Proteomics-Based Approach to Identify Novel Blood Biomarker Candidates for Differentiating Intracerebral Hemorrhage From Ischemic Stroke—A Pilot Study

Background: A reliable distinction between ischemic stroke (IS) and intracerebral hemorrhage (ICH) is required for diagnosis-specific treatment and effective secondary prevention in patients with stroke. However, in resource-limited settings brain imaging, which is the current diagnostic gold standard for this purpose, is not always available in time. Hence, an easily accessible and broadly applicable blood biomarker-based diagnostic test differing stroke subtypes would be desirable. Using an explorative proteomics approach, this pilot study aimed to identify novel blood biomarker candidates for distinguishing IS from ICH.Material and Methods: Plasma samples from patients with IS and ICH were drawn during hospitalization and were analyzed by using liquid chromatography/mass spectrometry. Proteins were identified using the human reference proteome database UniProtKB, and label-free quantification (LFQ) data were further analyzed using bioinformatic tools.Results: Plasma specimens of three patients with IS and four patients with ICH with a median National Institute of Health Stroke Scale (NIHSS) of 12 [interquartile range (IQR) 10.5–18.5] as well as serum samples from two healthy volunteers were analyzed. Among 495 identified protein groups, a total of 368 protein groups exhibited enough data points to be entered into quantitative analysis. Of the remaining 22 top-listed proteins, a significant difference between IS and ICH was found for Carboxypeptidase N subunit 2 (CPN2), Coagulation factor XII (FXII), Plasminogen, Mannan-binding lectin serine protease 1, Serum amyloid P-component, Paraoxonase 1, Carbonic anhydrase 1, Fibulin-1, and Granulins.Discussion: In this exploratory proteomics-based pilot study, nine candidate biomarkers for differentiation of IS and ICH were identified. The proteins belong to the immune system, the coagulation cascade, and the apoptosis system, respectively. Further investigations in larger cohorts of patients with stroke using additional biochemical analysis methods, such as ELISA or Western Blotting are now necessary to validate these markers, and to characterize diagnostic accuracy with regard to the development of a point-of-care-system for use in resource-limited areas.

Coagulation Factor-XII induces interleukin-6 by primary lung fibroblasts: A role in idiopathic pulmonary fibrosis?

Background The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Methods Plasma levels of FXII were measured by ELISA in patients with IPF and age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Findings Compared to 35 healthy donors, plasma levels of FXII were not higher in IPF (n=27, p>0·05). Tissue FXII was elevated in IPF (n=11) and increased numbers of FXII+ cells were found in IPF (n=8) lung tissue compared to non-diseased controls (n=6, p<0·0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII-induced IL-6 production via PAR-1 and NF-kB. FXII induced migration of fibroblasts in a concentration-dependent manner. Interpretation FXII is normally confined to the circulation but leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis. Funding National Health and Medical Research Council CRE in Lung Fibrosis and CSL Ltd.

Altered fibrin clot structure and dysregulated fibrinolysis contribute to thrombosis risk in severe COVID-19

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in critically ill COVID-19 patients in comparison to patients with severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19 as opposed to ARDS-influenza. Using confocal and electron microscopy, we showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to ARDS-infleunza subjects. Dysregulatated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19 patients. Together, compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to high incidence of thrombotic events in COVID-19.

Inhibition of Factor XII Attenuates Prothrombotic Complications in Sickle Cell Mice

