Human milk triggers coagulation via tissue factor-exposing extracellular vesicles.

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.

Extracellular vesicles and coagulation in blood from healthy humans revisited.

Background: In 2001, we studied the presence and coagulant properties of "microparticles" in the blood of healthy humans. Since then, multiple improvements in detection, isolation and functional characterization of the now called "extracellular vesicles" (EVs) have been made, and shortcomings were identified. Aim: To revisit the presence and function of EVs in blood from healthy humans. Methods: Blood was collected from 20 healthy donors. EV-containing plasma was prepared according to new guidelines, and plasma was diluted to prevent swarm detection. Single EVs were measured by flow cytometry with known sensitivity of fluorescence and light scatter. The haemostatic properties of EVs were measured by thrombin-, fibrin-, and plasmin generation. Plasma concentrations of thrombin-antithrombin complexes and prothrombin fragment 1 + 2 were measured to assess the coagulation status in vivo. Results: Compared to 2001, the total concentrations of detected EVs increased from 190- to 264-fold. In contrast to 2001, however, EVs are non-coagulant which we show can be attributed to improvements in blood collection and plasma preparation. No relation is present between the plasma concentrations of EVs and either TAT or F1 + 2. Finally, we show that EVs support plasmin generation. Discussion: Improvements in blood collection, plasma preparation and detection of EVs reveal that results from earlier studies have to be interpreted with care. Compared to 2001, higher concentrations of EVs are detected in blood of healthy humans which promote fibrinolysis rather than coagulation.

Extracellular vesicles exposing tissue factor for the prediction of venous thromboembolism in patients with cancer: A prospective cohort study.

INTRODUCTION: The procoagulant activity of extracellular vesicles (EV) exposing tissue factor (TF) is a promising biomarker for venous thromboembolism (VTE) in cancer patients. We evaluated an in-house EV-TF activity assay (the fibrin generation test) for the prediction of cancer-associated VTE. We also compared the results with the fibrin generation tests to an EV-TF-dependent factor Xa generation assay in samples from pancreatic cancer patients. MATERIALS AND METHODS: Data collected in a multinational, prospective cohort study were used. Patients with various types of advanced cancer were enrolled if chemotherapy was scheduled or started in the previous 3 months. Patients were followed for 6 months for the occurrence of VTE. The fibrin generation test was performed at baseline to measure EV-TF procoagulant activity. RESULTS: The fibrin generation test was performed in 648 patients with advanced cancer. The mean age was 62 years; 58% had distant metastasis. Forty patients (6.1%) developed VTE. Overall, a high fibrin generation test result was associated with a two-fold increased risk for VTE (HR 2.0; 95%-CI, 1.1-3.6). The association was stronger in patients with pancreatic cancer (HR 4.1; 95%-CI, 0.91-19) than in those with other tumor types (HR 1.5; 95%-CI, 0.72-3.1). Correlation between the FGT and the TF-dependent factor Xa generation assay in patients with pancreatic cancer was poor (Spearman's R = 0.35). CONCLUSION: This study shows that a high EV-TF procoagulant activity as measured by the fibrin generation test is associated with an increased risk of VTE in cancer patients, in particular in those with pancreatic cancer. Future studies should aim to further improve the feasibility and accuracy of EV-TF activity assays.

Coagulation activation and microparticle-associated coagulant activity in cancer patients. An exploratory prospective study.

Cancer increases the risk of venous thromboembolism (VTE). Here, we investigated the contribution of microparticle (MP)-dependent procoagulant activity to the prothrombotic state in these patients. In 43 cancer patients without VTE at study entry and 22 healthy volunteers, markers of in vivo and MP-dependent coagulation were measured and patients were prospectively followed for six months for the development of VTE. Procoagulant activity of MPs was measured in vitro using a tissue factor (TF)-independent phospholipid dependent test, a factor Xa-generation assay with and without anti-TF, and a fibrin generation test (FGT) with and without anti-factor VII(a). Markers of in vivo coagulation activation and total number of MPs at baseline were significantly elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/l, D-dimer 0.76 vs. 0.22 mg/l and 5.53 x 106 vs. 3.37 x 106 MPs/ml). Five patients (11.6%) developed VTE. Patients with VTE had comparable levels of coagulation activation markers and phospholipid-dependent MP procoagulant activity. However, median TF-mediated Xa-generation (0.82 vs. 0.21 pg/ml, p=0.016) and median VIIa-dependent FGT (13% vs. 0%, p=0.036) were higher in the VTE group compared with the non-VTE group. In this exploratory study the overall hypercoagulable state in cancer patients was not associated directly with the MP phospholipid-dependent procoagulant activity. However, in the patients who developed VTE within six months when compared to those who did not, an increased MP procoagulant activity was present already at baseline, suggesting this activity can be used to predict VTE.

