Molecular and Functional Differences Induced in Thrombospondin-1 by the Single Nucleotide Polymorphism Associated with the Risk of Premature, Familial Myocardial Infarction
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m311090200
ISSN1083-351X
AutoresNatalya V. Narizhneva, V Byers-Ward, Martin Quinn, Frank Zidar, Edward F. Plow, Eric J. Topol, Tatiana V. Byzova,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoA serine (Ser-700) amino acid rather than an asparagine (Asn-700) at residue 700 of thrombospondin-1 has been linked to an increased risk for development of premature, familial heart attacks. We now have identified both functional and structural differences between the Ser-700 and Asn-700 thrombospondin-1 variants. The Ser-700 variant increased the rate and extent of platelet aggregation and showed increased surface expression on platelets compared with the Asn-700 variant. These differences could be ascribed to an enhanced interaction of the Ser-700 variant with fibrinogen on the platelet surface and are consistent with a prothrombotic phenotype in Ser-700 individuals. The Ser-700 variant thrombospondin-1 was conformationally more labile than the Asn-700 variant as demonstrated by increased susceptibility to proteolytic digestion and enhanced susceptibility to unfolding by denaturants. These data suggest a potential molecular and cellular basis for a genetic risk factor associated with early onset myocardial infarction. A serine (Ser-700) amino acid rather than an asparagine (Asn-700) at residue 700 of thrombospondin-1 has been linked to an increased risk for development of premature, familial heart attacks. We now have identified both functional and structural differences between the Ser-700 and Asn-700 thrombospondin-1 variants. The Ser-700 variant increased the rate and extent of platelet aggregation and showed increased surface expression on platelets compared with the Asn-700 variant. These differences could be ascribed to an enhanced interaction of the Ser-700 variant with fibrinogen on the platelet surface and are consistent with a prothrombotic phenotype in Ser-700 individuals. The Ser-700 variant thrombospondin-1 was conformationally more labile than the Asn-700 variant as demonstrated by increased susceptibility to proteolytic digestion and enhanced susceptibility to unfolding by denaturants. These data suggest a potential molecular and cellular basis for a genetic risk factor associated with early onset myocardial infarction. Genome-wide scan and high throughput single nucleotide polymorphism (SNP) 1The abbreviations used are: SNP, single nucleotide polymorphism; MI, myocardial infarction; r, recombinant; TSP, thrombospondin protein; PRP, platelet-rich plasma; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; GdnHCl, guanidine HCl. 1The abbreviations used are: SNP, single nucleotide polymorphism; MI, myocardial infarction; r, recombinant; TSP, thrombospondin protein; PRP, platelet-rich plasma; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; GdnHCl, guanidine HCl. association studies have begun to identify specific genes and polymorphisms that underlie complex diseases such as acute myocardial infarction (MI) (1Weiss E.J. Bray P.F. Tayback M. Schulman S.P. Kickler T.S. Becker L.C. Weiss J.L. Gerstenblith G. Goldschmidt-Clermont P.J. N. Engl. J. 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This rarity leads to a low likelihood of successful replication in small studies (7Yamada Y. Izawa H. Ichihara S. Takatsu F. Ishihara H. Hirayama H. Sone T. Tanaka M. Yokota M. N. Engl. J. Med. 2004; 347: 1916-1923Crossref Scopus (602) Google Scholar, 8Boekholdt S.M. Trip M.D. Peters R.J. Engelen M. Boer J.M. Feskens E.J. Zwinderman A.H. Kastelein J.J. Reitsma P.H. Arterioscler. Thromb. Vasc. Biol. 2002; 22: e24-e27Crossref PubMed Google Scholar), but in one subsequent preliminary report (9Peyvandi F. Palla R. Ardissino D. Foco L. Bernardinelli L. Bauer K. Mannucci P.M. Blood. 2003; 102 (Abstr. 1129): 11Google Scholar) this disease association of the Ser-700 variant was corroborated. TSP-1 is synthesized by a variety of cells and is a component of the extracellular matrix (10Adams J.C. Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (99) Google Scholar). It is stored within secretory granules of platelets (11Hagen I. Biochim. Biophys. 