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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1138-1146

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© 1996 American Heart Association, Inc.

Articles

Arterial Smooth Muscle Cell Heparan Sulfate Proteoglycans Accelerate Thrombin Inhibition by Heparin Cofactor II

Rebecca A. Shirk; Frank C. Church; William D. Wagner

the Department of Comparative Medicine, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem (R.A.S., W.D.W.), and the Department of Pathology and Laboratory Medicine and Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill (F.C.C.), NC.

Correspondence to William D. Wagner, PhD, Department of Comparative Medicine, The Bowman Gray School of Medicine of Wake Forest University, Medical Center Blvd, Winston-Salem, NC 27157-1040. E-mail Wwagner@bgsm.edu.


*    Abstract
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 *Abstract
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 down arrowResults
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Heparin cofactor II (HCII) is a potent thrombin inhibitor in the presence of heparin and dermatan sulfate, glycosaminoglycans that accelerate the inhibition reaction. HCII is postulated to be an extravascular thrombin inhibitor that is stimulated physiologically by dermatan sulfate proteoglycans. To understand how thrombin activity may be downregulated within the artery wall, cultured monkey aorta smooth muscle cell (SMC) proteoglycans were tested for their ability to accelerate thrombin inhibition by HCII. Early confluent SMC monolayers increased thrombin-HCII inhibition rates 2-fold to 4-fold compared with reactions in cell-free control wells (7.3±0.5 versus 2.7±0.2x104 mol·L-1·min-1, with and without SMCs, respectively; n=7 experiments). Extracellular matrix obtained by cell monolayer removal also accelerated the thrombin-HCII inhibition reaction 3-fold to 5-fold. Rate increases were abolished by Polybrene or protamine sulfate. Pretreatment of monolayers with heparitinase I (and of extracellular matrix with HNO2) to degrade heparan sulfate blocked the thrombin-HCII inhibition rate increase. In contrast, pretreatment with chondroitinase ABC in the presence of proteinase inhibitors had no effect. "Pericellular" (cell surface– and extracellular matrix–derived) SMC heparan sulfate proteoglycans (HSPGs) were purified and fractionated by charge on DEAE-Sephacel. At a concentration of 1 µg/mL hexuronic acid, high-charge HSPG stimulated a 7-fold thrombin-HCII inhibition rate increase relative to reactions without proteoglycan, whereas low-charge HSPG induced a 2-fold rate increase. In comparison, an 18-fold rate increase was observed with 1 µg/mL dermatan sulfate proteoglycan purified from SMC culture media. These results indicate that SMC HSPG could contribute significantly to thrombin inhibition by HCII in the artery wall.


Key Words: proteoglycans • smooth muscle cells • thrombin • serpin • heparin cofactor II


*    Introduction
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 *Introduction
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Thrombin, a serine proteinase generated by proteolytic activation of its plasma precursor prothrombin, is a central player in hemostasis and thrombosis owing to its ability to generate fibrin and activate platelets. Increasing evidence suggests that thrombin may also play a role in postclotting cellular events involved in the host response to vascular injury, such as inflammation, healing, and the less advantageous processes of atherogenesis and restenosis. In vitro studies with cultured cells have demonstrated that thrombin is a mitogen for vascular SMCs1 2 3 and fibroblasts,4 5 6 a chemoattractant for monocytes,7 8 an inducer of interleukin-1 production by macrophages,9 and a stimulator of EC synthesis of platelet-derived growth factor10 and platelet-activating factor.11 Indirect evidence for a pathologic role for thrombin within the artery wall is provided by the presence of a promoter of thrombin generation (tissue factor)12 13 and the "tethered-ligand" thrombin receptor14 15 on cells of fatty streaks and advanced atherosclerotic lesions; in contrast, these two proteins are normally absent from the subendothelial intima of nonatherosclerotic arteries. Additionally, the specific thrombin inhibitors D-Phe-Pro-Arg chloromethyl ketone and recombinant hirudin significantly reduce SMC proliferation in animal models of restenosis.16 17 The potential regulation of thrombin within the artery wall may therefore alter the progression of an atherosclerotic lesion.

Thrombin activity in vivo is regulated through inhibition by proteinase inhibitors. In plasma, thrombin activity is inhibited primarily by AT18 19 20 21 (historically referred to as AT III) and secondarily by {alpha}2-macroglobulin,22 23 HCII,24 25 and {alpha}1-proteinase inhibitor.22 26 27 However, it is unclear whether thrombin activity below the EC surface and underlying basement membrane is inhibited and if so, by what mechanism. In vitro studies suggest that thrombin bound to the ECM is protected from inhibition by AT.28 HCII has been postulated to be an extravascular thrombin inhibitor.29 30 31 32 HCII and AT are plasma glycoprotein members of the serpin (serine proteinase inhibitor) superfamily of proteins,33 which inhibit target proteinases by acting as pseudosubstrates and forming inactive bimolecular complexes. HCII and AT are heparin-binding serpins. Inhibition of thrombin by both HCII and AT is accelerated by heparin and some similar GAGs,31 34 which are long, unbranched polysaccharides composed of negatively charged repeating disaccharide units. In vivo, GAGs are covalently linked to a core protein to form PGs. The therapeutic action of heparin administered as an anticoagulant is mediated by AT,21 whereas activation of AT in vivo without exogenous heparin is thought to be due to HSPGs associated with the vascular endothelium.35 36 37 38 HCII activity is stimulated in vitro to some extent by a variety of GAGs and polyanions, but thrombin inhibition by HCII is greatly accelerated by heparin and DS.31 HCII has been proposed to have a role in the inhibition of "extravascular" thrombin (released or generated outside the circulation due to vascular damage) and to be activated physiologically by DS in the form of DSPGs.29 30 31 32 DSPGs are found in the ECM of the artery wall,39 40 41 42 skin,43 cartilage,43 44 bone,45 46 and other tissues. Evidence for DSPG activation of HCII is provided by the observations that purified skin and cartilage DSPGs accelerate HCII inhibition of thrombin to the same extent as do their respective free DS chains and that the DSPGs have comparable activity whether in solution or bound to immobilized collagen type V.32 In addition, McGuire and Tollefsen30 reported that cultured fibroblasts accelerated the rate of thrombin inhibition by HCII and that a DSPG was primarily responsible. These authors also observed activation of HCII by cultured porcine arterial SMCs,30 but the mechanism of activation was not investigated.

