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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:252-257.)
© 1995 American Heart Association, Inc.

Articles

Characterization of Serum Amyloid P Component From Human Aortic Atherosclerotic Lesions

X. A. Li; K. Hatanaka; H. Ishibashi-Ueda; C. Yutani; A. Yamamoto

From the Departments of Etiology/Pathophysiology (X.A.L., K.H., A.Y.) and Pathology (H.I.-U., C.Y.), National Cardiovascular Center, Suita City, Osaka, Japan.

Correspondence to Dr Kaoru Hatanaka, Developmental Research Laboratories, Shionogi & Co, Ltd, 3-1-1 Futaba-cho, Toyonaka, Osaka 561, Japan.

	Abstract

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Abstract Serum amyloid P component (SAP) is a glycoprotein in human plasma. We recently showed the localization of SAP in human atherosclerotic lesions by immunohistochemical staining. In this study, the presence of SAP in atherosclerotic lesions was confirmed, and the biochemical character of SAP in atherosclerotic intima was investigated and compared with that of native SAP. Atherosclerotic intima was sequentially extracted with 2 mmol/L CaCl₂–Tris-buffered saline (TBS), 10 mmol/L EDTA-TBS, 3 mol/L guanidine-TBS, and collagenase digestion. The character of SAP in each extract was studied with double immunodiffusion, electroimmunoassay, crossed immunoelectrophoresis, and Western immunoblotting. The total amount of SAP in atherosclerotic intima was 190±64 µg/g wet tissue with an SAP-albumin ratio of 1:22.7, which is 44 times higher than the relative plasma ratio of 1:1000. This suggests that SAP is specifically localized in atherosclerotic lesions. SAP from the intima was indistinguishable from plasma or purified SAP with respect to immunological character and molecular weight. However, electrophoretic mobility and the binding of SAP to atherosclerotic intima appeared heterogeneous. Of total extractable SAP, about 43% appeared in the CaCl₂-TBS fraction, 25% in the EDTA-TBS fraction, and 32% in the collagenase digestion fraction. SAP is one of the two pentraxins in human plasma; the other is C-reactive protein, which has also been reported to locate in atherosclerotic lesions. Our findings suggest a role for SAP in atherogenesis and encourage efforts to determine more precisely the physiological contributions of the pentraxin family to the development of atherosclerosis.

Key Words: amyloid P component • atherosclerosis • characterization • pentraxin • human aorta

	Introduction

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Serum amyloid P component (SAP) is a plasma glycoprotein that is synthesized by the liver and circulates in normal human blood at a concentration of 30 to 45 mg/L.¹ It is universally present in amyloid deposits,² including the cerebral amyloid of Alzheimer's disease.³ SAP is also present in the renal glomerular basement membrane⁴ and the peripheral microfibrillar mantle of elastic fibers in blood vessels.⁵ SAP has been shown to bind to a variety of ligands in a calcium-dependent manner, including fibronectin,⁶ C4b-binding protein,^{6 7} C-reactive protein (CRP),^{8 9} heparin,¹⁰ heparan sulfate and dermatan sulfate,¹¹ zymosan,¹² agarose,^{13 14} immobilized human DNA,¹⁵ chromatin,^{16 17} amyloid fibrils,¹⁸ mannose-terminated glycoproteins,¹⁹ complement component Clq^{20 21} and C3bi,²² saccharides,²³ and aggregated IgG.²⁴ No deficiency of SAP has been reported in humans. The association of SAP with the above ligands and its stable evolutional conservation imply that it has important functions. However, the biological role is not clear. We recently showed the localization of SAP in human atherosclerotic lesions with immunohistochemical staining, suggesting SAP as a potential modulator of atherosclerosis (K.H. et al, unpublished data, 1994). In this study, the presence of SAP in atherosclerotic lesions was confirmed, and the biochemical character of SAP in atherosclerotic intima was investigated and compared with that of native SAP.

	Methods

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Materials
Purified human SAP and bovine serum albumin (BSA) were purchased from Sigma Chemical Co; rabbit anti-human SAP antiserum from UCB-Bioproducts, SA, B; standard normal human plasma (FACT) from George King Bio-Medical; purified human IgG from Organon Teknika Corp; bacterial collagenase from Wako Chemical Industries; protein A column kit (Ampure PA kit) from Tosoh Ltd; acrylamide, bisacrylamide, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate, sodium dodecyl sulfate (SDS), and protein low-molecular-weight standards from Bio-Rad Laboratories; and agarose (Seakem ME) from FMC BioProducts. All other reagents were of the best grade available.