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Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar), the region harboring the N700S SNP is devoid of such known functional assignments with respect to platelet and other vascular cell responses (14Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (323) Google Scholar, 16Lawler J. Curr. Opin. Cell Biol. 2000; 12: 634-640Crossref PubMed Scopus (372) Google Scholar). Moreover, TSP-1 has not been previously regarded as a significant contributor to thrombotic diseases. Although TSP-1 has been identified as a constituent of human atherosclerotic plaques (17Riessen R. Kearney M. Lawler J. Isner J.M. Am. Heart J. 1998; 135: 357-364Crossref PubMed Scopus (92) Google Scholar), mice in which the TSP-1 gene has been inactivated do not exhibit an overt hemostatic phenotype (18Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Investig. 1998; 101: 982-992Crossref PubMed Scopus (382) Google Scholar). The goal of the current study was to determine how the structure and function of N700S SNP in TSP-1 are altered and, thereby, to identify potential mechanisms by which this substitution predisposes individuals to familial, premature MI. Protein Purification and Radioiodination—Asn-700 and Ser-700 variants of TSP-1 were purified from the platelets of individual donors homozygous for each variant, and samples from at least three different donors of each genotype were used. The TSP-1 from these donors was isolated according to methods described previously (13Lawler J.W. Slayter H.S. Coligan J.E. J. Biol. Chem. 1978; 253: 8609-8616Abstract Full Text PDF PubMed Google Scholar) but using 0.5-1 unit of blood as starting material. Platelets were washed by centrifugation in the presence of prostaglandin E1 and activated with thrombin to induce secretion. TSP-1 in the releasate was bound onto a heparin-Sepharose column (Amersham Biosciences) and eluted with an NaCl gradient (see Fig. 3). The TSP-1 variants were radioiodinated with 125I using a modified chloramine-T procedure (19Wolff R. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1986; 261: 6840-6846Abstract Full Text PDF PubMed Google Scholar). The purity of the TSP-1 preparations was assessed by SDS-PAGE (see under "Results"). In addition, the presence of active transforming growth factor-β1, which is bound and activated by TSP-1 (20Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 21Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar), was assessed as a likely contaminant with biological activity using a commercially available enzyme-linked immunosorbent assay (Promega). The levels of the growth factor were: Asn-700 rTSP-1, 1 pg/μg protein; Ser-700 rTSP-1, <0.5 pg/μg; Asn-700 TSP-1, 95% pure, based on SDS-PAGE, as shown in Fig. 4b) enhanced the aggregation response to 80% of maximum (Fig. 1a). Addition of the same amount (in moles) of fibrinogen or bovine serum albumin failed to enhance the response. Observed microscopically, TSP-1 increased the size of the platelet aggregates by 3-4-fold. This effect was evident when washed platelets were stimulated (20 μm ADP), treated with paraformaldehyde to fix the fibrinogen receptor, αIIbβ3, in an activated state (25Du X.P. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (417) Google Scholar), and naturally occurring TSP-1 was added. Aggregation was more extensive when induced by fibrinogen with TSP-1 than when TSP-1 was omitted (Fig. 1b). The use of fixed platelets excludes an effect of TSP-1 or possible contaminants on platelet activation and indicates that TSP-1 exerts an influence on the post-stimulation stage of platelet-platelet cohesion. With conditions established under which TSP-1 exerted a notable influence on platelet aggregation, we analyzed the effect of isolated Asn-700 and Ser-700 TSP-1 from the platelets of individual donors. Under the conditions used, maximum aggregation of stimulated, fixed platelets responding to addition of fibrinogen was 23.4 ± 1.3% with Asn-700 TSP-1 (Fig. 1c) compared with 34.6 ± 3.9% and 32.8 ± 2.8% with Ser-700 TSP-1 from the two different donors (p = 0.018 and 0.03, respectively, compared with the Asn-700 variant). Of note, platelet aggregation in the presence of Ser-700 TSP-1 began immediately on stirring, whereas a 40-60-s lag was observed with the Asn-700 TSP-1 (Fig. 1d). These data indicate that Ser-700 TSP-1 enhances platelet aggregation, consistent with the prothrombotic phenotype of these individuals.Fig. 1TSP-1 variants and platelet aggregation. a, platelet aggregation in PRP in the absence (curve 1) or presence (curve 2) of rTSP-1 (Asn-700) was induced by 20 μm ADP. PRP was preincubated with 10 μg/ml rTSP-1 for 10 min. Aggregation was monitored by the decrease of light transmission, with the minimum and maximum set with platelet-poor and platelet-rich plasma, respectively. b, micrographs show aggregates of ADP-stimulated platelets fixed with 0.5% paraformaldehyde, washed, and then aggregated by addition of fibrinogen (300 nm) in the absence (panel 1) or presence (panel 2) of purified rTSP-1 at 