In the present study, cultured monkey aortic SMCs were used to simulate the SMC environment within an artery wall to investigate a potential role for HCII as an intramural thrombin inhibitor. SMC monolayers and the underlying ECM material were characterized for their ability to accelerate thrombin inhibition by HCII, and the specific PG responsible for enhancing HCII activity was identified. Next, PGs were purified from cultured SMCs and assayed in thrombin-HCII inhibition reactions. The results reported herein suggest a model in which more than one class of PG can contribute significantly to thrombin inhibition by HCII in the artery wall.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Materials
Tissue culture reagents were obtained from JRH BioSciences, MEM from Media Tech, and neonatal calf serum from Atlantic Biologicals. [35S]Na2SO4 (1050 to 1600 Ci/mmol) was purchased from DuPont–New England Nuclear. The following items were from Sigma: collagen type V (from human placenta), Triton X-100, PMSF, pepstatin A, N-ethylmaleimide, BSA, HEPES, and hexadimethrine bromide (Polybrene). HCII,47 AT,47 and active site–titrated thrombin48 were purified as described. The chromogenic substrate for thrombin, tosyl-Gly-Pro-Arg-p-nitroanilide (Chromozym-TH), was purchased from Boehringer Mannheim. Chondroitinase ACII (from Arthrobacter aurescens, EC 4.2.2.5), chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4), and heparitinase I (Flavobacterium heparinum, EC 4.2.2.8) were obtained from Seikagaku America Inc. Ultrapure guanidine HCl and urea were from ICN Biomedicals Inc. DEAE-Sephacel and CL-4B Sepharose were from Pharmacia Biotech, and the BioSep SEC-S2000 high-performance liquid chromatography column from Phenomenex. Butyl nitrate was purchased from Aldrich Chemical Co, polyethylene glycol 8000 from US Biochemical Corp, and protamine sulfate from Elkins Sinn Corp. Ninety-six–well Easy Wash enzyme-linked immunosorbent assay microtiter plates were from Corning.

Cell Culture
Primary cultures of SMCs were derived from explants of adult monkey (Macaca fascicularis) thoracic aortas as described previously.49 50 Subcultures were maintained at 37°C under a 5% CO2 atmosphere in MEM containing Earle's base salts and MEM essential vitamins, 10% heat-inactivated neonatal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 200 mmol/L L-glutamine (complete MEM). For monolayer and ECM experiments, SMCs were plated in Nunc 24-well plates (190 mm2 surface area) at 30 000 cells per well by hemacytometer count. In some experiments wells were first coated with 100 µg/mL collagen type V at 4°C overnight, washed, and irradiated with UV light prior to seeding. Monolayers became visibly confluent on day 5 or 6 after plating and were either used on day 7 as early confluent monolayers or fed and maintained to day 14 or 15 for use as late confluent monolayers or subcellular ECM preparations. SMCs from passages 10 to 20 were used.

Human dermal fibroblasts were the kind gift of Dr R.A. Briggaman (Department of Dermatology, University of North Carolina School of Medicine, Chapel Hill). The fibroblasts were maintained in Dulbecco's MEM (GIBCO BRL) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin and plated in Costar 24-well plates (200 mm2 surface area) for monolayer experiments. Fibroblasts from passage 7 were used.

Preparation of Wells Coated With Subcellular ECM
To prepare SMC subcellular ECM material, day-14 or -15 monolayers were first washed three times with HBS (20 mmol/L HEPES, pH 7.4, and 150 mmol/L NaCl). Cells were removed by incubation with freshly prepared 25 mmol/L NH4OH and 5 mg/mL Triton X-100 in HBS for 3 minutes at ambient temperature,28 followed by two washes with distilled water and two with HBS. Light microscopy verified removal of the cell debris and integrity of the ECM.