Collection of Arterial Specimens
Samples of atherosclerotic lesions consisting of atheromatous and fibrous plaques derived from human abdominal aortas were obtained from autopsies of atherosclerotic patients within 2 hours of death at the National Cardiovascular Center, Osaka, Japan. Calcified lesions were omitted. The tissues were rinsed briefly in several changes of 0.1 mol/L phosphate-buffered saline (PBS), pH 7.5, to remove any adherent blood components. The tunica intima was separated from the underlying tunica media along the natural cleavage plane of the internal elastic lamina. The tissues were blotted to remove excess moisture, weighed, and stored at -40°C. The tissues were analyzed within 2 weeks. The data in this report were obtained from 58- and 75-year-old men and a 74-year-old woman.

All quantitative experiments were performed with three replicates from which a mean value (mean±SD) was obtained.

Extraction of SAP From Atherosclerotic Intima
The intimas were cut into small pieces, and 1 g of each was homogenized in a blender with 12 mL of 0.1 mol/L Tris-buffered saline (TBS), pH 7.5, containing 2 mmol/L CaCl₂ for 5 minutes. The homogenate was incubated for 30 minutes, and the extract was collected by centrifugation at 20 000g for 30 minutes. The pellets were washed by rehomogenization with CaCl₂-TBS and recentrifugation. The wash was discarded. The pellets were then extracted with 10 mmol/L EDTA-TBS, pH 7.5, for 30 minutes, and the extract was collected by centrifugation at 20 000g for 30 minutes. The pellets were washed and further extracted with 3 mol/L guanidine–10 mmol/L EDTA-TBS, pH 7.5, for 30 minutes. The extract was collected by centrifugation at 20 000g for 30 minutes and dialyzed against 3 L of 10 mmol/L EDTA-TBS, pH 7.5, overnight. All extraction buffers contained 5 mmol/L benzamidine as a protease inhibitor, and all procedures were performed at 4°C or in an ice bath. In preliminary experiments, the intima was extracted with the above buffers for 2 hours, which gave no obvious difference from 30-minute incubation. To elute covalent-bound SAP from the pellets, the extracted pellets were washed four times with 50 mmol/L Tris–5 mmol/L CaCl₂, pH 7.5, and suspended in 6 mL of 50 mmol/L Tris–5 mmol/L CaCl₂, pH 7.5, containing 200 U/mL bacterial collagenase. The digestion was performed at 37°C for 1 hour. The sequential extracts were used for immunochemical and biochemical studies as described below.

Immunochemical Identification and Quantification of SAP From Atherosclerotic Intima
Immunologic properties of the sequential extracts were studied with the double immunodiffusion technique using rabbit antiserum against human SAP following standard methods.²⁵ The antiserum is monospecific against whole human serum, as shown by crossed immunoelectrophoresis (CIE).¹⁰ A 1% agarose gel was made in 75 mmol/L barbital buffer (pH 8.6) containing 10 mmol/L EDTA. Electrophoretic behaviors of the sequential extracts were studied with CIE as previously described.¹⁰ The first dimension was run at 10°C for 80 minutes in the presence of 5 mmol/L EDTA–25 mmol/L barbital, pH 8.6, or 110 minutes in the presence of 2 mmol/L CaCl₂–37.5 mmol/L barbital, pH 8.6. The second dimension was run at 15°C for 18 hours in the presence of 0.18% rabbit anti-SAP antiserum and 5 mmol/L EDTA–25 mmol/L barbital, pH 8.6. The amounts of SAP in extracts were measured by electroimmunoassay following the method of Laurell.²⁶ Barbital buffer (25 mmol/L), pH 8.6, containing 5 mmol/L EDTA was used as the electrophoretic buffer. Serial dilutions of normal human plasma were used as standards that were calibrated against purified SAP. The area of the rocket was used to make the standard curve.

