© 1997 American Heart Association, Inc.
Articles |
Human Brain Pericytes Differentially Regulate Expression of Procoagulant Enzyme Complexes Comprising the Extrinsic Pathway of Blood Coagulation
the Cell and Molecular Biology Program (B.A.B., M.A.S., P.B.T.) and the Department of Biochemistry (M.A.S., P.B.T.), University of Vermont College of Medicine, Burlington.
Correspondence to Paula B. Tracy, PhD, Department of Biochemistry, University of Vermont College of Medicine, Given Bldg C409, Burlington, VT 05405.
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Abstract |
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After vascular injury, pericytes may function in blood coagulation events that lead to thrombin formation due to their subendothelial location in the microvasculature. Pericytes from human cerebral cortex microvessels were isolated and characterized, and their ability to express and regulate procoagulant enzyme complexes was determined. Tissue factor was detected on the cell surface of cultured human brain pericytes by immunocytochemistry and was shown to form a functional complex with factor (F) VIIa to effect both FIX and FX activation. Treatment of pericytes with the calcium ionophore A23187 increased the observed tissue factor activity twofold to fivefold, which was shown to be due to an enhancement of cofactor activity and not the release of endogenous antigen stores. Pericytes also provided the appropriate membrane surface required for the assembly of a functional prothrombinase complex, so that in the presence of FVa and FXa, they effected thrombin formation 50 to 100 times faster than any other cell examined to date. In marked contrast to observations in other cell systems, pericyte expression of prothrombinase activity remained unaltered after treatment with A23187. As has been shown for platelets, the membrane receptor on pericytes for FXa assembly into the prothrombinase complex appears to at least partially consist of the FXa receptor effector cell protease receptor-1. These combined data indicate that pericytes can activate and propagate the coagulant response through the extrinsic pathway and that the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation. These observations support the concept that pericytes may play an important role in regulating coagulation events after cerebrovascular injury.
Key Words: pericytes • tissue factor • prothrombinase • effector cell protease receptor-1 • A23187
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Two cell types predominate in the microvasculature: ECs and pericytes. Because the primary role of the endothelium is to serve as a barrier between the blood and surrounding tissues, much research has focused on the importance of the endothelium in cardiovascular physiology and homeostasis.1 In contrast, the roles played by pericytes, the subendothelial cells of the microvasculature, in these processes are less well understood. Pericytes have been proposed to function in angiogenesis,2 vasoproliferative disorders,2 wound healing,2 and atherosclerosis,3 processes that often occur at extravascular sites and are characterized by extensive fibrin deposition4 and an active fibroproliferative response.5 The serine protease thrombin serves as a potential effector of both fibrin deposition6 and mesenchymal cell proliferation.7 Although normally not in contact with flowing blood due to their subendothelial location, pericytes are uniquely situated, so that after vascular injury they may participate in thrombin-associated intravascular processes. The best documented of these processes, thrombosis and hemostasis, are characterized by the formation of an insoluble fibrin clot as a result of thrombin cleavage of fibrinogen.6 Although the outcomes of these two processes are distinct, the coagulation events that lead to thrombin generation are believed to be identical.
The extrinsic pathway of blood coagulation is initiated by the association of cell membrane–associated TF with plasma FVIIa at sites of vascular injury to proteolytically activate the zymogens FIX and FX to their active enzyme species FIXaß and FXa, respectively.8 Cells in contact with flowing blood, such as peripheral blood monocytes and ECs, normally do not express TF, thereby forming a TF-deficient barrier between FVIIa and extravascular sources of TF.8 The observation that TF antigen is constitutively expressed by subendothelial cells in situ8 suggests that blood coagulation is initiated by the association of FVIIa with subendothelial TF. The TF/FVIIa complex activates FIX to FIXaß by two consecutive proteolytic events, which initially form the inactive intermediate FIX
that is again cleaved to form the active enzyme FIXaß.9 10 Generation of FXa leads to propagation of the coagulant response through the assembly and function of the prothrombinase complex, a stoichiometric (1:1) complex that consists of the nonenzymatic cofactor FVa and the serine protease FXa bound to an appropriate membrane (cell) surface, which effects the proteolytic conversion of prothrombin to thrombin.6 Furthermore, monocyte-bound FXa cleaves FIX to the nonenzymatic intermediate FIX11 and may therefore function to modulate TF/FVIIa activity. Because of their unique anatomic location, pericytes have easy access to the plasma proteins involved in blood coagulation after vascular injury. Therefore, we hypothesized that one important function of pericytes might be the regulation of intravascular and extravascular thrombin concentrations via activation of the extrinsic pathway of blood coagulation.
