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From www.bloodjournal.org by guest on June 14, 2017. For personal use only. Rapid Purification and Characterization of Human Platelet Glycoprotein V: The Amino Acid Sequence Contains Leucine-Rich Repetitive Modules as in Glycoprotein Ib By Takeshi Shimomura, Kingo Fujimura, Shuji Maehama, Motoyoshi Takemoto, Kenji Oda, Tetsuro Fujimoto, Rieko Oyama, Masami Suzuki, Keiko Ichihara-Tanaka, Koiti Titani, and Atsushi Kuramoto Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment. We have developed a rapid and simple method for isolation of t h e protein from platelet extracts using a combination of gel permeation, anion-exchange. and lectin affinity chromatography. The partial amino acid sequence was determined by analysis of peptides generated by digestion of t h e S-carboxyamidomethylated protein with Achromobacter protease I or cyanogen bromide. The sequence shows a remarkable periodicity of leucine residues, which is homologous to t h e consensus sequence of a highly diversified protein super- family with a common repetitive module. Thrombin cleava g e site w a s determined to be located at t h e C-terminal region of GPV by analysis of t h e products separated by sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, t h e existence of mucintype carbohydrate chains w a s indicated, as well as t h e existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis. From t h e s e data, we report a structural model of GPV t h a t is analogous to glycoprotein Ib. 0 1990 b y The American S o c i e t y of Hematology. G 1,200g for 15 minutes to sediment platelets, which were subsequently washed three times in 10 mmol/L HEPES, pH 7.6 (1 mmol/L in EDTA, 0.15 mol/L in NaCI). GPV was extracted from platelet plasma membrane by incubating platelets (2 x lo9platelets/ Bernard-Soulier syndrome, a rare inherited bleeding disormL) at 37OC for 20 hours in 10 mmol/L HEPES, pH 7.6 (1 mmol/L der, this glycoprotein is absent, as are GPIb and GPIX.3-6 in EDTA, 0.3 mol/L in NaCI, 1 mmol/L in p-aminophenylmethylPlatelets from these patients can be activated by thrombin, sulphonyl fluoride). The extract from two donors' platelets was but a long lag time is required to start the a g g r e g a t i ~ n . ~ brought to 40% saturation with solid ammonium sulfate, stirred for Binding of a-thrombin to a high or moderate affinity site 30 minutes and centrifuged, and then brought to 60% saturation located in the glycocalycin portion of G P I b is considered to with ammonium sulfate and treated similarly. The pellet precipibe a step necessary for platelet a~tivation.'.~However, tated in 60% saturation was taken up in a minimum volume (10 mL) of 50 mmol/L potassium phosphate, pH 6.8 (1 mmol/L in EDTA) measured equilibrium binding of a-thrombin to platelets (buffer A), dialyzed against the same buffer for about 4 hours at appears to be unrelated to platelet activation," and binding 4OC, concentrated with hygroscopic polymer (Aquacides, Calbioof thrombin inactivated by blocking the active site causes no chem, USA) in a dialyzing bag, and clarified by centrifuging at activation. GPV has been proposed to be a thrombin 10,OOOg for 30 minutes at 4OC for the subsequent purification receptor." However, rate or extent of GPV hydrolysis seems process. not to be correlated directly with platelet activation.I2 In We applied 500 pL of the dialyzed sample to a Superose 12 addition, blocking of GPV hydrolysis by anti-GPV antibodcolumn equilibrated with buffer A. The column was eluted with the ies does not prevent thrombin tim mu la ti on.'