Glycanase and Chemical Pretreatment of SMC Monolayers and Subcellular ECM
SMC cultures in 24-well plates were metabolically labeled with 30 µCi/mL [35S]Na2SO4 for 48 hours before use and washed three times with HBS; in some cases ECMs were prepared. Cells and ECM were then incubated with 50 mU chondroitinase ACII, 50 mU chondroitinase ABC, or 4 mU heparitinase I for 2 hours at 37°C. Incubations were performed in either complete MEM alone (for monolayers) or 100 mmol/L HEPES and 30 mmol/L sodium acetate, pH 8.0 or pH 7.4 (for chondroitinase ABC or chondroitinase ACII, respectively), with the proteinase inhibitors 10 mmol/L EDTA, 1 mmol/L PMSF, 10 mmol/L N-ethylmaleimide, and 0.36 mmol/L pepstatin A (for ECMs).51 Untreated monolayers and subcellular ECMs were incubated with solvent but without glycanase. Low-pH HNO2 treatment was performed by incubating ECMs for 2 hours at ambient temperature with freshly prepared 5% butyl nitrate in 0.25 mol/L HCl with gentle rotation.50 After pretreatment the supernatants were collected, and all monolayers and ECMs were washed three times with HBS and verified microscopically to be intact. The final wash of HNO2- treated ECMs was confirmed with pH paper to be the same as that for the glycanase-pretreated wells. After thrombin inhibition assays were completed, monolayers and ECMs were washed three times with HBS and solubilized with 1 mol/L NaOH at 60°C for 30 minutes. Radioactivity in the NaOH lysates and pretreatment supernatants was measured in a Packard 1500 Tri-Carb liquid scintillation counter with Ready Safe (Beckman) as the fluor. Protein measurements were made on the NaOH lysates by the method of Lowry et al52 with BSA in 1 mol/L NaOH as the standard.

Thrombin Inhibition Assays on Cell Monolayers and Subcellular ECMs
A standard assay to measure serpin inhibition of thrombin chromogenic activity53 54 was modified for use with cultured cells, as suggested.55 Assays were performed at ambient temperature in HNP buffer and final concentrations of 50 nmol/L HCII, 5 nmol/L thrombin, 1 mg/mL BSA, and where indicated 1 mg/mL Polybrene. Cell monolayers were washed three times with HBS, and subcellular ECM was prepared and washed as described above. Immediately after the final aspiration, monolayers and ECM were overlaid with 100 µL BSA in HNP alone (100% thrombin control) or 100 µL of 100 nmol/L HCII in BSA/HNP. Reactions were started by adding 100 µL of 10 nmol/L thrombin. In selected experiments the reaction mixtures also contained 1 mg/mL Polybrene. After incubation for 90 minutes, residual thrombin chromogenic activity was measured by adding 100 µL of 0.3 mmol/L Chromozym-TH containing 2 mg/mL Polybrene. After 2 minutes substrate hydrolysis was quenched and measured by transferring 135 µL of the reaction mixture to a microtiter plate containing 50 µL of 8.7 mol/L glacial acetic acid and reading the absorbance at 405 nm on a Vmax microplate reader (Molecular Devices). Assays were performed with gentle rotation (100 rpm) to ensure adequate monolayer or ECM coverage. Monolayers and ECM remained intact for the duration of the experiment, as judged by light microscopy. Basal thrombin inhibition rates were measured without cells or ECM in 24-well plates.

Second-order inhibition rate constants were calculated as k2=-ln(a)/t[I], where a is the fractional thrombin activity remaining relative to the uninhibited (100%) thrombin control ( ie, a=[A405 of HCII+thrombin]/A405 of 100% thrombin), t is incubation time in minutes, and I is the inhibitor concentration in moles per liter. Therefore, k2 units are reported as mol-1·L·min-1 (abbreviated as M-1·min-1 in the figures and tables). Each assay consisted of triplicate determinations, and the mean±SEM is reported. Incubation times were chosen to ensure at least 20% residual thrombin activity (ie, a>=0.2). Control experiments confirmed that substrate hydrolysis remained linear during the 2-minute color development period and that monolayers had no chromogenic activity in the absence of exogenous thrombin. Thrombin chromogenic activity on monolayers in the absence of HCII retained >=95% of the activity of thrombin in empty BSA-coated wells, implying that there was negligible loss of thrombin activity due to interaction with or internalization by SMCs. Thrombin inhibition rates obtained in empty BSA-coated wells and complete MEM (10% neonatal calf serum) -coated wells were not statistically different.

AT-thrombin inhibition assays on SMC monolayers were performed with 50 nmol/L AT, 5 nmol/L thrombin, and an incubation period of 20 minutes. HCII-thrombin inhibition assays on fibroblast monolayers differed from subsequent SMC assays by the addition of 1 mmol/L CaCl2 to maintain monolayer integrity30 and a smaller reaction volume (100 µL of 75 nmol/L HCII and 50 µL of 15 nmol/L thrombin).

