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Cardiovascular Research 2006 71(3):486-495; doi:10.1016/j.cardiores.2006.04.010
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Copyright © 2006, European Society of Cardiology

Localization and characterization of a novel secreted protein SCUBE1 in human platelets

Cheng-Fen Tua, Yueh-Hsing Sua, Ya-Ni Huanga,c, Ming-Tzu Tsaia, Li-Tzu Lib, Yuh-Lien Chend, Chien-Jui Chenge, Dao-Fu Daif,g and Ruey-Bing Yanga,h,*

aInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
bInstitute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
cGraduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
dInstitute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
eGraduate Institute of Medical Sciences and Department of Pathology, School of Medicine, Taipei Medical University, Taipei, Taiwan
fSection of Cardiology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
gSection of Cardiology, Department of Internal Medicine, Taoyuan General Hospital Department of Health Executive Yuan, Taoyuan, Taiwan
hInstitute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan

* Corresponding author. Institute of Biomedical Sciences, Academia Sinica, 128, Academia Road, Sec. 2, Taipei 11529, Taiwan. Tel.: +886 2 2652 3943; fax: +886 2 2785 8847. Email address: rbyang{at}ibms.sinica.edu.tw

Received 10 January 2006; revised 22 March 2006; accepted 12 April 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1.

Methods and results Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the {alpha}-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively.

Conclusion These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.

KEYWORDS Atherosclerosis; Electron microscopy; Extracellular matrix; Histo(patho)logy; Platelets

This article is referred to in the Editorial by S. Lindemann and M. Gawaz (pages 414–415) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
We have previously utilized a combination of comprehensive library sequencing and genome-wide microarray expression profiling to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells (HUVECs) [1,2]. One gene family identified by this approach encoded secreted proteins harboring a signal peptide at the amino terminus followed by 9 copies of EGF-like repeats and one CUB domain at the carboxyl terminus. Therefore, these genes were named SCUBE for signal peptide-CUB-EGF-like domain containing proteins [2,3]. In addition, a spacer region containing multiple potential N-linked glycosylation sites is located between the EGF-like repeats and the CUB domain in these proteins. To date, 3 distinct gene members have been identified and designated SCUBE1 to 3, in the order of their discoveries [2,3]. Overexpression of SCUBE proteins in human embryonic kidney (HEK)-293T cells resulted in secreted glycoproteins that can form oligomers, tethered to the cell surface [2,3].

The SCUBE genes are present in the human, mouse and zebrafish, which suggests that these proteins are evolutionarily conserved and may have critical biological functions. In the mouse, Scube1 and Scube2 are expressed in a variety of embryonic tissues [4,5]; thus, the Scube gene family may play critical roles during development [4,5]. Consistent with this finding, recent genetic studies have demonstrated that a number of nonsense mutations in the SCUBE2 gene causes developmental defects in zebrafish [6,7]. However, the molecular and biochemical mechanisms underlying the function of SCUBE2 are poorly understood. In addition to its developmental expression, SCUBE1 mRNA appears to be expressed in the endothelium in the adult [2]. However, little is known about the protein expression and the biological functions of mammalian SCUBE1 in adult tissues.

In the present study, we have produced a number of protein reagents, including monoclonal antibodies (mAb) and recombinant protein fragments, to further explore the protein localization and function of SCUBE1 in the vascular system. Although its mRNA was originally found in the endothelium [2], SCUBE1 protein is predominantly expressed in platelet and could be proteolytically modified to release a smaller, active fragment to be associated with thrombus and localized with the subendothelial matrices in atherosclerotic plaque. Furthermore, molecular and biochemical analyses suggest that SCUBE1 is a novel adhesive molecule that mediates platelet–matrix interaction and ristocetin-induced platelet agglutination.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Studies of animals and human subjects
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with the principles outlined in the Declaration of Helsinki [8].

2.2 Preparation of GST fusion proteins
Fusion proteins with glutathione S-transferase (GST) were constructed in the expression vector pGEX-4T (Amersham Bioscience). PCR fragments encoding SCUBE1 amino acids 26–285, 157–412 or 789–988 were ligated in the EcoRI/XhoI sites in the vector to generate GST-E1–6, E4–9 or -CUB fusion protein, respectively. Fusion proteins were expressed in E. coli and purified from the soluble fraction of the bacterial lysate with glutathione-Sepharose beads according to the manufacturer's instructions (Amersham).

