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Table S1: Mean change in sVEGF according to selected variables - Simple linear regression models Table S2: Mean change in sVEGF* according to selected variables - "Multiple Model 1" Table S3: Mean change in sVEGF according to selected variables - "Multiple Model 2" Table S4: sVEGF and the risk of death-from-any-cause - Multivariable Cox regression model

Purpose: Cancer patients are at an increased risk of venous thromboembolism (VTE). VEGF promotes the growth of highly thrombogenic tumor vessels. Here, we explored the utility of soluble plasma VEGF-A (sVEGF) as a biomarker for the prediction of VTE in patients with cancer.Experimental Design: Eight hundred four patients with newly diagnosed cancer or progression after remission were prospectively followed for 2 years until the occurrence of VTE or death [tumor sites: brain (n = 87), breast (n = 137), lung (n = 120), gastrointestinal (n = 143), pancreas (n = 53), prostate (n = 95), kidney (n = 22), myeloma and lymphoma (n = 99), and others (n = 48)]. Primary endpoint was symptomatic or fatal VTE. sVEGF was measured by immunoassay in baseline plasma.Results: Fifty-five patients developed VTE (6.8%) and 364 patients (45.3%) died. Five-hundred and forty-two (68.3%) participants had sVEGF levels above the detection limit of 0.5 pg/mL. The median sVEGF level (25th–75th percentile) was 8.1 pg/mL (0–17.7). The cumulative 2-year incidence of VTE was 10.2% [95% confidence interval (CI), 6.4–14.9] in patients with sVEGF greater than the 75th percentile of the sVEGF distribution (Q3, cutoff: 17.7 pg/mL), and 5.9% (95% CI, 4.2–7.9) in patients with lower levels (P = 0.03). The corresponding 2-year risk of death was 52.8% (95% CI, 46.0–60.0) and 43.9% (95% CI, 40.0–48.0), respectively (P = 0.02). In univariable time-to-VTE regression, elevated sVEGF was associated with VTE [subhazard ratio (SHR) per 10 pg/mL increase, 1.04; 95% CI,1.00–1.09; P = 0.04)]. The association between sVEGF and risk of VTE prevailed after multivariable adjustment for high-risk tumor sites, age, gender, factor VIII, thrombin generation potential, and soluble P-Selectin (adjusted SHR, 1.04; 95% CI, 1.00–1.09, P = 0.05).Conclusions: Elevated sVEGF is associated with an increased risk of VTE in patients with cancer. Clin Cancer Res; 22(1); 200–6. ©2015 AACR.

BACKGROUND: Cases of ChAdOx1 nCoV-19 (AstraZeneca) vaccinated patients with thrombocytopenia, elevated D-dimer, and elevated platelet factor 4 (PF4) antibody levels with- and without thrombosis have been reported. No recommendations regarding the duration of anticoagulation have been made, because data on the long-term course beyond the first weeks is lacking. OBJECTIVE: To report on the treatment, medical course, and longitudinal follow-up of laboratory parameters in patients with vaccine-induced prothrombotic immune thrombocytopenia (VIPIT). PATIENTS: We followed VIPIT patients with- (n = 3) and without (n = 3) venous thromboembolism fulfilling the aforementioned laboratory criteria. RESULTS: Elevated D-dimer (median: 35.10 µg/ml, range: 17.80-52.70), thrombocytopenia (42 G/l, 20-101), and strong positivity in the platelet factor 4 (PF4)/heparin-enzyme-immunoassay (2.42 optical density [OD], 2.06-3.13; reference range

Objective: Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response. Approach and Results: Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle–tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05–5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16–3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2–6.5] and 3.0 [1.5–3.9], compared with 13.4 [9.7–16.6] and 7.5 [5.9–9.8] in patients without VTE (both P Conclusions: VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle–tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy. Graphic Abstract: A graphic abstract is available for this article.

