Tissue factor (TF)-initiated extrinsic pathway plays an integral role in the revised coagulation cascade (1). The physiologic function of TF is to serve as a cellular receptor for factor VII (FVII) and/or its activated form FVIIa, which has been proposed to be the focal event of the extrinsic pathway (2,3). FVIIa, a resulting active serine protease of the proteolytic cleavage between Arg152 and Ile153 on FVII binding to TF (4), leads to the subsequent proteolytic activation of FX (1). The activated FX (FXa) in prothrombinase converts prothrombin to thrombin, which in turn polymerizes fibrinogen to fibrin clots. The accelerated or uncontrolled blood coagulation could contribute to occlusive thrombosis and its manifestation, presenting threats to the cardiovascular functions (1-6).
TF is a plasma membrane-bound single-chain glycoprotein with 43 kDa expressed on the circulating monocytes on tissue injury or stimulation including inflammations, sepsis, and endotoxemia with bacterial endotoxin (LPS) infection (7). It has been reported that the upregulation of TF activity induced by LPS was associated with the elevated TF mRNA (8) presumably enhancing TF synthesis. In a recent clinical study dealing with whole blood samples under acute infection, we showed that LPS primarily stimulated peripheral blood monocyte TF activity without a direct effect on prothrombin time, including FVII, FX, and thrombin activities per se in the extrinsic coagulation (9).
Antagonism to LPS-inducible TF upregulation could be achieved by a wide variety of blockades of LPS cell signaling. For instance, our previous studies using two model human monocytic cell lines (THP-1 and U937) demonstrated that pretreatment with ethanol (10), n-3 polyunsaturated fatty acids (11), and interleukin-4 (IL-4) (12) blocked CD14-dependent LPS recognition, resulting in the depression in LPS-inducible TF upregulation. Our preliminary data also showed that the inducible upregulation was significantly suppressed by the inhibitor of protein kinase C (calphostin C) or tyrosine kinase (geinstein), both of which blocked LPS intracellular signaling (13,14). However, these preventive approaches often failed to rescue monocytic TF hypercoagulation induced by LPS.
Our laboratory is the first to demonstrate the novel anticoagulant activity of compound 48/80 (48/80; Fig. 1) (9,15,16), despite its diverse biologic properties including the activations of Gi protein (17), Na+/K+-pump activity (18), Ca+2-independent NOS activity in gastric mucosa (19), and the release of arachidonic acid (20) and histamine (18,21) in mast cells. The 48/80 also elevated intracellular Ca+2(22) and Ins(1,4,5)P3 content (21), or enhanced Ca+2 release from intracellular stores (23). In addition, 48/80 inhibited calmodulin (24), adenosine diphosphate (ADP)-ribosylation (25), and human platelet phospholipase C (26). The release of histamine by 48/80, however, has never been demonstrated in vivo. Our previous study showed that 48/80 directly depressed monocytic TF upregulation independent of LPS signaling (15). A recent study concerning the acute LPS-infected whole blood also showed that 48/80 effectively inhibited monocytic TF procoagulation (9): 48/80 exhibited a broad spectrum of inhibition on monocytic TF upregulation in response to various stimuli other than LPS (15). The 48/80 might have a clinical application in the management of monocytic hypercoagulation.
In the present study, we showed that FVII activation was mainly responsible for the monocytic TF initiation of the extrinsic coagulation. The current data also demonstrate that 48/80 instantaneously inhibited monocyte TF-initiated clotting activity in a dose-dependent fashion, which was mediated by the preferential inhibition of FVII activation with the diminished FVIIa formation.
MATERIALS AND METHODS
The 48/80, CaCl2, NaCl, Tris-HCl, monoclonal anti-human FVII mAb, FITC goat anti-mouse immunoglobulin G (IgG), and horseradish peroxidase-conjugated goat anti-sheep IgG were supplied by Sigma Chemical Co. (St. Louis, MO, U.S.A.). Human factor VII (FVII), FVIIa, FX and FXa were from Enzyme Research Laboratories, Inc. (South Bend, IN, U.S.A.). S-2288 and S-2222 were supplied by DiaPharma Group, Inc. (West Chester, OH, U.S.A.). Sheep anti-human FVII (anti-hFVII Ab) and sheep anti-human TF (anti-hTF Ab) were from Accurate Chemical & Scientific (Westburg, NY, U.S.A.). Pooled normal human plasma and FVII-deficient plasma were from George King Biomedical Inc. (Overland, KS, U.S.A.). Rabbit brain thromboplastin (rbTF) was from Dade Diagnostics International (Miami, FL, U.S.A.).
