Yale Nemerson

Tissue Factor Modulates the Thrombogenicity of Human Atherosclerotic Plaques

BACKGROUND The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of tissue factor in the thrombogenicity of different types of atherosclerotic plaques and their components. METHODS AND RESULTS Fifty human arterial segments (5 foam cell-rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for tissue factor by use of an in situ binding assay for factor VIIa. Tissue factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P = .0002). The lipid-rich core also exhibited the most intense tissue factor staining (3 +/- 0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and tissue factor staining score (r = .35, P < .01). CONCLUSIONS Our results show that tissue factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.

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

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVIIa 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.

Macrophages, Smooth Muscle Cells, and Tissue Factor in Unstable Angina Implications for Cell-Mediated Thrombogenicity in Acute Coronary Syndromes

Background Macrophage expression of tissue factor may be responsible for coronary thrombogenicity in patients with plaque rupture. In patients without plaque rupture, smooth muscle cells may be the thrombogenic substrate. This study was designed to identify the cellular correlations of tissue factor in patients with unstable angina. Methods and Results Tissue from 50 coronary specimens (1560 pieces) from patients with unstable angina and 15 specimens from patients with stable angina were analyzed. Total and segmental areas (in square millimeters) were identified with trichrome staining. Macrophages, smooth muscle cells, and tissue factor were identified by immunostaining. Tissue factor content was larger in unstable angina (42±3%) than in stable angina (18±4%) ( P =.0001). Macrophage content was also larger in unstable angina (16±2%) than in stable angina (5±2%) ( P =.002). The percentage of tissue factor located in cellular areas was larger in coronary samples from patients with unstable angina (67±8%) than in samples from patients with stable angina (40±5%) ( P =.00007). Multiple linear stepwise regression analysis showed that coronary tissue factor content correlated significantly ( r =.83, P r =.98, P Conclusions Tissue factor content is increased in unstable angina and correlates with areas of macrophages and smooth muscle cells, suggesting a cell-mediated thrombogenicity in patients with acute coronary syndromes.

Macrophages, Smooth Muscle Cells, and Tissue Factor in Unstable Angina

BACKGROUND Macrophage expression of tissue factor may be responsible for coronary thrombogenicity in patients with plaque rupture. In patients without plaque rupture, smooth muscle cells may be the thrombogenic substrate. This study was designed to identify the cellular correlations of tissue factor in patients with unstable angina. METHODS AND RESULTS Tissue from 50 coronary specimens (1560 pieces) from patients with unstable angina and 15 specimens from patients with stable angina were analyzed. Total and segmental areas (in square millimeters) were identified with trichrome staining. Macrophages, smooth muscle cells, and tissue factor were identified by immunostaining. Tissue factor content was larger in unstable angina (42 +/- 3%) than in stable angina (18 +/- 4%) (P = .0001). Macrophage content was also larger in unstable angina (16 +/- 2%) than in stable angina (5 +/- 2%) (P = .002). The percentage of tissue factor located in cellular areas was larger in coronary samples from patients with unstable angina (67 +/- 8%) than in samples from patients with stable angina (40 +/- 5%) (P = .00007). Multiple linear stepwise regression analysis showed that coronary tissue factor content correlated significantly (r = .83, P < .0001) with macrophage and smooth muscle cell areas only in tissue from patients with unstable angina, with a strong relationship between tissue factor content and macrophages in the atheromatous gruel (r = .98, P < .0001). CONCLUSIONS Tissue factor content is increased in unstable angina and correlates with areas of macrophages and smooth muscle cells, suggesting a cell-mediated thrombogenicity in patients with acute coronary syndromes.

Role of Risk Factors in the Modulation of Tissue Factor Activity and Blood Thrombogenicity

Background—Several studies suggest a role for an increased circulating pool of tissue factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating tissue factor activity. We have extended our study to smokers and hyperlipidemic subjects. Methods and Results—Poorly controlled patients with type 2 diabetes mellitus (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by factor Xa (FXa) generation in the presence of factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber. Patients with improvement in glycemic control showed a reduction in circulating TF (362±135 versus 243±74 pmol/L per min FXa, P =0.0001). A similar effect was observed in BT (15 445±1130 versus 12 072±596 &mgr;m/mm2, P =0.01). Two hours after smoking 2 cigarettes, TF was increased (217±72 versus 283±106 pmol/L per min FXa, P =0.003). Hyperlipidemic subjects showed higher TF (237±63 versus 195±44 pmol/L per min FXa, P =0.035) than healthy volunteers. Conclusions—These findings suggest that high levels of circulating TF may be the mechanism of action responsible for the increased thrombotic complications associated with the presence of these cardiovascular risk factors. These observations strongly emphasize the usefulness of the management of the patients based on their global risk assessment.

