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Originally published In Press as doi:10.1074/jbc.M101299200 on June 13, 2001

J. Biol. Chem., Vol. 276, Issue 34, 32129-32135, August 24, 2001
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A Protein with Characteristics of Factor H Is Present on Rodent Platelets and Functions as the Immune Adherence Receptor*

Jessy J. AlexanderDagger§, Bradley K. Hack, Patrick N. CunninghamDagger, and Richard J. Quigg

From the Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, Illinois 60637

Received for publication, February 9, 2001, and in revised form, May 9, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Complement-coated particles interact with specific immune adherence receptors (IAR). In primates, this function is served by complement receptor 1 (CR1) on erythrocytes. In contrast, rodent platelets bear IAR distinct from CR1, the identity of which was studied here. A 150-kDa C3b-binding protein was isolated from rat platelets, which had immunochemical and biochemical identity to plasma factor H. Immunofluorescence microscopy and flow cytometry demonstrated that factor H was present on the surface of rat and mouse platelets, which could be removed by treatment with neuraminidase. Sheep erythrocytes bearing C3b underwent immune adherence with rat and mouse platelets, which was blocked with anti-factor H F(ab')2 antibodies, but not with antibodies binding to the complement regulator, Crry, on the platelet surface. By reverse transcription-polymerase chain reaction using rat platelet RNA and primers designed from mouse factor H, a 472-base pair product was generated that was identical in sequence to that produced from rat liver RNA. The translated protein product was 85% similar to mouse liver factor H. The 3'-nucleotide sequence from platelets predicted a soluble factor H protein. By Northern analysis, liver and platelets had identically sized factor H mRNA. Thus, rat and mouse platelets have a membrane protein with characteristics of factor H that is linked via sialic acid residues and functions as the IAR. Whether platelet factor H is acquired by passive adsorption from sera and/or is produced by platelets remains to be determined.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The complement system has three pathways containing over 30 plasma and membrane-bound proteins that play a key role in immune defense (1). These proteins cause the rapid destruction of invading microorganisms and mediate the solubilization and clearance of immune complexes. Immune complexes bearing the activated complement component C3b bind to cells with specific membrane receptors, which transport them to the mononuclear phagocyte system in the liver and spleen. This phenomenon is called immune adherence (IA)1 (2, 3) and is responsible for the disposal of immune complexes from the body (4, 5). The in vitro correlate of this is rosetting of IAR-bearing cells with C3b-containing particles (3, 6).

The IAR of primates is CR1, which is present on erythrocytes (7). In addition to erythrocytes, CR1 is also present on blood cells, including monocytes, lymphocytes, neutrophils, and eosinophils and resident cells such as podocytes in the renal glomerulus, follicular dendritic cells in lymphoid organs and astrocytes in the brain (8-10). CR1 is a member of the regulators of complement activation gene cluster located on chromosome 1q32, all of which have ligand specificity for activation products of C3 and/or C4 and contain repetitive units known as short consensus repeats (SCRs) (11, 12). In contrast, IA function in non-primates, including rodents, is not served by erythrocytes, but rather by platelets bearing an undefined IAR (13, 14).

Several attempts have been made to identify this non-primate IAR. Mouse platelets form rosettes with C3b-containing erythrocytes, which can be disrupted by incubating cells with factor I (15), implying that the IAR provides factor I cofactor activity. The obvious candidate for this is mouse CR1; yet, CR1 is clearly absent from mouse platelets, as well as erythrocytes and unstimulated neutrophils (16). A 150-kDa C3b/iC3-binding protein isolated from rabbit platelets was absent on rabbit erythrocytes, but was not characterized further (17). In our previous studies (18, 19), we identified rodent platelet proteins that bound C3 fragments and hence were candidates similar to the IAR. However, at that time, we could only characterize them on the basis of their size and reactivities with available antibodies to mouse CR1 and CR2.

The purpose of this study was to identify the rodent platelet IAR. By functional, biochemical, immunochemical, and molecular biological approaches, we conclusively show rodent platelets can produce and bear a protein with characteristics of factor H, a member of the regulators of complement activation family. This platelet-associated factor H functions as the IAR.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies (Abs)-- Goat anti-human factor H was purchased from Quidel (San Diego, CA), and cross-reacts with mouse and rat factor H (see below), which is not surprising given that factor H is highly conserved among species (20). Sheep Abs were raised to rat rCrry (21) and rDAF (22) (a gift of Dr. B. Paul Morgan, University of Wales College of Medicine, Cardiff). Rabbit anti-mouse rCrry was supplied by Dr. V. Michael Holers (23) (University of Colorado Health Sciences Center, Denver). A goat anti-rat thrombocyte Ab was purchased from Accurate Scientific (Westfield, NY). Although rodent platelets are known to not bear Fc receptors (24), all experiments were performed with F(ab')2 Ab fragments, which were prepared by standard techniques (25).

C3 Purification-- The purification of rat and mouse C3 from plasma have been described previously (19, 26). In brief, rat C3 was purified by a 5-11% polyethylene glycol precipitation followed by Mono Q anion-exchange chromatography (Amersham Pharmacia Biotech). A similar technique was used to purify mouse C3, except that a 5-10% polyethylene precipitation was used, and following Mono Q chromatography, size exclusion chromatography on Sephacryl S-300 HR was performed. The preparations were noted as pure with SDS-PAGE.

Animals-- Outbred Harlan Sprague Dawley rats and CD1 mice used for the isolation of blood and tissue were obtained from Harlan Bioproducts for Science (Indianapolis, IN). Mice with targeted deletion of CR1 have been generated previously, which have been backcrossed over 7 generations onto the C57BL/6 strain (27). Normal C57BL/6 mice were used as wild-type controls for CR1-/- mice. The animals were maintained and experiments performed in accordance with the guidelines set by the University of Chicago Institutional Animal Care and Use Committee.

