Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions

Endothelial cell-mediated coagulation and leukocyte adhesion are processes that might be connected by the generation of thrombin. To examine the interaction of procoagulant and proadhesive activity, cultures of endothelial cells were stimulated with tumor necrosis factor-alpha, which resulted in the surface expression of tissue factor. Subsequent exposure to human nonanticoagulated blood at a shear rate of 100 s-1 in a parallel plate perfusion device led to the deposition of polymerized fibrin, which covered 63% of the endothelial surface. In addition, numerous platelet aggregates (71 per 10 mm cross- section) and leukocytes (53 +/- 6/mm2) were deposited on stimulated endothelial cells, whereas no fibrin and only a few platelet aggregates (4 +/- 1 per 10 mm cross-section) and leukocytes (6 +/- 1/mm2) were detected on control cells. A significant portion of the adherent leukocytes bound to fibrin and platelets. However, when the deposition of fibrin and platelet aggregates was inhibited with the anti-tissue factor antibody HTFI-7B8 by 100% and 86%, respectively, leukocyte adherence remained unchanged (68 +/- 6/mm2). This indicated that leukocytes could efficiently adhere to endothelial cells through direct cell-cell contact independent of both thrombin and deposited fibrin. Moreover, this direct adhesion of leukocytes to the endothelial surface was reduced twofold to threefold when fibrin deposition occurred. These data suggest a relationship between endothelial procoagulant and proadhesive properties in that tissue factor-initiated coagulation may contribute to leukocyte adhesion through the formation of an adhesive fibrin/platelet meshwork but concurrently prevents the adhesive endothelial surface to bind leukocytes at its full capacity.     

Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.     

Loss of blood platelet adhesion after heating native and cultured human subendothelium to 100 degrees Celcius

STUDY OBJECTIVE--Balloon angioplasty produces mechanical vessel wall injury that leads to substantial blood platelet deposition at the angioplasty site. The aim of the study was to determine whether thermal angioplasty might, by contrast, reduce platelet adhesion by denaturation of subendothelial adhesive proteins. DESIGN--Native and cultured human subendothelium was briefly heated to greater than or equal to 100 degrees C by laser irradiation or to 50-100 degrees C by immersion in preheated phosphate buffered saline (PBS). Subsequently, the subendothelium was exposed for 5 min to flowing human blood at a shear rate of 1300 s-1. Blood platelet adhesion to the subendothelium was determined quantitatively. EXPERIMENTAL MATERIAL--Human umbilical arteries were used and the subendothelial matrix derived from cultured umbilical vein endothelial cells. MEASUREMENTS AND RESULTS--After heating arterial subendothelium by laser irradiation to greater than or equal to 100 degrees C, zero platelet adhesion was found v 36(SD 2)% adhesion to the non-heated surface (p less than 0.001). After laser heating of the subendothelial matrix to greater than or equal to 100 degrees C, platelet adhesion was absent in 10/10 experiments (p less than 0.01). After heating the matrix to 100 degrees C by immersion in PBS, platelet adhesion was reduced to 5(5)% v 31(7)% at 37 degrees C (p less than 0.001). CONCLUSIONS--These in vitro results, if extrapolated to catheter interventions, suggest that thermal injury to the vessel wall by laser angioplasty or other thermal angioplasty methods may provide a basic and clinically relevant advantage over mechanical angioplasty modalities, because of a potentially reduced risk of complications related to platelet adhesion.     

Generation of procoagulant activity by mononuclear phagocytes: a possible mechanism contributing to blood clotting activation within malignant tissues

This study investigated the procoagulant activity (PCA) of mononuclear phagocytes from rabbits bearing the V2 carcinoma. Macrophages harvested from either intraperitoneally or subcutaneously growing tumors were found to express a very strong procoagulant activity as compared with peritoneal macrophages and circulating mononuclear cells from the same animals. On the other hand, when incubated with or without endotoxin in short-term cultures, circulating mononuclear cells from tumor-bearing animals generated significantly more procoagulant activity than those from control animals. In all instances, procoagulant activity was identified as tissue factor by using assay systems with plasmas selectively deficient in the various clotting factors. These results indicate that, besides cancer cells, mononuclear phagocytes might also play an important role in the activation of blood coagulation and in the deposition of fibrin at the host-tumor interface.     

Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS- induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF- PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.     

The production and availability of tissue thromboplastin in cellular populations of whole blood exposed to various concentrations of endotoxin. An assay for detection of endotoxin

These studies were undertaken to determine the type and availability of the procoagulant activities generated in blood incubated with endotoxin. The shortening of the recalcification time of blood incubated with endotoxin was directly correlated with the increase in synthesis of tissue thromboplastin in the monocytes. The procoagulant activity which resulted in the shortening of the clotting time was shown to be almost totally blocked by tissue thromboplastin antibodies. Thus, no additional procoagulant activity was generated in platelets during the 5 h incubation of blood with endotoxin. However, lysed platelets enhanced the synthesis of tissue thromboplastin in blood monocytes in the presence of endotoxin. Lysed red blood cells or granulocytes had no such effect. In endotoxin stimulated monocytes the main part of the newly synthesized tissue thromboplastin appeared to be exposed on the cellular surface. Thus, only 25% of the tissue thromboplastin activity was recovered when tissue thromboplastin antibodies had been present during the stimulation. Unstimulated monocytes were also found to possess tissue thromboplastin activity, but this low activity was not affected by tissue thromboplastin antibodies unless the monocytes were disrupted by sonication. The high percentage of tissue thromboplastin exposed on the surface of the endotoxin stimulated monocytes in whole blood may contribute significantly to the rapid induction of disseminated intravascular coagulation in gram negative sepsis.     

Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood

Activated platelets provide assembly sites for coagulation enzyme complexes and in this way can mediate coagulation during hemostasis and thrombosis. In this study, we examined the procoagulant activity of platelets adhering directly to fibrillar collagen, a main thrombogenic constituent of subendothelium. For this purpose, we used a human ex- vivo thrombosis model in which collagen-coated coverslips were exposed to flowing nonanticoagulated blood (shear rate, 65/s) for 5.5 minutes, which led to the deposition of adherent platelets, platelet thrombi, and fibrin. To examine the procoagulant activity of adherent platelets only, a selective antagonist of the platelet GPIIb-IIIa complex, Ro 44- 9883, was infused via a mixing device, resulting in a complete abrogation of platelet thrombus formation but leaving the collagen- adherent platelet layer intact. This platelet layer generated increased postchamber fibrinopeptide A (FPA) levels (203 +/- 33 ng/mL) as compared with control experiments without infusion of inhibitor (95 +/- 13 ng/mL). Concomitantly, fibrin deposition measured by morphometric analysis of cross-sections was also increased, as was the platelet adhesion to collagen. An immunochemical staining of fibrin fibers further showed that the adherent platelets formed the nuclei for fibrin fiber formation. This increase in fibrin deposition was mediated by the intrinsic factor X (F.X) activation complex on adherent single platelets, because almost complete inhibition of FPA generation (9 ng/mL) and fibrin deposition (0.4% +/- 0.2% coverage) was achieved upon coinfusion of the GP IIb-IIIa antagonist and active site-inhibited F.IXa. The large platelet thrombi that were deposited in control experiments contained no significant amounts of immunodetectable fibrin except at the thrombus base, where adherent platelets anchored the thrombi to the collagen surface. These results suggest that the collagen-adherent platelets are important promoters of coagulation during the initial phase of thrombogenesis by providing assembly sites for the F.X activation complex.     