2.3 Generation of anti-SCUBE1 monoclonal antibodies (mAb)
Splenocytes from BALB/c mice immunized with recombinant GST-CUB protein were fused with P3X myeloma cells to produce hybridomas. Hybridomas positive for SCUBE1 but not to GST, hSCUBE2 or hSCUBE3 were identified and cloned. Ascites fluids were prepared, and purified IgG was obtained using protein G chromatography. Three independent clones (mAb 33, 701, 712) were obtained. These mAbs could specifically recognize the recombinant full-length SCUBE1 protein expressed in human embryonic kidney (HEK)-293T cells but not SCUBE2 or SCUBE3 by Western blot or flow cytometrical analyses, respectively. On the basis of its higher specificity, the mAb 701 (IgG1 isotype) was used throughout this study.

2.4 Preparation of washed platelets
Peripheral blood was drawn from healthy donors into 1/6 volume of acid–citrate–dextrose anticoagulant (85 mM sodium citrate, 111 mM dextrose and 71 mM citric acid), then supplemented with PGI2 (50 ng/ml) and apyrase (0.67 U/ml). Platelet-rich plasma was prepared by centrifugation at 160 g for 20 min at ambient temperature. Platelets were sedimented by centrifugation at 1100 g for 10 min, washed once with CGS buffer (13 mM sodium citrate, 30 mM glucose and 120 mM sodium chloride, pH 7.0) and resuspended in Tyrode's-HEPES buffer (138 mM sodium chloride, 2.9 mM potassium chloride, 12 mM sodium bicarbonate, 5.5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4) and adjusted to 1 x 109/ml.

2.5 Quantitative real-time RT-PCR (TaqMan) analysis
The assays were performed by use of the Applied Biosystems PRISM 7700 sequence detection system with a panel of platelet cDNAs derived from healthy donors. Normalization involved use of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA levels as controls in parallel TaqMan reactions. The thermal cycler conditions were as follows: hold for 2 min at 50 °C and 10 min at 95 °C, followed by two-step PCR for 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min. The forward and reverse primers and a fluorescence-labeled probe were as follows: SCUBE1 forward, 5'-AAC ACA CGG GTA CCG CCT CTT; SCUBE1 reverse, 5'-GTA TTG TAG TGG TGT CCG GGA GA; SCUBE1 probe, 5'-CCA GGA CTG CGA GGC CAA AGT GCA T; GAPDH forward, 5'-TGA AGG TCG GAG TCA ACG G; GAPDH reverse, 5'-AGA GTT AAA AGC AGC CCT GGT G; and GAPDH probe, 5'-TTT GGT CGT ATT GGG CGC CTG G. The relative expression ratio of SCUBE1 transcript in comparison to GAPDH transcript was calculated on the basis of a mathematical model as described [9].

2.6 Confocal immunofluroscence microscopy
Glass coverslips were coated with 20 µg/ml fibrinogen in PBS (pH 7.4) at 37 °C for 2 h and blocked with 1% bovine serum albumin for 1 h. Washed platelets (100 µl of 108/ml) were allowed to adhere for 5 min, then fixed with 1% paraformaldehyde. Platelets were left untreated or permeabilized with 0.5% Trixton X-100 for 10 min. A control experiment with the secondary antibody showed no autofluorescence or nonspecific fluorescence. All images were acquired on Bio-Rad MRC-1000 confocal microscopy.

2.7 Flow cytometry
Washed platelets were incubated at 37 °C for 30 min before activation. Platelets were stimulated with 0.2 U/ml of human thrombin at 37 °C for 20 min without stirring. Unstimulated or activated platelets were fixed with 1% paraformaldehyde, washed and reacted with platelet-specific GPIIIa mAb (IgG1 isotype, BD PharMingen), anti-SCUBE1 mAb 701 (IgG1, 10 µg/ml), or isotype control antibody. Platelets were then washed and stained with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories). Gating to select the majority of platelets was based on forward- and side-scatter and the expression of GPIIIa. Histograms were generated from measurements of 10,000 platelets, and data were analyzed by use of the CellQuest software on a FACScaliber system.