Cases of unusual thrombosis and thrombocytopenia after administration of the ChAdOx1 nCoV-19 vaccine (AstraZeneca) have been reported. The term vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) was coined to reflect this new phenomenon. In vitro experiments with VIPIT patient sera indicated that high-dose intravenous immunoglobulins (IVIG) competitively inhibit the platelet-activating properties of ChAdOx1 nCoV-19 vaccine induced antibodies. Here, we report a case of a 62-year-old woman who had received this vaccine and developed VIPIT. She visited the emergency ward because of petechiae and hematomas. In the laboratory work-up, thrombocytopenia, low fibrinogen, elevated D-dimer, and positivity in the platelet factor 4/heparin-enzyme-immunoassay were present. Signs and symptoms of thrombosis were absent. Upon immediate therapy with non-heparin anticoagulation, high-dose IVIG, and prednisolone, laboratory parameters steadily improved and the patient was discharged from hospital without thrombotic complications. We conclude that early initiation of VIPIT treatment results in a swift response without thrombotic complications.

Almost a century ago, it was discovered that human milk activates the coagulation system, but the milk component that triggers coagulation had until now been unidentified. In the present study, we identify this component and demonstrate that extracellular vesicles (EVs) present in normal human milk expose coagulant tissue factor (TF). This coagulant activity withstands digestive conditions, mimicking those of breastfed infants, but is sensitive to pasteurization of pooled donor milk, which is routinely used in neonatal intensive care units. In contrast to human milk, bovine milk, the basis of most infant formulas, lacks coagulant activity. Currently, the physiological function of TF-exposing vesicles in human milk is unknown, but we speculate that these vesicles may be protective for infants. Another explanation could be nipple skin damage, which occurs in most breastfeeding women. Milk-derived TF-exposing EVs may seal the wound and thereby reduce bleeding and breast inflammation.

Background and aimsNon-O blood type (BT) is a risk factor for thromboses, which has been attributed to its effects on von Willebrand factor (VWF)/factor VIII (FVIII) levels. Although high VWF/FVIII may be risk factors for portal vein thrombosis (PVT) in patients with advanced chronic liver disease (ACLD), the impact of BT on PVT is unknown. We aimed to assess (I) whether non-O-BT is a risk factor for PVT and (II) whether non-O-BT impacts VWF/factor VIII in patients with ACLD.MethodsRetrospective analysis comprising two cohorts: (I) "US" including all adult liver transplantations in the US in the MELD era and (II) "Vienna" comprising patients with a hepatic venous pressure gradient (HVPG) >= 6 mmHg.Results(I) The "US cohort" included 84 947 patients (non-O: 55.43%). The prevalence of PVT at the time of listing (4.37% vs 4.56%; P = .1762) and at liver transplantation (9.56% vs 9.33%; P = .2546) was similar in patients with O- and non-O-BT. (II) 411 patients were included in the "Vienna cohort" (non-O: 64%). Mean HVPG was 18(9) mmHg and 90% had an HVPG >= 10 mmHg. Patients with non-O-BT had slightly increased VWF levels (318(164)% vs 309(176)%; P = .048; increase of 23.8%-23.9% in adjusted analyses), but this difference was driven by patients with less advanced disease. However, non-O-BT explained only 1% of the variation in VWF and had no effect on FVIII.ConclusionsAlthough non-O-BT impacts VWF in patients with early stage ACLD, its contribution to VWF variation is considerably smaller than in the general population. Moreover, non-O-BT had no impact on FVIII. These findings may explain the absence of an association between non-O-BT and PVT in patients with advanced cirrhosis.

Milk supports the growth and development of infants. An increasing number of mostly recent studies have demonstrated that milk contains a hitherto undescribed component called extracellular vesicles (EVs). This presents questions regarding why milk contains EVs and what their function is. Recently, we showed that EVs in human milk expose tissue factor, the protein that triggers coagulation or blood clotting, and that milk-derived EVs promote coagulation. Because bovine milk, which also contains EVs, completely lacks this coagulant activity, important differences are present in the biological functions of human milk-derived EVs between species. In this review, we will summarize the current knowledge regarding the presence and biochemical composition of milk EVs, their function(s) and potential clinical applications such as in probiotics, and the unique problems that milk EVs encounter in vivo, including survival of the gastrointestinal conditions encountered in the newborn. The main focus of this review will be human milk-derived EVs, but when available, we will also include information regarding non-human milk for comparison.