Purification of SMC Pericellular HSPG and Media DSPG
Trypsin-releasable cell surface– and subcellular ECM–derived ("pericellular") SMC HSPGs were purified from a total of 11 flasks (8000 mm2 surface area per flask) of confluent day-14 SMC monolayers. After removal of the culture media, cell monolayers were washed twice with PBS and incubated in 0.05% trypsin and 0.02% EDTA in PBS for 5 to 10 minutes. Trypsin was inactivated by adding complete medium to the flasks. Cell suspensions were collected and pooled with a wash of fresh medium in each flask and pelleted by centrifugation at 200g for 5 minutes at 4°C. The supernatants were decanted and stored at -20°C until purification. Three "tracer" flasks of cells were metabolically labeled with 30 µCi/mL [35S]Na2SO4 for 48 hours before harvest, and the labeled trypsinates were pooled with thawed, unlabeled trypsin fluids. Solid guanidine HCl was added to the pool to a final concentration of 4 mol/L and the volume reduced to 40 mL in an Amicon stirred-cell concentrator with a YM30 membrane (Mr cutoff of 30 kD). The concentrate was dialyzed into 50 mmol/L Na2SO4 in urea/Tris buffer (see below) to remove free 35S radiolabel and then into urea/Tris buffer alone (6 mol/L urea in 50 mmol/L Tris, pH 7.2, 0.1% [vol/vol] Triton X-100, and 150 mmol/L NaCl). The PGs were partially purified and separated into high- and low-charge fractions by DEAE-Sephacel chromatography as follows. The dialysate was loaded onto a 10-mL DEAE-Sephacel column equilibrated in urea/Tris buffer, washed with 5 bed volumes of urea/Tris buffer, and eluted stepwise with 0.35 mol/L NaCl followed by 1 mol/L NaCl in urea/Tris buffer. Eluate fractions containing significant 35S radioactivity were pooled. Low-charge (0.35 mol/L NaCl eluate) and high-charge (1 mol/L NaCl eluate) pools were dialyzed into 100 mmol/L Tris, pH 8.0, and 30 mmol/L sodium acetate and digested with 0.5 to 1 U chondroitinase ABC for 18 hours at 37°C with proteinase inhibitors (described above).51 Digests were adjusted to 6 mol/L urea and 150 mmol/L NaCl. HSPGs were separated from the digested disaccharides and core proteins by binding to a 1- to 2-mL DEAE-Sephacel column equilibrated in urea/Tris buffer with 30 mmol/L sodium acetate and elution with 1 mol/L NaCl in the same buffer. High- and low-charge HSPG pools were further concentrated with Amicon CF25 concentrator cones and dialyzed into HBS. The HS concentration was determined by the uronic acid assay method of Blumenkrantz and Asboe-Hansen56 with D-glucuronic acid as the standard. Aliquots reserved for functional studies were adjusted to 1 mg/mL BSA and stored at -20°C.

DSPGs secreted into the media of cultured SMCs were purified from 11 flasks of confluent day-7 cells. The media were collected, cleared of cell debris by centrifugation at 200g for 15 minutes at 4°C, and stored at -20°C until fractionation. One tracer flask of cells was metabolically labeled for 48 hours before collection with 30 µCi/mL [35S]Na2SO4. Radiolabeled and unlabeled media were pooled, concentrated to 60 mL, and dialyzed into urea/Tris buffer as described above for pericellular HSPGs. The dialysate was first partially purified from serum proteins after being loaded onto a 3-mL DEAE-Sephacel column equilibrated in urea/Tris buffer, washed with 10 bed volumes of the same buffer, and eluted with 1 mol/L NaCl in urea/Tris buffer. The eluate was diluted to 0.15 mol/L NaCl with urea/Tris buffer (no NaCl) and reloaded onto a 3-mL DEAE-Sephacel column equilibrated in urea/Tris buffer. The column was washed with 2 bed volumes of the same buffer followed by 3 bed volumes of urea/Tris buffer with no NaCl and eluted at a flow rate of 0.5 mL/min with a 40-mL linear gradient of 0 to 1 mol/L NaCl in urea/Tris buffer. Fractions (0.5 mL) were collected and analyzed for radioactivity and conductivity. DSPG and CSPG coeluted as a single peak at 0.36 to 0.64 mol/L NaCl, which was preceded by a small "shoulder" of HSPG at 0.15 to 0.35 mol/L NaCl. The DSPG-CSPG fractions were pooled and dialyzed into 4 mol/L guanidine HCl in 50 mmol/L sodium acetate, pH 5.8 (guanidine/acetate buffer), and the volume was reduced to 3.5 mL in an Amicon stirred-cell concentrator. DSPGs and CSPGs were fractionated on a CL-4B gel filtration column (16x760 mm) at a flow rate of 20 mL/h in guanidine/acetate buffer and collected in 2-mL fractions that were analyzed for radioactivity. CSPGs eluted first at a Kav of 0.0 to 0.19 and DSPGs at a Kav of 0.38 to 0.65, when Kav is defined as

and the void volume (Vo), total volume (Vt), and elution volume (Ve) are the elution positions of blue dextran, tryptophan, and the analysate, respectively. Pooled DSPG fractions were dialyzed into distilled water, lyophilized, and resuspended in a small volume of distilled water. DSPGs were assayed for uronic acid concentration, and aliquots for functional analysis were adjusted to 1 mg/mL BSA for storage at -20°C.

The purity of high- and low-charge HSPGs was established by their susceptibility to HNO2 depolymerization. DSPG purity was analyzed by its susceptibility to chondroitinase ABC degradation and resistance to HNO2. Aliquots were either treated with nitrous acid as described above and neutralized with NaOH or digested with 0.3 U chondroitinase ABC (or chondroitinase ACII) for 18 hours at 37°C with proteinase inhibitors.51 The samples were diluted in 0.25 mol/L Tris phosphate, pH 7.6, and chromatographed on a high-performance liquid chromatography gel filtration BioSep SEC-S2000 column (300x7.5 mm) at a flow rate of 0.5 mL/min in Tris phosphate buffer. Fractions (0.5 mL) were collected and measured for radioactivity by liquid scintillation counting. All untreated PGs eluted at the Vo. Degradation of GAG chains was assessed by movement of the radiolabel into the included volume typical of charged and uncharged disaccharides (Kav=0.52). Most (96%) of the low- and high-charge HSPG 35S radioactivity shifted from Vo to Kav=0.48 after HNO2 treatment, indicating >95% purity. Ninety-five percent, 55%, or 12% of DSPG radioactivity shifted from Vo to Kav=0.48 after chondroitinase ABC, chondroitinase ACII, or HNO2 treatment, respectively. Cell culture–derived DSPGs are know to be enriched in glucuronic acid residues and to be partially degraded by chondroitinase ACII, which cleaves at the glycosidic linkages to glucuronic acid. Therefore, DSPG was interpreted to be {approx}92% pure.