100 μm. c, aggregation of fixed platelets stimulated by 10 μm ADP/20 μm epinephrine plus 20 μg/ml fibrinogen in the absence or presence of 10 μg/ml TSP-1 isolated from one Asn-700 donor (black bar) or from two different Ser-700 donors (striped bars). The maximum extent of aggregation, recorded at 5 min after addition of ADP/epinephrine, is presented as the means ± S.D. of five separate experiments using platelets from five Asn-700 donors. d, representative aggregation curves of platelets prepared as in c in the presence of Ser-700 (curve 2) and Asn-700 (curve 1) TSP-1 (10 μg/ml).View Large Image Figure ViewerDownload (PPT) When it is secreted from platelet α granules, a significant portion of the TSP-1 becomes associated with the surface of stimulated platelets (30Aiken M.L. Ginsberg M.H. Plow E.F. Semin. Thromb. Hemostasis. 1987; 13: 307-316Crossref PubMed Scopus (17) Google Scholar, 31Legrand C. Thibert V. Dubernard V. Begault B. Lawler J. Blood. 1992; 79: 1995-2003Crossref PubMed Google Scholar). We assessed basal and thrombin-stimulated TSP-1 surface expression by FACS, using a TSPI-1 mAb that was verified to react equally with Asn-700 and Ser-700 TSP-1 and platelets from three Ser-700 and three Asn-700 donors. Little Asn-700 TSP-1 was detected on the surface of resting platelets, but stimulation of the cells with thrombin, a strong agonist, resulted in a substantial increase in surface-bound TSP-1. Under identical conditions, the signal with Ser-700 TSP-1 platelets was 2.4-fold higher (Fig. 2a), and even basal Ser-700 TSP-1 expression was higher. These differences were observed despite the Ser-700 donor patients' daily dosage of aspirin, administered to blunt platelet reactivity. The observed differences in TSP-1 surface expression were not the results of different amounts of the TSP-1 variants released from or within the platelets as determined by enzyme-linked immunosorbent assay (data not shown). Furthermore, exposure of P-selectin, another α granule release marker (32Stenberg P.E. McEver R.P. Shuman M.A. Jacques Y.V. Bainton D.F. J. Cell Biol. 1985; 101: 880-886Crossref PubMed Scopus (736) Google Scholar), was not elevated in the Ser-700 individuals, with or without platelet stimulation (Fig. 2b). In fact, P-selectin expression was slightly lower with the Ser-700 versus the Asn-700 platelets and was similar to that obtained when an Asn-700 individual ingested aspirin for 3 consecutive days prior to the analysis (Fig. 2b). To directly assess whether the differences in surface expression of the variants arise from differential interaction between the TSP-1s and platelets, we measured the binding of naturally occurring and recombinant TSP-1 variants to platelets isolated from healthy donors by FACS analysis. TSP-1 variants were added to platelets stimulated with 10 μm ADP and fixed with paraformaldehyde to limit the measured reaction to the direct binding of the TSP-1 variants and to avoid potential platelet stimulation by the TSP-1 preparations (19Wolff R. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1986; 261: 6840-6846Abstract Full Text PDF PubMed Google Scholar). After a 40-min incubation of the ADP-fixed platelets with the TSP-1 variants, the amount of bound TSP-1 was assessed with anti-TSP-1 labeled with FITC. Under identical conditions, the Ser-700 TSP-1 variant exhibited significantly increased binding to platelets compared with the Asn-700 variant (Fig. 2c). Significant differences (p = 0.05) were observed also in the interaction of the recombinant TSP-1 variants with platelets, although the extent of the difference was less dramatic than with the naturally occurring proteins (Fig. 2d). As an independent approach to examining the interaction of the TSP-1 variants with platelets, we iodinated the purified, naturally occurring variants of TSP-1 and measured their direct binding to platelets. Under the conditions used, minimal interaction was observed unless fibrinogen was present (Fig. 3a). This finding supports previous data showing that fibrinogen bound to integrin αIIbβ3 provides a primary pathway for TSP-1 binding to platelets (33Bonnefoy A. Hantgan R. Legrand C. Frojmovic M.M. J. Biol. Chem. 2001; 276: 5605-5612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), a reflection of the high affinity of TSP-1 for fibrinogen. In the presence of fibrinogen (300 nm, ∼Kd for αIIbβ3), the amount of Asn-700 TSP-1 bound to platelets increased by 8-fold, whereas the amount of bound Ser-700 TSP-1 increased by 16-fold. The interaction between TSP-1 and fibrinogen-occupied platelets was almost completely inhibited by heparin (Fig. 3a), which binds with high affinity to the N-terminal region of TSP-1 (34Legrand C. Morandi V. Mendelovitz S. Shaked H. Hartman J.R. Panet A. Arterioscler. Thromb. 