Genetic predispositions to venous thromboembolism (VTE) are relatively frequent in the general population and comprise a heterogeneous group of disorders. Whereas the most frequent congenital risk factors for thrombosis only moderately increase the risk, a deficiency in antithrombin (AT), one of the most important natural inhibitors of blood coagulation, carries a higher risk. Congenital AT deficiency is an infrequently encountered genetic risk factor for VTE, and different subtypes vary with regard to their thrombotic risk. Patients with congenital AT deficiency, especially those with quantitative deficiency (type 1), may develop thrombosis early in life and often have a conspicuous family history of first- and second-degree relatives with VTE. Women are particularly affected because of the risk potentiation by combined estrogen/progestogen oral contraceptive use or pregnancy. The lack of controlled trials or even observational studies of large cohorts does not allow therapeutic decisions to be based on scientific evidence. In this review, we will discuss cases with thrombotic manifestations and the tailored management of patients with this congenital thrombosis risk factor.

The clinical and scientific interest in extracellular vesicles (EVs) is growing exponentially. The term EVs is an umbrella term for various types of vesicles that are present in body fluids and other (bio)fluids. This umbrella term is used because clear hallmarks to distinguish different types of EVs from each other are lacking. Thus, the term “EVs” encompasses earlier “microparticles” or “microvesicles” and exosomes, which are vesicles released directly from the plasma membrane or by secretion of intraluminal vesicles stored in multivesicular endosomes, respectively.1, 2 There is evidence that EVs play a role in intercellular communication and contribute to coagulation and likely inflammation.3, 4, 5, 6 The oldest‐known function of “platelet dust,” now known as platelet‐derived EVs, is their ability to support coagulation by exposing negatively charged phospholipids, such as phosphatidylserine (PS). Such PS exposing EVs facilitates formation of tenase and prothrombinase complexes. Furthermore, different subtypes of EVs, such as leukocyte, endothelial, or tumor‐derived EVs, can also trigger coagulation by exposing tissue factor (TF).7 Tissue factor‐exposing EVs (TF‐EVs) are present in body fluids, such as saliva and urine, under physiological conditions. The presence of TF‐EVs in saliva may explain the reflex to lick a wound, thereby exposing blood to extravascular TF and accelerating hemostasis and reducing the risk of infection.8 Although Tissue factor was initially thought to be exclusively present outside the vasculature (“envelope model”); there is increasing evidence that during medical intervention and in various clinical conditions, such as surgery, or in patients suffering from sepsis or cancer, the presence of coagulant TF‐EVs is associated with disseminated intravascular coagulation and venous thrombosis.9, 10 There are two reasons why a proposed standardization is timely and relevant. First, there is a growing interest to improve the reproducibility of results in science in general, and this also holds true for the new field of EV research. During the last few years, “minimal requirements” have been published by the International Society of Extracellular Vesicles (ISEV) regarding the reporting on studies involving EVs,11, 12, 13 as well as a structure to record and score reporting of preanalytical variables 14, 15, 16, 17 In addition, guidelines and position papers have been published18, 19 and an increasing number of standardization studies have been and are being performed involving various aspects of EV detection and characterization.2, 14, 20, 21 At present, various in‐house and commercially available assays have been developed to measure the EV‐associated TF (EV‐TF) activity, but hitherto the results of these methods have not been easily compared and required standardization. Second, to identify cancer patients at risk of developing venous thromboembolism, an EV‐TF‐based factor Xa generation assay and an EV‐TF‐based plasma clotting test have been developed and applied in clinical trials and have shown promising results for the prediction of VTE in pancreatic cancer patients. This underscores the relevance of studying TF‐EVs as a potential clinically relevant biomarker.22, 23 Taken together, we provide a summary of the outcomes of the questionnaire and discussion with the goal to improve future standardization of studies measuring the TF activity of EVs.

Development of ascites is the most common form of decompensation of cirrhosis. We aimed to investigate the coagulation system in ascitic fluid and plasma of patients with cirrhosis. We determined coagulation parameters and performed clotting and fibrinolysis experiments in ascitic fluid and plasma of thoroughly characterized patients with cirrhosis and ascites (n = 25) and in plasma of patients with cirrhosis but without ascites (n = 25), matched for severity of portal hypertension. We also investigated plasma D-dimer levels in an independent cohort of patients (n = 317) with clinically significant portal hypertension (HVPG ≥ 10 mmHg), grouped according to ascites severity. Ascitic fluid was procoagulant in a clotting assay. The procoagulant potential of ascitic fluid was abolished by depletion of extracellular vesicles from ascitic fluid by filtration or by addition of a tissue factor-neutralizing antibody. Compared with plasma, extracellular vesicle-associated tissue factor activity was high in ascitic fluid, while activities of other coagulation factors were low. The extracellular vesicle-depleted fraction of ascitic fluid induced fibrinolysis, which was prevented by aprotinin, indicating the presence of plasmin in ascitic fluid. Plasma peak thrombin generation and parameters reflecting fibrinolysis were independently associated with the presence of ascites. Finally, plasma D-dimer levels were independently linked to ascites severity in our second cohort comprising 317 patients. In conclusion, coagulation and fibrinolysis become activated in ascites of patients with cirrhosis. While tissue factor-exposing extracellular vesicles in ascitic fluid seem unable to pass the peritoneal membrane, fibrinolytic enzymes get activated in ascitic fluid and may re-enter the systemic circulation and induce systemic fibrinolysis.