Thrombin-HCII Inhibition Assays With Purified SMC PGs
Inhibition assays were performed at ambient temperature in BSA-coated 96-well microtiter plates as described previously.53 54 They contained 50 nmol/L HCII, 5 nmol/L thrombin, 2 mg/mL BSA, and where indicated, 1 µg uronic acid per milliliter purified PG in HNP buffer containing 3 mmol/L NaN3. Inhibition reactions (50 µL) were started by adding thrombin. After incubation for 90 minutes for HSPG or no PG or 20 minutes for DSPG, residual thrombin activity was measured by adding 135 µL of 0.2 mmol/L Chromozym-TH containing 2 mg/mL Polybrene. After 4 minutes color development was quenched with 50 µL of 8.7 mol/L glacial acetic acid and the absorbance read at 405 nm. Rate constants were calculated as described above. The mean±SEM of least three separate determinations, each performed in triplicate, is reported.

Statistical Analysis
Differences between means were determined to be significant (P<.05) by ANOVA followed by Tukey's analysis for means separation.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Initial experiments were performed with cultured human dermal fibroblasts to confirm the earlier observation that these cells accelerate thrombin inhibition by HCII30 and to validate the assay approach. A mixture of purified thrombin and HCII was incubated on extensively washed confluent cell monolayers grown in 24-well plates. To measure residual thrombin activity a chromogenic substrate was added and absorbance measured on an aliquot of the reaction transferred to a microtiter plate. Fibroblast monolayers increased thrombin-HCII inhibition rate 8-fold compared with that obtained in cell-free, BSA-coated wells (27.3±1.5 versus 3.6±0.3x104 mol-1·L·min-1, respectively). Eighty-one percent of the rate increase was blocked by pretreatment of the monolayers with the glycanase chondroitinase ABC, which selectively removes DS and CS GAGs. These results are consistent with but alone do not confirm the report by McGuire and Tollefsen that cell surface DSPGs are primarily responsible for the {approx}3-fold increase in HCII activity stimulated by IMR-90 fibroblasts.30

Accelerated Thrombin Inhibition by HCII on SMC Monolayers and Subcellular ECM
Thrombin inhibition by HCII on SMC monolayers occurred in a time-dependent manner, as illustrated in Fig 1Down. In multiple experiments the inhibition rate increased 2-fold to 4-fold in the presence of early confluent SMC monolayers (day-7 postseeded) relative to that obtained in cell-free, BSA-coated wells (Fig 2Down). Late confluent monolayers (day-15 postseeded) promoted a 7-fold acceleration of the thrombin-HCII inhibition reaction (16.2±0.8 versus 2.3±0.1x104 mol-1·L·min-1, with and without SMCs, respectively). The thrombin-HCII inhibition rate increase stimulated by SMC monolayers was blocked by addition of 1 mg/mL Polybrene (Fig 2Down), a highly positively charged compound commonly used to chelate heparin and other GAGs. Protamine sulfate, traditionally used to rapidly reverse the effects of therapeutic heparin administration, was tested at 10 µg/mL and also found to have the same effect as Polybrene (data not shown). The ability of these polycations to block cell surface–mediated inhibition rate increases suggested that the increases may have been mediated by cell surface GAGs present as PGs.



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  		Figure 1. Time course of thrombin inhibition by HCII with ({bullet}) or without ({square}) arterial SMC monolayers. HCII (50 nmol/L), 5 nmol/L thrombin, and 1 mg/mL BSA were incubated for the indicated times and residual thrombin activity was measured. The percent residual thrombin activity was calculated as (thrombin activity with HCII÷thrombin activity without HCII)x100% at each time point. Note that the log of thrombin activity is plotted as a function of time. The means of n=2 (no cells) or n=3 (SMCs) determinations are reported.



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  		Figure 2. Arterial SMC monolayers accelerate thrombin inhibition by HCII. The rate of thrombin inhibition by HCII was measured with or without confluent SMC monolayers. Inhibition assays contained 50 nmol/L HCII, 5 nmol/L thrombin, 1 mg/mL BSA, and where indicated 1 mg/mL Polybrene (Pb). The inhibition rate constant (k2) was determined as described in "Methods." The mean±SEM of multiple experiments is reported (n=7, SMCs; n=7, no cells; n=4, SMCs+Pb). Means not having an identical superscript (a, b) are significantly different (P<.05).

SMCs of the artery wall play a major role in deposition of arterial ECM. SMCs in culture secrete ECM proteins and PGs50 57 58 59 60 61 to sites below the cell layer and into the culture medium above the monolayer. The thin layer of ECM beneath cultured SMCs was obtained by removal of cells from late confluent (day 14) SMC monolayers with Triton X-100 and NH4OH.28 In multiple experiments SMC subcellular ECM was found to increase the rate of thrombin inhibition by HCII 3-fold to 5-fold compared with reactions in empty BSA-coated wells (Fig 3Down). Polybrene blocked the reaction rate increase, implicating a PG(s) as a possible mediator. Because cultured monkey arterial SMCs secrete DSPGs primarily into the medium50 61 and purified decorin and biglycan DSPGs bind to adsorbed collagen type V,32 SMCs were also grown in wells coated with collagen type V. Subcellular ECM on collagen type V also increased the thrombin-HCII inhibition rate compared with those in empty collagen type V–coated wells (Fig 3Down). However, subcellular ECM on collagen type V did not show a greater rate enhancement than did subcellular ECM in uncoated plastic wells, suggesting that DSPGs may not be responsible for the rate increase observed. Thus, further SMC ECM experiments were performed without collagen type V.