Identification of Active Tissue Factor in Human Coronary Atheroma

BACKGROUND Recent observations suggest that thrombosis in vivo is initiated via the tissue factor (TF) pathway. The TF activity of human coronary atheroma has not been reported. METHODS AND RESULTS Directional coronary atherectomy (DCA) specimens from 63 lesions were analyzed with the use of a quantitative TF-specific activity assay. The median content of TF was 10 ng/g plaque (95% CI, 6 to 13 ng/g; range, 0 to 47 ng/g). After homogenization of the specimens, TF activity was detected in 28 of 31 lesions (90%). With a polyclonal anti-human TF antibody, the use of immunohistochemistry detected TF antigen in 43 of 50 lesions (86%); TF antigen was expressed in cellular and acellular areas of the plaque. Histologically defined thrombus was present in 19 of the 43 lesions with detectable TF antigen and in none of the 7 lesions without detectable TF antigen (19 of 43 versus 0 of 7; P < .02). TF antigen was undetectable with immunohistochemistry in 4 of 13 restenotic lesions (31%) and in 3 of 37 de novo lesions (8%) (P < .05). CONCLUSIONS TF contributes to the procoagulant activity of most atherosclerotic lesions treated with DCA. The association of immunohistochemically detectable TF with plaque thrombus suggests that TF plays a role in coronary thrombosis. Diminished TF expression in restenotic lesions may in part account for the lower complication rate that has been associated with DCA of restenotic versus de novo lesions. Inhibition of TF may represent a therapeutic goal for the prevention of thrombotic complications associated with percutaneous coronary interventions.

Tissue factor is rapidly induced in arterial smooth muscle after balloon injury.

Tissue factor (TF) is a major activator of the coagulation cascade and may play a role in initiating thrombosis after intravascular injury. To investigate whether medial vascular smooth muscle provides a source of TF following arterial injury, the induction of TF mRNA and protein was studied in balloon-injured rat aorta. After full length aortic injury, aortas were harvested at various times and the media and adventitia separated using collagenase digestion and microscopic dissection. In uninjured aortic media, TF mRNA was undetectable by RNA blot hybridization. 2 h after balloon injury TF mRNA levels increased markedly. Return to near baseline levels occurred at 24 h. In situ hybridization with a 35S-labeled antisense rat TF cRNA probe detected TF mRNA in the adventitia but not in the media or endothelium of uninjured aorta. 2 h after balloon dilatation, a marked induction of TF mRNA was observed in the adventitia and media. Using a functional clotting assay, TF procoagulant activity was detected at low levels in uninjured rat aortic media and rose by approximately 10-fold 2 h after balloon dilatation. Return to baseline occurred within 4 d. These data demonstrate that vascular injury rapidly induces active TF in arterial smooth muscle, providing a procoagulant that may result in thrombus initiation or propagation.

Tissue Factor Is Induced by Monocyte Chemoattractant Protein-1 in Human Aortic Smooth Muscle and THP-1 Cells

Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine thought to play a major role in recruiting monocytes to the atherosclerotic plaque. Tissue factor (TF), the initiator of coagulation, is found in the atherosclerotic plaque, macrophages, and human aortic smooth muscle cells (SMC). The exposure of TF during plaque rupture likely induces acute thrombosis, leading to myocardial infarction and stroke. This report demonstrates that MCP-1 induces the accumulation of TF mRNA and protein in SMC and in THP-1 myelomonocytic leukemia cells. MCP-1 also induces TF activity on the surface of human SMC. The induction of TF by MCP-1 in SMC is inhibited by pertussis toxin, suggesting that the SMC MCP-1 receptor is coupled to a Gi-protein. Chelation of intracellular calcium and inhibition of protein kinase C block the induction of TF by MCP-1, suggesting that in SMC it is mediated by activation of phospholipase C. SMC bind MCP-1 with a K d similar to that previously reported for macrophages. However, mRNA encoding the macrophage MCP-1 receptors, CCR2A and B, is not present in SMC, indicating that they possess a distinct MCP-1 receptor. These data suggest that in addition to being a chemoattractant, MCP-1 may have a procoagulant function and raise the possibility of an autocrine pathway in which MCP-1, secreted by SMC and macrophages, induces TF activity in these same cells.

Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.

The expression of the placental anticoagulant protein, annexin V, by villous trophoblasts: Immunolocalization and in vitro regulation

We evaluated the histological and ultrastructural localization of the potent anticoagulant protein, annexin V, at the light and electron microscopic levels, using immunohistochemistry and an immunogold method. Annexin V was found to localize to the microvillar surface of the villous syncytiotrophoblasts. Isolated villous-derived trophoblasts were then utilized to evaluate the expression of annexin 1 protein mRNA in response to syncytialization in vitro, as well as to exposure to adenylate cyclase and protein kinase C agonists. Levels of immunoreactive annexin V released into the conditioned media and associated with cell protein were assessed by ELISA while levels of annexin V mRNA were evaluated by Northern analysis. No significant change in either media or cell-associated annexin V concentrations were detected over time in culture or in response to 1.5 mM 8-bromo-cyclic-adenosine-monophosphate (8-b-cAMP) or 0.15 nM phorbol ester myristic acid (PMA). These results indicate that annexin V is ideally positioned to inhibit intervillous thrombosis and maintain the fluidity of the intervillous circulation. Moreover, the absence of trophoblast annexin V regulation by intracellular second messenger regulators suggests that this crucial placental anticoagulant factor is constitutively produced.