2.8 Immunolocalization of SCUBE1
Preparation of monkey tissue samples was as described previously [2]. Abdominal aortic aneurysms were obtained from patients undergoing surgery. All patients gave informed consent to participate in studies by the Institutional Review Board at the Veterans General Hospital–Taipei. The investigation conforms with the principles outlined in the Declaration of Helsinki. Immunohistochemistry and immunogold labeling were performed as described previously with anti-SCUBE1 mAb 701 [10].

2.9 Platelet adhesion assay
Coverslips were incubated with a suspension (100 µg/ml) of GST, recombinant SCUBE1 proteins (GST-E1-6,-E4-9,-CUB), fibrinogen or BSA overnight at 4 °C. Surface were washed twice with PBS and blocked with denatured BSA (5 mg/ml) for 1 h at room temperature. Platelets showed minimal binding and failed to become activated to surfaces coated with denatured BSA. For the adhesion experiments, washed human platelets (1 x 107) labeled by fluorescent methods as described [11] were incubated on coated coverslips at 37 °C for 1 h in a humid environment. After 2 washings with PBS, adherent platelets were fixed and examined under an inverted fluorescent microscope (Olympus IX71) and photographed. Total numbers of adherent platelets were counted in 5 randomly selected fields (x 200), with counters blinded to the fusion protein coated. Experiments were repeated 3 times with similar results. Values are mean±SEM. Differences between GST-SCUBE1 fusion proteins and GST were evaluated by use of unpaired Student's t-test.

2.10 Platelet agglutination/aggregation assay
Briefly, platelet-rich plasma (PRP) from healthy donors were pre-incubated with GST, GST-E1-6, GST-E4-9 or GST-CUB (5 µg/ml) in a 4-channel aggregometer (BioData, Horsham, PA) for 1 min at 37 °C, then left unstimulated (protein only) or stimulated with ristocetin (1 mg/ml), ADP (10 µM), or collagen (2.5 µg/ml) with constant stirring. The agglutination/aggregation was measured turbidimetrically after the addition of ristocetin or agonists for 10 min.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Monoclonal antibody (mAb) production and immunolocalization of SCUBE1
We have previously demonstrated by in situ hybridization that SCUBE1 mRNA is expressed in the endothelium lining the arteries, veins or capillaries [2]. To further localize SCUBE1 protein expression, we first generated mAbs against SCUBE1. The spleen cells from the BALB/c mice immunized with the GST-CUB domain fusion protein containing residues 789–988 of SCUBE1 were used to prepare the mAb with use of a standard hybridoma technique. After screening by ELISA and subcloning, three specific mAbs (#33, #701, and #712) against SCUBE1 were obtained. The specificity of these mAbs was tested by Western blot analysis with recombinant SCUBE1, SCUBE2, or SCUBE3 proteins expressed in HEK-293T cells. As shown in Fig. 1A, this mAb detected only SCUBE1 and did not cross-react with SCUBE2 or SCUBE3. In addition, the deletion-mutant SCUBE1-D1 lacking the CUB domain showed no immunoreactivity, further confirming the specificity of mAb and the epitope binding site.


Figure 1
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  		Fig. 1 MAb production and immunostaining of SCUBE1. (A) Specificity of anti-SCUBE1 mAb. Three independent hybridoma clones (mAb 33, 701, and 712) were generated in BALB/c mice immunized with the carboxyl-terminal CUB domain of SCUBE1. All of these mAbs could specifically recognize the recombinant full-length (FL) SCUBE1 protein expressed in HEK-293T cells but not SCUBE2 or SCUBE3. Note that the deletion-mutant SCUBE1-D1 (see Fig. 6A for domain organization) lacking the carboxyl-terminal CUB domain showed no immunoreactivity, further supporting the specificity of these mAbs. As a control for the amount of protein loaded, protein expression of the Flag-tagged SCUBE proteins was confirmed by anti-FLAG antibody (bottom panel). (B–D) Expression of SCUBE1 in platelet-rich thrombus. In panel B, a section of monkey lung was stained with anti-SCUBE1 mAb 701. While weak staining is seen within the microvascular endothelium (arrows), SCUBE1 immunoreactivity is strongly associated with intravascular thrombus (arrowhead). Thrombus-associated SCUBE1 staining has been consistently observed across a broad range of vascular tissue sections, including brain, heart, intestine and kidney (not shown). In panel C, immunohistochemistry staining of SCUBE1 in early thrombus in human vessel revealed positive staining adjacent to endothelium of vessel, but no staining was found away the endothelium. In panel D, subsequent section stained with fibrinogen revealed similar staining pattern to SCUBE1 (original magnification: 400 x).

