Intravascular Tissue Factor Research Articles

Kininogen supports inflammation and bacterial spreading during Streptococccus Pyogenes Sepsis.

BackgroundHigh-molecular-weight kininogen is a cofactor of the human contact system, an inflammatory response mechanism that is activated during sepsis. It has been shown that high-molecular-weight kininogen contributes to endotoxemia, but is not critical for local host defense during pneumonia by Gram-negative bacteria. However, some important pathogens, such as Streptococcus pyogenes, can cleave kininogen by contact system activation. Whether kininogen causally affects antibacterial host defense in S. pyogenes infection, remains unknown.MethodsKininogen concentration was determined in course plasma samples from septic patients. mRNA expression and degradation of kininogen was determined in liver or plasma of septic mice. Kininogen was depleted in mice by treatment with selective kininogen directed antisense oligonucleotides (ASOs) or a scrambled control ASO for 3 weeks prior to infection. 24 h after infection, infection parameters were determined.FindingsData from human and mice samples indicate that kininogen is a positive acute phase protein. Lower kininogen concentration in plasma correlate with a higher APACHE II score in septic patients. We show that ASO-mediated depletion of kininogen in mice indeed restrains streptococcal spreading, reduces levels of proinflammatory cytokines such as IL-1β and IFNγ, but increased intravascular tissue factor and fibrin deposition in kidneys of septic animals.InterpretationMechanistically, kininogen depletion results in reduced plasma kallikrein levels and, during sepsis, in increased intravascular tissue factor that may reinforce immunothrombosis, and thus reduce streptococcal spreading. These novel findings point to an anticoagulant and profibrinolytic role of kininogens during streptococcal sepsis.FundingFull details are provided in the Acknowledgements section.

COVID-19-Associated Coagulopathy: An Exacerbated Immunothrombosis Response.

Since the onset of the global pandemic in early 2020, coronavirus disease 2019 (COVID-19) has posed a multitude of challenges to health care systems worldwide. In order to combat these challenges and devise appropriate therapeutic strategies, it becomes of paramount importance to elucidate the pathophysiology of this illness. Coronavirus disease 2019, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), is characterized by a dysregulated immune system and hypercoagulability. COVID-associated coagulopathy (CAC) was recognized based on profound d-dimer elevations and evidence of microthrombi and macrothrombi, both in venous and arterial systems. The underlying mechanisms associated with CAC have been suggested, but not clearly defined. The model of immunothrombosis illustrates the elaborate crosstalk between the innate immune system and coagulation. The rendering of a procoagulant state in COVID-19 involves the interplay of many innate immune pathways. The SARS-CoV2 virus can directly infect immune and endothelial cells, leading to endothelial injury and dysregulation of the immune system. Activated leukocytes potentiate a procoagulant state via release of intravascular tissue factor, platelet activation, NETosis, and inhibition of anticoagulant mechanisms. Additional pathways of specific relevance in CAC include cytokine release and complement activation. All these mechanisms have recently been reported in COVID-19. Immunothrombosis provides a comprehensive perspective of the several synergistic pathways pertinent to the pathogenesis of CAC.

Monocyte procoagulant responses to anthrax peptidoglycan are reinforced by proinflammatory cytokine signaling.

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis. We hypothesized that immune recognition of PGN by monocytes is critical for procoagulant responses to PGN and investigated whether and how PGN induces TF expression in primary human monocytes. We found that PGN induced monocyte TF expression in a large cohort of healthy volunteers similar to lipopolysaccharide stimulation. Both immune and procoagulant responses to PGN involve intracellular recognition after PGN internalization, as well as surface signaling through immune Fcγ receptors (FcγRs). In line with our hypothesis, blocking immune receptor function, both signaling and FcγR-mediated phagocytosis, significantly reduced but did not abolish PGN-induced monocyte TF expression, indicating that FcγR-independent internalization contributes to intracellular recognition of PGN. Conversely, when intracellular PGN recognition is abolished, TF expression was sensitive to inhibitors of FcγR signaling, indicating that surface engagement of monocyte immune receptors can promote TF expression. The primary procoagulant responses to PGN were further amplified by proinflammatory cytokines through paracrine and autocrine signaling. Despite intersubject variability in the study cohort, dual neutralization of tumor necrosis factor-α and interleukin-1β provided the most robust inhibition of the procoagulant amplification loop and may prove useful for reducing coagulopathies in gram-positive sepsis.