Simple Summary Disseminated intravascular coagulation (DIC) may occur in patients with advanced prostate cancer. In the present study, we detected elevated extracellular vesicle (EV)-associated tissue factor (TF) activity in the plasma of prostate cancer patients with DIC compared with matched prostate cancer patients without DIC and healthy individuals. TF-exposing EVs from DIC patients were highly coagulant in a clotting assay. In in vitro co-culture experiments, EV-TF activity was increased by interactions between a TF-exposing prostate cancer cell line (DU145), peripheral blood mononuclear cells (PBMCs), and platelets. Data from this study contribute to the understanding of the pathogenesis of prostate cancer-related DIC. Abstract Patients with advanced prostate cancer may develop fulminant disseminated intravascular coagulation (DIC). Circulating extracellular vesicles (EVs)-exposing tissue factor (TF), the initiator of the coagulation cascade, may play an important role. We included 7 prostate cancer patients with DIC, 10 age- and stage-matched cancer controls without DIC, and 10 age-matched healthy male individuals. EV-TF activity was highly elevated in prostate cancer patients with DIC (11.40 pg/mL; range: 4.34–27.06) compared with prostate cancer patients without DIC (0.09 pg/mL; range: 0.00–0.30, p = 0.001) and healthy controls (0.18 pg/mL; range: 0.09–0.54; p = 0.001). Only EVs from patients with DIC reduced fibrin clot formation time of pooled plasma in a TF-dependent manner. Next, we performed in vitro co-culture experiments including EVs derived from a prostate cancer cell line with high (DU145) and low (LNCaP) TF expression, peripheral blood mononuclear cells (PBMCs), and platelets. Co-incubation of DU145 EVs with PBMCs and platelets significantly increased EV-TF activity in conditioned medium and induced TF activity on monocytes. No such effects were seen in co-culture experiments with LNCaP EVs. In conclusion, the findings indicate that elevated EV-TF activity plays a role in the development of prostate-cancer-related DIC and may result from interactions between tumor-derived EVs, monocytes, and platelets.

Summary Background Non-malignant portal vein thrombosis (PVT) is common in patients with advanced liver disease. Anticoagulation (AC) increases the chances of recanalization and may improve liver function in patients with cirrhosis. Aim We retrospectively assessed the course of non-malignant PVT in patients receiving AC. Methods Parameters related to hepatic injury (aspartate aminotransferase [AST]/alanine aminotransferase [ALT]), severity of disease (ascites) and synthesis function (albumin) as well as AC, rates of PVT regression/progression and AC-associated complications were documented. Results Among 122 patients with PVT, 51 patients with non-malignant PVT (27 incomplete, 24 complete) were included, 12 patients (25%) received long-term AC therapy (≥9 months) as compared to 36 patients without long-term AC. We observed a trend towards higher regression rates with long-term AC of 58% (vs. 28% without AC; p = 0.08) and lower progression rates of 25% (vs. 42% without AC; p = 0.15). In the subgroup of patients with decompensation prior to PVT diagnosis (n = 39), long-term AC (n = 10, 25.6%) resulted in a significantly higher rate of PVT regression/resolution (70% vs. 24%, p = 0.031). Interestingly, AST/ALT tended to decrease (−19%/−16%) and the proportion of patients with ascites became lower (−33%) with long-term AC (without AC: ±0%). Furthermore, there was a significant improvement in albumin levels (+9%/+3.6 g/dl) when compared to patients without long-term AC (−2%/−0.8 g/dl; p = 0.04). Additionally, 10 patients were treated with direct oral anticoagulants (DOACs) for splanchnic vein thrombosis. Importantly, there were no AC-associated bleeding events in patients with conventional AC and one bleeding event in patients with DOAC treatment (10%). Conclusion Our findings support anticoagulation in patients with non-malignant PVT, since AC seems safe and associated with superior PVT regression rates and might also decrease hepatic injury and improve liver synthesis.