 
To investigate the protein expression of SCUBE1, we then utilized mAb 701 for immunohistochemistry of numerous vascular tissue samples. As shown in Fig. 1B–D, while a weak SCUBE1 staining was seen in the microvascular endothelial cells, immunoreactive signal was strongly localized to the platelet- and fibrin-rich areas within the organized thrombus. In addition to the lung sections, a similar staining pattern is also seen across a broad range of vascular tissues examined, including brain, heart, intestine, and kidney (data not shown). These data suggest that SCUBE1 protein synthesized in the endothelium could be effectively removed into the circulation, which is then entrapped in fibrin clots during thrombus formation. Alternatively, SCUBE1 could also be expressed in platelets, much like many endothelial membrane proteins, including P-selectin, von Willebrand factor (vWF), and thrombomodulin, which are present in platelets as well [12–14].

3.2 Expression of SCUBE1 mRNA and protein in human platelets
We then further investigated whether SCUBE1 is expressed in human platelets. As shown in Fig. 2A, quantitative real-time PCR analysis revealed abundant SCUBE1 mRNA in platelets from 3 independent healthy donors. As a positive control, the expression of glycoprotein (GP) V or IIb mRNAs, 2 platelet-enriched or -specific genes, was shown to be abundantly expressed in these platelet RNA samples but absent or at a low level in other RNA samples (data not shown).


Figure 2
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  		Fig. 2 Expression of SCUBE1 mRNA and protein in human platelets. (A) Expression of SCUBE1 mRNA in human platelets. Expression of SCUBE1 was measured by quantitative real-time RT-PCR analysis in a panel of platelet cDNAs derived from 3 healthy donors (platelets 1–3). The cDNAs from HEK-293T cells stably expressing SCUBE1 or human lung were used as a control. Expression levels were normalized to GAPDH. (B) SCUBE1 protein expression in platelets. Detection of SCUBE1 protein in lysates from human resting platelets or platelet-rich thrombi generated by a collagen-coated perfusion chamber [15] with use of mAb 701. Note that a proteolytic SCUBE1 fragment is associated with thrombus lysate (arrow). Recombinant Flag-tagged SCUBE1 (Flag.SCUBE1) expressed in HEK-293T cells was used as a control. Anti-GPIIIa (integrin β3) blotting was used to confirm the integrity and loading of samples (bottom). (C–F) Confocal immunofluorescence analysis. Panels C and E show the confocal images and the phase-contrast merged images are presented in Panels D and F. SCUBE1 was identified in unstimulated, paraformaldehyde-fixed non-permeabilized (C, D) or Trixton-permeabilized (E, F) human platelets with use of mAb 701 and FITC-labeled secondary antibody. Weak labeling is seen on the surface of platelets (C, D) or in the intracellular pool of granular compartments in resting platelets (E, F). Magnification: C–F, x 1000.

 
To confirm the SCUBE1 protein expression, western blot analysis with mAb 701 was performed on platelet protein extract. As shown in Fig. 2B, this antibody detected SCUBE1 in lysates from human platelets or platelet-rich thrombi generated by a collagen-coated perfusion chamber [15]. We then utilized immunofluorescence analysis to examine the protein localization in the platelet. The same antibody (mAb 701) detected a faint labeling in nonpermeabilized platelets (Fig. 2C and D), which indicates that only trace amounts of SCUBE1, if any, was targeted to the surface of the platelet. In contrast, after permeabilization with Trixton X-100, SCUBE1 staining revealed a spotted or granular pattern with intense fluorescence (Fig. 2E and F), which suggests the presence of an intracellular pool of SCUBE1, possibly in the platelet storage granules. Consistently, double immunofluorescence staining confirmed the colocalization of the SCUBE1 signal with that of P-selectin, an {alpha}-granule content protein (not shown).

