Plasma H3Cit-DNA Discriminates Between Cancer and Inflammation in a Cohort of Patients with Unspecific Cancer Symptoms.

Cancer detection is challenging, especially in patients with unspecific cancer symptoms. Biomarkers could identify patients at high risk of cancer. Prior studies indicate that neutrophil extracellular traps (NETs) are associated with cancer, but also with autoimmune and infectious diseases. The objective of this prospective study was to investigate markers associated with NET formation (nucleosomal citrullinated histone 3 [H3Cit-DNA], cell free DNA [cfDNA] and neutrophil elastase [NE]), and c-reactive protein (CRP) in patients with unspecific cancer symptoms, such as fatigue, weight loss or radiological sign of malignancy without an apparent primary tumor, referred to the Diagnostic Center at Danderyd Hospital in Sweden. Blood samples were drawn on admission, before cancer diagnosis. Out of 475 patients, 160 (34%) were diagnosed with cancer, 56 (12%) with autoimmune disease, 32 (7%) with infectious disease, 71 (15%) with other diseases and 156 (33%) received no diagnosis. H3Cit-DNA, cfDNA, NE and CRP were significantly higher in patients with cancer compared to patients without cancer (p < 0.0001, p < 0.0001, p = 0.004, and p = 0.0002 respectively). H3Cit-DNA, but not cfDNA, NE or CRP, was significantly elevated in patients with cancer compared to patients with autoimmune disease (p = 0.0001). H3Cit-DNA, cfDNA, NE or CRP did not differ between cancer and infectious disease. In conclusion, H3Cit-DNA is elevated in patients diagnosed with cancer compared to non-cancer patients with the same symptomatology. Further studies should evaluate if H3Cit-DNA could aid in selecting patients that would benefit the most from a rapid cancer diagnostic work-up.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Soluble Glycoprotein VI Predicts Abdominal Aortic Aneurysm Growth Rate and is a Novel Therapeutic Target.

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (JAQ1) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist.

4D intravital imaging identifies platelets as the predominant cellular procoagulant surface in a mouse hemostasis model.

Interplay between platelets, coagulation factors, endothelial cells (ECs) and fibrinolytic factors is necessary for effective hemostatic plug formation. This study describes a four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components in mice with high spatiotemporal resolution. Fibrin accumulation following laser-induced vascular injury was observed at the platelet plug-EC interface, controlled by the antagonistic balance between fibrin generation and breakdown. We observed less fibrin accumulation in mice expressing low levels of tissue factor (TFlow) or F12-/- mice compared to controls, whereas increased fibrin accumulation, including on the vasculature adjacent to the platelet plug, was observed in plasminogen-deficient mice or wild-type mice treated with tranexamic acid (TXA). Phosphatidylserine (PS), a membrane lipid critical for the assembly of coagulation factors, was first detected at the platelet plug-EC interface, followed by exposure across the endothelium. Impaired PS exposure resulted in a significant reduction in fibrin accumulation in cyclophilin D-/- mice. Adoptive transfer studies demonstrated a key role for PS exposure on platelets, and to a lesser degree on ECs, in fibrin accumulation during hemostatic plug formation. Together, these studies suggest that (1) platelets are the functionally dominant procoagulant cellular surface, and (2) plasmin is critical for limiting fibrin accumulation at the site of a forming hemostatic plug.

Cancer patients and ischemic stroke.

Patients with cancer have an increased risk of ischemic stroke compared to the general population. Additionally, these patients have a worse prognosis compared to stroke patients without cancer. Activation of coagulation appears to play a key role in the pathophysiology of ischemic stroke in patients with cancer. However, the underlying mechanisms remain unknown. Moreover, we do not have a way to identify cancer patients with a high risk of stroke and cannot develop prevention strategies. Therefore, there is an urgent need for neurologists and oncologists to develop screening and prevention strategies for stroke in patients with cancer. In this review, we summarize the characteristics of cancer patients at a high risk of stroke, the predictors for the development of stroke and survival in cancer patients, and possible treatments.

Plasma kallikrein supports FXII-independent thrombin generation in mouse whole blood.