The procoagulant activities of the TF and prothrombinase complexes have been demonstrated to be upregulated in response to total cell lysis or cell treatment with intracellular Ca2+-mobilizing agents, such as calcium ionophore A23187.12 13 14 15 16 17 18 19 20 21 In some cell systems, these increases have been attributed to exposure of anionic phospholipids on the outer leaflet of the plasma membrane12 18 21 and/or release of procoagulant membrane microvesicles.14 17 19 20 In contrast, other studies have clearly shown that these membrane alterations are not solely responsible for the observed increases in TF and prothrombinase procoagulant activity22 23 and that expression of specific membrane receptors regulates procoagulant enzyme complex function.24 25 26 Recently we demonstrated that the assembly and function of the platelet prothrombinase complex is substantially regulated by expression of the FXa receptor EPR-1 by activated platelets.26 On the basis of these collective studies, we hypothesized that the platelet membrane receptor for FXa assembly into the prothrombinase complex consists minimally of bound FVa, EPR-1, and anionic phospholipids.
In the present study, we examined the ability of pericytes from human brain microvasculature to assemble and regulate enzyme complexes that are involved in the extrinsic pathway of blood coagulation. We demonstrate for the first time that human brain pericytes constitutively express TF, which forms a functional complex with added FVIIa to activate FX to FXa. FXa can subsequently interact with added FVa to assemble a functional prothrombinase complex at the membrane surface. Furthermore, these activities are differentially regulated in response to agonist stimulation. Additional experiments indicate that EPR-1 expression by human brain pericytes regulates assembly of a functional prothrombinase complex. We hypothesize that pericytes possess discrete regulators of the enzyme complexes within the extrinsic coagulation pathway.
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Isolation and Culture of Human Brain Pericytes
Pericytes were derived from human brain microvessels as described previously.27 By collagenase digestion, microvessels were isolated from human cerebral cortex surgical specimens that consisted exclusively of gray matter and were assessed to be free from abnormal pathology. These specimens were obtained after informed consent was procured by the neurosurgical team at the Medical Center Hospital of Vermont, Burlington. Dissociated cells and vessel fragments were sieved through a 70-µm mesh, collected by centrifugation, and seeded into 100-mm dishes with modified medium M-199 (9.87 g/L medium M-199, 10 mL/L basal medium Eagle vitamin solution [100x], 1 g/L glucose, and 2.2 g/L NaHCO3) containing 20% heat-inactivated low-endotoxin fetal bovine serum (Salzmann Corp), 100 µg/mL neomycin, 20 U/mL nystatin, and 2 mmol/L L-glutamine (complete medium). After cell proliferation, pericytes were distinguished from ECs on the basis of their larger size, irregular morphology, and non–contact inhibited growth pattern and from SMCs by their slower growth rate, irregular morphology, and lack of "hill-and-valley" growth at confluence.28 Pericytes were isolated from the other cell types by conventional cloning techniques.
Human brain pericytes were cultured in complete medium (37°C, 5% CO2). Culture of the pericyte clones continued for 3 to 6 weeks, depending on the donor, until the cultures reached 
75% confluence; they were then subcultured for experiments (passage 1). Subculture was performed after passage with 0.25% trypsin and 1 mmol/L EDTA. The cultures were fed fresh medium 24 hours before experimentation. Experiments were performed on cells at 75% confluence before multicellular nodules could form (80 000 cells per 1.9 cm2). Because of our concern that pericytes like SMCs undergo dedifferentiation as a result of passage, experiments were performed on passage 1 to 3 cells only.2 For serum-deprivation experiments, complete medium was replaced with serum-free medium that consisted of modified medium M-199 supplemented with 0.35% BSA, 100 µg/mL neomycin, 20 U/mL nystatin, and 2 mmol/L L-glutamine. In other experiments, nystatin was omitted from the complete medium to examine its effect on pericyte procoagulant activities.