To further determine the subcellular localization of SCUBE1 in platelets, we used mAb 701 and gold-labeled secondary antibody to immunostain resting platelets, followed by electron microscopy. Most of the gold particles localized to membrane systems within the interior of the cells, with minimal surface labeling (Fig. 3). Gold particles were observed in membranes of {alpha}-granules of platelets (Fig. 3A and B, arrowheads), which indicates this organelle as a possible storage site for SCUBE1. Labeling was also observed in the open canalicular system (OCS) or associated with thin channels (Fig. 3B and C, arrows), thus suggesting some of SCUBE1 might be bound to or localized to the membrane. Control experiments with mAb 701 omitted or with an equivalent amount of an irrelevant mouse IgG1 used resulted in virtually no labeling (Fig. 3D).


Figure 3
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  		Fig. 3 Immunogold localization of SCUBE1 in unstimulated platelets. (A) A typical distribution of SCUBE1 labeling showed most gold particles localized to membrane systems within the interior of the cells, although surface labeling was also observed. (B) Higher-power magnification of labeled intracellular structures. Detection of SCUBE1 was found on the membrane (arrowheads) of the {alpha}-granules (G) and the OCS membrane channels in the platelet (arrows). (C) Intensive immunogold labeling of SCUBE1 is found in the channels of the open canalicular system (OCS) (arrows) and in the OCS connected to the plasma membranes (PM) of the 2 adjacent platelets. (D) Control experiments performed with an equivalent amount of an isotype-matched irrelevant antibody showed virtually no labeling. A–C: bar=0.25 µm; D: bar=0.3 µm.

 
3.3 Expression of SCUBE1 on activated platelets
Many membrane-anchored proteins stored in platelet {alpha}-granules are translocated to the surface upon platelet activation (e.g., P-selectin or CD40L) [13,16]. Therefore, we then utilized flow cytometry to determine whether SCUBE1 expression can be upregulated on platelet surfaces upon agonist stimulation. As shown in Fig. 4, while activation of platelets by thrombin (0.2 U/ml) resulted in surface expression of P-selectin, activation-dependent expression of SCUBE1 was also observed. In addition, an ELISA specific for SCUBE1 was used to estimate the concentration of SCUBE1 in the lysate of platelets obtained from healthy donors at ~150 ng/1 x 109 platelets (data not shown). Thrombin induced the secretion of approximately 50% of the total amount of SCUBE1 in platelets. Together, these data demonstrate that preformed platelet SCUBE1 is stored in platelets and is translocated to the cell surface as part of the ‘basic platelet reaction,’ in which rapid upregulation of P-selectin and several other proteins on the cell surface is accompanied by the release of soluble mediators from intracellular granules [17].


Figure 4
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  		Fig. 4 Surface expression of SCUBE1 on activated platelets. Washed human platelets were unstimulated or stimulated with 0.2 U/ml thrombin, fixed and analyzed by flow cytometry for expression of P-selectin (as a positive control) or SCUBE1 (mAb 701). The background signal obtained with the isotype control antibody is shown in black. Unstim., unstimulated.

 
3.4 Localization of SCUBE1 protein in human atherosclerotic lesions
It is well documented that activated platelets play an important role in the initiation and progression of atherosclerosis [18–20]. Since SCUBE1 is a platelet granule protein (Figs. 2 and 3Go) and can be translocated to the surface upon activation (Fig. 4), we then evaluated whether the SCUBE1 expression is associated with human atherosclerotic plaque. Immunohistochemical analysis with mAb 701 was performed on serial sections of the atherosclerotic vessel wall. In addition to a faint endothelial staining (Fig. 5A), strong SCUBE1 staining was mainly distributed in the subendothelial matrices of atherosclerotic lesions, whereas staining of SCUBE1 in the stroma of the media was virtually undetectable (Fig. 5E). The immunostaining of vWF, a platelet-endothelial protein deposited in the subendothelial extracellular spaces of atherosclerotic plaque [21], was similar to that of SCUBE1 but the signal was stronger (Fig. 5F). This widespread, diffuse SCUBE1 staining pattern suggests an extracellular localization of this protein in atherosclerotic plaque. To further identify the cells responsible for SCUBE1 production, we stained serial sections for either SCUBE1 or cell-type specific markers, namely, CD68 for macrophages or {alpha}-smooth muscle actin for smooth muscle cells, but failed to demonstrate the colocalization of SCUBE1 in these 2 cell types within the lesions (data not shown). As a negative control, the primary antiserum replaced by the isotype-matched irrelevant antibody showed no SCUBE1 immunostaining (Fig. 5C and G).