Differential regulation of tissue factor and plasminogen activator inhibitor by human mononuclear cells

Fibrin is a hallmark of immune-mediated tissue lesions. The presence of fibrin in such lesions implies both the formation of fibrin via coagulation and the accompanying restriction of fibrinolysis, allowing fibrin to persist. Previous work has shown that human monocytes exposed to an inflammatory stimulus such as lipopolysaccharide (LPS) produce both tissue factor (TF) and plasminogen activator inhibitor--type 2 (PAI-2). These two proteins favor fibrin deposition, and evidence implies that cellular production of these two molecules may be linked. Another proinflammatory process pertinent to immune-mediated tissue damage and fibrin deposition is the response to alloantigen. Peripheral- blood mononuclear cells (PBM), consisting of lymphocytes and monocytes together, responded to alloantigen stimulation with differential expression of TF and PAI-2. PBM exposed to alloantigen developed high levels of TF activity, with no concomitant increase in PAI-2 activity or antigen. Alloantigen-stimulated PBM did not accumulate intracellular PAI-2, nor did they degrade PAI-2 added to cultures. This lack of PAI-2 production was not due to inadequate stimulation, as tritiated thymidine uptake and TF production demonstrated recognition of, and a vigorous reaction to, alloantigen. The divergent TF and PAI-2 responses of PBM exposed to alloantigen was maintained over 5 days and was reflected by mRNA profiles. These results imply that under specific physiologically relevant conditions, the procoagulant and antifibrinolytic effectors of inflammatory mononuclear cells can be independently regulated. This would imply more flexibility to monocyte mechanisms that favor fibrin deposition than previously thought.     

Deranged blood coagulation equilibrium as a factor of massive liver necrosis following endotoxin administration in partially hepatectomized rats

Activated Kupffer cells provoke massive liver necrosis after endotoxin stimulation through microcirculatory disturbance caused by sinusoidal fibrin deposition in rats undergoing 70% hepatectomy. In these rats, serum activities of purine nucleoside phosphorylase (PNP) and alanine transaminase (ALT) were increased at 1 and 5 hours, respectively, following endotoxin administration. When 70% resected liver was perfused with Dulbecco's modified Eagle medium (DMEM) containing heat-inactivated fetal calf serum, the increase in both enzyme activities was not affected by addition of endotoxin during perfusion, suggesting that activated Kupffer cells injured neither sinusoidal endothelial cells nor hepatocytes. The activity of tissue factor, an initiator of blood coagulation cascade, was much higher in Kupffer cells isolated from partially hepatectomized rats than in those from normal rats. In contrast, mRNA expressions of tissue factor pathway inhibitor (TFPI) as well as thrombomodulin were almost undetectable in normal and partially resected livers. When recombinant human TFPI was injected intravenously in 70% hepatectomized rats, TFPI was markedly stained on the surfaces of sinusoidal endothelial cells and microvilli of hepatocytes on immunohistochemistry. In these rats, endotoxin-induced liver injury was significantly attenuated compared with rats given no TFPI. Similar attenuation was also found in rats receiving recombinant human thrombomodulin. These results suggest that fibrin deposition developing in 70% hepatectomized rats after endotoxin administration may be caused by deranged blood coagulation in the hepatic sinusoids through increasing tissue factor activity in Kupffer cells and minimal TFPI and thrombomodulin in endothelial cells. The destruction of sinusoidal endothelial cells as well as hepatocytes may occur as a result of microcirculatory disturbance caused by such sinusoidal fibrin deposition.     

Rapid release of active tissue factor from human arterial smooth muscle cells under flow conditions

Circulating tissue factor (TF) is an important determinant of coronary thrombosis. Among other cell types, such as monocytes, vascular smooth muscle cells (SMCs) are capable of releasing TF. When studied under static conditions, SMCs do release TF, but this process is slow and, thus, cannot explain the elevated levels of circulating TF, as observed in patients with acute coronary syndromes. The present study demonstrates that cultured human mammary artery SMCs very rapidly (minutes) release active, microparticle-bound TF when exposed to flow conditions. There was a clear log-linear correlation between the shear rate (range 10 s(-1) to 1500 s(-1)) and the procoagulant activity of SMC perfusates. Flow-dependent release of TF was transient (10 minutes) and did not measurably reduce cell surface TF content. Interestingly, a time-dependent (t(1/2) 30 minutes) re-exposure of releasable TF was detected after a no-flow period. These data demonstrate that SMCs may become a pathophysiologically relevant source of TF that can be rapidly released into the circulation in situations in which endothelial damage occurs and SMCs come into a close contact with the flowing blood.     