Cell cultures and treatments
Human leukemic promonocytes (THP-1 cells) purchased from American Type Culture Collection were maintained in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 μg/ml), 10 mM HEPES, and 5% fetal bovine serum (FBS) in a 95%/5% air/CO2 humidified atmosphere at 37°C. FBS was inactivated by heat at 56°C for 30 min. These nonadherent cells were fed with an equal volume of fresh medium weekly. Before experiments, cells were washed with 15 ml serum-free medium followed by centrifugation at 600 g for 10 min at 4°C. The cells were replated in fresh RPMI and challenged with LPS (100 ng/ml) or A23187 (20 μM). At the end of incubation, the cells were washed and resuspended in the TF assaying buffer containing 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. In some cases, the harvested cells were resuspended in the appropriate buffer for immunofluorescent approaches.
Single-stage clotting assay
TF-initiated extrinsic coagulation was characterized and its activity was determined by a single-stage clotting assay (9,15,16) on a BBL Fibrosystem (Becton Dickinson, Cockeyville, MD, U.S.A.). In brief, 50 μl of 25 mM CaCl2 (prewarmed to 37°C) and 50 μl cell suspension prepared in a buffer containing 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl were pipetted into a fibrin cup. The indicated concentration of 48/80 prepared in PBS or anti-hTF Ab was added, and the reaction was initiated by addition of 50 μl normal human plasma (prewarmed to 37°C). The clotting time in seconds was recorded when the current between two vibrating electric probes was cut off by clots to stop the timer. The stock rbTF (6.5 mg/ml) arbitrarily assigned as 100,000 units/ml was assayed to construct a calibration curve of clotting time (seconds) versus TF concentration on a log-log graph paper with a linear regression formula: logY = −0.290 logX + 2.64 (r2 = 0.981). TF activity was expressed as units per number of cells determined by the Trypan Blue exclusion test.
S-2288 was used as a preferential chromogenic substrate for the determination of FVIIa amidolytic activity (16,27). FVIIa was incubated with the cell suspension prepared in 100 μl Buffer A consisting of 50 mM Tris-HCl (pH 7.4), 5 mM CaCl2, and 150 mM NaCl with or without inclusion of 48/80 (20 μM). In some cases, FVII activation also was estimated in a two-stage chromogenic assay in which FVII was preincubated with the cell suspension at 37°C for 30 min in 100 μl Buffer A with or without 20 μM 48/80. For the determination of the effect of sheep anti-TF Ab, the cell suspension was incubated with this antibody before addition of FVII. In all the cases, Buffer A (900 μl) was added to reconstitute a 1-ml chromogenic assay containing 100 μM S-2288. The hydrolysis took place at 25°C for 30 min. Thereafter, all samples were centrifuged at 3,000 g for 10 min to pellet down cells. The supernate (900 μl) was transferred into a cuvette, and the color formation was monitored at O.D. 405 nm on a Roy Milton spectrophotometer (model: Genesis 5, Rochester, NY, U.S.A.).
Chromogenic substrate (S-2222) at 100 μM was used for the determination of FXa amidolytic activity (16,27). FXa was incubated with the cell suspension in 100 μl Buffer A with or without 48/80 (20 μM). In some cases, FX activation was also studied through the formation of FXa triggering S-2222 hydrolysis with the addition of authentic FVIIa, dissecting from its upstream FVII activation. The cell suspension and FVIIa were preincubated with FX in 100 μl Buffer A with or without 48/80 at 37°C for 30 min. Buffer B (900 μl), consisting of 50 mM Tris-HCl (pH 8.3), 250 mM NaCl, and 80 mM EDTA, was added to reconstitute a 1-ml chromogenic assay containing 100 μM S-2222. The hydrolysis took place at 25°C for 30 min. After the centrifugation at 3,000 g for 10 min, the supernate (900 μl) was transferred into a cuvette, and the color formation was monitored at O.D. 405 nm.