ABSTRACT: Plasma kallikrein (PKa) is an important activator of factor XII (FXII) of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin-generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway-initiated TG compared with that of wild-type controls and was comparable with that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared with wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway-initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway-initiated TG in the complex milieu of mouse WB, and a component of this contribution occurs in an FXII-independent manner.

Biomarkers of bleeding and venous thromboembolism in patients with acute leukemia.

BACKGROUND: Coagulopathy and associated bleeding and deep vein thrombosis (DVT) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated. OBJECTIVES: To evaluate the associations between biomarker levels and bleeding and DVT in acute leukemia patients. METHODS: We examined plasma levels of activators, inhibitors, and biomarkers of the coagulation and fibrinolytic pathways in patients aged ≥18 years with newly diagnosed acute leukemia compared with those of normal controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and DVT in acute leukemia patients. The study included 358 patients with acute leukemia (29 with acute promyelocytic leukemia [APL], 253 with non-APL acute myeloid leukemia, and 76 with acute lymphoblastic leukemia) and 30 normal controls. RESULTS: Patients with acute leukemia had higher levels of extracellular vesicle tissue factor (EVTF) activity, phosphatidylserine-positive extracellular vesicles, plasminogen activator inhibitor-1, plasmin-antiplasmin complexes, and cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared with normal controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among acute leukemia patients. There were 41 bleeding and 23 DVT events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding (subdistribution hazard ratio, 2.30; 95% CI, 0.99-5.31), whereas high levels of plasminogen activator inhibitor-1 were associated with increased risk of DVT (subdistribution hazard ratio, 3.00; 95% CI, 0.95-9.47) in these patients. CONCLUSION: Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and DVT.

Mucin 1 and venous thrombosis in tumor-bearing mice and patients with cancer.

INTRODUCTION: Mucins released from epithelial tumors have been proposed to play a role in cancer-associated thrombosis. Mucin1 (MUC1) is a transmembrane mucin that is overexpressed in a variety of human malignancies, including breast and pancreatic cancer. We analyzed the association of MUC1 and venous thrombosis in a mouse tumor model and in patients with cancer. MATERIALS AND METHODS: We used a human pancreatic cancer cell line HPAF-II that expresses a high level of MUC1. We grew HPAF-II tumors in the pancreas of Crl:NU-Foxn1nu male mice. MUC1 in plasma and extracellular vesicles (EVs) isolated from plasma was measured using an enzyme-linked immunosorbent assay. MUC1 in EVs and venous thrombi from tumor-bearing mice was assessed by western blotting. We measured MUC1 in plasma from healthy controls and patients with stomach, colorectal or pancreatic cancer with or without venous thromboembolism. RESULTS AND DISCUSSION: MUC1 was detected in the plasma of mice bearing HPAF-II tumors and was associated with EVs. MUC1 was present in venous thrombi from mice bearing HFAP-II tumors. Recombinant MUC1 did not induce platelet aggregation. Levels of MUC1 were higher in patients with pancreatic cancer compared with healthy controls. In contrast to the mouse model, MUC1 was present in EV-free plasma in samples from healthy controls and patients with cancer. There was no significant difference in the levels of MUC1 in cancer patients with or without VTE. Our data did not find any evidence that MUC1 contributed to VTE in patients with cancer.

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Evaluation of the ability of commercial enzyme-linked immunosorbent assays to measure mouse tissue factor.

Background: Tissue factor (TF) is the primary cellular initiator of the blood coagulation cascade. Increased levels of TF expression on circulating monocytes or on extracellular vesicles (EVs) are associated with thrombosis in a variety of diseases, including sepsis and COVID-19. Objectives: Here, we aimed to evaluate the ability of 4 commercial TF enzyme-linked immunosorbent assays (ELISAs) to measure mouse TF in cells and plasma. Methods: We used 4 commercial mouse TF ELISAs (SimpleStep, R&D Systems, MyBioSource [sandwich], and MyBioSource [competitive]). We used recombinant mouse TF (rmTF; 16-1000 pg/mL), cell lysates from a TF-expressing mouse pancreatic cancer cell line, and plasma and EVs isolated from plasma from mice injected with vehicle or bacterial lipopolysaccharide (LPS). Results: The 2 MyBioSource kits failed to detect rmTF or TF in cell lysates. The SimpleStep and R&D kits detected rmTF in buffer or spiked into plasma in a concentration-dependent manner. These kits also detected TF in cell lysates from a mouse pancreatic cancer cell line. A higher signal was observed with the SimpleStep kit compared to the R&D kit. However, the SimpleStep and R&D kits failed to detect TF in plasma or EVs from LPS-treated mice. Conclusion: Our results indicate that some commercial ELISAs can be used to measure mouse TF levels in cell lysates but they cannot detect TF in plasma or EVs from endotoxemic mice.

