Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1

We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.     

A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P₂Y₁, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes.     

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.     

Alternatively spliced human tissue factor: a circulating,soluble, thrombogenic protein

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.     

Multiphysics and multiscale modeling of microthrombosis in COVID-19

Emerging clinical evidence suggests that thrombosis in the microvasculature of patients with Coronavirus disease 2019 (COVID-19) plays an essential role in dictating the disease progression. Because of the infectious nature of SARS-CoV-2, patients’ fresh blood samples are limited to access for in vitro experimental investigations. Herein, we employ a novel multiscale and multiphysics computational framework to perform predictive modeling of the pathological thrombus formation in the microvasculature using data from patients with COVID-19. This framework seamlessly integrates the key components in the process of blood clotting, including hemodynamics, transport of coagulation factors and coagulation kinetics, blood cell mechanics and adhesive dynamics, and thus allows us to quantify the contributions of many prothrombotic factors reported in the literature, such as stasis, the derangement in blood coagulation factor levels and activities, inflammatory responses of endothelial cells and leukocytes to the microthrombus formation in COVID-19. Our simulation results show that among the coagulation factors considered, antithrombin and factor V play more prominent roles in promoting thrombosis. Our simulations also suggest that recruitment of WBCs to the endothelial cells exacerbates thrombogenesis and contributes to the blockage of the blood flow. Additionally, we show that the recent identification of flowing blood cell clusters could be a result of detachment of WBCs from thrombogenic sites, which may serve as a nidus for new clot formation. These findings point to potential targets that should be further evaluated, and prioritized in the anti-thrombotic treatment of patients with COVID-19. Altogether, our computational framework provides a powerful tool for quantitative understanding of the mechanism of pathological thrombus formation and offers insights into new therapeutic approaches for treating COVID-19 associated thrombosis.     

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.     

Circadian rhythms in mouse blood coagulation

The circadian clock, influencing many biological processes, has been demonstrated to modulate levels of specific coagulation factors, but its impact on the coagulation efficiency is unknown. In a mouse model, the authors evaluated the temporal variations in the initial rate of activated factor X (FXa) and thrombin generation. Upon coagulation activation through the FVIIa-TF pathway (extrinsic activation), both parameters showed rhythmic variations with a significant peak at ZT 12, the light-to-dark transition. In mice subjected to a 6-h delayed light-dark cycle, the peak was shifted as expected. These cyclic oscillations were also observed in constant darkness, thus demonstrating, for the first time, the existence of strong circadian rhythms of the initial rate of either FXa or thrombin generation activity levels. These circadian variations overlapped with those that have been recently described in factor VII (FVII) activity. The peak of FXa generation activity was simulated by the addition of purified human FVII, thus indicating that circadian variations in FVII activity are important determinants of the circadian rhythm of the procoagulant cascade efficiency. These findings help to elucidate the complex control on the coagulation process and might contribute in explaining the temporal variations in the frequency of cardiovascular events observed in humans.     

III. Instantaneous inhibition by compound 48/80 of tissue factor-initiated extrinsic coagulation is mediated by the downregulation of factor VII activation

Our previous study has demonstrated a unique biological function of compound 48/80 (48/80) in the downregulation of monocytic tissue factor (TF)-initiated hypercoagulation in response to bacterial endotoxin (lipopolysaccharide, LPS) [A. J. Chu et al. (1999) Biochim. Biophys. Acta 1472, 386-395]. The inhibition was not due to the blockade of LPS cell signaling as evidenced by the unaffected LPS-induced TF synthesis. In the present study, we investigate the direct inhibitory action of 48/80 on the extrinsic coagulation cascade. TF-initiated coagulation was assayed by a single-stage clotting assay. Chromogenic assays dissected the extrinsic pathway to measure the activities of FVII, FX, and prothrombin by monitoring the hydrolyses of nitroaniline-conjugated substrates, identifying the inhibitory site(s). We report that 48/80 in vitro instantaneously inhibited rabbit brain thromboplastin (rbTF)-initiated coagulation in a dose-dependent manner. 48/80 preferentially inhibited FVII activation without any detectable effect on FVIIa, FXa, and thrombin activities. Neither FX activation nor prothrombin activation was affected. The significant inhibition on FVII activation was found to be noncompetitive with a fourfold reduction in the apparent Vmax of FVIIa formation from 7.1 to 1.7 nM/min, while the apparent Km (approximately 365 nM) remained unaffected. Western blotting analysis further confirmed that FVIIa formation derived from FVII was significantly diminished by 48/80, which was accompanied by blocked FVII binding to rbTF. In conclusion, 48/80 readily blocked FVII binding to rbTF, leading to diminished FVII activation and FVIIa formation. As a result, TF-initiated extrinsic coagulation was downregulated.     