Biochemical Characterization of Human Brain Pericyte Cultures
Human brain pericytes, microvascular ECs, SMCs, and fibroblasts were fixed with 100% methanol (10 minutes, 22°C) and incubated for 1 hour at 37°C with the following primary antibodies: (1) anti–vonWillebrand factor (Cappel Laboratories), (2) anti–-actin (Sigma Chemical Co), and (3) anti–ß-actin (Sigma). Excess antibody was removed and the cells incubated for 1 hour at 37°C with FITC-conjugated secondary antibodies (Sigma).
The anti–EPR-1 monoclonal antibodies B6, 2E1, and 13E5 were generously provided by Dr Dario Altieri (Yale University, New Haven, Conn). Two (B6 and 13E5) were provided as ascitic fluid and 2E1 was provided in a purified form. Their production29 30 and characterization26 30 31 have been described elsewhere. Anti-human TF IgG (clone TF9-6B4) was from American Diagnostica.
Unfixed human brain pericytes were incubated for 1 hour at 22°C with either 67 nmol/L anti-human TF IgG or anti–EPR-1 antibody 13E5 (1:10 dilution of ascites). Excess antibody was removed and the cells incubated with a goat anti-mouse IgG phycoerythrin conjugate (1:100 dilution, Sigma).
Immunostained cultures were visualized with a Zeiss IM 35 inverted fluorescence microscope. All cultures stained negativelywith isotype-matched nonspecific antibodies and ascitic fluid(Sigma).
Treatment of Human Brain Pericyte Cultures
Cultures were washed four times with HBS (20 mmol/L HEPES and 150 mmol/L NaCl, pH 7.4) at 37°C and maintained in HBS with 0.1% BSA and 5 mmol/L CaCl2 (37°C) for assay. Cultures were treated with 20 µmol/L A23187 (Calbiochem) or an equivalent volume of 100% ethanol (vehicle control) for 15 minutes at 37°C before assay.
Isolation and Modification of Coagulation Proteins
Coagulation proteins (FX, FIX, prothrombin, and FV) were isolated from human fresh-frozen plasma obtained from the American Red Cross (Burlington, Vt) and activated by well established techniques as described previously.11 32 Recombinant FVIIa was purchased from Novo BioLabs and reconstituted according to the manufacturer's instructions. FIX was radioiodinated and characterized as previously described.11
Western Blotting Analysis
For SDS-PAGE, pericytes were solubilized with 62.5 nmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, 20 mmol/L EDTA, and 0.001% bromophenol blue. After this mixture was heated (95°C, 3 minutes), SDS-PAGE was performed on 5% to 15% gradient slab gels as described by Laemmli.33 After electrophoresis, cell lysates were transferred at 500 mA (2 hours, 4°C) to nitrocellulose by the electroblotting techniques described by Towbin et al.34 Nitrocellulose was blocked with 5% nonfat dry milk in TBS-T (20 mmol/L Tris, 0.15 mol/L NaCl, and 0.05% Tween 20, pH 7.4). Cell lysates were probed with specific anti–EPR-1 antibodies, B6 or 2E1, nonspecific mouse IgG, or ascites fluid (Sigma) diluted 1:50 in TBS-T. The secondary antibody was a goat anti-mouse IgG coupled to horseradish peroxidase (1:5000 dilution in TBS-T). Detection of EPR-1 was performed by enhanced chemiluminescence (Western Blot Chemiluminescence Detection Kit, DuPont-NEN) by exposure of the blots to Kodak Scientific Imaging film (X-Omat) developed in a Kodak M35A X-Omat processor. Western blotting analyses of CMK megakaryocyte-like cells and platelets were performed as described.26
TF Activity Measurements
Cells were assayed for TF with 10 nmol/L FVIIa and either 100 or 500 nmol/L FX or 100 nmol/L FIX with trace amounts of 125I-FIX as previously described.32 FX activation was monitored by chromogenic assay.32 The initial rate of FXa generation was calculated by linear regression analysis and normalized to cell number by DNA assay.35 FIX activation was monitored by SDS-PAGE on 10% slab gels under reducing conditions as described by Laemmli33 followed by autoradiography.32 Ethanol treatment had no effect on cellular TF activity. FIX activation catalyzed by a relipidiated recombinant TF/FVIIa complex was assessed as described previously.11
For anti-TF antibody inhibition studies, washed brain pericyte cultures were incubated with buffer and 67 nmol/L anti-human TF IgG or 67 nmol/L nonspecific mouse IgG (15 minutes, 37°C) before treatment with ethanol or A23187 and assay. In experiments designed to determine the presence of intracellular TF stores, unreacted antibody was removed by washing (four times in HBS, 37°C) before ethanol or A23187 treatment and assay. In each experiment the TF activity of cultures incubated with control antibody equaled that of cultures incubated with buffer alone.
