Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Impact of Early Microparticle Release during Isolated Severe Traumatic Brain Injury: Correlation with Coagulopathy and Mortality.

BACKGROUND: Microparticles (MPs) have been implicated in thrombosis and endothelial dysfunction. Their involvement in early coagulopathy and in worsening of outcomes in isolated severe traumatic brain injury (sTBI) patients remains ill defined. OBJECTIVE: We sought to quantify the circulatory MP subtypes derived from platelets (PMPs; CD42), endothelial cells (EMPs; CD62E), and those bearing tissue factor (TFMP; CD142) and analyze their correlation with early coagulopathy, thrombin generation, and in-hospital mortality. MATERIALS AND METHODS: Prospective screening of sTBI patients was done. Blood samples were collected before blood and fluid transfusion. MP enumeration and characterization were performed using flow cytometry, and thrombin-antithrombin complex (TAT) levels were determined using enzyme-linked immunosorbent assay (ELISA). Circulating levels of procoagulant MPs were compared between isolated sTBI patients and age- and gender-matched healthy controls (HC). Patients were stratified according to their PMP, EMP, and TFMP levels, respectively (high ≥HC median and low < HC median). RESULTS: Isolated sTBI resulted in an increased generation of PMPs (456.6 [228-919] vs. 249.1 [198.9-404.5]; P = 0.01) and EMPs (301.5 [118.8-586.7] vs. 140.9 [124.9-286]; P = 0.09) compared to HCs. Also, 5.3% of MPs expressed TF (380 [301-710]) in HCs, compared to 6.6% MPs (484 [159-484]; P = 0.87) in isolated sTBI patients. Early TBI-associated coagulopathy (TBI-AC) was seen in 50 (41.6%) patients. PMP (380 [139-779] vs. 523.9 [334-927]; P = 0.19) and EMP (242 [86-483] vs. 344 [168-605]; P = 0.81) counts were low in patients with TBI-AC, compared to patients without TBI-AC. CONCLUSION: Our results suggest that enhanced cellular activation and procoagulant MP generation are predominant after isolated sTBI. TBI-AC was associated with low plasma PMPs count compared to the count in patients without TBI-AC. Low PMPs may be involved with the development of TBI-AC.

Unresolved hemostasis issues in haemophilia.

Despite rapid technological advancement in factor and nonfactor products in the prevention and treatment of bleeding in haemophilia patients, it is imperative that we acknowledge gaps in our understanding of how hemostasis is achieved. The authors will briefly review three unresolved issues in persons with haemophilia (PwH) focusing on the forgotten function that red blood cells play in hemostasis, the critical role of extravascular (outside circulation) FIX in hemostasis in the context of unmodified and extended half-life FIX products and finally on the role that skeletal muscle myosin plays in prothrombinase assembly and subsequent thrombin generation that could mitigate breakthrough muscle hematomas.

Detection of a Prethrombotic State in Patients with Hepatocellular Carcinoma, Using a Clot Waveform Analysis.

Background: Although hepatocellular carcinoma (HCC) is frequently associated with thrombosis, it is also associated with liver cirrhosis (LC) which causes hemostatic abnormalities. Therefore, hemostatic abnormalities in patients with HCC were examined using a clot waveform analysis (CWA). Methods: Hemostatic abnormalities in 88 samples from HCC patients, 48 samples from LC patients and 153 samples from patients with chronic liver diseases (CH) were examined using a CWA-activated partial thromboplastin time (APTT) and small amount of tissue factor induced FIX activation (sTF/FIXa) assay. Results: There were no significant differences in the peak time on CWA-APTT among HCC, LC, and CH, and the peak heights of CWA-APTT were significantly higher in HCC and CH than in HVs and LC. The peak heights of the CWA-sTF/FIXa were significantly higher in HCC than in LC. The peak times of the CWA-APTT were significantly longer in stages B, C, and D than in stage A or cases of response. In the receiver operating characteristic (ROC) curve, the fibrin formation height (FFH) of the CWA-APTT and CWA-sTF/FIXa showed the highest diagnostic ability for HCC and LC, respectively. Thrombosis was observed in 13 HCC patients, and arterial thrombosis and portal vein thrombosis were frequently associated with HCC without LC and HCC with LC, respectively. In ROC, the peak time×peak height of the first derivative on the CWA-sTF/FIXa showed the highest diagnostic ability for thrombosis. Conclusion: The CWA-APTT and CWA-sTF/FIXa can increase the evaluability of HCC including the association with LC and thrombotic complications.