Figure 5
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  		Fig. 5 Immunohistochemical staining for SCUBE1 and vWF protein expression in serial sections of normal (A–C) atherosclerotic arterial walls (E–F). The lumen is uppermost in all sections. In addition to endothelial staining, strong staining for SCUBE1 (E) and vWF (F) was seen in markedly atherosclerotic lesions. The negative control with use of the isotype-matched irrelevant antibody in atherosclerotic arterial walls showed no immunoreactivity (C, G). Hematoxylin and eosin staining of normal (D) and atherosclerotic plaque (H).

 
3.5 SCUBE1 EGF-like repeats support platelet adhesion
Because SCUBE1 appears to be an extracellular matrix (ECM) protein in human atherosclerotic plaque (Fig. 5) and because a number of ECM component proteins including fibrinogen, vWF and fibronectin also play important roles in platelet adhesion [22], we then investigated the adhesive capacity of SCUBE1. The amino-terminal EGF-like repeats and the carboxyl CUB fragments of SCUBE1 were expressed as fusion proteins of glutathione S-transferase (GST) (Fig. 6A) and purified from the soluble fraction of the bacterial lysate, respectively (Fig. 6B). To determine whether platelets bind to matrix-bound SCUBE1, we gently laid washed human platelets over surface-immobilized proteins on coverslips and subsequently measured adhesion through labeled fluorescent intensity as described [23]. As shown in Fig. 6C, we first confirmed a low, baseline binding of platelets to the BSA-coated surface. In contrast, the amino-terminal EGF-like repeats fragments, GST-E1–6 (EGF-like repeats #1–6) or GST-E4–9 (EGF-like repeats #4–9), supported platelet adhesion to a similar extent as that of the positive control of fibrinogen. However, platelet adhesion between the carboxyl SCUBE1 fragment (GST-CUB) and GST did not differ. Furthermore, the addition of the soluble SCUBE1 fragment (E1–6 or E4–9) could significantly compete for platelet binding, thus demonstrating the specificity of this assay (Fig. 6C).


Figure 6
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  		Fig. 6 Platelet adhesion to recombinant SCUBE1 fragments. (A) Domain structure of SCUBE1 with recombinant fragments. Diagram of the SCUBE1 protein illustrates the organization of signal peptide (SP), EGF-like repeats (E), spacer region, cysteine-rich repeats (CR) [6,7], and CUB domain and shows the location of the recombinant fragments (GST-E1–6, -E4–9, and -CUB). (B) Purification of recombinant SCUBE1 protein fragments. GST-SCUBE1 fusion proteins purified from the soluble fraction of bacterial lysates with glutathione-Sepharose beads were analyzed by SDS-PAGE and stained with Coomassie brilliant blue. (C) SCUBE1 EGF-like repeats support platelet adhesion. Washed platelets were allowed to adhere to glass coverslips coated with BSA, GST, GST-E1–6 (EGF-like repeats #1–6), GST-E4–9 (EGF-like repeats #4–9), GST-CUB or fibrinogen as a positive control for 1 h at 37 °C. To validate the binding specificity, a 10-fold excessive amount of soluble GST-E1–6 or -E1–9 was added to block the platelet adhesion. The number of adherent fluorescence-labeled platelets was counted in 5 randomly selected fields on fluorescence microscopy. Values are mean±SEM from 3 independent experiments. *p<0.01.

 
3.6 Soluble SCUBE1 protein fragments augment ristocetin-induced platelet agglutination
We then examined whether the soluble SCUBE1 protein fragments affect vWF-mediated platelet adhesion. The main function of vWF is to mediate adhesive interactions of platelets under high fluid shear stress [24,25]. The antibiotic ristocetin causes conformational change of vWF and initiates the binding of vWF to platelet GPIb, then induces platelet agglutination, which is commonly used as a surrogate assay to mimic the platelet–subendothelial adhesion in a fluid phase. Platelet-rich plasma (PRP) derived from healthy donors was incubated with 5 µg/ml of GST, GST-E1–6, GST-E4–9 or GST-CUB, then left unstimulated or induced to agglutination by adding ristocetin. Platelet agglutination was measured by a turbidimetric method with a platelet aggregometer. While incubation of soluble SCUBE1 protein fragment alone did not affect platelet aggregation, SCUBE1 EGF-like repeat-containing fragments indeed enhanced ristocetin-induced platelet agglutination, as compared with GST as a baseline control (Fig. 7).