For studying FVIIa formation, FVII (1.5 μg) was incubated with THP-1 monocytes (0.5 × 106 cells) resuspended in 100 μl Buffer A with or without 48/80 (20 μM) at 37°C for 30 min. An aliquot of the sample was diluted in SDS sample buffer and loaded onto a mini-gel subjected to 12.5% SDS-PAGE, according to the procedure of Schmidt et al. (28). The protein was transferred onto a nitrocellulose membrane, which was blocked by 2.5% dry milk/TBS. The membrane was further immunostained with a sheep anti-hFVII Ab (1:250 dilution) for 2 h and a horseradish peroxidase-conjugated goat anti-sheep IgG followed by ECL enhancer (Amersham Life Science) and exposure to x-ray film. For studying TF synthesis, THP-1 cells after a 4-h challenge with agonists were subjected to the similar procedure of Western blotting analysis with the exception of immunostaining the membrane with a sheep anti-hTF Ab.
FVII binding to THP-1 cells
FVII binding to monocytes was performed in a sandwiched-immunostaining technique (15). The harvested cells were incubated with FVII (1 mg/ml) for 30 min at 25°C in Buffer A in the presence or absence of 48/80 (20 μM). In some cases, the cell suspension was preincubated with a sheep anti-hTF Ab (60 μg/ml) for 5 min before addition of FVII. After washing with PBS, all samples were then incubated with an anti-hFVII mAb (1:20 dilution; Sigma Chemical) for 1 h in PBS containing 5 mM CaCl2. After immunostaining with FITC-conjugated goat anti-mouse IgG for 30 min, the fluorescence was detected on a FACScan analyzer. The cells stained with the secondary antibody alone served as a fluorescent background.
Inhibition of upregulated monocytic TF activity
TF activity of THP-1 monocytes was substantially up-regulated by either LPS (100 ng/ml) or Ca ionophore (A23187 at 20 μM) as a function of incubation time (Fig. 2). A 1-h lag phase was often seen after challenge with LPS but not with A23187. A 4-h LPS challenge drastically stimulated TF synthesis, as evidenced by the enhanced immunodetection of TF at 43 kDa on Western blotting (Fig. 2 inset; lane 2). In contrast, TF protein level remained nondetectable (Fig. 2 inset; lane 3) despite the profoundly upregulated functional activity induced by A23187. The unaffected TF synthesis in response to A23187 also was confirmed by flow cytometry using a FACScan analyzer (data not shown).
The upregulated TF activity induced by either agonist was instantaneously inhibited by 48/80 in a dose-dependent manner (Fig. 3). Similarly, agonist-induced TF activity was also dose-dependently depressed by a sheep anti-hTF Ab (Fig. 4), which was a polyclonal 20150A (Cedarlane Labs Ltd., Hornby, Ontario, Canada) directing to the extracellular domain of TF. Unlike 48/80, anti-hTF Ab could not instantaneously inhibit TF activity without a 5-min preincubation with the monocytes at 37°C before the clotting assay. In resting THP-1 monocytes, both 48/80 and anti-hTF Ab also caused substantial inhibition, leading to nondetectable monocytic TF activity with clotting time >400 s (data not shown).
Inhibition of FVII activation
Using S-2288 as a chromogenic substrate, we measured FVIIa amidolytic activity in monocyte suspension. The 48/80 (20 μM) showed no direct effect on S-2288 hydrolysis (data not shown). Although FVII alone did not hydrolyze S-2288 (16), preincubation of FVII with agonist-challenged THP-1 monocytes for 30 min led to a significant hydrolysis (Fig. 5). Inclusion of 48/80 in the preincubation suppressed the hydrolysis (Fig. 5), indicating the depressed FVII activation. The similar inhibitory patterns were obtained irrespective of the cells being challenged by either LPS or A23187.