Essentials Risk stratification for venous thromboembolism (VTE) in patients with brain tumors is challenging. Patients with IDH1 wildtype and high podoplanin expression have a 6-month VTE risk of 18.2%. Patients with IDH1 mutation and no podoplanin expression have a 6-month VTE risk of 0%. IDH1 mutation and podoplanin overexpression in primary brain tumors appear to be exclusive. Summary Background Venous thromboembolism (VTE) is a frequent complication in primary brain tumor patients. Independent studies revealed that podoplanin expression in brain tumors is associated with increased VTE risk, whereas the isocitrate dehydrogenase 1 (IDH1) mutation is associated with very low VTE risk. Objectives To investigate the interrelation between intratumoral podoplanin expression and IDH1 mutation, and their mutual impact on VTE development. Patients/Methods In a prospective cohort study, intratumoral IDH1 R132H mutation and podoplanin were determined in brain tumor specimens (mainly glioma) by immunohistochemistry. The primary endpoint of the study was symptomatic VTE during a 2-year follow-up. Results All brain tumors that expressed podoplanin to a medium-high extent showed also an IDH1 wild-type status. A score based on IDH1 status and podoplanin expression levels allowed prediction of the risk of VTE. Patients with wild-type IDH1 brain tumors and high podoplanin expression had a significantly increased VTE risk compared with those with mutant IDH1 tumors and no podoplanin expression (6-month risk 18.2% vs. 0%). Conclusions IDH1 mutation and podoplanin overexpression seem to be exclusive. Although brain tumor patients with IDH1 mutation are at very low risk of VTE, the risk of VTE in patients with IDH1 wild-type tumors is strongly linked to podoplanin expression levels.

Neutrophil extracellular traps (NETs) are decondensed chromatin fibers decorated with granular enzymes that are released from activated neutrophils. The antimicrobial properties of NETs were discovered first, showing that NETs immobilize and kill bacteria and fungi [1]. More recently, it has been shown that NETs also interact with the blood coagulation system [2]. Mechanisms include activation of factor XII [31, binding of von Willebrand factor [4], providing a scaffold for platelet adhesion and fibrin deposition [5], and inactivation of tissue factor pathway inhibitor [6]. In animal models, NETs accumulate early in growing thrombi [2], and treatment with DNAse 1 dismantles NETs, diminishing thrombus formation [3,7]. Patients with cancer have a high risk of developing venous thromboembolism (VTE) [8–10]. However, the mechanisms by which cancer induces a prothrombotic state leading to overt cancer-associated thrombosis are not yet fully understood. Experimental data indicate that NETs could play an important role in cancer-associated coagulation activation. In murine cancer models, leukocytes were primed by cancer cells to release NETs, and this was associated with spontaneous venous thrombus formation [11]. Recently, methods to indirectly quantify NETs in human plasma samples have been developed [12,13]. Citrullinated histone H3 (H3Cit) has been proposed as a target biomarker reflecting NET formation [11,12]. Other putative biomarkers for NETs include cell-free DNA (cfDNA) and nucleosomes, which may also have other sources than NET formation, such as cellular decay or apoptosis. In recent years, clinical and laboratory risk factors for prediction of VTE in cancer patients have been identified, in order to stratify patients according to their risk of VTE during the course of disease [14]. In this prospective cohort study, we hypothesized that biomarkers reflecting NET formation in patients with cancer could predict the individual propensity to develop future VTE. The clinical investigation of NET-specific and NET-related biomarkers could also lead to novel insights regarding the pathogenesis of cancer-related VTE. To examine this hypothesis, we quantified H3Cit, cfDNA and nucleosome levels in the plasma of cancer patients, and followed them for the occurrence of VTE over a period of 2 years.

Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients. We characterized androgen receptor-mediated tissue factor regulation in prostate epithelial cells. Androgen receptor is dampening tissue factor expression in prostate epithelial cells. Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates. Summary Background Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)-mediated TF gene expression. Objectives To characterize AR-mediated TF regulation in vitro and in vivo. Methods We used the androgen-dependent prostate cancer cell lines LNCaP and MyC-CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor-binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR-mediated TF regulation in vivo. Results TF is directly regulated by AR. In LNCaP cells, nuclear factor-κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells. Conclusion AR is crucial for dampening TF expression, which could be important for increased TF expression and TF-positive microvesicle release in androgen-deprived prostate cancer patients.

BACKGROUND Elevated plasma von Willebrand factor antigen (vWF) has been shown to indicate the presence of clinically significant portal hypertension, and thus, predicts the development of clinical events in patients with cirrhosis. AIM To investigate the impact of bacterial translocation and inflammation on vWF, as well as the association between vWF and procoagulant imbalance. Moreover, we assessed whether vWF predicts complications of cirrhosis, independent of the severity of portal hypertension. METHODS Our study population comprised 225 patients with hepatic venous pressure gradient (HVPG) ≥ 10 mm Hg without active bacterial infections or hepatocellular carcinoma. RESULTS vWF correlated with markers of bacterial translocation (lipopolysaccharide-binding protein [LBP; ρ = 0.201; P = 0.021]), inflammation (interleukin 6 [IL-6; ρ = 0.426; P

Neutrophil extracellular traps (NETs) are supposed to play a central role in atherothrombosis. We measured circulating citrullinated histone H3 (H3Cit) and cell-free DNA (cfDNA), which serve as surrogate markers of NET formation, in 79 patients with peripheral artery disease (PAD) following infrainguinal angioplasty with stent implantation. Analysis of cfDNA and H3Cit was performed using Quant-iT&trade, PicoGreen&reg, dsDNA Assay Kit or an ELISA, respectively. Within two years of follow-up, the primary endpoint defined as nonfatal myocardial infarction, stroke or transient ischemic attack, cardiovascular death, and &gt, 80% target vessel restenosis occurred in 34 patients (43%). Both H3Cit (HR per 1-SD: 2.72, 95% CI: 1.2&ndash, 6.3, p = 0.019) and cfDNA (HR per 1-SD: 2.15, 95% CI: 1.1&ndash, 4.2, p = 0.028) were associated with the primary endpoint in a univariate Cox regression analysis. Multivariate linear regression analyses showed associations between cfDNA and platelet surface expression of P-selectin (p = 0.006) and activated glycoprotein IIb/IIIa (p &lt, 0.001) in response to arachidonic acid (AA) after adjustment for age, sex, clinical risk factors, and inflammatory markers. H3Cit was also associated with P-selectin expression in response to thrombin-receptor activating peptide (p = 0.048) and AA (p = 0.032). Circulating H3Cit and cfDNA predict ischemic outcomes after peripheral angioplasty with stent implantation, and are associated with on-treatment platelet activation in stable PAD.

Microvesicles (MVs) are small membrane bound vesicles released from various cell types after activation or apoptosis. In the last decades, MVs received an increased interest as biomarkers in inflammation, coagulation and cancer. However, standardized pre-analytical steps are crucial for the minimization of artifacts in the MV analysis. Thus, this study evaluated the MV release in whole blood samples under the influence of different anticoagulants, storage time and various temperature conditions. Samples were collected from healthy probands and processed immediately, after 4, 8, 24 and 48 hours at room temperature (RT) or 4°C. To identify MV subpopulations, platelet free plasma (PFP) was stained with Annexin V, calcein AM, CD15, CD41 and CD235a. Analysis was performend on a CytoFLEX flow cytometer. Procoagulatory function of MVs was measured using a phospholipid dependent activity and a tissue factor MVactivity assay. Without prior storage, sodium citrate showed the lowest MV count compared to heparin and EDTA. Interestingly, EDTA showed a significant release of myeloid-derived MVs (MMVs) compared to sodium citrate. Sodium citrate showed a stable MV count at RT in the first 8 hours after blood collection. Total MV counts increased after 24 hours in sodium citrated or heparinzed blood which was related to all subpopulations. Interestingly, EDTA showed stable platelet-derived MV (PMV) and erythrocyte-derived MV (EryMV) count at RT over a 48 h period. In addition, the procoagulatory potential increased significantly after 8-hour storage. Based on both, this work and literature data, the used anticoagulant, storage time and storage temperature differently influence the analysis of MVs within 8 hours. To date, sodium citrated tubes are recommended for MV enumeration and functional analysis. EDTA tubes might be an option for the clinical routine due to stable PMV and EryMV counts. These new approaches need to be validated in a clinical laboratory setting before being applied to patient studies. © 2016 International Society for Advancement of Cytometry.