^ Therefore, the same buffer at a flow rate of 0.5 mL/min, and 0.5 mL fractions were role of GPV in platelet activation by thrombin remains to be collected. The whole sample was separated by multiple runs. The elucidated. GPV-rich fractionswere pooled and then directly applied to a Mono Q column equilibrated with buffer A. The passed-through fraction Knowledge of the structure of the GPV molecule is was collected and incubated with wheat germ agglutinin Sepharose essential for understanding the mechanisms involved in its 6MB (bed volume, 3 mL) for 3 hours at room temperature. The gels interaction with thrombin and in disorders related to this protein. In this regard, we established a rapid and simple purification procedure by a modification of the method From the Department of Internal Medicine. Research Institute reported by Berndt and Phillips,14 Zafar and W a l ~ , ' ~or .'~ for Nuclear Medicine and Biology, Hiroshima University. HiBienz et all3 and determined the partial amino acid sequence roshima: and the Division of Biomedical Polymer Science, Institute with the purified protein. for Comprehensive Medical Science, School of Medicine. Fujita Health University, Toyoake, Aichi, Japan. MATERIALS AND METHODS Submitted December 28.1989: accepted February 23,1990, Columns and buffers. Superose 12 HR 10/30 gel permeation Supported by Grants-in-Aid from the Japanese Ministry of and Mono Q HR 5/5 anion exchange columns, and wheat germ Education, Science and Culture for Scientific Research on Priority agglutinin Sepharose 6MB were purchased from Pharmacia (UppAreas to A.K. and K.T. and from the Fujita Health University. sala, Sweden). All the reagents used throughout the isolation Address reprint requests to Kingo Fujimura. MD, Department of procedure are of high performance liquid chromatography (HPLC) Internal Medicine, Research Institute for Nuclear Medicine and grade. Biology, Hiroshima University. Kasumi 1-2-3. Minami-ku, HiPurification of GPK Fresh healthy donor platelets were preroshima 734. Japan. pared using Hemonetics 30s (Haemonetics Corp, MA) and used The publication costs of this article were defrayed in part by page within 1 day of venipuncture. We obtained approximately 5 x 10" charge payment. This article must therefore be hereby marked platelets from a single donor. Donor platelets were centrifuged at "advertisement" in accordance with 18 U.S.C. section I734 solely to 120 g for 15 minutes at rmm temperature to remove the contamiindicate this fact. nated red blood cells. Acid-citrated dextrose (ACD) anticoagulant 0 1990 by The American Society of Hematologv. (1/6 volume) was added to platelet-rich plasma and centrifuged at 0006-4971/90/7512-0014$3.00/0 LYCOPROTEIN (GP) V is the only platelet membrane glycoprotein so far identified that is specifically hydrolyzed during activation by In patients with 8/&, Vol 7 5 , No 12 (June 15). 1990: pp 2349-2356 2349 From www.bloodjournal.org by guest on June 14, 2017. For personal use only. SHIMOMURA ET AL 2350 were packed in a column and washed with IO bed volumes (30 mL) of buffer A. The retained glycoproteins were eluted from this column with IO mL of buffer A containing 2.5% N-acetylglucosamine. The eluted proteins were dialyied against 50 mmol/L NH,HCO, and lyophilized. An aliquot at each purification step was digested with human thrombin (Green Cross. Japan) (5 U/mL) for I 5 minutes at 37°C and, along with an undigest aliquot. was subjected tosodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE; 7.5%) according to the method of Laemmli." Monitoring of GPV was based on susceptibility of GPV to thrombin. as assaKd using SDS-PAGE. The amount of protein at each purification step was quantitated by the method of Lowry et al." Isoelectric focusing was carried out using the lmmobiline system (Pharmacia). Amino acid sequcncc analysis o/GPV. The protein was d u c e d with dithiothreitol and S-carboxyamidomethylated (CMA) with iodoacetamide (Nacalai Tesque. Kyoto. Japan). The CMA-protein wasdigested with Achromobacfer protease I(a gift of Dr T. Masaki. Department or Agricultural Chemistry. lbaraki University, Mito. Japan) for 3 hours at 37% in 3.1 mol/L urea/SO mmol/L Tris-HCI. pH 9.0. using an enzyme:subslrate ratio of 1300 W (W /I .)I Cleavage at methionyl residues was carried