Methyl isocyanate inhalation induces tissue factor-dependent activation of coagulation in rats

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125–500 ppm) by nose-only vapor inhalation for 30 min. Arterial O2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO2) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.

Neutrophil extracellular traps coincide with a pro-coagulant status of microcirculatory endothelium in burn wounds.

Burn-induced tissue loss is partly related to secondary expansion of necrosis into vital dermis neighboring the initial burn injury. An important factor herein is the severe loss of perfusion of the burn wound, probably caused by microvascular damage induced by the intense local inflammatory responses as well as burn-induced hypercoagulation. We hypothesize that the formation of neutrophilic extracellular traps (NETs) play an important role in this. The purpose of this study was to investigate postburn intravascular thrombosis, NETs formation and the coagulant state in the microvasculature of burns in both animal models and patients. We used two in vivo burn wound models: rats and pigs. In rats, the entire wound was excised at day 14 postburn and in pigs burn wound biopsies were collected at different time points up to 60 days postburn. To confirm the data in patients, eschar from the burn wound was obtained from burn wound patients at different time points after wounding. The number of intravascular thrombi, the presence of intravascular NETs and the number of tissue factor (TF) positive blood vessels in the burn wound was determined. In rats, a significant increase in intravascular thrombi and TF expression was observed 14 days postburn, that in majority coincided with NETs. In pigs, a significant increase in intravascular thrombi and TF expression was found over time up to 60 days postburn, that in majority coincided with NETs too. Also in eschar of burn wound patients, a significant increase in intravascular thrombi was noted, that in majority coincided with NETs, already 0.5 days postburn and remained elevated up to 46 days postburn. This study shows the presence of NETosis in microcirculatory thrombosis of burn wounds and a switch in the microcirculatory endothelium toward a procoagulant phenotype.

Venous thromboembolism: thrombosis, inflammation, and immunothrombosis for clinicians.

Venous thromboembolism (VTE) is a worldwide disease related with mortality, cardiovascular disability, impaired quality of life and, cause major long-term complications. Clinicians related to the acute and long-term patients care must be involved in the molecular mechanisms of thrombosis. The vessel wall and its inner lining of the endothelium are critical to the maintenance of a patent vasculature. After endothelial disruption, collagen (first line of endothelial defense) and intravascular tissue factor (second line of endothelial defense) are exposed to blood flow, starting the formation of a thrombus. Anticoagulant endovascular proteins and endogenous fibrinolysis have an active role in hemostasis. Currently, the process of coagulation is a cell surface-based model that includes three overlapping phases: initiation, amplification, and propagation. From a simple view, inflammation is one of the first responses of the immune system to infection; inflammation is driven by eicosanoids and cytokines, which are released by injured or infected cells. Common cytokines, which regulate inflammatory response, include interleukins (mainly interleukin-6) that are responsible for communication among white blood cells, chemokines that promote chemotaxis, and interferons that have anti-viral effects. Acute infections have been associated with a transient increase in the risk of myocardial infarction, stroke and recently with venous thrombosis, supporting the notion that systemic and respiratory infections increase the risk of thromboembolic events. Recently, immunothrombosis, another thrombosis mechanism that includes innate immune mechanisms, the neutrophil extracellular genetic traps, and the immunothrombosis dysregulation, could explain some cases of "unprovoked" VTE especially in elderly, a high-risk population for thrombosis.

High intravascular tissue factor-but not extracellular microvesicles-in septic patients is associated with a high SAPS II score.

BackgroundSepsis is associated with coagulation abnormalities, and a high content of intravascular tissue factor (TF) may contribute to the development of multisystem organ failure. Circulating microvesicles (MVs) are increased during sepsis and characterized by their phosphatidylserine content. It is unclear whether MVs—as a part of the host response to the infection—are beneficial or rather contribute to systemic complications in sepsis. In the present prospective clinical pilot study, we investigated whether plasma TF and MVs are associated with the risk of multiple organ failure and mortality.MethodsThirty patients diagnosed with sepsis, severe sepsis, or septic shock were enrolled and classified as 19 survivors and 11 non-survivors. Blood samples were collected on the day of admission and then daily for up to 2 weeks. MVs and TF were quantified in plasma by ELISA.ResultsNon-survivors had significantly higher TF concentrations on day 3 compared to survivors. Logistic regression analysis revealed that patients with high amounts of TF had significantly increased risk for severity of disease, according to high Simplified Acute Physiology Score II (SAPS II) scores (odds ratio 18.7). In contrast, a higher content of phosphatidylserine-rich MVs were apparently associated with a lower risk for mortality and multiple organ failure, although this was only a trend and the odds ratios were not significant.ConclusionsThis study showed that a high amount of TF in septic patients is significantly associated with increased risk for disease severity, according to a high SAPS II score. Quantification of total MVs in plasma, independent from their cell origin, might be indicative for the outcome of patients in sepsis.