1994; 14: 1784-1791Crossref PubMed Google Scholar). 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Thus, our data showing that Ser-700 TSP-1 has increased binding to fibrinogen provides a potential explanation as to why this variant supports enhanced platelet aggregation and increases thrombus burden. In our experiments we used TSP-1 purified from platelets as well as the recombinant TSP-1 variants. The quality of TSP-1 preparations was assessed by SDS-PAGE immediately after purification, under nonreducing (Fig. 4a) and reducing (Fig. 4b) conditions with subsequent Western blotting (Fig. 4c). Only fractions with minimum contamination (18Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Investig. 1998; 101: 982-992Crossref PubMed Scopus (382) Google Scholar, 19Wolff R. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1986; 261: 6840-6846Abstract Full Text PDF PubMed Google Scholar, 20Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 21Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. 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Chem. 1985; 260: 3762-3772Abstract Full Text PDF PubMed Google Scholar, 36Chen H. Herndon M.E. Lawler J. Matrix Biol. 2000; 19: 597-614Crossref PubMed Scopus (340) Google Scholar) were used in experiments. Because it still was possible that TSP-1 preparations contained platelet activators that are effective at the relatively low concentrations, we performed the majority of experiments using platelets fixed with paraformaldehyde. The isolation procedure we used to purify TSP-1 takes advantage of a binding affinity to heparin (35Lawler J. Derick L.H. Connolly J.E. Chen J.H. Chao F.C. J. Biol. Chem. 1985; 260: 3762-3772Abstract Full Text PDF PubMed Google Scholar, 36Chen H. Herndon M.E. Lawler J. Matrix Biol. 2000; 19: 597-614Crossref PubMed Scopus (340) Google Scholar), but Asn-700 and Ser-700 TSP-1 behaved differently when eluted from heparin-Sepharose columns (Fig. 4d). The Ser-700 TSP-1 required substantially higher NaCl concentrations to elute from the heparin columns than Asn-700 TSP-1. For the Ser-700 TSP-1 from three different individuals, the concentration of NaCl that eluted 50% of the bound TSP-1 was 650 mm compared with 470 mm for Asn-700 TSP-1. Nevertheless, the products that were isolated were similar as shown by Western blotting (Fig. 4c). Differences in heparin binding may reflect overall changes in the organization of the variants, which, in turn, might alter protease susceptibility, and we explored this possibility. As evident from Fig. 5a, cleavage fragments of Asn-700 TSP-1, detected by blotting with a 6G6 mAb against TSP-1, were evident only after 60 min of digestion with trypsin. In contrast, substantial degradation of the Ser-700 TSP-1 was observed at 15-30 min. The intensities of the immunoblotting bands below 180 kDa were quantified by densitometry. The amounts of product at 60 and 120 min of trypsin digestion were 10- and 4-fold higher with Ser-700 than Asn-700 TSP-1, respectively (Fig. 5b). The intensities of these bands with the Ser-700 variants isolated from two different donors were similar. Based on the differences in heparin affinity and trypsin sensitivity, we implemented approaches to compare the conformations of the two TSP-1 molecules. The intrinsic tryptophan fluorescence spectra of the TSP-1 proteins (Fig. 6a) were identical with the broad maximum at 345 nm, indicative of a large number of tryptophan residues (22 are present in TSP-1) in environments differing slightly in exposure to solvent (37Stryer L. Science. 1968; 162: 526-533Crossref PubMed Scopus (620) Google Scholar); however, as the two purified TSP-1s were unfolded with denaturants guanidine HCl (GdnHCl) or urea, differences in their intrinsic fluorescence at the 345 -nm maximum became evident (Fig. 6, b and c). Asn-700 TSP-1 retained its fluorescence signal at low concentrations of the denaturants (up to 1 m GdnHCl and 3 m urea), whereas the Ser-700 TSP-1 began to lose fluorescent intensity at very low concentrations of the denaturants. These differences suggest that the conformation of Ser-700 TSP-1 is more labile. This interpretation is supported by CD measurements (Fig. 6d). In the absence of GdnHCl, the far UV CD spectra of the two TSP-1 molecules were complex, indicating multiple secondary structural elements (38Adler A.J. Greenfield N.J. Fasman G.D. Methods Enzymol. 1973; 27: 675-735Crossref PubMed Scopus (561) Google Scholar), but were essentially the same (Fig. 6d). At a high concentration (3 m)of the denaturant, the spectra of the two proteins were
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