out with 2% cyanogen bromide in 70% formic acid at room temperature as described by Gross." The native GPV was digested with thrombin in 100 mmol/L sodium phosphate. pH 7.4. at 37°C for 30 minutes. Peptides were partially separated by HPLC on TSK gel columns connected in series (G3000SW-G200OSW,each 7.5 x 600 mm.ToyoWa. Japan) in 6 mol/l. guanidine HCI/IO mmol/L sodium phosphate. pH 6.8. Further purification of peptides was achieved by reversed-phase HPLC on a column of Cosmosil 5C4-300 (4 x I50 mm. Nacalai Tesque) using an acetonitrile gradient made in dilute aqueous trifluoroacetic acid. Amino acid compositions were determined with a Hitachi L-8500 Amino Acid Analyizr (Tokyo, Japan) or by the phenylisothiocyanate Fdman degradations were done in an Applied Biosystems 470A Protein Sequencer connected to an on-line PTH Analym." colicin GPV -88' - Prtpararion ofrabbi1 antibodits. A female New Zealand white rabbit was immunized at 2-week intervals for 6 weeks with purified GPV (0.2 mg/mL) in phosphatebuffered saline, mixed ( I : I by volume) with complete Freund's adjuvant (Difco Laboratories. Detroit. MI). lgG was prepared from the rabbit antisera by treatment with DEAE-Sephacel. k c f i n 6/01analysis. Aliquots (5 pg) of GPV and GPV treated with thrombin as described above were incubated with or without Arfhrohacrer neuraminidase (5 U/mL; Bochringer Mannheim Biochemicals. Yamanouchi. Tokyo. Japan) in 50 mmol/L sodium acetate bulTer. pH 5.0. at 37OC for IS hours. They were then applied to SDS-PAGE (8%). blotted onto a nitrocellulose membrane and stained with horseradish peroxidase-conjugated peanut agglutinin (PNA; Seikagaku-Kogyo. Tokyo, Japan) following manufacturer's instructions. At the same time. the binding to the blot of the primary rabbit antibody against GPV was detected with '-"l-protein A. followed by autoradiography. RESULTS Puri/ication. At each purification step. existence of GPV was analyzed by applying an aliquot of the collected fraction to SDS-PAGE and could be recognized as a band stained with periodic acid-SchitT (PAS), which was moved to a smaller band by thrombin proteolysis. At the step of SuperOSE 12 gel permeation column chromatography, we obtained GPV-rich fractions at the fractions 26 through 28 (Fig I ) . After aninity chromatography on wheat germ agglutinin. SDS-PAGE showed a single band by silver staining. and its molecular weight (mol wt) was estimated at approximately 8 2 Kd. Purified GPV was hydrolyzed by thrombin to yield a 65 Kd protein. which was regarded as the large fragment of GPV produced by thrombin proteolysis. previously reported as GPVR' (Fig 2A). To examine homogeneity of purified GPV. FPLC rcverscd-phase chromatography (PepRPC HR 5 / 5 . Pharmacia) was used by an acetonitrile gradient in -200 -200 -116 - I 16 -92 qwg2 GPV- . Y [r -66 -45 O.OE 0 0 m a C IO x > 3 0 4 0 5 0 FRACTION NUMBER F b l . &pmr.tknofp(.t.kt extrm by gol porlnootkn FPLC. Fr.crionr from Supuoso 12 eobnns were w b j m e d to SDS-PAGE (7.6%)under reducing conditions end then to PAS rteining. The PAS-steinad 82 Kd bend (GPV) in fractions 26 through 28 (tho loft i n u t ) was ahifted to the 66 Kd bend IGPVfl) by thrombin proteolysis (the right inut: the right lane sample of oech fraction wm an eliquot of the fraction treated with thrombin os described in M e t r u l s and Methods). Glycocolicin was observed in frections 21 through 26. From www.bloodjournal.org by guest on June 14, 2017. For personal use only. PURIFICATION AND CHARACTERIZATIONOf GPV 2351 kDo -200 A GPV-o GPVfl - 8 - I I6 -92 * 6.55 -66 4 0 5.85 Fig 2. Purified GPV o l t r whoat g u m agglutinin (WGA).Qlinity chromatography. (A) SDS-PAGE (7.5%) Of m o d GPV undor roducingconditions. Lano 1. sitwr staining of GPV; l a n u 2 through 4. Coomasio brilliant bluo staining of GPV, GPV + thrombin. and thrombin. rospoctivoly. (61Iaooloctrk focusing of purifiod GPV by Immobilino system. -45 Ttmnnbin- I ’ 4-4. 