Innate Immunity, Coagulation, and Thrombosis

In evolutionarily ancient animals such as insects and crustaceans, the host responses to physical injury and to invading pathogens can be mediated by the same mechanism of coagulum formation of the hemolymph. During vertebrate evolution hemostasis has emerged as an independent process primarily involved in the rapid repair of blood vessel injuries. The core processes of hemostasis are blood coagulation (resulting in fibrin formation) and platelet activation. Both processes can independently interact with inflammatory responses as apparent in a pathological context such as during development of disseminated intravascular coagulation (DIC). Moreover, extravascular fibrin formation can promote the trapping of pathogens and thereby help to contain infections. Nonetheless, the connections between fibrin formation, platelet activation and innate immunity are incompletely understood. We have recently shown that during early systemic infection with E. coli microvascular thrombi are formed which capture bacteria together with innate leukocytes. These thrombi are fibrin-rich and are in general observed in less than 10% of vessels with diameters < 25 µm. Their formation is not accompanied by marked activation of inflammation since the levels of pro-inflammatory markers are unchanged. Microvascular thrombosis is almost completely suppressed in mice deficient for the neutrophil serine proteases elastase and cathepsin G (NE/CG-/-) which are major microbicidal effectors of neutrophils. In the microcirculation of NE/CG-/- mice, microbes are mostly tissue-associated. In contrast, they are mostly present inside blood vessels in wild type mice. The results of experimental changes in microvascular fibrin formation show that intravascular blood coagulation is causally involved in the capturing of bacteria and of myeloid cells and, additionally, promotes the bacterial killing. Overall this suggests that microvascular thrombosis supports recognition, containment and elimination of bacteria without inducing noticeable damage to the host. It thus fulfills the criteria for a comprehensive intravascular process of innate immunity. This mechanism of intravascular immunity, which was termed "immunothrombosis," is supported by tissue factor (TF), the overall initiator of blood coagulation, and by factor XII, the starter protein of the contact pathway. In particular, extracellular nucleosomes (eNUC)/neutrophil extracellular traps (NETs) are indispensable for immunothrombosis. eNUC/NETs promote thrombosis by critically enhancing degradation of TFPI, the major antagonist of the coagulation start, via neutrophil elastase and by factor XII activation. Release of eNUC/NETs by neutrophils and induction of intravascular coagulation essentially require interaction of activated platelets with neutrophils. Interestingly, intravascular TF, factor XII, eNUC/NETs and innate leukocytes are almost completely dispensable for hemostasis. Furthermore, immunothrombosis in contrast to hemostasis develops in largely intact blood vessels. Together this indicates that thrombosis can be a physiological mechanism of innate immunity that is distinct from hemostasis. We have recently developed a new model for deep vein thrombosis (DVT) which closely reproduces the pathological changes in the vessel wall observed in most patients with DVT. Using this model, we show that intravascular TF, factor XII, eNUC/NETs, innate leukocytes and their interactions with platelets all critically promote DVT. Thus, DVT shares similar triggers (especially pathogens) and identical molecular and cellular mediators with immunothrombosis. In case of DIC, the connections to immunothrombosis are most likely similarly strong or even stronger. Finally, our results also show that mediators of immunothrombosis such as eNUC/NETs and neutrophil serine proteases are main triggers of arterial thrombosis. Hence, together with hemostasis, immunothrombosis likely constitutes the major biological template process for both (pathological) microvascular thrombosis and large vessel thrombosis. DisclosuresNo relevant conflicts of interest to declare.

The role of carrier number on the procoagulant activity of tissue factor in blood and plasma