SDS–Polyacrylamide Gel Electrophoresis and Immunoblotting
Samples were treated with an equal volume of reduced SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer at 100°C for 5 minutes and subjected to 12% SDS-PAGE in a 1-mm-thick slab gel with 4% stacking gel according to the method of Laemmli.²⁷ The electrophoresis was run at 18 mA for 1 hour. After electrophoresis, the gel was soaked in a transfer buffer that contained 48 mmol/L Tris, 39 mmol/L glycine, 20% methanol, and 0.037% SDS, pH 8.6, for 2 minutes and then electrotransferred to a nitrocellulose membrane (pore size, 0.2 µm; Toyo Roshi) at 130 mA for 15 minutes in a Semi-Dry Electrophoretic Transfer Cell (Trans-Blot SD, Bio-Rad) with five sheets of filter paper on each side that had previously been well equilibrated with the transfer buffer. After transfer, the nitrocellulose membrane was soaked in a blocking buffer containing 200 µL goat serum in 10 mL of 0.1 mol/L Tris–0.9% NaCl–0.1% Tween 20 (TTBS), pH 7.5, at room temperature on a rocking platform. Subsequently, the nitrocellulose membrane was exposed to the first antibody (1:500 dilution of rabbit anti-SAP antiserum in 10 mL of 1 mol/L NaCl-TTBS) for 30 minutes at 25°C on a shaker with the nitrocellulose membrane freely floating. The nitrocellulose membrane was then washed three times with TTBS for a total of 30 minutes. The binding of the first antibody to the membrane was detected with a Vecta-stain ABC kit (2V-1000-10, Vector Laboratories) as previously described.²⁸

Quantification of Human Albumin From Atherosclerotic Intima
The albumin concentration in the intimal extracts was estimated as follows. After SDS-PAGE, the proteins in gel were stained with Coomassie brilliant blue (CBB). The density of the protein band at 66.2 kD was measured with a double-wavelength thin-layer chromatographic scanner (CS-930, Shimadzu Corp), and the amount of albumin was estimated from a standard curve made from serial dilutions of normal human plasma.

Deletion of SAP From Human Plasma by BaSO₄ Adsorption
BaSO₄ fine powder (100 mg) was added to 1 mL citrated normal human plasma, and the mixture was incubated at 4°C for 2 hours with gentle agitation. The supernatant was collected by centrifugation at 5000g for 5 minutes. SAP precipitated by BaSO₄ was eluted with 0.2 mol/L EDTA, pH 7.5. No SAP antigen was detected in the supernatant fraction, and more than 95% of SAP antigen was recovered in the EDTA eluate fraction when checked by electroimmunoassay (data not shown).

Deletion of IgG From Intimal Extracts and Normal Human Plasma by Protein A Affinity Chromatography
We added 35 µL of 0.3 mol/L EDTA to 1 mL intimal extract by CaCl₂-TBS to a concentration of 10 mmol/L EDTA in the extract to inhibit calcium-dependent interaction between SAP and human IgG. The extract was then passed through a protein A column (1 mL) equilibrated with 0.1 mol/L PBS, pH 7.5, containing 5 mmol/L EDTA. Normal human plasma (1 mL) diluted 10 times with 0.5% BSA-PBS or 1 mL of intimal extract by EDTA-TBS was also applied to the protein A column.

	Results

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Immunochemical Identification and Quantification of SAP From Atherosclerotic Intima
The intimal extracts by CaCl₂-TBS, EDTA-TBS, and collagenase digestion contained an antigen that gave a precipitation line in the gel of complete identity with that between anti-SAP antibody and purified SAP or normal human serum (Fig 1). Electroimmunoassay of the sequential extracts showed that of total extractable SAP, about 43% was eluted by CaCl₂-TBS, 25% by EDTA-TBS, a tiny amount by guanidine-TBS, and 32% by collagenase digestion (Fig 2). The amount of SAP in atherosclerotic lesions was about 190±64 (mean±SD) µg/g wet tissue. SDS-PAGE of intimal extracts showed that more than 90% of intimal serum albumin appeared in the CaCl₂-TBS fraction (Fig 4A). The amount of albumin in atherosclerotic intima was 4.28±0.95 mg/g wet tissue. Therefore, the ratio of SAP to albumin in atherosclerotic intima was about 1:22.7 (wt/wt), which is 44 times higher than the relative plasma ratio of 1:1000 (wt/wt). As Fig 2 shows, SAP extracted from the intima produced a more sharply shaped rocket of immunoprecipitation; therefore, the area of the rocket was measured to quantify the amount of SAP.