Prothrombinase Activity Measurements
Cells were assayed for prothrombinase activity with 5 nmol/L FVa and FXa and 1.39 µmol/L prothrombin as described previously.32 Thrombin formation was monitored by chromogenic assay.32 The initial rate of thrombin generation was calculated by linear regression analysis and normalized to cell number by DNA assay.35 Ethanol treatment had no effect.
For anti–EPR-1 antibody inhibition studies, cultures were incubated with either buffer, anti–EPR-1 antibody B6 (1:50 dilution of ascitic fluid), or a 1:50 dilution of control ascites (15 minutes, 37°C). Cells were assayed for prothrombinase activity after sequential addition of prothrombin (1.39 µmol/L) and a mixture of FVa and FXa (0.5 nmol/L each). The prothrombinase activity of cultures incubated with control ascites equaled that of cultures incubated with buffer alone.
DNA Assays
DNA assays were performed as described.35 A value of 9.2 pg DNA per cell was used as the DNA content of human brain pericytes in culture.35
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Morphological and Biochemical Characteristics of Cultured Human Brain Pericytes
Pericytes isolated from human cerebral microvessels were first identified by their distinctive appearance in culture (Fig 1
). As assessed by phase-contrast microscopy, pericytes spread on the culture dish and formed large, polymorphic cells (Fig 1, arrow) that were slow growing and formed multicellular nodules rather than a confluent monolayer at high cell densities (Fig 1, arrowhead). Similar characteristics for cultured retinal pericytes of bovine origin have been described previously.2 28 36
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The purity of the human brain pericyte cultures was defined on the basis of certain biochemical markers that were first described in animal studies and the absence of contaminating cell types.2 Owing to the lack of an entirely pericyte-specific marker, isoactin antibodies are routinely used for identification of cultured pericytes.2 28 The immunocytochemical staining pattern of human brain pericytes was compared with that of the most likely contaminating cells, ie, ECs, SMCs, and fibroblasts. Human brain pericyte cultures stained positively with antibodies against both SM - and nonmuscle ß-actin isoforms (Fig 2A and 2B, respectively). When compared with brain microvascular ECs, no staining was observed with an anti–vonWillebrand factor antibody. In contrast, SMCs stained positively for -actin only, whereas fibroblasts stained positively for ß-actin only. Every cell in culture stained positively for - and ß-actin and negatively for von Willebrand factor, thus confirming that the cultures consisted of pericytes only.2 28
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TF Expression by Cultured Human Brain Pericytes
TF expression by cultured human brain pericytes was demonstrated by both indirect immunocytochemistry and functional assays for cofactor activity (Fig 3, Table 1). Without agonist stimulation, cultured human brain pericytes stained positively with a specific anti-human TF antibody (Fig 3A). Intense immunostaining was localized along the pericyte membrane surface (Fig 3A, left), and no staining was observed with an isotype-matched control antibody (Fig 3A, right). Membrane-expressed TF was able to form a functional enzyme complex with FVIIa. Without agonist stimulation, FX was activated to FXa at a mean rate equal to 1.5±0.42x10-17 moles FXa·min-1·cell-1 (9.0±2.5x106 molecules FXa·min-1·cell-1 at a substrate concentration of 500 nmol/L; Table 1) in a reaction that absolutely depended on the presence of added FVIIa, since no FX activation was detected without it (data not shown). A lower yet still substantial amount of FX was activated (0.59±0.16x10-17 moles FXa·min-1·cell-1; 3.5±1.0x106 molecules FXa·min-1·cell-1) when used at its plasma concentration (100 nmol/L; Table 1). When pericyte cultures were preincubated with a monoclonal antibody against human TF, cleavage of FX to FXa was undetectable at 30 minutes by an assay capable of detecting as little as 0.25 nmol/L FXa (data not shown). These data confirm that FXa generation resulted from the formation of a specific TF/FVIIa complex on human brain pericytes.