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  		Figure 3. ECM beneath arterial SMC monolayers accelerates thrombin inhibition by HCII. The rate of thrombin inhibition by HCII was measured with or without subcellular SMC ECM. SMCs were cultured in either untreated wells [(-) col V] or in wells coated with 100 µg/mL collagen type V [(+) col V], and underlying ECM was obtained by cell removal with 25 mmol/L NH4OH and 0.5% Triton X-100. Thrombin inhibition assays were performed as described in the legend to Fig 2Up, and where indicated contained 1 mg/mL Polybrene (Pb). The mean±SEM of multiple experiments is reported [n=7, ECM or no ECM (black bars); n=3, ECM or no ECM (gray bars); n=2 (no SEM indicated); ECM+Pb (black and gray bars)]. Means not having an identical superscript (a, b, c) are significantly different (P<.05).

Effect of Enzymatic and Chemical Pretreatment of SMC Monolayers and Subcellular ECM on Accelerated Thrombin Inhibition by HCII
To determine which type(s) of PG was responsible for the SMC monolayer–mediated thrombin-HCII inhibition rate increase, 35[S]Na2SO4-radiolabeled SMC monolayers were enzymatically pretreated to remove specific classes of GAG chains from PG core proteins. Chondroitinase ACII and chondroitinase ABC were used to degrade CSPG and CSPG/DSPG, respectively. Heparitinase I treatment was used to depolymerize HSPG. Analysis of pretreated and untreated control monolayers demonstrated that glycanase pretreatment caused a greater release of 35S label into the pretreatment supernatant and a reduction of the 35S label remaining in the monolayer (Fig 4ADown). Pretreatment resulted in no protein loss as detected by protein measurements on pretreated and untreated control monolayers (data not shown). Chondroitinase ACII and chondroitinase ABC pretreatment of SMC monolayers had no significant effect on inhibition rate compared with untreated cells (Fig 4BDown). In contrast heparitinase I pretreatment blocked the SMC-mediated rate increase by 73%, suggesting involvement of cell surface HSPGs in the acceleration of thrombin inhibition by HCII (Fig 4BDown).



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  		Figure 4. Effect of glycanase pretreatment on arterial SMC–accelerated thrombin inhibition by HCII. Day-5 SMC monolayers were radiolabeled for 48 hours with 30 µCi/mL [35S]Na2SO4, washed extensively, and treated for 2 hours at 37°C with complete medium alone (untx) or complete medium containing either 50 mU chondroitinase ACII (ch ACII), 50 mU chondroitinase ABC (ch ABC), or 4 mU heparitinase I (hep I). Pretreatment supernatants were collected and thrombin-HCII inhibition assays performed on washed monolayers. After the assay was completed, SMC monolayers were lysed with 1 mol/L NaOH. A, To demonstrate glycanase activity, radioactivity in the pretreatment supernatant ("released" during incubation with glycanase or medium alone) and radioactivity remaining cell associated ("residual") were measured by scintillation counts of pretreatment supernatants and cell lysates, respectively. Note that glycanase pretreatment resulted in decreased residual 35S radiolabel in the SMC monolayer and increased release of 35S radiolabel into the supernatant compared with untreated SMC controls. B, The thrombin inhibition rate constant (k2) was determined as described in the legend to Fig 2Up. The mean±SEM of a representative experiment is shown (n=3 k2 determinations, n=6 radioactivity measurements). Means not having an identical superscript (a, b, c, etc) are significantly different (P<.05).

The effect of glycanase pretreatment of SMC subcellular ECM on thrombin-HCII inhibition rates was also investigated. Pretreatment with chondroitinase ACII, chondroitinase ABC, or heparitinase I without proteinase inhibitors (as in the SMC monolayer experiments) significantly reduced the rate increase for all three treatment groups, with the most pronounced effect exhibited by heparitinase I (data not shown). Pretreatment of subcellular ECM with chondroitinase ACII or chondroitinase ABC with proteinase inhibitors did not effect the rate increase (Table 1Down), suggesting that the effect without proteinase inhibitors was due to nonspecific PG removal by a contaminating proteinase in the chondroitinase preparations. Low-pH HNO2 pretreatment, which depolymerizes HS, blocked the inhibition rate increase by 82% (Table 1Down).


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  		Table 1. Effect of Pretreatment of SMC ECM on Thrombin-HCII Inhibition Rates

The ability of heparitinase I and HNO2 pretreatment to block SMC monolayer– and subcellular ECM–accelerated thrombin-HCII inhibition implied a major role for SMC-associated HSPG. Since thrombin inhibition by AT is accelerated by heparin and HS, thrombin-AT inhibition assays were performed on late confluent (day 15) monolayers. Thrombin-AT inhibition rates on SMC monolayers versus cell-free, BSA-coated wells were 3.9±0.3x105 and 3.5±0.2x105 mol-1·L ·min-1 ( n=6), respectively, demonstrating no SMC-mediated rate enhancement for the thrombin-AT interaction.