Cell-derived vesicles exposing coagulant tissue factor in saliva.

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease.

BACKGROUND: Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease. DESIGN AND METHODS: In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in 25 consecutive patients with sickle cell disease. RESULTS: The majority of microparticles originated from platelets (GPIIIa,CD61) and erythrocytes (glycophorin A,CD235), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=-0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.023). CONCLUSIONS: We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles.

Prolactin does not affect human platelet aggregation or secretion.

Platelets play an important role in the development of plaque formation and in the events after rupture of the atherosclerotic plaque, leading to atherothrombosis. Multiple hormones, either in excess or when deficient, are involved in the development of atherothrombotic disease, but, to which extent such hormones affect platelet function, is still controversial. It was the objective of this study to assess the ability of the pituitary hormone prolactin to affect platelet functions. Venous blood was collected from six healthy males. Platelet activation was studied by (i) flow cytometry in whole blood (exposure of P-selectin as a measure of platelet secretion, and binding of PAC-1 as a measure of ligand-binding conformation of alpha(IIb)beta(3)), and by (ii) optical aggregation and whole blood aggregation. All studies were performed without and with exposure to several concentrations of ADP (0.1, 0.5 and 1.0 microM) and prolactin (50 and 1,000 microg/l). The presence of the prolactin receptor was investigated by Western blot and flow cytometry. In response to either 50 or 1,000 microg/l prolactin, no evidence of platelet activation or aggregation was found. In addition, ADP-induced platelet activation or aggregation was not enhanced by prolactin. Finally, prolactin receptors could not be detected on the surface of platelets. The present data indicate that prolactin does not directly modulate platelet function.

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes.

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.

Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism.

OBJECTIVE: To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation. METHODS: Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n = 10) and patients with other forms of arthritis (non-RA; n = 10) and in plasma from healthy subjects (n = 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F(1+2) (by enzyme-linked immunosorbent assay [ELISA]) and thrombin-antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo. RESULTS: Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non-RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F(1+2) (median 66 nM) and TAT complex (median 710 microg/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 microg/liter, respectively) as well as the controls (1.0 nM and 2.9 microg/liter, respectively). CONCLUSION: Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII-dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.

Enhanced coagulation activation in preeclampsia: the role of APC resistance, microparticles and other plasma constituents.

Coagulation activation in pregnancy is further enhanced in preeclampsia. We investigated whether this results from increased thrombin generation by the plasma itself or its cell-derived microparticles. Plasma samples were obtained from preeclamptic, normal pregnant and nonpregnant women (each n = 10). Prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) concentrations were increased in pregnancy and further increased in preeclampsia. In pregnancy and preeclampsia, increased activated protein C resistance occuffed (APC sensitivity ratio: 3.3 +/- 0.8 and 2.5 +/- 0.8, both P <0.001 vs. nonpregnant). In normal pregnant microparticle-free plasma the thrombin generation correlated with TAT (r = 0.84, P = 0.005) and APC resistance correlated with F1+2 (r = 0.68, P = 0.04). In preeclampsia thrombin generation by plasma was increased (P = 0.005), independent of APC resistance. Thrombin generation by microparticles was similar in all groups, although different coagulation activation pathways were utilized, indicating that circulating microparticles are not directly involved in coagulation activation in pregnancy and preeclampsia. In contrast, APC resistance can explain coagulation activation in pregnancy, while enhanced coagulation activation in preeclampsia results, in part, from an increased thrombin generating capacity of plasma independent of APC resistance.