View larger version (102K): [in this window] [in a new window]   Figure 1. Immunochemical identification of serum amyloid P component (SAP) from atherosclerotic intima by double immunodiffusion. Sequential extracts of SAP from atherosclerotic intima by CaCl2–Tris-buffered saline (TBS), EDTA-TBS, guanidine-TBS, and collagenase digestion (wells 3 through 6, respectively) were applied. Rabbit anti-SAP antiserum was diluted to 1:32 with 10 mmol/L EDTA–75 mmol/L barbital buffer, pH 8.6. Wells 1 and 2 were purified SAP and human serum, respectively.

View larger version (115K): [in this window] [in a new window]   Figure 2. Quantification of serum amyloid P component (SAP) from atherosclerotic intima by electroimmunoassay. Sequential extracts of SAP from atherosclerotic intima by CaCl2–Tris-buffered saline (TBS), EDTA-TBS, guanidine-TBS, and collagenase digestion (wells 1 through 4, respectively) were applied to Laurell Rocket with 1% agarose gel containing 0.15% rabbit anti-SAP antiserum. Wells 5 through 8 were human plasma standards diluted to concentrations of 20, 10, 5, and 2.5 mg/L, respectively.

View larger version (48K): [in this window] [in a new window]   Figure 4. Biochemical characterization of serum amyloid P component (SAP) from atherosclerotic intima by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A, SDS-PAGE. Samples were reduced with 2% mercaptoethanol and applied to 12% SDS-PAGE. Lane 1, molecular weight standards; lane 2, purified SAP (100 mg/L); lane 3, 1% human IgG+4% bovine serum albumin (BSA) (diluted 60 times); lanes 4 through 7, sequential extracts of SAP from atherosclerotic intima by CaCl2–Tris-buffered saline (TBS), EDTA-TBS, guanidine-TBS, and collagenase digestion, respectively. B, Immunoblotting. Samples were reduced with 2% mercaptoethanol and applied to 12% SDS-PAGE. After electrophoresis, the gel was electrotransferred to a nitrocellulose membrane and immunoblotted with rabbit anti-SAP antiserum. Lane 1, purified SAP (5 mg/L); lane 2, 1% human IgG+4% BSA (diluted 60 times); lanes 3 and 12, normal human plasma (diluted 60 times); lanes 4 through 7, sequential extracts of SAP from atherosclerotic intima by CaCl2-TBS, EDTA-TBS, guanidine-TBS, and collagenase digestion, respectively; lanes 8 and 9, intimal extracts by CaCl2-TBS and EDTA-TBS preadsorbed with protein A; lanes 10 and 11, normal human plasma preadsorbed with protein A and BaSO4, respectively.

Biochemical Characterization of SAP From Atherosclerotic Intima
When CIE was performed in the presence of EDTA, SAP from normal human plasma appeared as an -globulin with an R_f of 0.83 (Fig 3Aa), the same as purified SAP.¹⁰ However, SAP from the intimal extracts showed heterogeneous electrophoretic mobility. Compared with plasma SAP, SAP from the CaCl₂-TBS and EDTA-TBS fractions was more cathodic, with an R_f of 0.47 and 0.69, respectively (Fig 3Ab and 3Ac). SAP from the collagenase digestion fraction was more anodal, with an R_f of 0.93 (Fig 3Ad). When CIE was performed in the presence of calcium, SAP from normal human plasma appeared as a broad peak with the front at the -globulin position (R_f=0.61, Fig 3Ba), the same as purified SAP.¹⁰ However, SAP from the intimal extracts again showed heterogeneous electrophoretic mobility. SAP from the CaCl₂-TBS and EDTA-TBS fractions migrated more slowly than normal human plasma, with an R_f of 0.46 and 0.59, respectively (Fig 3Bb and 3Bc); SAP from the collagenase digestion fraction migrated faster than normal human plasma (R_f=0.82, Fig 3Bd).