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Physical cell disruption or treatment with calcium ionophore has been demonstrated to increase the capacity of cultured cells to promote TF activity.12 13 14 15 21 Consistent with these observations, treatment of human brain pericyte cultures with A23187 (20 µmol/L) resulted in a twofold to fivefold increase in the rate of FX activation at either substrate concentration (Table 1
). In parallel experiments, A23187 treatment and total cell lysis induced equivalent fold increases in TF activity (2.6 and 3.1, respectively) compared with that in untreated cells (data not shown). The ability of A23187 to induce a twofold to fivefold increase in human brain pericyte TF activity was unaffected by cell passage and was duplicated in multiple (n=30) experiments (range of 0.30x106 to 4.8x106 molecules FXa·min-1·cell-1 for untreated cells and 0.78x106 to 10.2x106 molecules FXa·min-1·cell-1 for A23187-treated cells with 10 nmol/L FVIIa and 100 nmol/L FX).
The ability of human brain pericytes to assemble a TF/FVIIa complex was confirmed further by activation of the alternate substrate FIX. FIX was activated to FIXaß by the TF/FVIIa complex assembled on pericytes to form products that comigrated on polyacrylamide gels with those generated by a TF/FVIIa complex reconstituted into synthetic phospholipid vesicles (Fig 3B). Similar to what was observed for the TF/FVIIa-catalyzed activation of FX, A23187 treatment substantially increased the rate of FIX activation (data not shown).
We verified that the observed TF activity was not due to endotoxin contamination of the culture reagents. Peripheral blood monocytes, which express TF activity subsequent to endotoxin stimulation,8 were isolated, cultured, and assayed32 with these same reagents and expressed no TF activity before or after stimulation with A23187 (data not shown).
Calcium Ionophore Treatment Does Not Induce Mobilization of Intracellular TF Stores
To begin to define the mechanism whereby A23187 increases human brain pericyte TF activity, initial experiments were performed to assess the presence of intracellular TF stores. In these studies, surface-expressed TF on untreated pericytes was blocked by preincubation with an inhibitory anti-TF monoclonal antibody, followed by extensive washing to remove unbound antibody before treatment with A23187 and assay (10 nmol/L FVIIa and 100 nmol/L FX). Preincubation with control antibody had no effect on the TF activity of cells treated with vehicle alone (0.34±0.02x106 molecules FXa·min-1·cell-1) or A23187 (0.84±0.21x106 molecules FXa·min-1·cell-1) compared with that of cells preincubated with buffer alone. In contrast, preincubation with the anti-TF antibody completely abolished the TF activity expressed by vehicle- and A23187-treated cells so that no activity remained, indicating that the A23187-induced increase in TF activity was not due to release of intracellular TF stores.
Prothrombinase Complex Assembly and Function on the Membrane Surface of Human Brain Pericytes
Brain trauma is often accompanied by the consumption of FV (by activation to FVa by thrombin or FXa6 ), FX, and platelets; defibrination; and generation of fibrin degradation products, suggesting function of the prothrombinase complex in these events.37 38 39 However, the cells involved in thrombin generation in the brain have yet to be defined. To determine whether cultured human brain pericytes could propagate the procoagulant response by assembly of a functional prothrombinase complex, the activation of prothrombin to thrombin in the presence of added FVa and FXa and a plasma concentration of prothrombin (1.39 µmol/L) was determined. Thrombin was generated at a mean rate of 4.1±0.5x10-15 moles thrombin·min-1·cell-1 (2.5±0.3x109 molecules thrombin·min-1·cell-1; Table 1) in a reaction that completely depended on addition of both FVa and FXa.