Lupus anticoagulants (LA) are a heterogeneous group of antiphospholipid antibodies (aPLAs) that promote thrombosis. Tissue factor (TF)–bearing extracellular vesicles (EVs) might contribute to the prothrombotic state of patients with persistent LA and a history of thrombosis. To investigate if EV-associated TF activity is elevated in a well-defined group of LA-positive patients with a history of thrombosis in comparison to that of healthy controls. Adult patients (n = 94, median age 40.1 years, interquartile range (IQR) 29.9–53.4; 87% females) positive for LA and a history of thrombosis (78% venous thrombosis, 17% arterial thrombosis, 5% venous thrombosis and arterial thrombosis) and healthy age- and sex-matched controls (n = 30, median age 42.9 years, IQR 38.6–45.8, 77% females) were included in this study. EV-TF activity was determined with a factor Xa generation assay and anti-β2-glycoprotein (anti-β2GPI) and anticardiolipin (aCL) antibodies by enzyme-linked immunoassays. EV-TF activity did not differ between 94 LA-positive patients with a history of thrombosis (median 0.05 pg/mL, IQR 0.00–0.14) and 30 healthy controls (median 0.06, IQR 0.00–0.11, p = 0.7745). No correlation was found between EV-TF activity and lupus-sensitive activated partial thromboplastin time (aPTT-LA) (rho = 0.034), Rosner index (rho = − 0.056), anti-β2GPI IgG (rho = 0.05), anti-β2GPI IgM (rho = − 0.08), aCL IgG (rho = 0.12), and aCL IgM (rho = − 0.11) in LA-positive patients. We found low EV-TF activity levels in LA-positive patients and a history of thrombosis and no correlation with analyzed aPLAs. Our data indicate that circulating TF-bearing EVs do not contribute to the prothrombotic state of patients with LA.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts (P < .001) and higher D-dimer levels (P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens (P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P = .010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.

Embolization of amniotic fluid (AF) into the blood circulation leads to disseminated intravascular coagulation (DIC). Procoagulant phosphatidylserine (PS)- and tissue factor (TF)–exposing extracellular vesicles (EVs) might play an important role in AF embolism–induced DIC. It was the aim of the present study to perform analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry. We applied a prothrombinase assay (that quantifies PS exposure on EVs), an EV-associated TF activity assay, a fibrin generation assay, a thrombin generation assay, a whole blood clotting model, and flow cytometry in AF and control plasma. We found that PS exposure on EVs was 21-fold increased in AF compared with plasma. Also, EV-associated TF activity was highly increased in AF compared with plasma. AF-derived EVs activated the blood coagulation cascade via PS and TF in the fibrin and thrombin generation assays. In a whole blood clotting model, AF-derived EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor XII (FXII) was not affected. Applying flow cytometry, a subpopulation of PS + and TF + EVs was identified in AF but not in control plasma. In conclusion, we investigated the effect of AF on blood coagulation and found that PS + and TF + EVs determine their procoagulant potential. Taken together, our data further delineate the pathomechanisms underlying AF-induced coagulopathy.

Uderhardt et al. show that eosinophils accumulate in freshly formed thrombi, where they provide a procoagulant surface that is rich in oxidized phospholipids and allows assembly and activation of plasmatic coagulation factors. This mechanism stabilizes the thrombus and enables hemostasis but also contributes to thrombotic disease., Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase–derived hydroxyeicosatetraenoic acid–phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

Tissue factor (TF) is the main in vivo initiator of the blood coagulation cascade. Active circulating TF was detected on small, negatively charged membrane vesicles, the so-called microvesicles (MVs), which are released upon cell activation and apoptosis from a variety of cells. Increased coagulation activation was found in morbidly obese patients, and elevated levels of TF-bearing MVs may contribute to the prothrombotic state in these patients. To determine MV-associated TF activity levels in morbidly obese patients before and after weight loss due to bariatric surgery. MV-TF activity was measured with a factor Xa generation assay in morbidly obese patients before and 2 years after bariatric surgery. In addition, clinical parameters were determined. Seventy-four morbidly obese patients (mean age: 42 (±11) years; 61 females) were included in this study. After bariatric surgery, the body mass index decreased from (median, 25–75th percentile) 45.5 (42.3–50.2) to 30.5 (28.0–34.4 kg m−2; P