Impact of systematic variations in hematocrit and platelet count on thrombelastometry tissue factor activated assay parameters.

BACKGROUND: Thromboelastogram testing is increasingly being used to manage patients with massive bleeding. An earlier study found that the test results were influenced by the hematocrit (Hct) and platelet (PLT) concentrations. This study sought to determine if these factors confounded the results of a different manufacturer's thromboelastography testing. METHODS: Using freshly collected whole blood from volunteers and stored red blood cells (RBC) and plasma, the whole blood was manipulated to achieve different Hct values and PLT concentrations. Each reconstituted whole blood sample was tested in triplicate on the ROTEM Delta device and the ExTEM results were recorded. RESULTS: Many of the ExTEM results varied according to the Hct and PLT concentration. In particular, the ExTEM clot formation time (CFT) was abnormally long when the Hct was 45% and the PLT concentration was ≤75 × 109/L, normalizing only when the PLT count was ≥100 × 109/L. CFT samples with Hct 25% and 35% were also abnormal with low PLT concentrations but normalized at lower PLT concentrations compared to the Hct 45% samples. The ExTEM angle also demonstrated abnormal results when the Hct was 45% and the PLT concentration was ≤50 × 109/L. The ExTEM A10 and maximum clot firmness (MCF) tests tended to also be abnormal when the Hct was between 25% and 45% and the platelet concentrations were below 75 × 109/L. CONCLUSION: While thromboelastogram testing is gaining popularity for managing bleeding patients, clinicians should be aware of these confounding factors when making transfusion decisions based on their results.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Coagulation activity and thrombotic risk following high-volume endurance exercise in recreationally active cyclists.

Despite the prognostic effect of physical activity, acute bouts of high-volume endurance exercise can induce cardiac stress and postexercise hypercoagulation associated with increased thrombotic risk. The aim of this study was to explore the effect of high-volume endurance exercise on coagulation and thrombotic activity in recreational cyclists. Thirty-four recreational cyclists completed 4.8 ± 0.3 h of cycling at 45 ± 5% of maximal power output on a bicycle ergometer. Intravenous blood samples were collected preexercise, immediately postexercise, 24 and 48 h postexercise, and analyzed for brain natriuretic peptide (BNP), cardiac troponin (cTn), C-reactive protein (CRP), D-dimer, thrombin-antithrombin (TAT) complex, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF-to-TFPI ratio (TF:TFPI). An increase in cTn was observed postexercise (P < 0.001). CRP concentrations were increased at 24 and 48 h postexercise compared with preexercise concentrations (P ≤ 0.001). TF was elevated at 24 h postexercise (P < 0.031) and TFPI was higher immediately postexercise (P < 0.044) compared with all other time points. TF:TFPI was increased at 24 and 48 h postexercise compared with preexercise (P < 0.025). TAT complex was reduced at 48 h postexercise compared with preexercise (P = 0.015), D-dimer was higher immediately postexercise compared with all other time points (P ≤ 0.013). No significant differences were observed in BNP (P > 0.05). High-volume endurance cycling induced markers of cardiac stress among recreational cyclists. However, plasma coagulation and fibrinolytic activity suggest no increase in thrombotic risk after high-volume endurance exercise.NEW & NOTEWORTHY In this study, a high-volume endurance exercise protocol induced markers of cardiac stress and altered plasma coagulation and fibrinolytic activity for up to 48 h in recreationally active cyclists. However, analysis of coagulation biomarkers indicates no increase in thrombotic risk when appropriate hydration and rest protocols are implemented.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Crosstalk of human coronary perivascular adipose-derived stem cells with vascular cells: role of tissue factor.