As has been described for TF activity, cellular prothrombinase activity can also be increased in response to cell treatment with calcium ionophore.16 17 22 23 In marked contrast to observations in other cell systems, however, A23187 treatment of human brain pericyte cultures had no effect on their expression of prothrombinase activity (Table 1), even though cell disruption induced a twofold to threefold increase in thrombin (1.1±0.16x109 molecules·min-1·cell-1 and 2.5±0.48x109 molecules·min-1·cell-1 for intact cells and cell lysates, respectively). These data also contrast with the observed effect of A23187 on human brain pericyte TF activity, whereby treatment with A23187 consistently induced a twofold to fivefold increase in activity (Table 1). The inability of A23187 to induce an increase in human brain pericyte prothrombinase activity was not affected by cell passage (Table 1) and was duplicated in multiple (n=30) experiments (range of 0.60 to 2.4x109 molecules thrombin·min-1·cell-1 for untreated cells). In each experiment, A23187 treatment had no effect. Furthermore, A23187-treated cells remained unaffected by concentrations of FVa and FXa as high as 25 nmol/L, thus indicating that the enzyme complex was not limiting in the assay (data not shown).
Serum Deprivation Downregulates Pericyte Expression of TF Activity but Not of Prothrombinase, Subsequent to Treatment With A23187
To further assess the differential regulation of human brain pericyte procoagulant activities, pericytes were cultured in serum-free medium and assayed for TF and prothrombinase activities for 72 hours without addition of fresh medium. Although continuous culture for 72 hours in serum-free medium had no effect on basal TF activity level (ie, that expressed before calcium ionophore treatment), there was a time-dependent decrease in the fold stimulation of TF activity subsequent to A23187 treatment (Fig 4), indicating loss of pericyte responsiveness to A23187. A parallel decline in TF activity was observed in cells continuously cultured in serum-containing (complete) medium (Fig 4, inset), indicating that the loss of responsiveness to A23187 in both experimental protocols was most likely due to depletion of essential nutrients. In marked contrast, prothrombinase activity subsequent to A23187 treatment remained unaltered by continuous culture of pericytes in the absence of serum. The rate of prothrombin activation at 24 hours (7.7±2.1x108 molecules·min-1·cell-1) was not significantly different at 48 (7.6±0.32x108 molecules·min-1·cell-1) or 72 (9.6±0.36x108 molecules·min-1·cell-1) hours. These observations further support the notion that pericytes possess discrete regulators of these procoagulant activities.
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-) or 20 µmol/L A23187 (-
-), TF activity was determined with 10 nmol/L FVIIa and 100 nmol/L FX at the indicated times as described. Inset, TF activity of human brain pericytes cultured in complete (serum-containing) medium subsequent to treatment with ethanol (-
Expression of EPR-1 by Human Brain Pericytes Regulates the Assembly and Function of the Prothrombinase Complex on Their Membrane Surface
Our inability to affect pericyte prothrombinase activity by cell treatment with A23187 suggested that effector molecules other than anionic phospholipids might be important in regulating procoagulant activity. We recently demonstrated that EPR-1 in concert with bound FVa mediates FXa binding to activated platelets to form a functional prothrombinase complex.26 Therefore, we hypothesized that pericytes may also express EPR-1. By Western blotting analyses, a protein of Mrapp (65 kD) similar to that found in platelets and megakaryocytic cell lines26 was detected in pericyte cell lysates with anti–EPR-1 antibody B6 (Fig 5A, arrow). All other reactivity was nonspecific (data not shown). Similarly, a 65-kD protein was also detected with anti–EPR-1 antibody 2E1 (data not shown). Cell surface expression of EPR-1 was confirmed by indirect immunocytochemistry. Positive membrane immunostaining was observed when specific anti–EPR-1 ascitic fluid 13E5 (Fig 5B, left panel) was used. In contrast, no staining was observed when a nonspecific ascitic fluid (Fig 5B, right panel) was used. These combined data indicate that cultured human brain pericytes express EPR-1 at their membrane surface.