A deleterious effect of aspirin on the antithrombotic properties of endothelial cells is not observed with platelets previously exposed to aspirin: studies in a flow system

We have explored both the independent and combined effects of aspirin on cultured endothelial cells and platelets, and its influence on platelet deposition onto an extracellular matrix. Blood was circulated through a flat perfusion chamber with two coverslips placed sequentially with respect to blood flow. The first coverslip (upstream) was covered with a cultured endothelial cell monolayer, and the second (downstream) coated with extracellular matrix obtained after endothelial cell removal. Platelet interaction was measured on the second coverslip. Treatment of endothelial cells on the first coverslip with 100 μM aspirin strongly reduced 6-keto-PGF(1a) levels recovered in the perfusates (118.3 ± 35.8 vs 1038.0 ± 308.5 pg/ml) and significantly increased platelet deposition on the downstream coverslip (% covered surface: 38.6 ± 6.4% vs 14.6 ± 1.8%; P < 0.001). Increased platelet deposition (% covered surface: 24.9 f 3.1%; P < 0.01) was observed in perfusions performed with blood containing aspirinized platelets, in the presence of intact endothelial cells. Treatment with aspirin of both platelets and endothelial cells had no additional effect on platelet adherence. Pretreatment of cultured endothelial cells with aspirin did not influence the adhesive properties of their underlying extracellular matrix. Our results indicate that, although endothelial cell cyclooxygenase is important in regulating platelet adhesion, its blockade seemed to have minimal effect on platelet deposition once platelet-cyclooxygenase was already inhibited.     

Possible mechanisms of fibrin deposition in the hypereosinophilic syndrome

Patients with the hypereosinophilic syndrome (HES) are at increased risk of thrombosis and have signs of fibrin deposition in the myocardial cavity; the pathogenesis of these complications is still unknown. We have studied a 51-year-old man affected by HES with heart, lung, skin, and gastrointestinal involvement. Routine laboratory parameters of the hemostatic system were normal with the exception of blood fibrinolytic activity. The latter was evaluated by both diluted blood clot lysis time and euglobulin lytic activity on fibrin plates before and after 10 min venous occlusion. The fibrinolytic activity measured on four occasions during a 3-month period, was impaired both in basal conditions and following venous occlusion. Platelet studies on two different occasions before and during therapy showed spontaneous platelet aggregation, lowered threshold concentrations of various aggregating agents, reduced platelet regeneration time and increased plasma beta-thromboglobulin concentration. The patient's polymorphonuclear cells (more than 75% eosinophils) were devoid of any procoagulant activity (PCA). Instead, patient's mononuclear cells studied before therapy generated significantly higher PCA on stimulation by endotoxin than cells from control subjects. The procoagulant response to endotoxin decreased markedly during therapy. The observed abnormalities could, at least partially, contribute to fibrin deposition in HES.     

Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface.

Cultured endothelial cells can be induced by tumor necrosis factor/cachectin (TNF) and other cytokines to synthesize the procoagulant cofactor tissue factor (TF). Intact monolayers of TNF-treated endothelial cells showed only minimal TF activity. In contrast, after permeabilization of these monolayers with detergent (saponin, 0.02%), there was approximately 10- to 20-fold increase in TF-mediated, factor VIIa-dependent factor Xa formation. Extracellular matrix derived from TNF-treated endothelium, prepared after removing the cells by hypotonic lysis or ammonium hydroxide (0.1 N), also had similarly enhanced TF activity. Incubation with a blocking monoclonal antibody to TF inhibited the procoagulant activity of both TNF-stimulated endothelial cells, whether they were intact or permeabilized, and of their matrices. However, when the apical cell surface was pretreated with anti-TF antibody, washed, and then cells were lysed with water or permeabilized with saponin, similar augmentation of TF activity was still observed, suggesting the presence of a pool of TF to which the antibody did not initially gain access. Consistent with this concept, the presence of TF in the matrix of TNF-treated endothelial cells was shown by immunoblotting and morphologic studies; cultured endothelial monolayers and the native endothelium of aortic segments after exposure to TNF showed TF in extracellular matrix, associated with vesicles. In contrast, TF was virtually undetectable on the apical endothelial surface. Taken together, these findings suggest that endothelial TF can be present in a cryptic pool that only gains access to the blood after alteration in the integrity of the endothelial monolayer.     