Figure 7
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  		Fig. 7 Effects of soluble SCUBE1 protein fragments on ristocetin-induced platelet agglutination. PRP was incubated for 1 min at 37 °C with the indicated soluble SCUBE1 proteins (GST-E1–6, -E4–9, or -CUB) or GST as a negative control before the addition of ristocetin (1 mg/ml). Platelet agglutination was determined by the turbidimetric method with use of a platelet aggregometer. Note that soluble SCUBE1 protein fragments or GST alone do not induce platelet aggregation (protein only).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we produced a number of recombinant SCUBE1 protein fragments and monoclonal antibodies to examine the protein expression and function of a newly discovered gene, SCUBE1 [2]. Despite its original discovery in the endothelium, SCUBE1 is highly expressed in human platelets at both the mRNA and protein levels (Figs. 2 and 3Go). This finding suggests that the platelet-associated SCUBE1 is not simply a result of endocytosis from the circulation but instead could be synthesized in the platelet–megakaryocytic lineage. To explore whether SCUBE1 is expressed by other types of blood cells, flow cytometric analysis was performed on freshly isolated peripheral blood leukocytes (PBL) from healthy donors, without positive staining (data not shown). Likewise, we also failed to detect the SCUBE1 mRNA from the PBL sample by Northern blot analysis [2]. These data suggest that the platelet is probably the principal source of the SCUBE1 expressed in the vascular system.

Confocal immunofluorescence and immunogold labeling revealed only a negligible amount of this protein, if any, present at the platelet surface, whereas a predominant pool resides within the platelet. Specifically, SCUBE1 was found in membranes of {alpha}-granules and elements of the OCS (Fig. 3). Western blot analysis demonstrated that the apparent molecular mass of SCUBE1 in resting platelets is approximately 135 kDa (Fig. 2B), which agrees with the molecular mass of the full-length product [2]. In addition, a proteolytic fragment (~95 kDa) was reproducibly observed in thrombus lysate (Fig. 2B, arrow). Because 2 novel CUB domain-containing growth factors (PDGF-C or -D) require the proteolytic removal of the CUB domain to generate the biologically active growth factor core domain [26–29], the SCUBE1 fragment is likely derived from a proteolytic modification of the surface-bound or secreted platelet SCUBE1 via locally released fibrinolytic and coagulation proteases at the site of the growing thrombus. Although the precise identity of the SCUBE1 processing protease is currently elusive, our recent in vitro study revealed that a minimal recognition site (RRXR, residues 535–538) for the furin-like protease [30] located within the spacer region seems to be responsible for the serum-dependent proteolytic cleavage of SCUBE1. In support of this concept, a proteolytic SCUBE1 fragment carrying a similar molecular mass of 95 kDa has been reproducibly detected and shown to be highly elevated in plasma samples from patients with acute coronary syndrome (ACS) undergoing plaque rupture/erosion with subsequent platelet activation but virtually undetectable in normal controls (Yang et al., unpublished data). Thus, platelet SCUBE1 may be released or deposited at the lesion sites under acute ischemic conditions such as ACS or acute ischemic stroke. By nature of promoting platelet adhesion and agglutination, SCUBE1 may be involved in the pathogenesis or progression of these thrombotic diseases.

One important observation resulting from this report is that we have demonstrated the presence of SCUBE1 in human abdominal aortic atherosclerotic lesions (Fig. 5). Despite its strong staining in the lesion areas, SCUBE1 expression was not detected in macrophages and smooth muscle cells in any of the atheromas examined (Fig. 5 and data not shown). This finding raises a possibility that the major component of SCUBE1 associated with atherosclerotic plaques may be derived from an exogenous source. Because this protein is present in the platelet and because platelets and their granular contents (such as vWF, P-selectin or CD40L) are closely associated with atherosclerotic plaques and play a critical role in the development of this disease [18,20,31–34], SCUBE1 deposited within the subendothelial matrix of plaques might be of platelet and/or endothelial origin and involved in the pathogenesis of atherosclerosis. However, this suggestion remains to be further validated.