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The anti–EPR-1 antibody B6, which has been demonstrated to be directed against the FXa binding site on EPR-1,31 was used in experiments to assess the role of EPR-1 in pericyte prothrombinase activity. Preincubation of human brain pericyte cultures with B6 before the prothrombinase assay inhibited the initial rate of thrombin generation by 60% (Table 2). These data indicate that expression of EPR-1 by pericytes is an active participant in regulating the assembly and function of the prothrombinase complex on their membrane surface.
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Discussion |
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Thrombin generation subsequent to brain vessel injury would limit intracranial hemorrhage or alternatively could result in thrombotic stroke or disseminated intravascular coagulation.40 Furthermore, recent evidence suggests that thrombin present in the neuronal compartment of the brain may result in neural cell damage41 as well as the pathogenesis of Alzheimer's disease (reviewed in Reference 42) owing to its ability to retract neuronal and astrocytic processes. In the present study, we have demonstrated for the first time that pericytes derived from the subendothelium of human brain microvasculature can assemble and regulate the enzyme complexes comprising the extrinsic pathway of blood coagulation, thereby leading to thrombin generation. Without agonist stimulation, human brain pericytes express TF, as demonstrated both antigenically and functionally. Furthermore, the observed pericyte TF activity is quantitatively similar to that expressed by endotoxin-stimulated monocytes on a per-cell basis32 when identical substrate concentrations are used in the assays.
The coagulant response initiated through TF expression can be efficiently propagated, since human brain pericytes can also assemble a functional prothrombinase complex to effect rapid thrombin formation at their membrane surface. At plasma concentrations of prothrombin, the rate of thrombin generation expressed by human brain pericytes is 50 to 100 times greater on a per-cell basis than that expressed by any other cell studied to date, including monocytes, platelets, and human ECs of venous and arterial origin.6 If the FVa/FXa complex assembled on human brain pericytes expresses the same catalytic efficiency per site as that bound to platelets and monocytes (1800 molecules thrombin·min-1·site-1),6 then human brain pericytes will express 1x106 prothrombinase binding sites per cell, resulting in the generation of a high local thrombin concentration within the brain microvasculature. These combined data suggest that subsequent to cerebrovascular injury, pericytes could play a pivotal role in regulating the coagulant response.
These studies also allowed us to make the interesting, novel observation that pericytes differentially regulate these two procoagulant activities subsequent to agonist stimulation with calcium ionophore. Similar to observations made by Bach and Rifkin of bovine retinal pericytes,12 we have shown that human brain pericytes either treated with calcium ionophore or lysed by freezing/thawing express TF activity that is twofold to fivefold greater than that expressed by untreated or vehicle-treated cells, which reflects the well documented increase in cofactor specific activity (reviewed in Reference 15) rather than mobilization of intracellular stores. Similar observations have been made in other cell culture systems, including bovine fibroblasts, endotoxin-stimulated human monocytes and ECs, J82 bladder carcinoma cells, and human fibroblasts.12 13 14 15
Similar studies have demonstrated that the assembly and function of the prothrombinase complex can be increased in response to cell disruption or calcium ionophore treatment,16 17 19 20 resulting in as much as a 130-fold increase in the rate of thrombin generated via prothrombinase.17 In marked contrast to those observations, we have demonstrated that although prothrombinase activity expressed by human brain pericytes is not affected by calcium ionophore treatment, it could be increased twofold to threefold after total cell lysis. In those cell systems that respond positively to A23187 as an agonist, greater procoagulant activity after cell disruption or calcium ionophore treatment has been attributed to (1) redistribution of membrane lipids, such that phosphatidylserine is expressed in the outer leaflet of the plasma membrane,12 18 21 and (2) the release of procoagulant microvesicles,14 17 19 which can contain TF14 20 and support prothrombinase activity.17 19 Other studies have demonstrated that these membrane changes are a result of inhibition of aminophospholipid translocase, the enzyme responsible for maintaining the asymmetric distribution of membrane phospholipids.44 45 However, our