ischemic brain injury. The therapeutic potential of this cooperative effect was confirmed in chapter 9 with a bispecific diabody, constructed with the variable domains of the anti-PAI-1 and anti-TAFI monoclonal antibodies. Importantly, bleeding complications were absent in all of the treated mice. While very promising, future preclinical testing in other relevant animal models is necessary to further evaluate the safety and efficacy of this novel therapeutic strategy. To conclude, in this PhD project we have identified VWF as novel thrombolytic target and ADAMTS13 as a novel thrombolytic agent capable of cleaving VWF inside a thrombus. Furthermore, we provide evidence of a link between thrombus composition and stroke etiology. Finally, we demonstrated the detrimental role of platelet VWF in cerebral reperfusion injury and identified TAFI and PAI-1, as novel targets to reduce cerebral ischemia/reperfusion injury.status: publishe

Targeting Glycoprotein VI for Thromboembolic Disorders All Gain With No Pain?

status: publishe

von Willebrand Factor and Platelet Glycoprotein Ib: A Thromboinflammatory Axis in Stroke

von Willebrand factor (VWF) and platelets are key mediators of normal hemostasis. At sites of vascular injury, VWF recruits platelets via binding to the platelet receptor glycoprotein Ibα (GPIbα). Over the past decades, it has become clear that many hemostatic factors, including VWF and platelets, are also involved in inflammatory processes, forming intriguing links between hemostasis, thrombosis, and inflammation. The so-called “thrombo-inflammatory” nature of the VWF-platelet axis becomes increasingly recognized in different cardiovascular pathologies, making it a potential therapeutic target to interfere with both thrombosis and inflammation. In this review, we discuss the current evidence for the thrombo-inflammatory activity of VWF with a focus on the VWF-GPIbα axis and discuss its implications in the setting of ischemic stroke.status: Published onlin

Studies on the native conformation of alpha synuclein

status: publishe

Investigation of Neutrophil Extracellular Traps as Potential Mediators in the Pathogenesis of Non-Acute Subdural Hematomas: A Pilot Study

Non-acute subdural hematomas (NASHs) are a cause of significant morbidity and mortality, particularly with recurrences. Although recurrence is believed to involve a disordered neuroinflammatory cascade involving vascular endothelial growth factor (VEGF), this pathway has yet to be completely elucidated. Neutrophil extracellular traps (NETs) are key factors that promote inflammation/apoptosis and can be induced by VEGF. We investigated whether NETs are present in NASH membranes, quantified NET concentrations, and examined whether NET and VEGF levels are correlated in NASHs. Samples from patients undergoing NASH evacuation were collected during surgery and postoperatively at 24 and 48 h. Fluid samples and NASH membranes were analyzed for levels of VEGF, NETs, and platelet activation. NASH samples contained numerous neutrophils positive for NET formation. Myeloperoxidase-DNA complexes (a marker of NETs) remained elevated 48 h postoperatively (1.06 ± 0.22 day 0, 0.72 ± 0.23 day 1, and 0.83 ± 0.33 day 2). VEGF was also elevated in NASHs (7.08 ± 0.98 ng/mL day 0, 3.40 ± 0.68 ng/mL day 1, and 6.05 ± 1.8 ng/mL day 2). VEGF levels were significantly correlated with myeloperoxidase-DNA levels. These results show that NETs are increasing in NASH, a finding that was previously unknown. The strong correlation between NET and VEGF levels indicates that VEGF may be an important mediator of NET-related inflammation in NASH

Neutrophil extracellular traps in ischemic stroke thrombi

Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular, no information is yet available on the presence of NETs in cerebral occlusions. Such information is, however, essential to improve current thrombolytic therapy with tissue plasminogen activator (t-PA). This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi.status: publishe

Reduced ADAMTS13 levels in patients with acute and chronic cerebrovascular disease

Von Willebrand Factor (VWF) plays a major role in thrombosis and hemostasis and its thrombogenicity is controlled by ADAMTS13. Whereas increasing evidence shows a clear association between VWF levels and acute ischemic stroke, little is known about a correlation with ADAMTS13. Therefore, the aim of this study was to compare plasma levels of ADAMTS13 between 85 healthy volunteers (HV), 104 patients with acute ischemic stroke and 112 patients with a chronic cerebrovascular disease (CCD). In this case-control study, plasma ADAMTS13 antigen levels were measured by ELISA and plasma VWF levels, measured previously, were next used to calculate VWF:ADAMTS13 ratios. ADAMTS13 levels and VWF:ADAMTS13 ratios were subsequently correlated with key demographic and clinical parameters. ADAMTS13 levels were significantly lower in acute ischemic stroke patients (82.6 ± 21.0%) compared with HV (110.6 ± 26.9%). Also, CCD patients (99.6 ± 24.5%) had significantly lower ADAMTS13 levels compared with HV however these were still higher than in acute stroke patients. Furthermore, when assessing the VWF:ADAMTS13 ratios, an even greater difference was revealed between stroke patients (2.7 ± 1.9), HV (1.1 ± 0.5) and CCD patients (1.7 ± 0.7). The VWF:ADAMTS13 ratio was significantly associated with stroke severity and modality. In conclusion, both in acute and chronic cerebrovascular disease patients, ADAMTS13 levels were significantly decreased, with the lowest ADAMTS13 levels found in acute stroke patients. This difference was even more distinct when the ratio of VWF:ADAMTS13 was considered. These results demonstrate the potentially important involvement of the VWF/ADAMTS13 axis in ischemic stroke.status: publishe