The coronary perivascular adipose tissue (cPVAT) has been associated to the burden of cardiovascular risk factors and to the underlying vessel atherosclerotic plaque severity. Although the "outside to inside" hypothesis of PVAT-derived-adipokine regulation of vessel function is currently accepted, whether the resident mesenchymal stem cells (ASCs) in PVAT have a regulatory role on the underlying vascular arterial smooth muscle cells (VSMCs) is not known. Here, we investigated the interactions between resident PVAT-ASCs and VSMCs. ASCs were obtained from PVAT overlying the left anterior descending (LAD) coronary artery of hearts removed at heart transplant operations. PVAT was obtained both from patients with non-ischemic and ischemic heart disease as the cause of heart transplant. ASCs were isolated from PVAT, phenotypically characterized by flow cytometry, functionally tested for proliferation, and differentiation. Crosstalk between ASCs and VSMCs was investigated by co-culture studies. ASCs were detected in the adventitia of the LAD-PVAT showing differentiation capacity and angiogenic potential. ASCs obtained from PVAT of non-ischemic and ischemic hearts showed different tissue factor (TF) expression levels, different VSMCs recruitment capacity through the axis ERK1/2-ETS1 signaling and different angiogenic potential. Induced upregulation of TF in ASCs isolated from ischemic PVAT rescued their angiogenic capacity in subcutaneously implanted plugs in mice, whereas silencing TF in ASCs decreased the proangiogenic capacity of non-ischemic ASCs. The results indicate for the first time a novel mechanism of regulation of VSMCs by PVAT-ASCs in angiogenesis, mediated by TF expression in ASCs. Regulation of TF in ASCs may become a therapeutic intervention to increase cardiac protection.

Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

First-in-Class Humanized Antibody against Alternatively Spliced Tissue Factor Augments Anti-Metastatic Efficacy of Chemotherapy in a Preclinical Model of Pancreatic Ductal Adenocarcinoma.

Alternatively spliced tissue factor (asTF) promotes the progression of pancreatic ductal adenocarcinoma (PDAC) by activating ß1-integrins on PDAC cell surfaces. hRabMab1, a first-in-class humanized inhibitory anti-asTF antibody we recently developed, can suppress PDAC primary tumor growth as a single agent. Whether hRabMab1 has the potential to suppress metastases in PDAC is unknown. Following in vivo screening of three asTF-proficient human PDAC cell lines, we chose to make use of KRAS G12V-mutant human PDAC cell line PaCa-44, which yields aggressive primary orthotopic tumors with spontaneous spread to PDAC-relevant anatomical sites, along with concomitant severe leukocytosis. The experimental design featured orthotopic tumors formed by luciferase labeled PaCa-44 cells; administration of hRabMab1 alone or in combination with gemcitabine/paclitaxel (gem/PTX); and the assessment of the treatment outcomes on the primary tumor tissue as well as systemic spread. When administered alone, hRabMab1 exhibited poor penetration of tumor tissue; however, hRabMab1 was abundant in tumor tissue when co-administered with gem/PTX, which resulted in a significant decrease in tumor cell proliferation; leukocyte infiltration; and neovascularization. Gem/PTX alone reduced primary tumor volume, but not metastatic spread; only the combination of hRabMab1 and gem/PTX significantly reduced metastatic spread. RNA-seq analysis of primary tumors showed that the addition of hRabMab1 to gem/PTX enhanced the downregulation of tubulin binding and microtubule motor activity. In the liver, hRabMab1 reduced liver metastasis as a single agent. Only the combination of hRabMab1 and gem/PTX eliminated tumor cell-induced leukocytosis. We here demonstrate for the first time that hRabMab1 may help suppress metastasis in PDAC. hRabMab1's ability to improve the efficacy of chemotherapy is significant and warrants further investigation.

The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles.

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Reduced Monocyte and Neutrophil Infiltration and Activation by P-Selectin/CD62P Inhibition Enhances Thrombus Resolution in Mice.