Tissue factor (TF) is a transmembrane glycoprotein cofactor of activated blood coagulation factor VII (FVIIa) that is required for hemostatic thrombin generation at sites of blood vessel injury. Membrane-associated TF detected in circulating blood of healthy subjects, referred to as intravascular or circulating TF has been shown to contribute to experimental thrombus propagation at sites of localized vessel injury. Certain disease states, such as metastatic cancer, are associated with increased levels of intravascular TF and an elevated risk of venous thromboembolism. However, the physiological relevance of circulating TF to hemostasis or thrombosis, as well as cancer metastasis, is ill-defined. This study was designed to assess whether the spatial separation of intravascular TF carriers in blood, demonstrated with TF-inducible human monocytic cell line U937 or TF-coated polymer microspheres, affected procoagulant activity and hence thrombogenic potential. Experiments were performed to characterize the effects of TF-carrier number on the kinetics of clot formation in both open and closed systems. The procoagulant activity of TF carriers was found to correlate with spatial separation in both closed, well-mixed systems and open, flowing systems. TF carriers enhanced the amidolytic activity of FVIIa toward the chromogenic substrate, S-2366, as a function of carrier count. These results suggest that TF-initiated coagulation by circulating TF is kinetically limited by mass transport of TF-dependent coagulation factors to the TF-bearing surface, a constraint that may be unique to circulating TF. Spatial separation of circulating TF carriers is therefore a critical determinant of the procoagulant activity of circulating TF.

High intravascular tissue factor expression in dogs with idiopathic immune-mediated haemolytic anaemia

A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.

Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.

Cell-derived vesicles exposing coagulant tissue factor in saliva

AbstractOn 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.

Intravascular but Not Extravascular Tissue Factor Is Required for Fibrin Generation During Thrombus Formation in Cremaster Arterioles in Living Mice Subjected to Laser Injury.

Abstract 332▪▪This icon denotes an abstract that is clinically relevant.Tissue factor (TF) is required for the initiation of thrombin formation and fibrin generation in vivo. However, the functional role of intravascular tissue factor in thrombus formation is the subject of ongoing controversy. To address this question, we compared fibrin generation and platelet thrombus formation between mice lacking tissue factor in either the intravascular or the extravascular compartment using the laser injury model of thrombosis in the mouse cremaster muscle. Laser injury initiation was chosen for three reasons: (i) thrombus initiation in this model has been shown to follow endothelial activation as measured by an increase in intracellular calcium in the endothelium following laser injury; (ii) thrombus formation in this model is dominated by activation of platelets by thrombin rather than collagen; (iii) endothelial disruption by laser injury histologically appears minimal compared with thrombus initiation by ferric chloride, Rose Bengal or mechanical disruption. In this study, TF was selectively deleted in tissues of intravascular versus extravascular origin using the Cre-lox method of targeted gene deletion. TFflox/flox mice were crossed with Tie2Cre+/o mice for combined endothelial and hematopoietic TF deletion, and sm22αCre+/o mice for smooth muscle cell TF deletion. In the Tie2Cre+ TFflox/flox mice, TF DNA was reduced by 93% in the whole blood compared with Cre- controls. In the aortic media of sm22αCre+ TFflox/flox mice, TF mRNA was 96% decreased and TF activity was reduced by 95% compared with control, while there was no difference in circulating TF activity (Wang et al, Blood. 2009;113). Using the laser injury model of thrombosis in the cremaster arteriole of a live mouse, thrombus and fibrin generation were evaluated using intravital multichannel fluorescence and brightfield widefield microscopy. Platelet accumulation was measured using either an Alexa-649 labeled anti-mouse GP1bβ antibody, or Fab fragments of an Alexa-647 labeled anti-mouse GPIIbIIIa antibody directed against CD41. Fibrin formation was measured using an Alexa-488 labeled fibrin-specific antibody. TF deletion in endothelial and hematopoietic tissue resulted in a marked decrease in maximal fibrin generation (82%, p=0.004) compared with Cre- control mice. Maximal thrombus size was decreased by 55% (p=0.04). In contrast, TF deletion in the smooth muscle cell compartment resulted in no change in fibrin formation compared with Cre- control. We conclude that when endothelial disruption is minimized, such as in the laser injury model of thrombosis, intravascular TF plays a significant role in fibrin formation, while smooth muscle cell derived TF does not. Although these models of thrombus formation have no direct parallel in human disease, it is likely that the pathogenesis of thrombosis in humans include some mechanisms in which extravascular tissue factor plays a dominant role and others in which intravascular tissue factor plays a dominant role. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Tissue Specific Expression of Alternatively Spliced Forms of Tissue Factor Pathway Inhibitor (TFPI) in Mice

TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

Tissue factor in the antiphospholipid syndrome

Antiphospholipid (aPL) antibodies are clinically important acquired risk factors for thrombosis and pregnancy loss and are thought to have a direct prothrombotic effect in vivo. Data suggest that a major mechanism by which aPL antibodies contribute to thrombophilia is the upregulation of tissue factor (TF) (CD142) on blood cells and vascular endothelium. TF is the physiological trigger of normal blood coagulation and thrombosis in many hypercoagulable conditions. This article reviews the physiology of TF, the molecular regulation of TF expression and the effects of aPL antibodies on intravascular TF regulation and expression. Inhibition of TF and the pathways by which aPL antibodies induce TF expression are potentially attractive therapeutic targets in the antiphospholipid syndrome.

High Availability of Intravascular Tissue Factor in Neonates

In the present study, we compared the levels of intravascular tissue factor (TF) present in cord versus adult whole blood (WB) prior and after lipopolysaccharide (LPS) stimulation. High levels of intravascular TF might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors. Quantitative reverse transcription-polymerase chain reaction revealed same (basal) TF mRNA expression levels in both native cord and adult WB, and approximately same increase in TF mRNA expression owing to LPS incubation in both cord and adult WB (normalized to the housekeeping gene beta-actin). Flow-cytometric (fluorescence activated cell sorting) analysis revealed significantly higher surface TF antigen exposure on the neonatal monocyte membrane in native WB samples, and approximately same ability of neonatal and adult monocytes to express TF upon LPS-stimulation. Thrombelastography revealed significantly shorter clotting times of native cord versus adult WB (527+/-41 vs. 592+/-23 s, P<0.05). Moreover, shortening of clotting times owing to LPS-stimulation was significantly more pronounced in cord versus adult WB (29.65+/-3.35% vs. 12.03+/-6.23%, P<0.05). Because both quantitative reverse transcription-polymerase chain reaction and fluorescence activated cell sorting analysis revealed same capability of both neonatal and adult monocytes to express TF upon LPS-stimulation, this efficient shortening effect in cord WB might be explained by the constitutively high number of monocytes present in neonates. We suggest that the high levels of intravascular TF present in neonates (prior and after LPS-stimulation) might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors.

New locations of intravascular tissue factor

Tissue factor (TF) is the major initiator of blood coagulation, and a mediator of inflammation, angiogenesis, and carcinogenesis. According to recent evidence, preformed TF and inducible expression of the protein is observed in several blood components. TF is apparently constitutively expressed on circulating microparticles, and can be exposed within minutes on the cell membrane of activated eosinophils and platelets, and, potentially, on neutrophils. Expression in monocytes and neutrophils largely requires transcriptional activation of the TF gene. Eosinophils appear to harbour the highest concentration of preformed TF among all blood components under resting conditions. TF expression in eosinophil progenitor cells is substantially higher than in precursors of other granulocyte fractions. Eosinophil TF promotes transendothelial migration, which documents that presynthesized TF in blood supports functions apart from coagulation. It is still an open question how the intravascular TF is activated to trigger initiation of coagulation. TF activation in different blood components is likely to be differentially regulated according to the (patho)physiologic context.

Signaling Pathways in HIT Immune Complex Stimulation of Monocyte Tissue Factor.

In heparin-induced thrombocytopenia (HIT), platelet-fibrin thrombi form in vivo due to generation of thrombin initiated by intravascular tissue factor (TF). Stimulation of monocytic cells in vitro by the HIT immune complex (IC), consisting of heparin/PF4 complexes and IgG anti-hep/PF4 antibodies, triggers TF expression. The HIT IC binds to Fcγ receptors on monocytic cells to initiate a signal cascade which results in new TF transcription. Though much is known about monocyte TF transcription as a consequence of LPS stimulation of TLR4, the mechanism by which HIT IC engagement of FcγR leads to TF transcription is unknown. Transcription of pro-inflammatory and anti-inflammatory cytokines as a consequence of IC engagement of FcγRs on monocytes has been reported to involve the ras/Erk and/or PI3K/Akt pathways downstream of syk. We tested the hypothesis that HIT IC- FcγR engagement signals via a PI3K/Akt pathway to trigger TF transcription. We created a model HIT IC consisting of an optimal ratio of heparin and recombinant human PF4, bound by well-characterized monoclonal anti-hep/hPF4 antibody KKO. Using human monocytic cell line THP-1, we observed that the HIT IC induction of new TF mRNA, assessed by qRT-PCR after treatment for 2 h at 37°C, was reduced approximately 80% by pretreatment of the cells with PI3K inhibitor wortmannin (100 nM; n = 3, p < 0.05). In addition, the same effect was observed with another IC, heat-aggregated human IgG, namely 80% inhibition of stimulated TF expression by wortmannin (pretreatment with 100 nM, 37°C, 30 min, n = 4, p< 0.05). Of note, the LPS-mediated increase in TF expression was enhanced over 2-fold (1 ug/ml, 2 h, p <0.05) by the same wortmannin pretreatment, in direct contrast to the result with IC stimulation. In preliminary studies with Akt inhibitor SH5, we have observed ~80% inhibition of IC-stimulated TF expression. We have verified the effects of PI3K inhibition of IC-stimulation of TF (75–80% inhibition by wortmannin pretreatment) by using primary peritoneal macrophages derived from FcγRIIa-transgenic mice, which express a similar FcγR pattern as human cells and our HIT mouse model. Immunoblot analysis of phospho-Akt using anti-Ser473 Akt antibody(generously provided by Donna Woulfe, PhD, Thomas Jefferson University) verified the phosphorylation of Akt by HIT IC treatment and marked reduction when PI3K was inhibited. In summary, HIT IC stimulation of monocytic cell TF involves a PI3K/Akt pathway in a way clearly distinct from LPS-stimulated TF expression. Further studies are in progress to elucidate the upstream activators and downstream effectors, the role of other signaling pathways and the identity of the responsible transcription factor complexes.

Functional and Antigenic Detection of Intravascular Tissue Factor (TF).

Recently, intravascular TF has been the focus of tremendous research activity in the fields of hematology, oncology, and cardiovascular medicine. The biological relevance of this so-called blood-borne TF, however, remains controversial. This may, at least in part, be due to a lack of controlled studies comparing functional and antigenic detection systems. The following study was conducted to provide insight into this area of uncertainty. Incubation of citrated blood with LPS (4 h) increased monocyte TF antigen from 0–2 to 7–35% and reduced plasma recalcification times (RTs) from 648±87 to 388±23 s (n=10), an effect that was completely reversed by inhibitory TF mAb. In all donors, monocyte-platelet-conjugates, as assessed by CD41 labeling of CD14+ events, decreased following LPS stimulation (47±15 vs. 58±16%, P<0.01), suggesting that microparticles (MPs) shed from monocytes into plasma were associated with platelet membrane components. Plasma TF antigen, as measured by a commercial ELISA using non-lipidated, full-length recombinant human TF (rhTF1–263) as standard and 0.1% Triton X-100 for sample dilution, was increased from 21±33 to 160±110 pg/ml (P<0.01). TF antigen levels were unaffected by ultracentrifugation or 0.2-μm filtration, both of which reduced cellular MPs by >90% and significantly prolonged clotting times. After 18 h, LPS further shortened plasma RTs to 170±31 s, whereas a difference in plasma TF antigen was no longer detectable (80±66 vs. 127±103 pg/ml, P=0.48). We used a highly sensitive clotting assay ± TF mAb to determine the TF-specific procoagulant activity (PCA) of washed and concentrated plasma MPs. Clotting times were calibrated against serial dilutions (1/10–1/105) of relipidated rhTF1–263, showing a linear correlation in a log-log plot with R2>0.99. MP-associated TF PCA was increased >10fold after LPS stimulation. However, by flow cytometry using PE-conjugated TF mAb (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization, we could not reliably detect an increase in TF+ MPs. Furthermore, a chromogenic assay, which was 10fold less sensitive than the clotting assay, failed to detect significant TF-VIIa-dependent Xa generation by LPS-induced MPs. Similarly, incubation of strongly TF-expressing adenocarcinomatous HT29 cells in citrated blood under stirring conditions (1 h) shortened plasma RTs from 694±26 to 152±2 s and increased MP-associated TF PCA >100fold, whereas plasma TF antigen was elevated by only 18%. In an 84-year-old patient with metastatic adenocarcinoma and Trousseau's syndrome, who presented with DIC, bilateral deep vein thrombosis, pulmonary embolism, intracardiac thrombi, and extensive cutaneous necrosis due to thrombotic microangiopathy, we found significant expression of TF PCA on tumor cells derived from pleural effusion. Whereas TF-specific PCA of MPs isolated from the patient's plasma on three different occasions was 8–97fold higher than in five healthy controls, plasma TF antigen was only slightly increased (110 vs. 45±26 pg/ml). In summary, functional appear superior to antigenic assays in detecting intravascular TF, with clotting assays being most sensitive and reproducible. Particularly, commercial ELISAs may not adequately reflect MP-associated TF in plasma. Instead, increased TF antigen after short-term exposure of whole blood monocytes to LPS may indicate preferential detection of soluble variants such as alternatively spliced TF.

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenecity of human platelets

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.