Our adhesion studies demonstrated that matrix-immobilized SCUBE1 protein serves as an adhesive molecule to support platelet adhesion (Fig. 6). However, this adhesive capacity is restricted only to the amino-terminal EGF-like repeats, and the CUB domain seems to be absent or only minimally involved in the adhesion and agglutination effects of SCUBE1 (Figs. 6 and 7Go). Together, these data suggest that SCUBE1 is organized in a modular fashion and has a distinct function associated with each domain. Although the CUB domain lacks adhesive function, recent genetic studies demonstrated that the CUB domain is essential for the zebrafish SCUBE2 to function as a mediator of hedgehog signaling during embryogenesis. Furthermore, the SCUBE1–platelet interactions appear to be specific, since incubation with an excessive amount of the soluble SCUBE1 fragments GST-E1–6 or -E4–9 abolished platelet adhesion (Fig. 6C).

Interestingly, instead finding the classic integrin-binding RGD motif that is critical in mediating adhesion to integrin receptors on the platelet surface, we found 2 CGD sequences residing in the EGF-like repeats #1 and #8. Some RGD-derived, integrin-binding motifs, such as AGDV or KGD sequences, have been described in the {gamma}-chain of fibrinogen [35] or soluble CD40 ligand [36], respectively. To test whether the CGD tripeptide represents a type of integrin/platelet-binding motif, 2 peptides containing the CGD motif (GKQCGDIDE or TTHCGDVDE, derived from the SCUBE1 EGF-like repeat #1 or #8, respectively) were synthesized and used to examine their effects on platelet adhesion. However, both CGD-based peptides (up to 100 µM) were unable to block the platelet adhesion on the SCUBE1-coated surface. Thus, these CGD motifs may not be directly involved in the SCUBE1-mediated platelet adhesion.

Alternatively, the EGF-like repeats may form homophilic platelet–platelet or platelet–matrix adhesions, since we have previously demonstrated that 9 copies of EGF-like repeats alone are sufficient to form oligomers, tethered to the cell surface [2]. In support of this finding, the binding between the EGF-like repeats has been reported for the intercellular interactions between the transmembrane Notch receptor and their ligands Delta and Serrate [37], as well as direct the receptor–receptor trans-interactions between separate cells [38]. Interestingly, a novel EGF-like repeat-containing transmembrane receptor has been recently reported to participate in platelet contact-induced activation [39]. It is noteworthy that the binding assay used in this study was under static conditions, which may explain the moderate platelet adhesion (Fig. 6C). Since hemodynamic forces have been demonstrated to play an important role in modulating the platelet–matrix adhesion, we will further evaluate the effects of fluid shear stress on the SCUBE1-mediated adhesion in our future studies.

As shown in Fig. 7, soluble SCUBE1 fragments could enhance ristocetin-induced platelet agglutination, which is a fluid-phase analogy to platelet–subendothelial adhesion. Because true platelet aggregation activates the platelet to bind fibrinogen to GPIIb–IIIa, the insensitivity of ADP or collagen-induced aggregation to added SCUBE1 fragments (not shown) further implies a role for SCUBE1 in platelet adhesion, as opposed to platelet aggregation. It is of interest to further investigate whether SCUBE1 fragments enhance vWF binding to GPIb-IX–V complex or whether these fragments cooperate with ristocetin to agglutinate the platelets via self-associated SCUBE1.

In summary, this study demonstrated that SCUBE1, mainly derived from platelets, is a biologically significant molecule in the cardiovascular system. Its matrix-bound or soluble forms may play an adhesive role in mediating the platelet–platelet or platelet–matrix interactions. Further studies of SCUBE1 are required to unravel its roles in hemostasis or thrombosis or the pathogenesis of cardiovascular diseases.


   Acknowledgements
 
We thank Dr. Shyh-Chyi Lo for advice and help on platelet aggregation assay. This study was supported by Grant 91-B-FA09-2-4 from the Ministry of Education Program for Promoting Academic Excellence of Universities and National Science Council Grants NSC 93-2320-B-001-048, NSC 95-2752-B-006-003-PAE, and NSC 95-2752-B-001-002-PAE.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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 J Am Coll CardiolHome page
D.-F. Dai, P. Thajeb, C.-F. Tu, F.-T. Chiang, C.-H. Chen, R.-B. Yang, and J.-J. Chen
Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
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C.-F. Tu, Y.-T. Yan, S.-Y. Wu, B. Djoko, M.-T. Tsai, C.-J. Cheng, and R.-B. Yang
Domain and Functional Analysis of a Novel Platelet-Endothelial Cell Surface Protein, SCUBE1
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