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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2600-2609
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Anatomy/Cell Biology, Biochemistry and
Medicine/Renal Division, SUNY Downstate Medical Center, Brooklyn, NY;
Program in Neuroscience and the Departments of Chemistry and Biology,
CUNY/CSI, Staten Island, NY; and the Department of
Medicine/Division of Experimental Medicine and Hematology, Brigham and
Women's Hospital, Harvard Medical School, Boston, MA.
This study demonstrates that the human platelet F11 receptor (F11R)
functions as an adhesion molecule, and this finding is confirmed by the
structure of the protein as revealed by molecular cloning. The F11R is
a 32-/35-kd protein duplex that serves as the binding site through
which a stimulatory monoclonal antibody causes platelet aggregation and
granule secretion. A physiological role for the F11R protein was
demonstrated by its phosphorylation after the stimulation of platelets
by thrombin and collagen. A pathophysiological role for the F11R was
revealed by demonstrating the presence of F11R-antibodies in patients
with thrombocytopenia. Adhesion of platelets through the F11R resulted
in events characteristic of the action of cell adhesion molecules
(CAMs). To determine the structure of this protein, we cloned the F11R
cDNA from human platelets. The predicted amino acid sequence
demonstrated that it is an integral membrane protein and an
immunoglobulin superfamily member containing 2 extracellular C2-type
domains. The structure of the F11R as a member of a CAM family of
proteins and its activity in mediating adhesion confirm each another.
We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is
antibody-induced platelet aggregation and secretion that are dependent
on both the Fc
The pathophysiologic significance of circulating
autoantibodies and alloantibodies directed against platelet membrane
proteins has been observed in patients diagnosed with immune
thrombocytopenia and posttransfusion purpura.1-11 These
studies have pointed at the clinical importance of elucidating the
mechanism of action of stimulatory antibodies that bind to the platelet
surface and cause platelet activation. Among these are antibodies
directed against the major platelet glycoproteins GPIIb/IIIa (CD42),
CD9, CD69, and GPIV (CD36).12-20 Our studies have
identified and characterized a monoclonal antibody (mAb),
mAb.F11, that stimulates human platelets and acts as an
agonist that induces platelet aggregation and granule secretion.21-25 The first step in the biochemical pathway
by which mAb.F11 induces platelet aggregation and secretion was shown
to be the recognition by mAb.F11 of a platelet surface protein duplex named the F11 receptor (F11R). The F11R was identified as 2 membrane proteins on the platelet surface with molecular masses of 32 kd and 35 kd.22 After the purification of the F11 receptor by ion exchange and affinity chromatography, it was found that
N-deglycosylation of either the 32 kd or the 35 kd protein produces a
core protein of 29 kd, recognizable by mAb.F11.23
Subsequently, approximately one third the length of this core protein
has been sequenced, including 26 amino acids of the N-terminus of this
protein (SVTVHSSEPEVRIPENNPVKLSXAYS) and several fragments
including 43 amino acids of an endoproteinase proteolytic fragment
(WKFDQGDTTRLVEYNNKITASYEDRVTFLPTGITFKSVTRED)23 (NCB
accession number S56749). Extensive database searches performed at that
time determined that the F11 receptor represented a novel protein not
reported previously.
In the process that induces platelet aggregation, the initial
recognition of the F11R by mAb.F11 was shown to be coupled to the
cross-linking of the antibody through the Fc domain to the platelet
Fc The elucidation of the biochemical pathways outlined above determined
that the activation of platelets by mAb.F11 involved enzymatic
mechanisms that are activated by physiological platelet agonists.
However, by themselves, these studies did not reveal a physiological
role for the F11 receptor, nor did they demonstrate directly the
clinical significance of this platelet surface protein. The study
reported here has been designed to provide information relevant to
these important questions. Using mutually complementary approaches,
functional studies of platelet activation, and molecular biologic
techniques of gene cloning, we report here that the platelet F11
receptor is a cell surface adhesion molecule (CAM) of the immunoglobulin superfamily. The F11R appears to be involved in the
activation of platelets by physiological agonists and by stimulatory antibodies; in addition, it participates in mechanisms underlying the
adhesion of platelets and other hematopoietic cells. Finally, the
finding in the plasma of patients with thrombocytopenia of antibodies
that recognize the F11R points to the potential clinical importance of
the investigation of this protein and suggests that peptides with
sequences of critical sites in the F11R protein should be tested as
novel therapeutic agents for the treatment of certain thrombocytopenias.