0 3.50 1 2 3 4 dilute aqueous trifiuoroacetic acid. A single sharp peak was obtained at 45% of acetonitrile concentration (data not shown). Purified GPV showed multiple forms, ranging from 5.6 to 6.6on isoelectric focusing (Fig 2B). All these characteristics. including 8 2 Kd carbohydrate-rich protein, thrombin susceptibility. and pl. are concomitant to those previously reported“ for GPV. Yields of GPV at various purification steps are summarized in Table 1. Amino ucids composition. Amino acid composition of CMA-GPV is shown in Table 2 on a molar basis. The calculation was based on the data of Berndt and Phillips“ that polypeptide moiety comprises 52% of the total mass of GPV (mol wt of 82 Kd). corresponding to a protein with mol wt of 43 Kd. GPV appears to have some characteristic enrichment of leucine residues. Generution of peptides. A digest of the CMA-protein (about 2 nmol each) with Achromohuctcr protease I (Fig 3A. B. and C ) or cyanogen bromide (data not shown) was separated by size exclusion HPLC. and pooled fractions were further purified by reversed-phase HPLC. Two primary sets of peptides, KI-K7 and MI-M8 (cyanogen bromide fragments). were thus isolated and subjected to amino acid and sequence analyses. The digest of the intact protein with thrombin was separated in a similar manner as described above by a combination of size-exclusion (Fig 4) and reversed-phase HPLC (data not shown). Two fragments. Thl and Th2, were isolated. Fragment Thl seemed to be identical to the previously reported GPVfl on the basis of the apparent mol wt (69 Kd). Sequence analysis. Sequence analysis of the intact protein (100 pmol) yielded no phenylthiohydantoins (PTHs) in three cyclcs of Edman degradations. indicating that the amino terminus of the protein is blocked. The aminoTable 2. Amino A d d compodtknof CMA-GW CM-cya Asx Thr 7 31 16 28 37 34 30 16 sa Glx GC Ala Vd Met Ib 6 8 67 4 16 Lw Platdot a r a c t from 10’’plat~ Ammonium Mate fractionation (40% to 60%) supaoso 12 Mono0 WGA-S@WOM I5 TW Phl3 100 26 18 2 0.2 LW His 3 0.3 0.2 100 10 6.6 .Amounts of GPV prosant a dtrsrent isdatim stops w w roughly esfimated from dansitomotricscan8 of SDS-PAGE gals. tThe % y d d w w bwed on the a ” t of GPV prosent in W - r i c h fractions from !3mroae 12 column. V ~ 11 R O 2 22 27 Trp NO m ~ e s l n o b mdof l Gpv(mdwt. ~ ~ ~ 82 Kdl. which her apromincontont of 52% carnpondno to43 Kd. bawdon the data of &mdt md Phinii.’. m i : NO. not dotorminod. w From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 2352 SHIMOMURA ET AL K1 B I I - 0 - I 0 I I v I EC E (0 C 0 cu 8 cu Q I Y z % I C a 0 tl , JJ 20 I A 30 2 +-+- 3 4 c I 40 50 1-0 VOLUMEImll 2-0 3b RETENTION TIME lminl Fig 3. Separationof peptides generated by digestion of the CMA-protein with Achromobacferprotease 1. (A) Primary separation of the digest (2 nmol) on tandem TSK columns (G3000SW-G2000SW, each 7.5 x 600 mm) equilibrated in 6 mol/L guanidine HCI/10 mmollL sodium phosphate, pH 6.8, at a flow rate of 0.5 mL/min. Peptides were monitored at 230 nm (0.16 AUFS) and collected manually. (6 and C) Purification of pools 1 and 3 by reversed-phase HPLC on a Cosmosil 5C4-300column (4 x 150 mm) using a trifluoroacetic acid-an acetonitrile system. Purified peptides are identified by prefix K. 0.1 E C 8 cv a OW/ 20 30 40 50 VOLUMElmll Fig 4. Primary separation of thrombin digest of intact GPV by sizing HPLC, as described in Fig 3. Void volume (V0),total elution volume (Vt), and the elution positions of standard proteins with known molecular weights are shown by vertical arrows. terminal sequences of seven K peptides (Kl-7), eight M peptides (Ml-8), and two Th peptides (Thl and 2) are shown in Table 3. The link of K5 to K6 was established by analysis of M2. Peptide M8 was considered to be included within the sequence of K5. Peptides M3 and M4 were initially isolated as a mixture, but could be differentiated by referring to the sequence of Th2 that provided one of