View larger version (71K): [in this window] [in a new window]   Figure 3. Biochemical characterization of serum amyloid P component (SAP) from atherosclerotic intima by crossed immunoelectrophoresis (CIE). Normal human plasma (a) and sequential extracts of SAP from atherosclerotic intima by CaCl2–Tris-buffered saline (TBS) (b), EDTA-TBS (c), and collagenase digestion (d) were applied to CIE with the first dimension in the presence of 5 mmol/L EDTA (A) or 2 mmol/L CaCl2 (B) and with the second dimension in the presence of 5 mmol/L EDTA and 0.18% rabbit anti-SAP antiserum.

When purified SAP was subjected to immunoblotting, a single SAP band at 29.5 kD was detected (Fig 4B, lane 1), agreeing with the result of CBB staining (Fig 4A, lane 2). When the sequential extracts of the intima were subjected to immunoblotting, three darkly stained bands at 28.5, 29.5, and 50 kD were detected for the CaCl₂-TBS fraction; two darkly stained bands at 29.5 and 50 kD for the EDTA-TBS and guanidine-TBS fractions; and one band at 29.5 kD for the collagenase digestion fraction (Fig 4B, lanes 4 through 7). Many positively stained bands, including 28.5-, 29.5-, and 50-kD bands, were detected for normal human plasma (Fig 4B, lanes 3 and 12). As shown in our previous study,²⁸ for immunoblotting, false-positive staining may be caused by nonspecific cross interactions between human plasma proteins and antiserum proteins. To distinguish the false-positive staining, SAP-deficient plasma, prepared by adsorption of normal human plasma with BaSO₄ (Fig 2, well 9), was subjected to immunoblotting. The 29.5-kD band disappeared, but other positively stained bands were still there (Fig 4B, lane 11). To further characterize the 28.5- and 50-kD bands, normal human plasma or the intimal extracts by CaCl₂-TBS and EDTA-TBS were passed through a protein A affinity column to remove human IgG and then subjected to immunoblotting. As a result, the 28.5- and 50-kD bands disappeared, but the 29.5-kD band was still there (Fig 4B, lanes 8 through 10). To confirm the nature of the 28.5- and 50-kD bands, a mixture of 1% human IgG and 4% BSA was subjected to SDS-PAGE. Three darkly stained bands at 28.5, 50, and 66.2 kD were detected (Fig 4A, lane 3), which corresponded to the heavy and light chains of human IgG and BSA, respectively. The 28.5- and 50-kD bands, not the BSA band, were intensively stained when the mixture was subjected to immunoblotting stained with the antiserum against human SAP (Fig 4B, lane 2). These results indicate that the 28.5- and 50-kD bands produced by intimal extracts and human plasma were made by cross interactions between human IgG and anti-SAP antiserum proteins.

	Discussion

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Atherosclerotic intima contained comparable amounts of SAP antigen and serum albumin. The SAP-albumin ratio was 1:22.7, which is 44 times higher than the relative plasma ratio of 1:1000. This suggests that SAP is specifically localized in atherosclerotic lesions, not just nonspecifically retained there. Although SAP from atherosclerotic intima was indistinguishable from plasma SAP or purified SAP with respect to immunologic character and molecular weight, the electrophoretic behavior of SAP from the intima and the binding of SAP to atherosclerotic intima appeared heterogeneous. Of total extractable SAP, about 43% appeared in the CaCl₂-TBS fraction, 25% in the EDTA-TBS fraction, and 32% in the collagenase digestion fraction. Different from intimal SAP, nearly all intimal albumin appeared in the CaCl₂-TBS fraction. Accordingly, we assume that three types of binding contribute to the presence of SAP in atherosclerotic intima. About 43% of SAP locates there by weak noncovalent interaction or binding (calcium-dependent or calcium-independent) to the local component(s) of atherosclerotic intima, and the SAP or the SAP-ligand(s) complex can be eluted by CaCl₂-TBS. About 25% of SAP is held there through calcium-dependent binding to the local component(s) of the intima, and the SAP or SAP-ligand(s) complex can be eluted by EDTA. The rest (32%) of the SAP is covalently bound to the matrix component(s), such as collagen in atherosclerotic intima, and can be eluted only by collagenase digestion.