Reagents
Platelet isolation
Cell preparation and cell culture HEL cells were purchased from the American Type Culture Collection (Rockville, MD). Megakaryocytic cell lines (CMK, CMK11-5, and CMK6) were the generous gift of Dr Hava Avraham (Beth Israel Deaconess Medical Center, Boston, MA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Bovine aortic endothelial cells (BAEC) were obtained from Dr George Tuszynski (MCP Hahnemann University, Philadelphia, PA). The human endothelial hybrid cell line (Ea.hy 926) was obtained from Dr Cora-Jean S. Edgell (University of North Carolina, Chapel Hill, NC). The Eahy926 cells were cultured in DMEM containing 10% FBS and 100 µmol hypoxanthine, 0.4 µmol aminopterin, and 16 µmol thymidine. The K562 cell line was maintained in 15% FBS-RPMI medium.Preparation of antibodies mAb.F11 was identified and characterized as previously described.21,22 A polyclonal antibody directed to the 21-amino acid N-terminal sequence of the F11 receptor23 (SVTVHSSEPEVRIPENNPVKL-Cys) was prepared in rabbits by Quality Controlled Biochemicals (Hopkinton, MA) and was subsequently purified by affinity chromatography.Electrophoresis and immunoblotting Samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as follows: red blood cell ghosts were prepared as described.28 Cell extracts of HEL, K562, CMK, CMK6, and CMK11-5 cells were harvested, washed, and solubilized in Laemmli buffer. Eahy926, HUVEC, and BAEC cells were suspended in 10 mmol/L EDTA-phosphate-buffered saline (PBS), harvested, washed, suspended in Laemmli buffer, and processed for SDS-PAGE. Nitrocellulose membranes were incubated with the primary monoclonal (mAb.F11), rabbit polyclonal F11 receptor antibodies, or purified human antibodies, followed by incubation with secondary, rabbit or goat antimouse/human Ig-AP conjugates 1:4000, or donkey antirabbit antibodies conjugated with HRP 1:2000 (Amersham Pharmacia Biotech, Piscataway, NJ).Labeling of human platelet with [32P]-orthophosphate Washed platelets were labeled with [32P]-orthophosphate at 37°C for 60 minutes, then washed and resuspended in Tyrode's buffer as described previously.22Flow cytometry The CMK, CMK11-5, CMK6, HEL, and K562 cell lines were washed with PBS and resuspended in 0.1% BSA/PBS. HUVEC, Eahy926, and BAEC cells were treated with 10 mmol/L EDTA/PBS, washed, and resuspended in 0.1% BSA/PBS. To isolate the buffy coat fraction, whole blood was mixed with equal amounts of Hanks' balanced salt solution (HBS), overlaid on Ficoll, and centrifuged, and the buffy coat layer and RBC were collected separately. Cells were resuspended in 0.1% BSA/PBS (4 × 105 cells/sample) and incubated with mAb.F11 (5 µg/mL) or with an isotype-identical nonreactive IgG (control). After a 1-hour incubation at 22°C, the cells were washed with 0.1% BSA/PBS, treated with 50 µL 1/100 diluted goat antimouse Ig-FITC (GAM), incubated for 30 minutes on ice, washed, and resuspended in 0.1% BSA/PBS. The samples were analyzed using an Immunocytometry Systems flow cytometer (FAC Sort, Becton Dickinson, San Jose, CA). Flow cytometric analysis of isolated human platelets was performed in the presence of PGE1 (1 mg/mL).Detection of platelet antibodies detecting the F11R in patients Serum was obtained from a 64-year-old man with a history of hypertension after 2 separate cerebrovascular accidents within a 2-week period. Although workup revealed no obvious source of the infarcts (results of carotid Doppler imaging were normal, and no thrombus was detected), aspiration pneumonia developed and the patient was treated with antibiotics. After acute renal failure developed, dialysis was initiated. At this time, thrombocytopenia developed accompanied by a decrease in platelet count from an admission level of 266 000/mL to 50 000/mL. Bilateral gangrene then developed in both legs. Although the patient was administered heparin, it was only begun after the gangrene developed, thus excluding a diagnosis of heparin-induced thrombocytopenia. Severe thrombocytopenia continued, and the patient died 3 weeks after hospital admission because of overwhelming sepsis, respiratory failure, and cardiac failure. Another patient, a 40-year-old man with thrombocytopenia and end-stage renal disease secondary to hypertension, was examined for F11R antibodies in his circulatory system. He underwent cadaveric kidney transplantation and was administered the immunosuppressive drug tacrolimus (FK506). The patient is alive and has persistent thrombocytopenia.RNA isolation and processing Platelets were solubilized in lysis buffer (4 mol/L guanidine isothiocyanate, 25 mmol/L sodium citrate, 0.5% sodium sarcosine, and 100 mmol/L -mercaptoethanol) dissolved in DEPC-treated water and
processed according to standard purification techniques.29