the two sequences. The cause of unexpected cleavage between Asp-Ser within the Th2 peptide is unknown. The amino terminus of T h l was blocked, clearly indicating that GPVfl (69 Kd fragment of GPV) was derived from the N-terminal region of GPV, and the thrombin cleavage site was located in a close proximity to the C-terminus of GPV. We have not yet obtained the entire sequence of GPV, but most of peptides ( K l , K2, K4, K5-K6, M1, M6, and M7) show a remarkable periodicity of leucine residues (Fig 5). This periodicity appears to be homologous to that observed with a group of proteins, including integral membrane or membrane-associated proteins (GP Ib,22-24 adenylate cyclase of yeast,2s toll gene product,26 ~haoptin,~’) proteoglycans or connective tissue proteins (PGI,28 PGII,29 fibrom~dulin,~~), and plasma proteins (LRP,3’ RA13*) (Fig 6). Aligning the peptides so as to correspond to the consensus sequences reported in this article made it possible to obtain highly homologous tandem repeats and to propose consensus sequence of GPV. GPV is likely to contain 10 or more of these repeats (Fig 5 ) . Lectin blot analysis. After treatment of purified GPV with neuraminidase, the apparent mol wt was reduced to 70 Kd (Fig 7) and it bound PNA, clearly showing the presence of sialic acid and mucin-type carbohydrate chain. Thrombin- From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 2353 PURIFICATION AND CHARACTERIZATION OF GPV Table 3. Amino Acid Sequence of Three Primary Sets of Peptide Fragments Generated by Digestion With Achromobacter Protease 1, Cyanogen Bromide, or Thrombin Peotide Sequence K1 K2 K3 K4 K5 K6 KJ LRQVSLRRNRLRALPRALFRILSSLESVQLD~NGLLGAQAKLERLLLHSNRLVSlDsgCVFRDAAQCSGGDVARlSALGLPT.LT LLDLSGN*LTHLPK MVLLEQLFLDHNALRGIDQNMFQK LVNLQELALNQNQLDFLPASFTNLENL- M1 GGLQEL~r RTQLRnPAAAFRrLSRLRMGVTLSPRI-a- M2 M3 M4 M5 M6 MJ M8 pqgafw FQKLVNLQELaLNQNQLDFLPAslfSS-EAPVHPALAP-S-EP-V Y NTPDReLAtYGGFN-blocked ISDSHlSAVAPGTFSDLlKLKTLRLsrNTGRGVLQSQSFSGTkVLQrVLL Th 1 Th2 N-blocked GPPRPAADSS-EAPVHPALAP-S-EP-V-AQD Three sets of peptides are prefixed by K, M, and Th. respectively. Sequences written in lower-case letters indicate tentative identification. Those not identified are shown by dashes. Asterisks indicate potential asparginelinked glycosylation sites. cleavaged fragment, GPVfl, seems to bind P N A less intensively than GPV after neuraminidase treatment, in comparison with the intensity of immunoblotted GPVfl, suggesting that the mucin-type carbohydrate chains mostly exist in the C-terminal region of GPV (Fig 8). DISCUSSION In the present study, we attempted to purify the platelet membrane glycoprotein V to homogeneity by modification of the method reported by Berndt and P h i l l i p ~ or ’ ~ Zafar and W a l ~ . ” The ~ ’ ~modification included use of FPLC system at the size-exclusion and anion-exchange chromatography steps and a new lectin-affinity chromatography at the final step, and eliminated the hydroxyapatite and cation-exchange chromatography steps. The previous method^'^“^ required many dialysis steps using various buffers before each chromatography. W e used only one buffer system through all chromatographies, and all the procedures can be easily done within 4 days. As to the yield, we can easily purify 200 pg of GPV from 10l2platelets. This high yield also seems to have an advantage over the method reported by Berndt and Phillips or Zafar and Walz (100 pg from 10l2 platelets), although the yield seems to be dependent on freshness of platelet samples (data not shown) as reported by Berndt and Phillips.I4 The apparent molecular weight, thrombin sensitivity, PI, and amino acid composition of GPV purified in the present study are very similar to those of GPV isolated by the method previously reported. Bienz et all3 have purified GPVs, a fragment of GPV cleaved by calpain, from platelets sonicated in a buffer containing calcium ion, using wheat germ agglutinin and anion-exchange