Coagulation Factor Binding Orientation and Dimerization May Influence Infectivity of Adenovirus-Coagulation Factor Complexes

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus.     

The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.     

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.     

Antimicrobial peptide buforin I inhibits tissue factor-initiated coagulation

The enhanced extrinsic blood coagulation following septic shock often manifests cardiovascular complications. The upregulated monocytic tissue factor (mTF) was shown to be a primary contributor to the extrinsic hypercoagulation following acute bacterial endotoxin (LPS) infection. A single-stage clotting assay monitors TF-initiated coagulation. We herein demonstrate a novel anticoagulant activity of antimicrobial peptide Buforin I (BF I) in offsetting LPS-induced mTF hypercoagulation in THP-1 cells, which was confirmed in a cell-free in vitro model, showing that BF I effectively blocked rabbit brain thromboplastin (rbTF) procoagulant activity. Upon inclusion of 25 microM BF I into human plasma, the prolonged prothrombin time (PT) was consistent with the depressed TF-initiated coagulation. In a two-stage chromogenic assay monitoring S-2288 hydrolysis, BF I significantly inhibited not only mTF- but also rbTF-catalyzed FVII activation accompanied by the diminished FVIIa formation. The inhibition by BF I of FVII activation accounted for its novel anticoagulant activity in offsetting mTF-initiated hypercoagulation.     

Gene Targeting of Tissue Factor, Factor X, and Factor VII in Mice: Their Involvement in Embryonic Development

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5.FX^–/– mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII^–/– neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease activated receptors (PARs) may have important roles in embryonic development.     

Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation

P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are vascular adhesion molecules that play an important role in leukocyte-endothelial and leukocyte-platelet interaction during the inflammatory response. Their functions are now known to include a role in thrombus formation, specifically in relation to fibrin generation and propagation. Recent findings have demonstrated that leukocyte-derived microparticles, bearing both tissue factor and PSGL-1, circulate in the blood and accumulate in the developing platelet-rich thrombus following vessel wall injury, thus concentrating tissue factor at the site of vascular injury and initiating blood coagulation.     

Progesterone utilizes distinct membrane pools of tissue factor to increase coagulation and invasion and these effects are inhibited by TFPI

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.     

Sulfatide can markedly enhance thrombogenesis in rat deep vein thrombosis model

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.     

Effects of Thrombin on the Neurovascular Unit in Cerebral Ischemia

Cerebral ischemia is a cerebrovascular disease with high morbidity and mortality that poses a significant burden on society and the economy. About 60% of cerebral ischemia is caused by thrombus, and the formation of thrombus proceeds from insoluble fibrin, following its transformation from liquid fibrinogen. In thrombus-induced ischemia, increased permeability of the blood–brain barrier (BBB), followed by the extravasation of blood components into the brain results in an altered brain microenvironment. Changes in the brain microenvironment affect brain function and the neurovascular unit (NVU), the working unit of the brain. Recent studies have reported that coagulation factors interact with the NVU and its components, but the specific function of this interaction is highly speculative and warrants further investigations. In this article, we reviewed the role of coagulation factors in cerebral ischemia and the role of coagulation factors in thrombosis. Additionally, the influence of thrombin on the NVU is introduced, as well as in the function of NVU, which may help to explore part of brain injury mechanism during ischemia. Lastly, we propose some novel therapeutic approaches on ischemic stroke by reducing the risk of coagulation.

     

Effects of warfarin and l-carnitine on hemostatic function and oxidative stress in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and l-carnitine on those parameters. Forty male Sprague–Dawley rats were divided into four groups: control, DM, and DM received warfarin or l-carnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, l-carnitine decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While l-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress.