Intravascular Tissue Factor Research Articles

Initiation of coagulation by tissue factor carriers in blood

Fibrin can be generated in blood vessels in the absence of substantial damage to the vessel wall. According to novel observations, fibrin formation within the vessel lumen could be initiated by intravascular (blood borne) tissue factor (TF), a central starter protein of blood coagulation. We have recently detected TF in platelets, principally allowing coagulation to be initiated within the developing thrombus. While TF is stored in intraplatelet compartments under resting conditions, it is rapidly translocated to the cell surface in response to platelet activation, accumulating on filopodia. Platelet TF might be acquired from extracellular sources via transfer of TF positive microparticles and/or, potentially, be generated through translational mechanisms. Exocytotic microparticles, regular blood components, share the ability of the activated platelets to coexpose TF and phosphatidylserine, allowing the assembly of the entire coagulation system on a single membrane surface. Nonetheless, the procoagulant activity of the microparticles, when present alone, is limited. However, the vesicular TF might be uncovered by platelet–microparticle interactions. Thereby, the recruitment of platelets and microparticles to the site of vascular injury could synergistically trigger fibrin generation. In summary, by utilizing differential pathways, activated platelets are mandatory for the TF-mediated coagulation start in blood.

Mo-P2:212 Intravascular tissue factor in cord vs. adult whole blood

Acute coronary syndrome (ACS) is a challenging issue in cardiovascular medicine. Given platelet role in atherothrombosis, we sought to determine whether platelet indices can be used as diagnostic tests for patients who suffered from an acute chest discomfort.We prospectively enrolled 862 patients with an acute chest pain and 184 healthy matched controls. They were divided into four groups: 184 controls, 249 of non-ACS, 421 of unstable angina (UA), and 192 of myocardial infarction (MI) cases. Blood samples were collected at admission to the emergency department for routine hematologic tests.The mean platelet volume (MPV), platelet distribution width (PDW), and platelet large cell ratio (P-LCR) were significantly greater in patients with MI compared with those of non-ACS or control subjects. Negative and significant correlations existed between MPV, PDW, and P-LCR values and platelet count (P < 0.001). Receiver operating characteristic (ROC) curves showed that the MPV, PDW, and P-LCR with cut-off values of 9.15 fL, 11.35 fL, and 20.25% and with area under the curves of 0.563, 0.557, and 0.560, respectively, detected MI patients among those who had chest discomfort. The sensitivities and specificities were found to be 72% and 40%, 73% and 37%, and 68% and 44% for MPV, PDW, and P-LCR, respectively.An elevated admission MPV, PDW, and P-LCR may be of benefit to detect chest pain resulting in MI from that of non-cardiac one, and also for risk stratification of patients who suffered from an acute chest discomfort.

Tissue Factor Encryption

Tissue factor (TF) encryption is the post-translational suppression of TF procoagulant activity (PCA) on the cell surface. There is emerging evidence of encrypted TF in normal blood associated with monocytes and platelets. Expression of this latent TF PCA during the propagation phase of blood coagulation may contribute to hemostasis. One pathway leading to the decryption of TF PCA begins with an increase in cytosolic calcium. A large calcium influx triggers both the exposure of phosphatidylserine and the expression of TF PCA on cell surfaces. The connections between these events are reviewed along with evidence that lipid raft association may also contribute to TF encryption. The last step in the decryption of TF PCA is the proteolytic activation of zymogen factor VII. This event may be a key to understanding the different roles of intravascular and extravascular TF in the process of blood coagulation.

A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Novel Initiation Mechanism of Blood Coagulation by Intravascular Tissue Factor.

Bernd Engelmann Vascular Biology and Hemostasis, Inst. of Clinical Chemistry, Ludwig-Maximilians-Universität, Munich, Germany Following addition of fibrillar collagen to whole blood in order to mimick the starting process of hemostasis, we unexpectedly observed that tissue factor (TF), the central initiator of coagulation, was exposed within 3–5 min in association with CD15 and CD14 positive blood cells. A series of experiments revealed that the TF presentation was restricted to conjugates of neutrophils (and monocytes) with platelets. To verify the source of the TF, isolated neutrophils and platelets were evaluated for the presence of TF. Using a double sandwich Elisa, the washed platelets were found to contain TF. Conversely, TF was undetectable in the neutrophils. When searching for the intraplatelet location of TF by immunoelectron microscopy (IEM), TF was observed to reside in the alpha-granules and in the surface connected system. No TF was present in the cytoplasma and the dense granules. In response to activation, platelet TF was translocated to the cell surface by fusion of the alpha-granules with the plasma membrane. The externalized TF was found to cluster on platelet filopodia. Inspection of rapidly isolated buffy coat preparations confirmed the absence of TF from the neutrophils. Stimulation of TF-dependent factor Xa formation by the activated platelets was markedly amplified by the isolated neutrophils. This required neutrophil-platelet conjugate formation, as evident from inhibition by antibodies targeting PSGL-1 and CD18. To assess whether the TF triggered coagulation was connected to the platelet recruitment, we evaluated the participation of the ADP system. Disrupting the interaction of ADP with its platelet receptors P2Y12 and P2Y1 suppressed the TF activity in the neutrophil-platelet conjugates. Since the TF exposing filopodia represent preferential sites for the formation of microparticles (MP), we isolated the total pool of circulating MP from whole blood, known to be mainly derived from the platelets. Then, the MP were separated by cell sorting. In MP positive for the platelet specific CD42b, TF could be detected and quantified by western blotting and Elisa. Moderate increases in MP number excessively stimulated blood based TF activity in the presence of platelets and in whole blood. Since activated platelets are known to secrete tissue factor pathway inhibitor (TFPI), an anti-TFPI antibody targeting the Kunitz-2 domain of TFPI was included into the suspensions of the activated platelets. Thereby the TF activity of the isolated platelets was enforced, while the activity in the presence of the neutrophils remained unaffected, suggesting that TFPI partially masks the functional competence of the platelet TF. The potential contribution of platelet-collagen interactions for the activation of coagulation in vivo was analyzed by injecting collagen into the venous blood of mice. Local fibrin formation was documented in pulmonary vessels by EM, and systemic thrombin generation was revealed by increased thrombin-antithrombin complexes. In mice deficient for the P2Y1 ADP receptor, the thrombin generation was markedly reduced, indicating a basic role for the platelet-triggered coagulation during thrombus growth. In conclusion, the intravascular tissue factor enables the entire coagulation system to proceed on the plasma membrane of a single blood component, the surface of the activated platelets. Consequently the coagulation start can be regulated within the platelet aggregate, allowing fibrin formation to be flexibly adjusted to the size of the thrombus and the duration of its development.

Intravascular Tissue Factor (TF) Is Predominantly Platelet-Associated during the Aplastic Phase of Hematopoietic Stem Cell Transplantation (HSCT).

TF is a 47 kDa cell-bound glycoprotein that initiates coagulation upon contact with factor VII(a). Both monocytes and cells derived from the vessel wall (such as endothelial cells) are capable of TF synthesis, and both intact cells and microparticles (MPs) derived from them are considered to be the principal source(s) of intravascular TF. Data from in vitro systems and animal models suggest that MP-borne TF may be transferred to activated platelets via a P-selectin dependent pathway. We previously demonstrated that during the aplastic phase of HSCT, about 55% of baseline (pre-transplant) levels of circulating whole blood TF procoagulant activity (PCA) remained detectable (Thromb & Haemost 2001; 85:250). This study was therefore undertaken to identify whether this residual TF activity is accounted for by intact cells or MPs, and to determine whether cells derived from the vessel wall or the hematopoietic compartment are responsible. MP-associated TF PCA was measured using a recently described MP capture assay (Blood 2004; 103:4545). MP-associated TF PCA declined by 89% at day 0 and by 77% at day +7 compared to baseline levels measured at day −7, with a subsequent normalization of activity paralleling engraftment. TF PCA was assayed on fractionated platelets and mononuclear cells obtained on days −7,0,7,14 and 21 from 11 patients undergoing HSCT. Mononuclear cell and platelet fractions were obtained by differential centrifugation and exposed to calcium ionophore to maximally ‘de-encrypt' TF PCA. Following pre-incubation with FVIIa, the clotting time of added plasma was recorded, and the amount of TF calculated by reference to a standard curve generated using Innovin™. In parallel with the drop in circulating monocyte counts, a significant decline in TF PCA in both the mononuclear cell (1855±690 at day −7 vs. 74±37 pg/ml at day +7, p=0.003) and platelet (260±45 at day −7 vs. 51±16 pg/ml at day +7, p=0.05) fractions was noted. TF PCA in these fractions subsequently returned towards baseline levels with engraftment. Notably, while platelet-associated TF PCA accounted for only 12% of the total cell-associated TF PCA at baseline (day −7), it accounted for fully 59% at day 0 and 41% at day +7. This led us to postulate that transfused platelets might contribute to circulating TF during the aplastic phase. Within 1 hour of receiving leuco-depleted platelet transfusion, total cell-associated (platelet + mononuclear cell) TF PCA had increased significantly (142 ± 43 pg/ml to 317 ± 55 pg/ml, p=0.015), confirming that transfused platelets are indeed a source of TF. While this augmentation of cell-associated TF PCA was primarily seen in the platelet fraction, an increase in mononuclear cell-associated TF PCA was also noted. Conclusion: The majority of cell-associated TF PCA is associated with the mononuclear cell fraction in whole blood. However, with the onset of severe leucopenia during HSCT, the platelet pool becomes the dominant location for TF. Our data are in agreement with recent transgenic mouse model data demonstrating that the bone marrow - not the vessel wall - is the dominant source of both cell- and MP-associated intravascular TF.