Virulent Treponema pallidum activates human vascular endothelial cells.

Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium.     

Activated protein C inhibits thrombus formation in a system with flowing blood

We studied the effect of increasing concentrations of protein C (PC) and activated protein C (APC) on haemostasis in an in vitro thrombosis model. Blood from healthy donors was anticoagulated with citrate–phosphate–dextrose (final citrate concentration 19 m m ) or a low molecular weight heparin (LMWH, 20 IU/ml). Enzymatically denuded rabbit aorta segments were exposed to flowing blood for 10 min in an annular perfusion chamber. PC and APC were added to the perfusate immediately prior to exposure. In citrated blood at a shear rate of 800/s, PC and APC induced a statistically significant decrease in platelet deposit at 16 μg/ml and 32 μg/ml. In perfusions performed with blood anticoagulated with LMWH, there was no effect on platelet deposition at 16 and 32 μg/ml either at shear rates of 300/s or 800/s. Addition of PC showed no effect on fibrin deposition at a shear rate of 300/s; in contrast, a non-statistically significant 40% reduction was seen at a shear rate of 800/s, compared to controls. Addition of APC caused a 100% reduction in fibrin formation at 16 and 32 μg/ml at both shear rates studied. PC and APC inhibited platelet deposition on the exposed subendothelial surface, in a dose-dependent manner. Effects of PC and APC on platelet function might be mediated through inhibition of thrombin generation at the platelet microenvironment.     

The effect of cultured endothelial cells on factor VIII procoagulant activity.

Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N-benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p-nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.     

Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture

The microfibrils of anchoring filaments, a typical ultrastructural feature of initial lymphatic vessels, consist mainly of fibrillin and are similar to the microfibrils of elastic fibers. As we previously demonstrated, they radiate from focal adhesions of lymphatic endothelium to the perivascular elastic network. Although present in large blood vessels, fibrillin microfibrils have never been detected in blood capillaries. Here we report immunohistochemical evidence that cultured bovine aortic and lymphatic endothelial cells express fibrillin microfibrils. These microfibrils form an irregular web in lymphatic endothelial cells, whereas in blood vessel endothelial cells they are arranged in a honeycomb pattern. Cultured lymphatic and blood vessel endothelial cells also produce focal adhesion molecules: focal adhesion kinase, vinculin, talin, and cytoskeletal beta-actin. Our data suggest that anchoring filaments of initial lymphatic vessels in vivo may be produced by endothelium. Through their connection with focal adhesions, they may form a mechanical anchorage for the thin wall of initial lymphatic vessels and a transduction device for mechanical signals from the extracellular matrix into biochemical signals in endothelial cells. The complex anchoring filaments-focal adhesions may control the permeability of lymphatic endothelium and finely adjust lymph formation to the physiological conditions of the extracellular matrix. The different deposition of fibrillin microfibrils in blood vessel endothelial cells may be related to the necessity of withstanding shear forces. Thus, in our opinion, differences in fibrillin deposition imply a different role of fibrillin in blood vessel and lymphatic endothelium.     