In a separate protocol, the cell suspension was first incubated with a sheep anti-TF Ab (60 μg/ml) for 5 min at 37°C. The inhibition of FVII activation was observed (Fig. 5) without the direct effect on FVIIa amidolytic activity in monocyte suspension (data not shown).
With S-2222 as a chromogenic substrate, FXa amidolytic activity and FX activation were determined in monocyte suspension, dissecting from its upstream FVII activation. There was no detectable 48/80 effect on FXa amidolytic activity, nor did 48/80 show any significant effect on the dissected FX activation catalyzed by the authentic FVIIa in THP-1 cell suspension (data not shown). It was true for the cells challenged with either LPS or A23187.
Inhibition of FVIIa formation
The detection of FVIIa formation by Western blotting analysis served as a confirmation of FVII activation. FVII did not undergo cleavage without the incubation with monocyte suspension. After a 30-min incubation of FVII with LPS- or A23187-challenged monocytes (0.5 × 106) at 37°C, FVIIa formation appeared at 33- and 21-kDa protein bands (Fig. 6; lanes 3 and 5), both of which were immunodetectable by the sheep anti-hFVII Ab. Only trace amounts of FVIIa formation were detected after the incubation with resting THP-1 monocytes (Fig. 6; lane 1).
The presence of 48/80 (20 μM) in the incubation significantly diminished FVIIa formation (Fig. 6; lanes 4 and 6); the appreciable FVII accumulation also was noted at lanes 4 and 6.
Blockade of FVII binding to THP-1 cells
In view of the physiologic function of TF being a cellular receptor for FVII (2,3,29,30), we showed the ability of FVII binding to monocytes to demonstrate the functionally active TF expression on the monocyte surface. After an incubation of FVII with cell suspension in Buffer A for 30 min, FVII binding was monitored by immunostaining with a monoclonal anti-hFVII mAb and an FITC-conjugated mouse IgG. FACScan analysis showed significant enhancement of FVII binding to agonist-challenged monocytes (Fig. 7). The 48/80 inhibited FVII binding to the resting, LPS-challenged, and A23187-challenged cells by 35, 50, and 60%, respectively.
In a separate approach, the cells were preincubated with the sheep anti-hTF Ab (directing to the extracellular domain of TF) at 37°C for 5 min before the FVII-binding assay. FVII binding to resting, LPS-, or A23187-challenged cells detected by an anti-hFVII mAb was depressed by 45, 55, or 75%, respectively.
FVII activation dictating monocytic TF-initiated coagulation
In the present study, we showed FVII activation with FVIIa formation consistently dictating monocytic TF-initiated coagulation. LPS drastically enhanced monocytic TF synthesis (Fig. 2; inset), which was in agreement with the enhanced TF mRNA reported by others (8), being a prerequisite for the extrinsic hypercoagulation. It was likely that the enhanced TF synthesis led to the elevated FVII binding to THP-1 monocytes (Fig. 7B). As a result, FVII activation (Fig. 5A) was upregulated, which was consistent with the elevated FVIIa formation observed in Fig. 6 (lane 3).
However, TF synthesis did not always account for its upregulated functional activity. Unlike LPS, A23187 drastically induced functional TF activity without any significant increase in TF synthesis observed on Western blotting analysis (Fig. 2, inset). A23187 even more potent than LPS substantially induced FVII binding to THP-1 monocytes (Fig. 7C), which could possibly be responsible for the upregulation of the extrinsic coagulation (Figs. 2, 3B, and 4B).
The posttranslational TF regulation by cytosolic Ca (31) has been proposed by the Bach group, who also demonstrated that TF could undergo transformation from its inactive dimer to active monomer form in HL-60 cells under such conditions (32). The similar TF regulation by Ca ionophore was also reported in human brain pericytes (33). Based on the physiologic function of TF being a cellular receptor for FVII (2,3), we directly showed FVII binding to demonstrate the functionally active TF expression on the monocyte surface. Analogous to those findings (31,32), our current data revealed that the existing "latent" TF might undergo transformation into its active form on A23187 treatment, resulting in the enhanced FVII binding to THP-1 monocytes (Fig. 7C). As a result, the enhanced FVII activation (Fig. 5B)[i.e., FVIIa formation (Fig. 6, lane 5] could account for such profound stimulation by A23187 on monocytic TF-initiated extrinsic coagulation.