Plasma Proteome Profiling: Widespread Oxidative Changes in Endotoxemia Correlate with Intravascular Tissue Factor Response.

The initial response to systemic infection in sepsis syndromes is mediated by the innate immune system and results in widespread activation of inflammation and coagulation. Some of these changes are recapitulated in the human Endotoxemia model. Proteomics represents a novel approach to study the pathophysiology of disease processes, and offers the potential for identification of biomarkers that may be used for diagnostic and/or prognostic purposes. Therefore, plasma proteome profiling was performed to study the response to intravenously administered low-dose lipopolysaccharide (LPS; 2ng/kg) in 28 healthy adult male volunteers. Interleukin 6 (IL 6), which mediates the host inflammatory responses in endotoxemia and sepsis, and circulating tissue factor (TF), which initiates coagulation, were also measured as markers of these respective processes. Plasma was obtained at baseline and at 1, 2, 3, 4, 8, and 24 hours following LPS administration. Protein profiling was performed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. All individuals showed qualitative profile changes that peaked at 8 hours after LPS and reverted towards baseline at 24 hours. The spectra obtained consisted of 15 non-redundant peaks that were seen consistently in all samples, and ratios of these peaks were used in data analysis. These peaks consisted of Apolipoproteins CI, CII and CIII, and Transthyretin (TTr) and their various isoforms. Serum Amyloid A appeared at later time points following endotoxin. Six of the 28 subjects demonstrated severe oxidation of plasma proteins detected by the loss of free sulfhydryls of TTr and addition of oxygen to many proteins. Increased proteolysis of plasma proteins was also noted in these subjects, along with the appearance of new protein peaks. These 6 individuals were designated ‘high responders'. Microparticle (MP)-associated TF procoagulant activity (PCA) was measured using a recently described assay [Blood 2004 103: 4545]. The TF PCA peaked between 3-4 hours following endotoxin challenge with a 10 fold increase, on average, compared to baseline (p < 0.001). The mean increase in MP-associated TF PCA at 4 hours after LPS was significantly higher among those subjects classified as high responders by protein profiling (p < 0.05). IL 6 levels, measured in 24 of the 28 subjects, peaked at 2–3 hours following LPS with a mean increase > 175 fold compared to baseline (p < 0.01). This study demonstrates significant increases in the MP-associated TF PCA and IL 6 levels in all subjects. The increase in TF PCA was significantly higher among those with extreme changes in their proteome profile despite the small number of individuals in this category. The magnitude of changes observed among the high responder subset supports the hypothesis that some individuals have a predisposition to increased responsiveness to endotoxin. This response may reflect either a protective effect or an increased susceptibility. Further, based on the findings of this study, this phenomenon can be rapidly determined by a novel technique using a proteomics approach. This approach offers the potential to assess early events that may precede sepsis and warrants further exploration in the clinical setting.

Tissue factor promotor polymorphism −603 A/G is associated with myocardial infarction

Tissue factor (TF), the main initiator of the extrinsic coagulation cascade is expressed in atherosclerotic lesions and contributes to coronary thrombus formation in myocardial infarction (MI). Circulating TF reflects intravascular TF activation but also adds to prothrombotic activation. Because the G allele of the TF promotor polymorphism −603 A/G is associated with monocytic mRNA expression we evaluated its association with myocardial infarction, based on a recessive deleterious effect assumption. Patients with MI (MI; n = 793) and age and sex matched control subjects without coronary artery disease (C; n = 340) undergoing coronary angiography were included. In patients with MI, the −603 G (MI: 76%, C: 70%) allele was prevalent compared to the control group ( P = 0.04). Multivariate analysis revealed an odds ratio of 1.44 (confidence interval 1.07–1.93). Carriage of the −603 G allele is associated with an increased risk for myocardial infarction. Because higher plasma TF concentrations are found in −603 G carriers enhanced TF expression may be the mechanism underlying this association.

Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia

AbstractThe precise role of intravascular tissue factor (TF) remains poorly defined, due to the limited availability of assays capable of measuring circulating TF procoagulant activity (PCA). As a model of inflammation-associated intravascular thrombin generation, we studied 18 volunteers receiving an infusion of endotoxin. A novel assay that measures microparticle (MP)-associated TF PCA from a number of cellular sources (but not platelets) demonstrated an 8-fold increase in activity at 3 to 4 hours after endotoxin administration (P < .001), with a return to baseline by 8 hours. TF antigen-positive MPs isolated from plasma were visualized by electron microscopy. Interindividual MP-associated TF response to lipopolysaccharide (LPS) was highly variable. In contrast, a previously described assay that measurestotal (cell and MP-borne) whole-blood TF PCA demonstrated a more modest increase, with a peak in activity (1.3-fold over baseline; P < .000 01) at 3 to 4 hours, and persistence for more than 24 hours. This surprisingly modest increase in whole-blood TF activity is likely explained by a profound although transient LPS-induced monocytopenia. MP-associated TF PCA was highly correlated with whole-blood TF PCA and total number of circulating MPs, and whole-blood TF PCA was highly correlated with TF mRNA levels. (Blood. 2004;103:4545-4553)

Platelet ADP receptors contribute to the initiation of intravascular coagulation

AbstractWhile the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

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.

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets.

Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet-derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full-length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the alpha-granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood-based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P-selectin and neutrophil counterreceptor (PSGL-1, CD18 integrins) interactions. Moreover, neutrophil-secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF-mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface.

Intravascular tissue factor pathway – a model for rapid initiation of coagulation within the blood vessel

The loss of blood through vessel wall ruptures is initially prevented by the rapid adhesion of platelets to the subendothelium, and the formation of a thrombus consisting of platelets and different types of leukocytes. Concomitantly, the coagulation process is thought to be activated by vascular wall tissue factor (TF). Here, a new model for the initiation of coagulation is presented, based on unexpected findings on the presence and functional activation of TF within the blood itself. TF was recently found to be stored in the alpha-granules of resting platelets under physiological conditions. Activation by collagen exposes TF on the platelet cell membrane and on platelet derived microvesicles. Adhesive interactions of the TF bearing platelets and microvesicles to neutrophils and monocytes support the functional activation of the blood based TF. The intravascular TF pathway is proposed to play a significant role during hemostasis by enabling the generation of fibrin at the site of the developing thrombus.

Nuclear factor κB mediates a procoagulant response in monocytes during extracorporeal circulation

Objective: The objective of this study was to examine the mechanism of procoagulant activity and inhibition in whole blood during extracorporeal circulation. Methods: In this study we examine the development of procoagulant activity and monocyte activation in heparinized whole blood passing through a closed circuit consisting of a pump and silicone envelope membrane oxygenator for 6 hours. Results: Anaphylatoxins, C3a and C5a, determined by means of enzyme-linked immunosorbant assay, appeared in the blood within 30 minutes of circulation. Circulated blood developed a marked potential for coagulation demonstrated in a 1-step clotting assay that reached maximal activity by 4 hours of circulation. This procoagulant activity was neutralized by anti-tissue factor antibody, suggesting a prominent role for the extrinsic pathway in pump-induced intravascular coagulation. Isolation of monocytes from circulated blood revealed that tissue factor expression is upregulated on the cell surface. Furthermore, we observed nuclear factor κB nuclear translocation in monocytes from blood passing through the circuit, suggesting that tissue factor expression was due to monocyte stimulation and transcriptional activation of the tissue factor gene. Tissue factor expression resulted in an approximately 30-fold increase in thrombin generation. Monocyte nuclear factor κB activation, monocyte tissue factor expression, thrombin generation, and the procoagulant activity of blood in extracorporeal circulation were all blocked by the proteasome inhibitor MG132. Conclusions: We conclude that intravascular tissue factor expression during extracorporeal circulation of blood is due to nuclear factor κB-mediated activation of monocytes (possibly by complement), which can be controlled pharmacologically.J Thorac Cardiovasc Surg 2003;125:165-71

Collagen stimulation rapidly activates intravascular tissue factor

Collagen stimulation rapidly activates intravascular tissue factor

Platelet-Associated Tissue Factor Contributes to the Collagen-Triggered Activation of Blood Coagulation

The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.