Primers The sequence of the degenerate primers was based on the published23 GLµ-C(4) fragment sequence: F11/f-6 (GARTAYAAYAAYAARATHAC), F11/r-5(A) (TTRAANGTDATNCCNGTAGG), F11/r-5(C) (TTRAANGTDATNCCNGTCGG), F11/r-5(G) (TTRAANGTDATNCCNGTGGG), and F11/r 5(T) (TTRAANGTDATNCCNGTTGG). Primers used for 3' and 5'-RACE were GSP1 (AGCTTCCTATGAGGACCGGG), GSP2 (GTCACGGACTTGAAGGT), GSP3 (TTRAANGTDATNCCNGTTGG), and GSP4 (GGCAAGAAGGTCACCCGGTCC). The universal adaptor primer, the adaptor abridged universal amplification primer (AUAP), and the abridged anchor primer were provided with the RACE systems (GIBCO BRL).Reverse transcription-polymerase chain reaction with degenerate primers to obtain unique internal complementary DNA (cDNA) sequences Reverse transcription (RT) was carried out using 1 µg total RNA and the oligo d(T)16 primers (Perkin Elmer [currently PE Biosystems], Foster City, CA) as detailed in the manufacturer's protocol. Polymerase chain reaction (PCR) was carried out using degenerate primer pairs derived from the published23 partial amino acid sequence of the F11 receptor, termed the GLµ-C(4) fragment (final concentration of each primer, 2 pmol/µL). The dNTP and Mg2+ were present at 200 µmol/L and 2 mmol/L, respectively. The PCR mix was subjected to hot start at 99°C/10 minutes followed by addition of the Taq polymerase at 85°C. Thermocycling consisted of 5 cycles at 95°C/1 minute, 48°C/2 minutes, and 72°C/2 minutes, followed by 45 cycles at 94°C/1 minute, 48°C/2 minutes, and 72°C/2 minutes, followed by a final extension at 72°C/5 minutes.DNA analysis by polyacrylamide gel electrophoresis The DNA samples (PCR products or plasmid DNA) were separated by electrophoresis in 15% polyacrylamide gels and analyzed according to standard procedures.30Purification and cloning of the amplified PCR products After PAGE, the DNA fragment was eluted by the standard crush-and-soak method as described.30 The eluted DNA was digested with T4 DNA polymerase (Promega), and the resultant blunt-end product was phosphorylated at its 5' ends using T4 polynucleotide kinase. The insert was then cloned into pBLUESCRIPT SK(+) (Stratagene, La Jolla, CA). The molar ratio of insert to vector was approximately 5:1. The subsequent construct was transformed into DH5a-competent cells (GIBCO BRL) as suggested by the manufacturer. The plasmid DNA was isolated using the Wizard Midipreps DNA purification system (Promega), and the presence of the cloned insert was confirmed by KpnI and SacI digestion of the purified plasmid DNA.DNA sequencing Isolated and purified plasmid DNA was sequenced by ACGT (Northbrook, IL). The fidelity of the data was confirmed by sequencing both strands using M13 universal forward and reverse primers.3' Rapid amplification of cDNA ends 3'-RACE was used for the amplification of nucleic acid sequences from an mRNA template between the internal site of the GLµ-C(4) fragment determined above and the 3' end of the mRNA. Total platelet RNA (0.5 µg) was prepared as described above. RT was primed with the adaptor primer as described in the 3'-RACE system (GIBCO BRL). PCR was carried out using GSP1 and AUAP primers (35 cycles at 94°C/5 seconds, 58°C/15 seconds, and 72°C/3 minutes, followed by a final extension at 72°C/3 minutes). The PCR product was cloned into pT-Adv vector (Clontech) as described by the manufacturer. Two clones were sequenced in both directions, and the remaining 4 clones were sequenced on 1 strand.5' Rapid amplification of cDNA ends Total RNA was isolated from freshly prepared human platelets using a RNeasy Mini Kit (Qiagen) as described in the manufacturer's RNeasy Mini Handbook. The 5'-RACE system for rapid amplification of cDNA ends (GIBCO BRL) was used to amplify the 5' end of the F11 cDNA following procedures detailed in the manufacturer's manual. In brief, the first-strand cDNA synthesis was primed using GSP2 antisense. The cDNA was then purified using a GlassMax DNA Isolation Spin Cartridge (GIBCO BRL, Rockville, MD). A homopolymeric C-tail was subsequently added to the 3'-cDNA end, and the cDNA was amplified by PCR using GSP3 and AUAP (45 cycles at 94°C/30 seconds, 48°C/1 minute, and 72°C/3 minutes, followed by a final extension at 72°C/3 minutes). The 100-fold diluted primary products were reamplified using a nested GSP4 primer and AUAP (35 cycles at 94°C/30 seconds, 55°C/1 minute, and 72°C/3 minutes, followed by a final extension at 72°C/3 minutes). The resultant PCR products were processed as described above for the 3'-RACE system. Two clones were sequenced in both directions, and 1 clone was sequenced on 1 strand.Data analysis The translated amino acid sequence of the platelet F11 receptor was analyzed using the following on the Internet: TMpred, FASTA, ScanProsite, PfamHMM, PSORT, SSPRED, ProfileScan, ProtScale, PatScan, pI/Mw, and Motif (available through Pedro's BioMolecular Research Tools at http://www.public.iastate.edu/~pedro/ rt_1.html), SignalP (http://www.cbs.dtu.dk/services/SignalP/output.html), Pfam (The Sanger Centre, http://www.sanger.ac.uk), and Simple Modular Architecture Research Tool (SMART) (http://coot.emblheidelberg.de/predict protein).