Activity of Purified SMC HSPG in Thrombin-HCII Inhibition Reactions
To confirm SMC HSPG activity in thrombin-HCII inhibition reactions, HSPG produced by cultured SMCs was isolated. Trypsin-releasable cell surface– and ECM-derived (pericellular) SMC HSPGs were purified and fractionated by DEAE-Sephacel chromatography into high- and low-charge species ({approx}80% and {approx}20% of total HSPG by hexuronic acid content, respectively). For comparison, DSPG secreted by SMCs into overlying culture media was purified by sequential DEAE-Sephacel and CL-4B gel filtration chromatography. The PGs were analyzed in a standard thrombin-HCII inhibition assay in a microtiter plate. At a concentration of 1 µg hexuronic acid per milliliter, the high-charge pericellular HSPG elicited a 7-fold inhibition rate increase relative to that without exogenous PG, but low-charge pericellular HSPG increased the inhibition rate only 2-fold (Table 2Down). Unfractionated, free HS chains from SMC media elicited a response intermediate to that observed for the two pericellular HSPG fractions. In comparison media-derived SMC DSPG, the PG species generally thought to activate HCII in vivo, stimulated an 18-fold rate increase (Table 2Down).


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  		Table 2. Acceleration of Thrombin-HCII Inhibition Reactions by Purified SMC PGs


*    Discussion
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 *Discussion
down arrowReferences
 
DS in the form of DSPG has long been thought to be the preferred putative physiological activator of HCII, at least in the extravascular environment. This assumption is based on the evidence that DS uniquely activates only HCII29 among the heparin-binding serpins and on the observation that while both heparin and DS greatly accelerate the rate of thrombin inhibition by HCII,29 31 heparin is not a normal constituent of the circulation or extravascular spaces. In addition studies with fibroblast cell surface–associated DSPG30 and DSPG purified from skin and cartilage32 have demonstrated HCII activation by intact DSPGs. The DS structural requirements for the interaction with HCII have been the subject of much investigation, and a highly sulfated, high-affinity HCII-binding hexasaccharide has been identified in skin DS.62 A similarly specific high-affinity HCII binding site in heparin has not been identified.63 An early comparison of several GAGs showed that heparin, DS, and the unusually "oversulfated" HS from bovine liver (in order of decreasing activity) greatly accelerated thrombin inhibition by HCII.29 HS from bovine aorta had little activity in the assay system used,29 but the assay was not sensitive to modest rate increases.

We report a new finding that HSPG on the surface of SMCs in monolayer culture and in the ECM below cultured SMCs accelerates thrombin inhibition by HCII. Unexpectedly, DSPG did not play a detectable role in the cell assay system. These conclusions were drawn from experiments that first confirmed the report by McGuire and Tollefsen30 that surfaces of SMCs in culture accelerate inhibition of thrombin by HCII and then demonstrated for the first time that ECM below cultured SMCs also accelerates the reaction. The identity of the PG mediator was established by showing that specific removal of HS chains from the cell surface and subcellular ECM by heparitinase I or HNO2 treatment blocked the rate increase. CS/DS removal by chondroitinase treatment had no significant effect. The activity of SMC HSPG was confirmed by using HSPG purified from cell surfaces and subcellular ECM (pericellular HSPG). DSPG was purified from SMC culture media for comparison. At equal concentrations of hexuronic acid in thrombin-HCII inhibition reactions, high-charge HSPG and DSPG stimulated comparable rate increases.

HS in the form of HSPG is associated with most mammalian cell types and is present on cell surfaces or in the surrounding basement membrane.64 65 HS polysaccharide chains are structurally heterogeneous, being composed of repeats of hexuronic acid–glucosamine disaccharides that exhibit variable degrees of N-acetylation or N-sulfation, O-sulfation, and conversion of ß-D-glucuronic acid to its epimer {alpha}-L-iduronic acid.66 HS and heparin are structurally related GAGs. Heparin, produced only by mast cells and thus prepared commercially from mast cell–rich intestinal mucosa or lung tissue, is distinguished from HS by complete secondary modification of the nascent polysaccharide chain, which results in a much higher iduronic acid and sulfate group content.67 The HS produced by arterial SMCs,68 ECs,69 and fibroblasts70 has been shown to have a more varied structure, consisting of stretches of oligosaccharides rich in iduronic acid and N-sulfation that are separated by less modified stretches of glucuronic acid and N-acetylation. The high-affinity interaction of HS and several ligands (eg, AT,71 basic fibroblast growth factor,72 and lipoprotein lipase73 ) has been attributed to unique and specific oligosaccharide sequences in the sulfate-rich domains. The SMC pericellular HSPG isolated in the current study was fractionated into low- and high-charge HSPGs. These charge differences may be due to different degrees of sulfation per chain, a different number of HS chains per molecule, or a combination of both. Regardless, the high-charge HSPG exhibited greater HCII activation than did the low-charge HSPG. These findings agree with fractionation studies of commercial heparin, which have shown that high-charge fractions possess more sulfate groups per molecule and exhibit greater HCII activity than do low-charge fractions.74