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice. First, Townes HbSS mice (SS) and non-sickle Townes HbAA controls (AA) (male and female, 16 weeks) received anti-FXII antibody or control IgGκ1 (10 mg/kg, IV) 30 minutes prior to subjecting them to venous thrombosis, initiated by applying positive current (3 volts, 90 sec) to the femoral vein. To visualize platelet and fibrin accumulation, mice were injected with rhodamine 6G and anti-fibrin antibody 59D8 labeled with Alexa Fluor 647, respectively. The femoral vein thrombi were imaged by intravital fluorescence microscopy using time-lapse capture every 10 seconds, to acquire images of fibrin and platelets over 60 min. The accumulation of platelets and fibrin was quantified for relative intensity of each fluorophore over the region of the observed thrombus. As previously shown, thrombi of SS/IgG mice showed an increased fibrin and platelet accumulation compared to AA/IgG group. Importantly, 15D10 treatment significantly attenuated both fibrin (p&lt;0.001) and platelet (p&lt;0.05) deposition over time in SS mice compared to SS/IgG group. The same effect of 15D10 treatment was observed in AA mice. At the end of experiment, clots were collected and stained with hematoxylin and eosin, and clot volume was assessed histomorphometrically (Nikon Ti-2, FIJI Software). Surprisingly, despite higher fibrin content, clots from SS/IgG mice had significantly smaller volume than clots from AA/IgG group (0.32 ± 0.04 versus 0.60 ± 0.11 mm 3, p&lt;0.05). Importantly, administration of 15D10 significantly reduced clot volume in both SS (0.086 ± 0.01 mm 3, p&lt;0.05) and AA mice (0.1 ± 0.02 mm 3, p&lt;0.05). Next, AA and SS mice (male and female, 8-10 weeks) were subjected to brain IR injury induced by middle cerebral artery occlusion for 60 minutes followed by 24 hours of reperfusion (mouse model of ischemic stroke). 15D10 or control IgGκ1 (10 mg/kg, IV) were injected 30 minutes before occlusion and again at 6 hours into the reperfusion period to generate 3 experimental groups: AA/IgG, SS/IgG and SS/15D10. All analyzed parameters of brain IR injury were significantly worse in the SS/IgG group compared to the AA/IgG group. Compared to IgG, pre-treatment of SS mice with 15D10 significantly attenuated neuronal damage determined by volume of brain infarction (11.7 ± 3.7 vs 24.9 ± 2.4%, p&lt;0.001) and improved behavioral deficit assessed by mean stroke score (9.0 ± 0.9 vs 14.6 ± 0.9, p&lt;0.01). These changes were accompanied by a significant increase in leukocytes rolling (1978.0 ± 93.5 vs 1517.0 ± 180.3 rolling leukocytes/sec/mm 2, p&lt;0.001), and significant reduction in the number of adherent leukocytes (367.2 ± 49.0 vs 723.4 ± 48.5, adherent leukocytes/mm 2, p&lt;0.001) observed in the brain microvasculature of SS mice treated with 15D10 compared to SS/IgG group. Together, our data indicates that in the mouse model of SCD FXII contributes to the experimental venous thrombosis and ischemic stroke. Given that targeting the intrinsic pathway can reduce thrombosis without affecting hemostasis, our data suggest that targeting FXII might be a beneficial treatment in reducing inflammatory and thrombotic complications in SCD patients without a risk of bleeding. Disclosures Wallisch: Aronora Inc,: Current Employment. Key: Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Uniqure: Consultancy, Other: Participation as a clinical trial investigator. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Severe High Molecular Weight Kininogen (HK) Deficiency: Clinical Characteristics, Deficiency-Causing KNG1 Variants in Reported and New Cases, and Estimated Prevalence