chromatography.” GPVs seems to have almost the same mol wt and PI as GPV purified in the present study. In our purification, there remains a possibility that purified GPV is a fragment generated by calpain. However, in order to activate calpain, it appears that their method consumes a half amount of platelets. Therefore, our method should have an advantage over theirs in terms of the final yield. Although we have not yet obtained the entire sequence of GPV, most of the peptides analyzed contained homologous leucine-rich repetitive sequences, which are observed with a number of proteins. Schneider et have proposed that these repetitive modules should be common structural features of a novel protein superfamily whose members exert their functions by highly specific protein-protein interactions. For example, PGII and fibromodulin bind to a specific type of collagen and cause inhibitory effects on collagen fibril f~rmation.~’-~’ RAI binds to angiogenin or RNase and abolishes both the angiogenic and ribonucleolytic a ~ t i v i t i e s . ~ ~ Yeast adenylate cyclase has been suggested to bind to the cytosolic side of the cell membrane with the repeat^.^' Drosophila toll gene product plays a role in embryonic dorsal-ventral patterning, hypothetically using the extracellular repeats for the cell adhesion.26 Drosophila chaoptin is an intercellular cytoadhesive protein for photoreceptor cells.27 K1 K L R Q R A L O R N a S S I E s v s 9’ V Q K2 K4 K5/6 Fig 5. Internal homology observed with peptides of GPV. Partial amino acid sequences so far determined are tentatively aligned to achieve maximum homology. Highly conserved residues (identified with a more than 40% frequency) are boxed. The tentative consensus sequence is indicated at the bottom; X is used where no clear homology is observed. K G L L G A Q A K L E R S G K L K K M V L L E Q L A M1 M6 M7 Tentative consensus L W L V M I L R D U J T S S N L D R R S S O M T P H T G Q ~ R T R L I S A V A R G V D Q S Q S l F ( S G T K V u Q R L X L X X N X L X X L P X X X F X X L X X L X X From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 2354 ilj,s SHIMOMURA ET AL GP V L X X L X X L P X X X r x x G P Ibd L X X L S X G P IbB L V N GP I X L L L A N N S LRG L D L O G N X PG I L X L X N N K L X X PG I1 L X L X N N K 4 X X H K X L X X FM L X L X H N Q L X X RAI L X X L X E YAdCY L X L S X N X L X X Toll L X L X X N X L X X Chaoptin L, I) , O X 2 X L X X "["I" L R T L Q T L X X Fig 6. Consonsus soquo" of OPlba//3, GPlX and othw proteins. LRO, Ieudnarkh u2-gfycoprotein: W I. protoogtycnn biglycan: PO II. proteogtycan dscarin: FM. 69 Kd connective t i u u a protein fibromodulin: RAI. ribonudwselangiogonin inhibitor module A: yAdcy, v s t adenylate cyclase: Toll, Drosophila Toll gene product: Chaoptin. Drosophila cytoadhesive protein of R coils. The Consensus sequences shown are defined by residues appearing with a more than 40% frequency. Lowercase a and b indicate hydrophobic amino acid residues (1. V. M. L, F. and AI and hydroxy amino acid residues IT and S).respectively. Boxes show identities or consorvotive repbcements within homologous amino acid residues: 1, V, M, L. F, and A, or T and S. and indicate deletion and gap, respectiveiy. - Neuraminidase - + -+ - + - + kDa 11697- F i g 7 . LoctInbbtano)y.h of GPV. Aliquotr of p u r h d OPV 66' 43- -1 31 2 3 4 5 6 7 8 llanos 1 through 4) and OPV treated with thrombin llanos 6 through 8 )were incubeted with ( + ) or without ( - 1 neuraminic k ~ WbjWed , t o SDS-PAGE (8%).and blotted onto nitrocellulose membranr. The membranes were either incubated with rabbit anti-GPV antibody followed by "%protein A treatment and autoradiographic imaging (lanes 1, 2, 6. and 8). or stained with horseradishperoxidase-conjugeted PNA using 4chloro-1-naphthol as a substrate lbnes 3. 4. 