The SMC-mediated inhibition rate increases observed in the current study, while statistically significant, may be viewed as modest compared with those obtained with high concentrations of commercial heparin and DS. However, HCII activation is characterized by a dose-dependent phenomenon that varies depending on the type of GAG.31 A concentration of 1000 µg/mL DS or 200 µg/mL heparin is required to elicit the maximal several-thousand–fold increase in the thrombin-HCII inhibition rate.53 75 Therefore, the rate increases observed in the current study would fall on the left shoulder of a dose-response curve, owing to the small amounts of PG present. Actual local concentrations of PG in vivo (ie, PG molecules per volume of extravascular fluid that bathes cells and ECM) are unknown but are expected to be much higher than those achieved in the present assay system and therefore to be capable of stimulating greater HCII activity. The effect of higher concentrations of isolated monkey SMC HSPG on HCII activity was not determined because the large amounts of material required were unavailable. However, a dose-dependent effect was indeed observed with HSPG isolated for a separate study from human arterial media-intima tissue (1.3-, 3-, 9-, and 25-fold inhibition rate increases were obtained with 0.1, 1, 10, and 30 µg/mL HSPG, respectively, compared with the rate without HSPG), demonstrating that higher concentrations of HSPG stimulate greater HCII activity. SMC HSPG was expected to be a considerably poorer HCII activator than DSPG on the basis of a previous comparison of HCII activity with various GAGs29 and the knowledge that HS has fewer sulfate groups per disaccharide than does DS.76 However, isolated arterial SMC HSPG and DSPG assayed at a fixed concentration of 1 µg/mL hexuronic acid in thrombin-HCII inhibition reactions were found to stimulate comparable rate increases. These results suggest that arterial SMC HSPG may contribute significantly to thrombin inhibition by HCII in the artery wall.

The identification of an SMC HSPG as an HCII activator is significant because it adds to a relatively small pool of data that addresses the question of the physiological activation of GAG-binding serpins, including HCII. Most studies to date have characterized mixtures of purified GAG, serpin, and proteinase; there are few studies that have assessed the physiological activation of serpins by intact PGs. AT activation in vivo by HSPG has been demonstrated in studies with rat hind limb perfusion36 and cultured ECs on microcarrier beads.35 An HSPG expressed by cultured fibroblasts was shown to accelerate the rate of thrombin inhibition by protease nexin-1,77 another serpin thought to inhibit thrombin in the extravascular environment, especially in brain tissue.78 79 Protein C inhibitor bound to kidney epithelial cell surface HSPG was shown to have increased inhibitory activity,80 and as cited above, cultured fibroblasts have been shown to accelerate thrombin inhibition by HCII via DSPG.30 Our finding that cultured SMCs accelerate the rate of thrombin inhibition by HCII verifies the earlier report by McGuire and Tollefsen30 and also identifies a PG mediator.

The HCII-activating potential of HSPG on the surfaces of cultured SMCs and underlying ECM and of purified HSPG and DSPG suggests that both SMC HSPG and DSPG regulate thrombin activity when thrombin and inhibitor simultaneously come into contact with PGs synthesized by vascular SMCs. Direct evidence for thrombin activity within the healthy arterial wall is lacking. However, after traumatic injury, plasma components (including HCII and thrombin) come into contact with cells of the artery wall. In atherosclerotic lesions, tissue factor12 13 and a thrombin receptor15 have been detected on cells throughout the lesion, suggesting the presence of thrombin within the diseased artery wall. A recent immunohistochemistry study detected HCII distributed diffusely throughout the intima below the endothelium of normal arteries,81 indicating that plasma-derived HCII can penetrate the endothelial layer and enter the healthy vessel wall.

HSPGs are found in the artery wall on SMC surfaces and in the basement membrane.82 In contrast DSPGs are located primarily in the type I and type III collagen-rich interstitial ECM of arteries.83 The DSPG secreted into the media of cultured monkey aortic SMCs very likely represents DSPG deposited in vivo in the interstitial collagenous ECM by SMCs. These cumulative findings suggest that intramural thrombin activity can be inhibited at two sites: by HCII stimulated by collagen-associated DSPG in the interstitial ECM and by a second "line of defense" at or near the SMC surface, as provided by pericellular HSPG–activated HCII. Because of its distribution, the HSPG-accelerated reaction could regulate thrombin activity at one of its sites of action, ie, near thrombin receptors on the SMC surface. This proposed physiological mechanism of thrombin inhibition is made more intriguing by the knowledge that HSPG content in atherosclerotic lesions is lower when compared with that in normal artery tissue.84 85 Thus, atherosclerosis progression is associated with higher thrombin generation at the same time that at least one potential thrombin regulatory mechanism is downregulated.


*    Selected Abbreviations and Acronyms
 
AT = antithrombin
(CS)PG(s) = (chondroitin sulfate) proteoglycan(s)
(DS)PG(s) = (dermatan sulfate) proteoglycan(s)
EC(s) = endothelial cell(s)
ECM = extracellular matrix
GAG(s) = glycosaminoglycan(s)
HBS = HEPES-buffered saline
HCII = heparin cofactor II
HNP = 20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, and 1 mg/mL polyethylene glycol
(HS)PG(s) = (heparan sulfate) proteoglycan(s)
MEM = (Eagle's) minimal essential medium
PG(s) = proteoglycan(s)
SMC(s) = smooth muscle cell(s)


*    Acknowledgments
 
This work was supported by training grant HL-017115 (to R.A.S.), research grants HL-25161 and HL-45848 (to W.D.W), and HL-32656 (to F.C.C.) from the National Institutes of Health, Bethesda, Md.

Received January 18, 1996; revision received May 30, 1996;
*    References
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