F11R-mediated platelet adhesion and spreading The dramatic change in the morphology of platelets after their adherence to an antibody mAb.F11-coated matrix is demonstrated in Figures 1A to 1C, showing video frames of the antibody-induced shape changes in a time-dependent manner under 3 different conditions. Figure 1A follows the adhesion of platelets to the antibody-coated surface in the absence of added inhibitors. It can be seen that after adhesion to the matrix, the platelets developed lamellipodia and filopodia, and the spreading of platelets through these structures could be observed within minutes. These morphologic changes were not inhibited by the fibrinogen receptor inhibitory peptide RGDS (Figure 1B) or by the presence of an antibody to the Fc RII receptor mAb.IV.3 (Figure 1C).
Platelet activation induces phosphorylation of the F11 receptor Figure 2 demonstrates that together with the mAb.F11-induced platelet shape change and aggregation, stimulation of the F11 receptor resulted in phosphorylation of the receptor protein duplex, shown in Figure 2D. Moreover, not only stimulation by the F11 antibody but also platelet stimulation by the physiological agonists thrombin (Figure 2B) and collagen (Figure 2C) resulted in an increase in the phosphorylation state of the F11 receptor, with enhanced phosphorylation of both the 32 kd and the 35 kd glycosylated forms of the F11R protein.
Cloning of the platelet F11R cDNA Based on the previously determined sequence of the F11R GLµ-C(4) fragment WKFDQGDTTRLVEYNNKITASYEDRVTFLPTGI- TFKSVTRED,23 4 degenerate oligonucleotide primers F11/r5(A), F11/r-5(C), F11/r5(G), and F11/r5(T) and the forward primer F11/f-6 were designed. The 3' part of the GLµ-C(4) cDNA was obtained as described in "Materials and methods." On analysis of its sequence, we determined that the translated amino acid sequence of the internal section of this product was identical to the internal amino acid sequence of the published23 GLµ-C(4) fragment, as detailed above.
Analysis of the platelet F11R amino acid sequence
Platelet F11R is a member of the immunoglobulin superfamily
Immunologic detection of the F11 receptor in platelet membranes
using antibodies directed to the N-terminal sequence
Cellular distribution of the platelet F11R
Identification of antibodies recognizing the F11R protein in the
circulation of patients with thrombocytopenia
The main new finding of this report is that F11R, a protein studied
extensively for its role in antibody-induced platelet aggregation,21-25 functions as a mediator of cell adhesion
processes and is a member of a family CAMs. These conclusions were
derived, respectively, from functional determinations and
gene cloning studies that confirmed each another, as discussed below.
Supported by grants from the American Heart Association/New York City
Affiliate/Heritage Foundation (E.K.), and from the Center for
Biotechnology, New York State Center for Advanced Technology, Grant
#X318Q, Stony Brook, NY; and by a Research Career Development Award
(E.K.) from the NHLBI of the National Institutes of Health.
Reprints: Elizabeth Kornecki, Department of Anatomy and
Cell Biology, Downstate, State University of New York, Box 5, 450 Clarkson Avenue, Brooklyn, NY 11203; e-mail:
ekornecki{at}netmail.hscbklyn.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Presented in part at the XVIIth Congress of the International Society
on Thrombosis and Haemostasis, Washington, DC, August 14-21, 1999.
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Top
Abstract
Introduction
and
from the cytoplasm to the membrane and a sustained, irreversible
association of the PKC isoenzymes 
, 
with the
plasma membrane.
an antibody-induced platelet
aggregation dependent on the involvement of both Fc