Tissue Factor and COVID-19 Associated Thrombosis.

Microbial infections activate the innate and adaptive immune systems.1 Pathogen-associated molecular patterns produced by microbes, such as double-stranded RNA, are detected by PRRs (pattern-recognition receptors), such as toll-like receptor 3, and this leads to the expression of interferons and cytokines.1,2.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Tissue factor pathway inhibitor is associated with risk of venous thromboembolism and all-cause mortality in patients with cancer.

Venous thromboembolism (VTE) is a common complication in patients with cancer. Data on the role of natural inhibitors of coagulation for occurrence of cancer-associated VTE are limited, thus, we investigated the association of tissue factor pathway inhibitor (TFPI) with risk of VTE and all-cause mortality in patients with cancer. Total TFPI antigen levels were measured with a commercially available enzyme-linked immunosorbant assay in patients included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study with the primary outcome VTE. Competing risk analysis and Cox regression analysis were performed to explore the association of TFPI levels with VTE and all-cause mortality. TFPI was analyzed in 898 patients (median age 62 years; interquartile range [IQR], 53-68; 407 (45%) women). Sixty-seven patients developed VTE and 387 died (24-month cumulative risk 7.5% and 42.1%, respectively). Patients had median TFPI levels at study inclusion of 56.4 ng/mL (IQR, 45.7-70.0), with highest levels in tumor types known to have a high risk of VTE (gastroesophageal, pancreatic and brain cancer: 62.0 ng/mL; IQR, 52.0-75.0). In multivariable analysis adjusting for age, sex, cancer type and stage, TFPI levels were associated with VTE risk (subdistribution hazard ratio per doubling =1.63, 95% confidence interval [CI]: 1.03-2.57). When patients with high and intermediate/low VTE risk were analyzed separately, the association remained independently associated in the high risk group only (subdistribution hazard ratio =2.63, 95% CI: 1.40-4.94). TFPI levels were independently associated with all-cause mortality (hazard ratio =2.36, 95% CI: 1.85-3.00). In cancer patients increased TFPI levels are associated with VTE risk, specifically in patients with high-risk tumor types, and with all-cause mortality.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Biomarkers of bleeding and venous thromboembolism in patients with acute leukemia.

Background: Coagulopathy and associated bleeding and venous thromboembolism (VTE) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated. Objectives: To evaluate the associations between biomarker levels and bleeding and VTE in acute leukemia patients. Patients/Method: We examined plasma levels of activators, inhibitors and biomarkers of the coagulation and fibrinolytic pathways in patients ≥18 years with newly diagnosed acute leukemia compared to healthy controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and VTE in acute leukemia patients. The study included 358 patients with acute leukemia (29 acute promyelocytic leukemia [APL], 253 non-APL acute myeloid leukemia [AML] and 76 acute lymphoblastic leukemia [ALL]), and 30 healthy controls. Results: Patients with acute leukemia had higher levels of extracellular vesicle (EV) tissue factor (TF) activity, phosphatidylserine-positive EVs, plasminogen activator inhibitor-1 (PAI-1), plasmin-antiplasmin complexes, cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared to healthy controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among the acute leukemia patients. There were 41 bleeding and 37 VTE events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding (sHR 2.30, 95%CI 0.99-5.31) whereas high PAI-1 was associated with increased risk of VTE (sHR 3.79, 95%CI 1.40-10.28) in these patients. Conclusions: Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and VTE.