Although differential cellular activation mechanisms might be involved, the enhanced FVII binding resulting in FVII activation with FVIIa formation was consistently able to dictate the upregulation of TF-initiated extrinsic coagulation. Thus, the data were in favor of the "rate-limiting" role of FVII activation in the extrinsic pathway.
Inhibition of FVII activation responsible for the downregulation of TF-initiated coagulation
Our observations were in agreement with a proposal of FVII activation being a focal event in triggering the extrinsic pathway (2,3,29,30). Accumulating evidence showed that the downregulation of FVII activation often led to the depression in the extrinsic coagulation. For instance, the inhibition of FVII binding by sphingosine (34) or ConA (35) resulted in the downregulation of the extrinsic coagulation. The anti-TF Ab complexing with its antigen could prevent the catalytic activity in initiation of blocking the coagulation (36) and exhibit anticoagulant activity in primates (37). Apart from ApoA-II (38) and ApoB (39) in lipoproteins showing inhibition on TF activity, the physiologic tissue factor pathway inhibitor in plasma formed a quaternary complex with TF/FXa/FVIIa, exerting the complicated feedback inhibition on FVII activation (40).
We demonstrated that an anti-hTF Ab directed to the extracellular TF domain significantly blocked monocytic TF-initiated coagulation, which was presumably due to the competitive binding with the nature ligand (FVII) of TF. In fact, FVII activation (Fig. 5) and the FVII binding to monocytes (Fig. 7) were blocked under such conditions. The data suggested that the FVII activation process could be not only rate-limiting but also regulatory in the extrinsic coagulation cascade.
It is of interest to note that 48/80, without any preincubation with monocytes, mimicked anti-hTF Ab to inhibit the extrinsic coagulation (Fig. 3). The 48/80 (Fig. 1), a water-soluble condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, showed instantaneous inhibition of TF-initiated coagulation in vitro (16). Its inhibitory ability in vitro ruled out the possible involvement of the cellular metabolism of 48/80 in the contribution to the inhibitory action. The preferential inhibition of FVII activation was accompanied by the diminished FVIIa formation and the blocked FVII binding to the partially purified TF protein (16). Similar observations were made in THP-1 monocytes. The depressed FVII activation (Fig. 5), which was confirmed by the appreciably diminished FVIIa formation (Fig. 6, lanes 4 and 6), could result from the blocked FVII binding to monocyte TF (Fig. 7). In addition, the relative accumulation of FVII (Fig. 6, lanes 4 and 6) in the presence of 48/80 seemed to be supportive to this notion of such blockade readily preventing FVII from proteolytic cleavage in monocytic suspension. In contrast, there was no detectable inhibition by 48/80 on FVIIa binding to monocytes (data not shown), which could be in line with the unaffected FVIIa amidolytic activity. Consistently we observed no detectable effect of 48/80 on the dissected FX activation in monocyte suspension (data not shown).
In summary, our current data together with the previous in vitro observations (16) consistently demonstrate that the ability of 48/80 to block FVII activation is the "regulatory point" of the extrinsic pathway. Thus, the diminished provision of FVIIa mediated the profound downregulation of the extrinsic hypercoagulation after a wide variety of monocyte activation (15). Apart from the previously reported anticoagulation by 48/80 in LPS acute infected human plasma samples (9), 3 μM 48/80 with a 2-day infusion could show its anticoagulant activity in a dog model (data not shown). The 48/80 could be of therapeutic value, presenting a clinical application as anticoagulant for effective management on monocytic hypercoagulation.
Acknowledgment: This study was sponsored in part by a grant from the American Heart Association. We thank Dr. J. K. Prasad for arranging the internal financial supports through the Detroit Medical Center and Surgery Department, School of Medicine, at Wayne State University.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
48/80; Factor VII; Tissue factor; Extrinsic coagulation; Endotoxin; Ca ionophore