Venous thromboembolism (VTE), a disease entity comprising deep vein thrombosis (DVT) and pulmonary embolism (PE), is a frequent and potentially life-threatening event. To date different agents are available for the effective treatment of acute VTE and the prevention of recurrence. For several years, the standard of care was the subcutaneous application of a low molecular weight heparin (LMWH) or fondaparinux, followed by a vitamin K antagonist (VKA). The so-called direct oral anticoagulants (DOAC) were introduced rather recently in clinical practice for the treatment of VTE. DOAC seem to have a favorable risk-benefit profile compared to VKA. Moreover, DOAC significantly simplify VTE treatment because they are administered in fixed doses and no routine monitoring is needed. Patients with objectively diagnosed DVT or PE should receive therapeutic anticoagulation for a minimum of 3 months. Whether a patient ought to receive extended treatment needs to be evaluated on an individual basis, depending mainly on risk factors determined by characteristics of the thrombotic event and patient-related factors. In specific patient groups (e.g., pregnant women, cancer patients, and elderly patients), treatment of VTE is more challenging than that in the general population and additional issues need to be considered in those patients. The aim of this review is to give an overview of the currently available treatment modalities of acute VTE and secondary prophylaxis. In particular, specific aspects regarding the initiation of VTE treatment, duration of anticoagulation, and specific patient groups will be discussed.

Overt disseminated intravascular coagulation (DIC) is a systemic process characterized by excessive coagulation activation and fibrinolysis that may occur in cancer patients. The underlying pathomechanisms are still poorly understood. Recent experimental studies found an important role for the interaction between procoagulant neutrophil extracellular traps (NETs) and tissue factor (TF) in the pathogenesis of thrombosis.To investigate whether NETs and TF-bearing microvesicles (MVs) play a central role in cancer-related overt DIC.Twenty-eight cancer patients with overt DIC (ISTH score ≥5, 14 females, median age: 62 years [range: 21-80], 13 with solid tumors, 15 with acute leukemia) and 28 matched healthy controls were included. NET formation parameters (plasma DNA and nucleosomes), MVassociated TF activity, and routine coagulation parameters were determined at study inclusion. In 11 patients with acute myeloid leukemia (AML), follow-up measurements were also performed.Plasma DNA, nucleosomes, and MV-TF activity were highly elevated in patients with cancer-related DIC compared to healthy individuals (all p-values0.001). Strong correlations were found between plasma DNA and nucleosomes (Spearman correlation-coefficient: r=0.68), nucleosomes and MV-TF activity (r=0.62), and DNA and MV-TF activity (r=0.57). In multivariate regression, altered routine coagulation parameters were highly associated with NET parameters and MV-TF activity. In detail, a doubling in plasma DNA was associated with a 7.6% decrease in fibrinogen (p=0.012), a 15.3% decrease in platelet count (p=0.002), a 3.9% decrease in prothrombin time (p=0.014), and a 41.0% increase in D-dimer (p0.001). A 10% increase in nucleosomes was associated with a 3.1% decrease in fibrinogen (p0.001), a 5.0% decrease in platelet count (p0.001), a 1.0% decrease in prothrombin time (p0.009), and a 112.7% increase in D-dimer (p0.001). A 10% increase in MV-TF activity was associated with a 4.9% decrease in fibrinogen (p0.001), a 7.1% decrease in platelet count (p0.001), a 1.3% decrease in prothrombin time (p0.001), and a 15.5% increase in D-dimer (p0.001). After initiation of chemotherapy in AML patients, NET parameters and MV-TF activity decreased significantly (nucleosomes: 3.3-fold decrease and normalization after 1 week; DNA: 1.2-fold decrease after 1 week and 1.5-fold decrease after 1 month; MV-TF activity: 10-fold decease after 1 week and normalization after 1 month) (Figure 1), and routine coagulation parameters improved.Our results add to experimental studies that have investigated the interaction between NETs and TF. Taken together, evidence indicates the presence of a liaison dangereuse between NETs and TF-bearing MVs, which could be the underlying cause of cancer-related overt DIC.

Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role. Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC. Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma. Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF. Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. Disclosures No relevant conflicts of interest to declare.