SAP from atherosclerotic intima showed a more sharply shaped rocket of immunoprecipitation in electroimmunoassay than that of native SAP. Besides, compared with native SAP, SAP from intimal CaCl₂-TBS and EDTA-TBS fractions showed retarded electrophoretic mobility; SAP from intimal collagenase digestion fraction showed enhanced electrophoretic mobility. There are two possible explanations. One is that SAP from the intima is cleaved; however, no cleaved form of SAP could be detected in our immunoblotting, which excludes such a possibility. The other is that SAP in the intima is complexed with some local components in the intima, as we assumed above.

We previously showed that immunoblotting may produce false-positive results.²⁸ In the present study, three deeply stained bands at 28.5, 29.5, and 50 kD were detected when intimal extracts were subjected to immunoblotting. The present experiments demonstrated that the bands at 28.5 and 50 kD were nonspecifically stained bands composed of light and heavy chains of human IgG, respectively.

SAP is a decameric protein composed of identical subunits noncovalently associated in two pentameric rings interacting face to face.²⁹ The molecular weight of each subunit was about 25 000.³⁰ However, we found a value of 29 500 on SDS-PAGE. This value is similar to that reported by Painter et al¹³ and is believed to be an artifact of the use of 0.1% SDS. The saccharide components of the SAP molecule may also be a factor in the higher apparent molecular weight of SAP on SDS-PAGE.

The source of SAP in atherosclerotic intima awaits further investigation. There are two possibilities. One is that intimal SAP is produced locally. This possibility is supported by the report that human fibroblasts cultured in vitro in the absence of human serum can be stained with anti–amyloid P component antibodies.³¹ The other is that intimal SAP derives from plasma SAP, is incorporated into atherosclerotic lesions, and deposits there through ligand binding. SAP has been shown to bind to a variety of ligands such as fibronectin,⁶ C4b-binding protein,^{5 7} CRP,^{8 9} heparin,¹⁰ heparan sulfate and dermatan sulfate,¹¹ DNA,¹⁵ chromatin,^{16 17} complement components C1q^{20 21} and C3bi,²² and human IgG²⁴ in a calcium-dependent manner. Among these ligands, fibronectin,³² CRP,^{33 34} dermatan sulfate,³⁵ complement components, and IgG^{33 36} have been shown to locate at the atherosclerotic lesions, which may act as ligands of circulating SAP in vivo. In this study, we found that about 25% of SAP was extracted with EDTA-TBS, which suggests that this portion of SAP may be held there by calcium-dependent ligand(s) binding. Identifying the SAP ligand(s) may provide more insight into the mechanism of how SAP localizes in atherosclerotic lesions.

The presence of SAP in atherosclerotic lesions raises a number of intriguing possibilities. First, SAP may modulate a complement system at the site of lesions; it has been shown that SAP can activate a complement system.^{21 37} Second, SAP may modulate a coagulating system at the site of lesions. It has been widely accepted that thrombosis makes a great contribution to the development of atherosclerosis. SAP has been shown to act as an effective anticoagulant in the presence of a tiny amount of heparin in vitro.³⁸ Recently, we showed that SAP can form a complex with heparin in human serum.¹⁰ Third, as mentioned, SAP in atherosclerotic lesions may make complexes with the local components and thereby affect the function of these components.

SAP is one of the two pentraxins in human plasma; the other is CRP, which also has been reported to locate in atherosclerotic lesions.^{33 34} The two pentraxins show great homologies in amino acid sequence and molecular configuration.²⁹ Our findings suggest that the pentraxin family may play a role in the development of atherosclerosis.

	Acknowledgments

 
We are indebted to Dr M. Imakita of the Department of Pathology, National Cardiovascular Center, Osaka, Japan, for collecting samples.

Received August 24, 1994; accepted November 10, 1994.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Pepys MB, Dash AC, Markham RE, Thomas HC, Williams BD, Petrie A. Comparative clinical study of protein SAP (amyloid P component) and C-reactive protein in serum. Clin Exp Immunol. 1978;32:119-124. [Medline] [Order article via Infotrieve]

2. Pepys MB, Baltz ML, de Beer FC, Dyck RF, Holford S, Breathnach SM, Black MM, Tribe CR, Evans DJ, Feinstein A. Biology of serum amyloid P component. Ann N Y Acad Sci. 1982;389:286-298. [Medline] [Order article via Infotrieve]