Background: Severe high molecular weight kininogen (HK) deficiency is an autosomal recessive defect of the contact system caused by mutations in KNG1. Limited scientific interest in HK deficiency due to the rarity of the seemingly asymptomatic condition may increase, as HK, the precursor of bradykinin, is now discussed as a therapeutic target e.g. in hereditary angioedema. Aims: We provide a comprehensive analysis of the diagnostic, clinical, and genetic features of HK deficiency and estimate its frequency. Methods: We identified a new case of HK deficiency, systematically review the literature, conduct new genetic studies of reported cases, and comprehensively analyze the clinical course and diagnostic criteria. Clotting activity of HK and prekallikrein (PK) (HK:C/PK:C) and antigen (HK:Ag/PK:Ag) were determined and genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. Characteristics deduced from the known HK deficiency-causing variants were used to estimate the frequency of HK deficiency from the KNG1 variants aggregated in GnomAD. Results: 677 studies were identified by systematic review of the literature for HK deficiency. 27 of these contained individual cases of HK deficiency including 6 studies not listed in PubMed. Little-noticed cases from the gray literature account for more than one-third (16/39) of the extracted, unrelated cases. We genotyped one newly diagnosed HK-deficient case and 2 cases described in the literature and additionally evaluated all 10 studies reporting genetic data in HK-deficiency (including one case previously misdiagnosed as having PK deficiency). A total of 10 KNG1 variants causing HK deficiency (one new) were found, the most frequent being c.586C&gt;T, p.Arg196* (4 unrelated families). Interestingly, all HK deficiency-causing variants are truncating, whereas two amino acid substitutions with presumed functional consequence, have been described as the cause of hereditary angioedema. Conservative prevalence estimates based on all known and putative HK deficiency-causing variants extracted from GnomAD (truncating variants in KNG1, including indels, nonsense and canonical splice site mutations located in that part of the gene, where relevant mutations have been described) revealed a frequency of 1 case of HK deficiency among 7,925,172 with slight differences in the analyzed ethnicities (see table). In addition, although not to the same extent as seen in PK deficiency, HK deficiency apparently is more prevalent in Africans. While it is already well known that HK deficiency causes decreased PK levels, our data indicate that factor XI levels are also frequently decreased, albeit to a lesser extent. The number of cases detected so far is too low for a more detailed analysis regarding bleeding, thrombotic, and cardiovascular events or immunological abnormalities. Conclusion: HK-deficiency is probably more frequent than previously thought. Suspected cases of contact phase defects should at least be analyzed for HK activity (besides factor XII, XI and PK activity) to facilitate conclusive evaluation of the clinical significance in the future. Figure 1 Figure 1. Disclosures Lämmle: Takeda: Membership on an entity's Board of Directors or advisory committees; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Baxter: Other: Travel Support, Speakers Bureau; Alexion: Other: Travel Support, Speakers Bureau; Siemens: Other: Travel Support, Speakers Bureau; Bayer: Other: Travel Support, Speakers Bureau; Roche: Other: Travel Support, Speakers Bureau; Sanofi: Other: Travel Support, Speakers Bureau.

Congenital hemophilia A with low activity of factor XII: a case report and literature review

Background Congenital hemophilia A is a recessive inherited hemorrhagic disorder. According to the activity of functional coagulation factors, the severity of hemophilia A is divided into three levels: mild, moderate and severe. The first bleeding episode in severe and moderate congenital hemophilia A occurs mostly in early childhood and mainly involves soft tissue and joint bleeds. At present, there are limited reports on severe congenital hemophilia A with low factor XII (FXII) activity during the neonatal period. Case presentation A 13-day-old neonate was admitted to the hospital with hematoma near the joints of both upper arms. Coagulation tests showed he had low activity of factor VIII (FVIII) and FXII. He was diagnosed with congenital hemophilia A and treated with human coagulation factor VIII (recombinant FVIII). Although the hematoma became smaller, FVIII activity was only increased to a certain extent and FXII activity decreased gradually. Unfortunately, the child responded poorly to recombinant human coagulation factor VIII and his guardian rejected prophylactic inhibitors and genetic testing and refused further treatment. Three months later, the child developed intracranial hemorrhage (ICH) due to low FVIII activity. Conclusions In hemophilia A, the presence of FVIII inhibitors, drug concentration and testing are three important aspects that must be considered when FVIII activity does not reach the desired level. Early positive disease treatment and prophylaxis can decrease the frequency of bleeding and improve quality of life. We recommend that pregnant women with a family history of hemophilia A undergo early prenatal and neonatal genetic testing.

Polyphosphate Expression by Cancer Cell Extracellular Vesicles Mediates Binding of Factor XII and Contact Activation

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EV are elevated in cancer, and studies suggest that EV may stimulate thrombosis in cancer patients through expression of tissue factor. However, limited data also implicates EV in activation of the contact pathway of coagulation through activation of factor XII (FXII) to factor XIIa (FXIIa). To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with activation of FXII, EV induced the cleavage of high molecular weight kininogen to cleaved kininogen. We also observed that EV from cancer patients stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or E. coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EV and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by deficiency of FXII, HK, or prekallikrein. Moreover, pre-treatment of EV with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates binding of contact factors to EV, and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.