observation that treatment with A23187 increased TF activity twofold to fivefold while having no effect on prothrombinase activity was reproduced in multiple experiments, demonstrating that the cells were most likely undamaged and therefore did not possess significant amounts of phosphatidylserine at their membrane surface before assay. Additionally, experiments that assessed the effects of cell culture on pericyte procoagulant activity (specifically the effects of nystatin, which binds to cholesterol in the cell membrane43 ) demonstrated that pericytes cultured in medium with or without nystatin expressed identical rates of FXa and thrombin generation, with or without A23187 stimulation (data not shown). In separate experiments, pericyte cultures that were extensively washed after A23187 treatment expressed rates of FXa and thrombin formation identical to those of unwashed cultures, indicating that the increase in TF activity was not a result of procoagulant microvesicle release (data not shown). Furthermore, Le and colleagues23 demonstrated that inhibition of aminophospholipid translocase with the sulfhydryl reagent N-ethylmaleimide led to an increase in fibroblast TF activity that could not be inhibited by the anionic phospholipid binding protein annexin V, although significant phosphatidylserine exposure was demonstrated. These studies combined with our observations suggest that plasma membrane alterations are not the single, plausible mechanism resulting in increases in cellular procoagulant activities.
The inability of A23187 treatment to alter pericyte expression of prothrombinase activity may be due to the significant role that the integral membrane protein EPR-1 plays in regulating thrombin generation, via prothrombinase, at the pericyte membrane surface. Our data clearly demonstrate that human brain pericytes express EPR-1 at their membrane surface. Function of the pericyte prothrombinase complex was substantially inhibited (60%) by the anti–EPR-1 antibody B6, a finding consistent with the recent observations of Ambrosini and Altieri,31 who epitope-mapped B6 to the FXa binding site on EPR-1.
Currently we can only speculate on the mechanism(s) whereby A23187 treatment affects TF cofactor activity on pericytes. In addition to inducing its well documented membrane alterations, A23187 treatment may actuate intracellular processes that lead to covalent modifications, dimerization, and/or conformational changes in the TF molecule to increase its cofactor activity in response to A23187 treatment. Agonist-induced surface expression of an additional protein cofactor cannot be ruled out either. It has been documented that TF is posttranslationally modified by both phosphorylation of a cytoplasmic serine as well as fatty acid esterification of a cytoplasmic cysteine.46 47 Other investigators have also suggested that TF may function as a dimer in vivo.48 49 Modulation of pericyte TF activity due to intracellular metabolic processes would be consistent with the results of the serum-deprivation experiments, wherein continuous pericyte culture with or without serum nearly abolished the increase in TF activity subsequent to A23187 treatment, although no changes in the basal constitutive level of activity were observed.
These combined data indicate that the events leading to thrombin generation at the membrane surface of human brain pericytes are complex and are regulated by multiple interactions between membrane phospholipids, integral membrane proteins, and added coagulation factors. However, the ability of human brain pericytes to express and differentially regulate the enzyme complexes comprising the extrinsic pathway of coagulation leading to thrombin generation will play a critical role in controlling cerebrovascular hemorrhage and infarct, neural damage, and neurodegenerative diseases.
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Acknowledgments |
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This work was supported by PO1 HL-46703 (project 4) and RO1 HL-52105 from the NHLBI, National Institutes of Health, to P.B.T., a University of Vermont College of Medicine Biomedical Research Support Grant to P.B.T. and M.A.S., the Collen Foundation; and the Department of Biochemistry, University of Vermont College of Medicine. We thank Dr Dario C. Altieri (Yale University) for generously providing us with the anti–EPR-1 monoclonal antibodies. Current address for Dr Bouchard: Division of Hematology and Oncology, NEMC, Box 832, 755 Washington St, Boston, MA 02111.
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Portions of this work were presented at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 12-17, 1994, and published in abstract form (Circulation. 1994;90[pt 2]:I-618.).
Received March 29, 1996;
revision received July 23, 1996;
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