3. Coria F, Castano E, Prelli F, Larrondo Lillo M, van Duinen S, Shelanski ML, Frangione B. Isolation and characterization of amyloid P component from Alzheimer's disease and other types of cerebral amyloidosis. Lab Invest. 1988;58:454-458. [Medline] [Order article via Infotrieve]

4. Dyck RF, Lockwood CM, Kershaw M, Mchugh N, Duance VC, Baltz ML, Pepys MB. Amyloid P-component is a constituent of normal human glomerular basement membrane. J Exp Med. 1980;152:1162-1174. [Abstract/Free Full Text]

5. Breathnach SM, Melrose SM, Bhogal B, de Beer FC, Dyck RF, Tennent G, Black MM, Pepys MB. Amyloid P component is located on elastic fibre microfibrils in normal human tissue. Nature. 1981;293:652-654. [Medline] [Order article via Infotrieve]

6. de Beer FC, Baltz ML, Holford S, Feinstein A, Pepys MB. Fibronectin and C4-binding protein are selectively bound by aggregated amyloid P component. J Exp Med. 1981;154:1134-1139. [Abstract/Free Full Text]

7. Schwalbe RA, Dahlbäck B, Nelsestuen GL. Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes. J Biol Chem. 1990;265: 21749-21757.

8. Swanson SJ, Christner RB, Mortensen RF. Human serum amyloid P-component (SAP) selectively binds to immobilized or bound forms of C-reactive protein (CRP). Biochim Biophys Acta. 1992;1160:309-316. [Medline] [Order article via Infotrieve]

9. Christner RB, Mortensen RF. Specificity of the binding interaction between human serum amyloid P-component and immobilized human C-reactive protein. J Biol Chem. 1994;269:9760-9766. [Abstract/Free Full Text]

10. Li X, Hatanaka K, Guo L, Kitamura Y, Yamamoto A. Binding of serum amyloid P component to heparin in human serum. Biochim Biophys Acta. 1994;1201:143-148. [Medline] [Order article via Infotrieve]

11. Hamazaki H. Ca²⁺-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate. J Biol Chem. 1987;262:1456-1460. [Abstract/Free Full Text]

12. Potempa LA, Kubak BM, Gewurz H. Effect of divalent metal ions and pH upon the binding reactivity of human serum amyloid P component, a C-reactive protein homologue, for zymosan: preferential reactivity in the presence of copper and acidic pH. J Biol Chem. 1985;260:12142-12147. [Abstract/Free Full Text]

13. Painter RH, De Escallon I, Massey A, Pinteric L, Stern SB. The structure and binding characteristics of serum amyloid protein (9.5S alpha 1-glycoprotein). Ann N Y Acad Sci. 1982;389:199-215. [Medline] [Order article via Infotrieve]

14. Hind CR, Collins PM, Renn D, Cook RB, Caspi D, Baltz ML, Pepys MB. Binding specificity of serum amyloid P component for the pyruvate acetal of galactose. J Exp Med. 1984;159:1058-1069. [Abstract/Free Full Text]

15. Pepys MB, Butler PJ. Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum. Biochem Biophys Res Commun. 1987;148:308-313. [Medline] [Order article via Infotrieve]

16. Breathnach SM, Kofler H, Sepp N, Ashworth J, Woodrow D, Pepys MB, Hintner H. Serum amyloid P component binds to cell nuclei in vitro and to in vivo deposits of extracellular chromatin in systemic lupus erythematosus. J Exp Med. 1989;170:1433-1438. [Abstract/Free Full Text]

17. Butler PJ, Tennent GA, Pepys MB. Pentraxin-chromatin interactions: serum amyloid P component specifically displaces H1-type histones and solubilizes native long chromatin. J Exp Med. 1990;172:13-18. [Abstract/Free Full Text]

18. Pepys MB, Dyck RF, de Beer FC, Skinner M, Cohen AS. Binding of serum amyloid P-component (SAP) by amyloid fibrils. Clin Exp Immunol. 1979;38:284-293. [Medline] [Order article via Infotrieve]

19. Kubak BM, Potempa LA, Anderson B, Mahklouf S, Venegas M, Gewurz H, Gewurz AT. Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins. Mol Immunol. 1988;25:851-858. [Medline] [Order article via Infotrieve]