7, and 8). Molecular weights - of merker proteins are indicated on the lamargin. From www.bloodjournal.org by guest on June 14, 2017. For personal use only. 2355 PURIFICATION AND CHARACTERIZATION OF GPV Thrombin N IIr m l t,, ,# C Fig 8. Schematic representation of the structural organization of GPV. It remains to be clarified whether or not GPV has a transmembrane domain. Aspargine-linked and mucin-type carbohydrate chains are shown by Y and I shape, respectively. Five aspargine-linked glycosylation sites have been identified so far by protein sequence analysis. Their numbers and locations are tentative. The leucine-rich repeats are shown by boxes. Arrow shows thrombin cleavage site. The N-terminal tryptic fragment of GPIba contains seven of these repeats, which appear to contain the binding sites for von Willebrand factor and t h r ~ m b i n . ~However, '.~~ Tanoue et a134have recently reported that the precise binding sites are located in the C-terminal region of the fragment without the repeats. GPV may also bind to other macromolecules. GPlbP, a major protein phosphorylated under the conditions that increase cytoplasmic concentration of CAMP in platelet^,^^.^' is linked to GPIba by disulfide bonds' and contains one leucine-rich module.24 GPIX forms a complex with GPIb38-40 and has been recently reported to contain one such m o d ~ l e . Therefore, ~' the platelet membrane glycoproteins, which are simultaneously defective in Bernard-Soulier syndrome, all contain more or less the leucine-rich modules. They must have evolved from the same ancestral gene and have some correlations in their functions. The apparent molecular weight of cyanogen bromide fragment M1 was estimated to be 36 Kd (almost half the mass of GPV) from the elution volume by size-exclusion chromatography (data not shown). The fragment appears to be derived from the C-terminal region of GPV on the basis of the amino acid composition, which contains no homoserine and which contains only one lysyl residue. Therefore, other cyanogen bromide fragments and most K peptides containing more or less the leucine-rich repeats were probably derived from the amino-terminal half of GPV. The sequence of fragment Th2 is very similar to that reported as the Nterminal sequence of GPVfl by Zafar and Walz.I6 However, our data clearly indicate that the N-terminus of the large fragment ( T h l ) generated by cleavage with thrombin is blocked. We assumed, therefore, that thrombin cleavage site is located in a close proximity to the C-terminus of GPV. From the data described herein, we propose the structural organization of GPV as shown in Fig 8, which shows a remarkable similarity to the structure of GPIbu reported by Titani et a122and Lopez et aLz3These findings should be useful for understanding the pathogenesis of Bernard-Soulier syndrome and also the mechanism of thrombin interaction with platelets. ACKNOWLEDGMENT We thank H. Sumida, Y. Oto, K. Yamamoto, S. Ohishi, and H. Ohta for their excellent secretarial assistance and typing of the manuscript. REFERENCES 1 . Phillips DR, Agin PP: Platelet plasma membrane glycoproteins: Identification of a proteolytic substrate for thrombin. Biochem Biophys Res Commun 75:940,1977 2. Mosher DF, Vaheri A, Choate JJ, Gahmberg CG: Action of thrombin on surface glycoproteins of human platelets. Blood 53:437, 1979 3. Nurden AT, Dupuis D, Kunicki TJ, Caen JP: Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Clin Invest 67:1431, 1981 4. Clemetson KJ, McGregor JL, James E, Dechavanne M, Luscher EF: Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis. J Clin Invest 70:304, 1982 5. 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For personal use only. 1990 75: 2349-2356 Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib T Shimomura, K Fujimura, S Maehama, M Takemoto, K Oda, T Fujimoto, R Oyama, M Suzuki, K Ichihara-Tanaka and K Titani Updated information and services can be found at: http://www.bloodjournal.org/content/75/12/2349.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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