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Background: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism., Objectives: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets., Methods: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied., Results: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold., Conclusion: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation., (© 2015 British Society for Immunology.)

Background: We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation., Methods: Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells., Results: In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC., Conclusions: Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Paroxysmal Nocturnal Hemoglobinuria is characterized by complement-mediated hemolysis and an increased thrombosis risk. Eculizumab, an antibody to complement factor C5, reduces thrombotic risk via unknown mechanisms. Clinical observations suggest that eculizumab has an immediate effect., Objectives: A better understanding of the mechanism via which eculizumab reduces thrombotic risk by studying its pharmacodynamic effect on coagulation and fibrinolysis., Methods: We measured microparticles (MP), tissue factor (TF) activity, prothrombin fragment 1+2 (F1+2), D-dimer and simultaneously thrombin and plasmin generation in 55 PNH patients. In 20 patients, parameters were compared before and during eculizumab treatment (at 1 and 2hours, 1, 4 and≥12weeks after commencement)., Results: Patients with a history of thrombosis had elevated D-dimers (p=0.02) but not MP. Among patients on anticoagulants, those with thrombosis had higher F1+2 concentrations (p=0.003). TF activity was undetectable in plasma MP. Unexpectedly, thrombin peak height and thrombin potential were significantly lower in PNH patients than in healthy controls. Fibrinolysis parameters were normal. During eculizumab treatment D-dimer levels significantly decreased after 1hour (p=0.008) and remained decreased at≥12weeks (p=0.03). F1+2 (p=0.03) and thrombin peak height (p=0.02) in patients not on anticoagulants significantly decreased at≥week 12. MP remained unchanged., Conclusions: Eculizumab induces an immediate decrease of D-dimer levels but not of other markers. The decrease in thrombin peak height and F1+2 suggests that eculizumab reduces thrombin generation. Elevated D-dimer levels in untreated PNH patients with a history of thrombosis suggest possible value in predicting thrombotic risk., (Copyright © 2015. Published by Elsevier Ltd.)

Tissue factor (TF) exhibits a distinct nonuniform tissue distribution. Thus, high levels are found in highly vascularized organs such as the lung, brain, and placenta; intermediate levels in the heart, kidney, intestine, testes, and uterus; and low levels in the spleen, thymus, and liver. Several cell types are known to express TF constitutively, such as astrocytes in the brain, epithelial cells enveloping organs and body surfaces, adventitial fibroblasts and pericytes, and cardial myocytes in the heart. Smooth muscle cells in the media of the vessel wall and monocytes/macrophages contain small amounts of TF, which is enhanced substantially upon activation of the cells. Endothelial cells probably do not express TF. The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed has been challenged recently by the observation that blood of healthy individuals may form TF-induced thrombi under conditions entailing shear stress and activated platelets, corroborating the notion of blood-borne TF. Accordingly, small amounts of decrypted TF activity is detected in calcium ionophore-stimulated monocytes, and microparticles from plasma of healthy subjects possess TF-like activity subject to partial inactivation by anti-TF antibody. In addition to microparticles, plasma TF also comprises the soluble alternatively spliced human TF and truncated TF, both of which probably require factor VIIa to be physiologically active. Although it has been suggested that activated platelets possess active TF, the notion of TF as an integral platelet component is contested by more recent data. Rather, platelets may be very important in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets.

The transmembrane glycoprotein TF (tissue factor) plays an essential role in haemostasis as the principal initiator of blood coagulation. In this paper, we describe how the circulating blood cells--monocytes, platelets, neutrophils and their microparticles--co-operate in regulating the expression, availability and activity of monocyte-derived TF.

The transmembrane glycoprotein tissue factor (TF) is the initiator of the coagulation cascade in vivo. When TF is exposed to blood, it forms a high-affinity complex with the coagulation factors factor VII/activated factor VIIa (FVII/VIIa), activating factor IX and factor X, and ultimately leading to the formation of an insoluble fibrin clot. TF plays an essential role in hemostasis by restraining hemorrhage after vessel wall injury. An overview of biological and physiological aspects of TF, covering aspects consequential for thrombosis and hemostasis such as TF cell biology and biochemistry, blood-borne (circulating) TF, TF associated with microparticles, TF encryption-decryption, and regulation of TF activity and expression is presented. However, the emerging role of TF in the pathogenesis of diseases such as sepsis, atherosclerosis, certain cancers and diseases characterized by pathological fibrin deposition such as disseminated intravascular coagulation and thrombosis, has directed attention to the development of novel inhibitors of tissue factor for use as antithrombotic drugs. The main advantage of inhibitors of the TF*FVIIa pathway is that such inhibitors have the potential of inhibiting the coagulation cascade at its earliest stage. Thus, such therapeutics exert minimal disturbance of systemic hemostasis since they act locally at the site of vascular injury.

Throughout Europe, including Norway, increased winter mortality from cardiovascular diseases (CVD) is well described. However, while there are associations between high CVD mortality and cold climate, the reason for the excess deaths is not entirely known. Recent epidemiological evidence suggests that brief outdoor exposure to cold conditions may be linked to increased winter mortality in the elderly. However, the question as to whether alterations in the haemostatic system following exposure to cold could be responsible for the increased winter risk has been little investigated in elderly subjects. In this study, we have compared the effect of exposing lightly clothed healthy elderly men and women (60-70 years) for 90 minutes to either a mild cold stress (16 degrees C) or thermoneutral conditions (28 degrees C). Measurements of a variety of autonomic and haematological parameters were made in order to compare to what extent exposure to cold stress affects production of thrombogenic risk factors. The overall autonomic responses clearly showed that the subjects were mildly cold exposed. The main changes in the blood system were a cold exposure increase in hemoconcentration and an increase in the fibrinolytic parameter, t-PA. This coupled with other changes support previous findings and it is concluded that short term mild cold exposure in the elderly initiates a mild inflammatory reaction and a tendency for an increased state of hypercoagulability.

Both the complement system and tissue factor ( TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor ( C1- INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1- INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide ( LPS) in the absence or presence of C1- INH or protease-inactivated C1- INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction ( RT- qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 ( PTF1·2) and long pentraxin 3 ( PTX3) were measured by enzyme-linked immunosorbent assay ( ELISA). Cytokines were analysed using a multiplex kit. C1- INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently ( P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1- INH ( P < 0·05). C1- INH reduced the release of PTX3 ( P < 0·05) and virtually all cytokines measured ( P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1- INH. C1- INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1- INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses. [ABSTRACT FROM AUTHOR]