20. Bristow CL, Boackle RJ. Evidence for the binding of human serum amyloid P component to C1q and Fab gamma. Mol Immunol. 1986;23:1045-1052. [Medline] [Order article via Infotrieve]

21. Ying SH, Gewurz AT, Jiang H, Gewurz H. Human serum amyloid P component oligomers bind and activate the classical complement pathway via residues 14-26 and 76-92 of the chain collagen-like region of C1q. J Immunol. 1993;150:169-176. [Abstract]

22. Hutchcraft CL, Gewurz H, Hansen B, Dyck RF, Pepys MB. Agglutination of complement-coated erythrocytes by serum amyloid P-component. J Immunol. 1981;126:1217-1219. [Abstract]

23. Loveless RW, Floyd O'Sullivan G, Raynes JG, Yuen CT, Feizi T. Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose, N-acetylgalactosamine and glucuronic acid. EMBO J. 1992;11:813-819. [Medline] [Order article via Infotrieve]

24. Brown MR, Anderson BE. Receptor-ligand interactions between serum amyloid P component and model soluble immune complexes. J Immunol. 1993;151:2087-2095. [Abstract]

25. Stollar D, Levine L. Two-dimensional immunodiffusion. In: Colowick SP, Kaplan NO, eds. Methods in Enzymology. New York, NY: Academic Press Inc; 1963;6:848-854.

26. Laurell CB. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem. 1966;15:46-50.

27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]

28. Li X, Hatanaka K, Guo L, Tsushima M, Kitamura Y, Yamamoto A. A sensitive peroxidase staining immunoblotting method for measuring total protein S in human plasma. Thromb Haemost. 1993;69:331-334. [Medline] [Order article via Infotrieve]

29. Emsley J, White HE, O'Hara BP, Oliva G, Srinivasan N, Tickle IJ, Blundell TL, Pepys MB, Wood SP. Structure of pentameric human serum amyloid P component. Nature. 1994;367:338-345. [Medline] [Order article via Infotrieve]

30. Pepys MB, Rademacher TW, Amatayakul-Chantler S, Williams P, Noble GE, Hutchinson WL, Hawkins PN, Nelson SR, Gallimore JR, Herbert J, Hutton T, Dwek RA. Human serum amyloid P component is an invariant constituent of amyloid deposits and has a uniquely homogeneous glycostructure. Proc Natl Acad Sci U S A. 1994;91:5602-5606. [Abstract/Free Full Text]

31. Spark EC, Shirahama T, Skinner M, Cohen AS. The identification of amyloid P-component (protein AP) in normal cultured fibroblasts. Lab Invest. 1978;38:556-559. [Medline] [Order article via Infotrieve]

32. Robert L, Jacob MP, Labat-Robert J. Cell-matrix interactions in the genesis of arteriosclerosis and atheroma. Ann N Y Acad Sci. 1992;673:331-341. [Medline] [Order article via Infotrieve]

33. Vlaicu R, Rus HG, Niculescu F, Cristea A. Immunoglobulins and complement components in human aortic atherosclerotic intima. Atherosclerosis. 1985;55:35-50.

34. Reynolds GD, Vance RP. C-reactive protein immunohistochemical localization in normal and atherosclerotic aortas. Arch Pathol Lab Med. 1987;111:265-269. [Medline] [Order article via Infotrieve]

35. Stevens RL, Colombo M, Gonzales JJ, Hollander W, Schmid K. Glycosaminoglycans of the human artery and their changes in atherosclerosis. J Clin Invest. 1976;58:470-481.

36. Hollander W, Colombo MA, Kirkpatrick B, Paddock J. Soluble proteins in the human atherosclerotic plaque. Atherosclerosis. 1979;34:391-405. [Medline] [Order article via Infotrieve]

37. Hicks PS, Saunero-Nava L, Du Clos TW, Mold C. Serum amyloid P component binds to histones and activates the classical complement pathway. J Immunol. 1992;149:3689-3694. [Abstract]

38. Williams EC, Huppert BJ, Asakura S. Neutralization of the anticoagulant effects of glycosaminoglycans by serum amyloid P component: comparison with other plasma and platelet proteins. J Lab Clin Med. 1992;120:159-167.[Medline] [Order article via Infotrieve]

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