Expression and kinetics of induced procoagulant activity in bovine pulmonary alveolar macrophages

Leukocytes, especially macrophages, are important cellular mediators of fibrin deposition and removal at tissue sites of inflammation. Pulmonary fibrin deposition is a prominent feature of bovine acute lung injury; therefore, we studied the resting and stimulated procoagulant responses of bovine pulmonary alveolar macrophages (PAM) and peripheral blood neutrophils (PMN). Freshly isolated normal PAM and PMN expressed negligible procoagulant activity. PAM stimulated with endotoxin lipopolysaccharide (LPS), 4 beta-phorbol 12-myristate 13-acetate (PMA) and bovine recombinant interleukin-1 beta (rBIL-1 beta) exhibited protein synthesis- and dose-dependent enhancement of procoagulant activity in 8-h cultures. Bovine recombinant granulocyte macrophage-colony stimulating factor (rBGM-CSF) and recombinant human gamma-interferon (rHIFN-gamma) did not induce procoagulant activity. The kinetics of LPS- and PMA-enhanced PAM procoagulant activity differed: LPS-induced enhancement developed earlier and more rapidly than PMA-induced enhancement. Pasteurella haemolytica LPS was more potent than Escherichia coli LPS in enhancing PAM procoagulant activity, while dexamethasone decreased both baseline and LPS- or PMA-stimulated activity by approximately 50%. PAM procoagulant activity resulted from tissue factor expression. Bovine PMN produced negligible procoagulant activity when stimulated, and are thus unlikely to be major contributors to procoagulant activity in bovine lung. Activity inhibitory to bovine tissue factor was present in both calf and adult sera, and was partly dependent on the presence of factor X for activity. Rapid induction of bovine PAM procoagulant activity by inflammatory mediators, and subsequent resistance to degradation, may thus combine to promote an alveolar microenvironment permissive to fibrin deposition in bovine acute lung injury.     

Lack of ability to synthesize tissue factor by endothelial cells in intact human saphenous veins

It is well established that cultured endothelial cells are induced to generate tissue factor activity when incubated with either endotoxin or thrombin. In this study a perfusion system was used on 3-4 cm long human saphenous veins. The veins were perfused with thrombin (2.5 U/ml), endotoxin (30 ng/ml) or just medium for 3 h at 37 degrees C. After the perfusion, the veins were treated with collagenase, and EC were collected and subjected to tissue factor activity measurements. Some perfused veins were examined for tissue factor activity on the vessel wall by allowing factor VII and factor X to interact with the lumen of the intact vessels, followed by quantitation of generated factor Xa in a chromogenic assay. No formation of tissue factor activity could be found after perfusion in either collagenase-dissolved endothelial cells or in the coupled chromogenic assay for tissue factor activity performed in the lumen of the vessel. Our data strongly suggest that endothelial cells in intact endothelium may behave quite differently from isolated endothelial cells stimulated in cell cultures.     

A deleterious effect of aspirin on the antithrombotic properties of endothelial cells is not observed with platelets previously exposed to aspirin: studies in a flow system

We have explored both the independent and combined effects of aspirin on cultured endothelial cells and platelets, and its influence on platelet deposition onto an extracellular matrix. Blood was circulated through a flat perfusion chamber with two coverslips placed sequentially with respect to blood flow. The first coverslip (upstream) was covered with a cultured endothelial cell monolayer, and the second (downstream) coated with extracellular matrix obtained after endothelial cell removal. Platelet interaction was measured on the second coverslip. Treatment of endothelial cells on the first coverslip with 100 μM aspirin strongly reduced 6-keto-PGF_1a levels recovered in the perfusates (118.3 ± 35.8 vs 1038.0 ± 308.5 pg/ml) and significantly increased platelet deposition on the downstream coverslip (% covered surface: 38.6 ± 6.4% vs 14.6 ± 1.8%; P < 0.001). Increased platelet deposition (% covered surface: 24.9 f 3.1%; P < 0.01) was observed in perfusions performed with blood containing aspirinized platelets, in the presence of intact endothelial cells. Treatment with aspirin of both platelets and endothelial cells had no additional effect on platelet adherence. Pretreatment of cultured endothelial cells with aspirin did not influence the adhesive properties of their underlying extracellular matrix. Our results indicate that, although endothelial cell cyclooxygenase is important in regulating platelet adhesion, its blockade seemed to have minimal effect on platelet deposition once platelet-cyclooxygenase was already inhibited.