BACKGROUND: Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions. METHODS: Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed. RESULTS: Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus, and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin. CONCLUSIONS: Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis.

Prothrombin conversion and thrombin decay in patients with cirrhosis-role of prothrombin and antithrombin deficiencies.

BACKGROUND: Thrombin generation (TG) in the presence of thrombomodulin (TG-TM) in the plasma of patients with cirrhosis (PWC) is tilted toward a hypercoagulable phenotype. Low protein C and elevated factor VIII levels play a role, but other determinants, such as the prothrombin/antithrombin pair, must also be studied. OBJECTIVES: The objectives were (i) to quantitatively assess the subprocesses (prothrombin conversion and thrombin decay) and (ii) to understand the underlying mechanism by studying TG dynamics after prothrombin and antithrombin plasma level correction in PWC. METHODS: We studied TG-TM in plasma samples of 36 healthy controls (HCs) and 41 PWC with prothrombin and antithrombin levels of <70% and after their correction. We initiated coagulation with an intermediate picomolar concentration of tissue factor. We determined the overall thrombin potential, prothrombin conversion, and thrombin decay. RESULTS: TG-TM was increased in PWC compared with HC due to impaired thrombin inhibition. Indeed, thrombin decay capacity (min-1) decreased from 0.37 (0.35-0.40) in HC to 0.33 (0.30-0.37) in the Child-Turcotte-Pugh A (CTP-A; P = .09), 0.27 (0.26-0.30) in the CTP-B (P < .001), and 0.20 (0.19-0.20) in the CTP-C (P < .001) group. Concomitant correction of prothrombin and antithrombin increased endogenous thrombin potential with prothrombin conversion surpassing thrombin decay. By contrast, when we corrected only antithrombin, TG-TM was normalized and even consistent with a hypocoagulable phenotype in the CTP-C group. CONCLUSION: Our results highlight that in PWC, hypercoagulability (evidenced in the presence of TM) is due to impaired thrombin decay, whereas low prothrombin levels do not translate into decreased prothrombin conversion, likely due to altered TM-activated protein C negative feedback.

Dysregulated hemostasis in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with a high incidence of early death, which occurs within 30 days of diagnosis. The major cause of early death in APL is severe bleeding, particularly intracranial bleeding. Although APL is known to be associated with activation of coagulation, hyperfibrinolysis, and thrombocytopenia, the precise mechanisms that cause bleeding have not yet been elucidated. I propose that a combination of four pathways may contribute to bleeding in APL: (1) tissue factor, (2) the urokinase plasminogen activator/urokinase plasminogen activator receptor, (3) the annexin A2/S100A100/tissue plasminogen activator, and (4) the podoplanin/C-type lectin-like receptor 2. A better understanding of these pathways will identify new biomarkers to determine which APL patients are at high risk of bleeding and allow the development of new treatments for APL-associated bleeding.

The pathogenesis of cancer-associated thrombosis.

Patients with cancer have a higher risk of venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), compared to the general population. Cancer-associated thrombosis (CAT) is a thrombotic event that occurs as a complication of cancer or cancer therapy. Major factors determining VTE risk in cancer patients include not only treatment history and patient characteristics, but also cancer type and site. Cancer types can be broadly divided into three groups based on VTE risk: high risk (pancreatic, ovarian, brain, stomach, gynecologic, and hematologic), intermediate risk (colon and lung), and low risk (breast and prostate). This implies that the mechanism of VTE differs between cancer types and that specific VTE pathways may exist for different cancer types. This review summarizes the specific pathways that contribute to VTE in cancer patients, with a particular focus on leukocytosis, neutrophil extracellular traps (NETs), tissue factor (TF), thrombocytosis, podoplanin (PDPN), plasminogen activator inhibitor-1 (PAI-1), the intrinsic coagulation pathway, and von Willebrand factor (VWF).

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.

TF-FVIIa PAR2-ß-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

BACKGROUND: Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. METHODS: Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. RESULTS: We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in ß-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of ß-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. CONCLUSIONS: These data provide insights into a TF-FVIIa signaling axis through PAR2-ß-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.