Isolation of Platelets-- Platelets were isolated as described earlier (18, 19). In brief, blood was drawn by cardiac puncture from rats or mice into plastic syringes containing phosphate-buffered saline (PBS, 0.1 M NaCl, 0.02 M sodium phosphate, pH 7.2) with 0.086 M EDTA. The blood was centrifuged at 300 × g for 20 min at room temperature. The top two-thirds of the supernatant was transferred into fresh tubes and further centrifuged at 2000 × g for 10 min at room temperature. The pellet was washed and suspended in PBS. By light microscopy, the preparation was >99% pure and viable by virtue of trypan blue exclusion.

C3b Affinity Chromatography-- C3b was generated by trypsinization of C3 and bound via its free sulfhydryl group to thiopropyl-Sepharose (Amersham Pharmacia Biotech). Platelet membranes were solubilized with PBS containing 1% Nonidet P-40, 10 mM EDTA, 10 mM iodoacetamide, and 5 mM diisopropyl fluorophosphate (Sigma Aldrich). Solubilized platelet proteins were diluted to 0.05 M NaCl and 0.1% Nonidet P-40 and subsequently loaded on the C3b-Sepharose column. The column was washed with 10-column volumes of 0.05 M NaCl, 0.01 M sodium phosphate, pH 7.2, 0.1% Nonidet P-40, and the specifically bound proteins were eluted with 0.5 M NaCl, 0.01 M sodium phosphate, pH 7.2, 0.1% Nonidet P-40. Collected fractions were subjected to SDS-PAGE.

Anti-factor H Affinity Chromatography-- 40 mg of anti-factor H IgG was completely bound to 5 ml of CNBr-Sepharose (Amersham Pharmacia Biotech) by standard techniques (28). Rat plasma was brought to 0.5 M NaCl and loaded onto the column. After washing with 10-column volumes of 0.5 M NaCl, 0.02 M sodium phosphate, pH 7.2, factor H was eluted from the column with 0.1 M glycine-HCl, pH 2.5. 1-ml fractions were collected into tubes containing 50 µl of 1 M Tris base to immediately neutralize the pH.

Western Blotting-- Proteins recovered by C3b affinity chromatography were separated by SDS-PAGE and electrophoretically transferred overnight to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 5% (w/v) nonfat dry milk in 0.1 M NaCl, 0.02 M Tris, pH 7.2, 0.5% Tween 20 (TBST) for 2 h. The membrane was incubated overnight with goat anti-factor H, washed three times with TBST and then incubated with peroxidase-conjugated anti-goat IgG (Jackson Immunoresearch, West Grove, PA). Subsequently, the membrane was washed in TBST and developed with the ECL detection kit (Amersham Pharmacia Biotech).

Amino Acid Sequencing-- Proteins were separated by SDS-PAGE and stained with Coomassie Blue. Relevant bands were excised, destained thoroughly, and subjected to amino acid sequencing. Sequence analysis was performed at the Harvard Microchemistry Facility (Cambridge, MA) by trypsin digestion followed by microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry (µLC/MS/MS) on a Finnigan LCQ Quadrupole Ion Trap mass spectrometer (ThermoQuest, San Jose, CA) (29).

Immunofluorescence Microscopy (IF)-- Rat and mouse platelets were processed for standard indirect IF. Platelets were fixed in buffered formalin and then incubated for 1 h with goat anti-factor H followed by FITC-conjugated rabbit anti-goat IgG (Cappel, Durham, NC) and viewed with a BX-60 IF microscope (Olympus Optical Co., Ltd., Tokyo, Japan). In parallel, platelets were stained with anti-rat or -mouse Crry or anti-rat thrombocyte F(ab')2 as positive controls or with nonimmune F(ab')2 as a negative control.

Flow Cytometry-- Purified platelets were incubated for 1 h at room temperature with anti-factor H, anti-Crry, or anti-DAF as positive controls, or nonimmune F(ab')2 as a negative control. After washing with PBS, the platelets were incubated with FITC-conjugated anti-goat or anti-sheep IgG (Cappel Laboratories, West Chester, PA). Thereafter, the platelets were washed and analyzed by flow cytometry (FACScan, BD PharMingen).

Additional experiments were designed to determine whether factor H was adsorbed on the platelet membrane or whether it was bound via glycosylphosphatidyl inositol (GPI) or sialic acid residues. Aliquots of platelets were washed with high salt (PBS + 0.4 M NaCl) or pretreated with neuraminidase (5 units in 100 µl of 100 mM NaCl, 50 mM sodium acetate, 5 mM CaCl2, pH 6.5, Ref. 30) or phosphatidylinositol-specific phospholipase C (PIPLC, 1 unit in 100 µl of PBS) for 30 min at 37 °C. After washing, platelets were then stained with anti-factor H Abs and analyzed by flow cytometry. Control assays using anti-Crry and anti-DAF Abs were run in parallel.

Rosette Formation-- Purified rat or mouse C3 was incubated with sheep E bearing human C4b and oxidized C2b (EC42, National Jewish Laboratories, Denver, CO) to form EC423b (21). A total of 2 × 107 EC423b cells were mixed with ~1 × 108 rat or mouse platelets in 200 µl of 0.14 M NaCl, 0.02 M sodium barbital, 0.015 mM CaCl2, 0.05 mM MgCl2, pH 7.4, and rotated at 37 °C for 20 min. As viewed by light microscopy, clumps of 3 or more EC423b cells were considered to be positive. In all instances, the source of C3b and platelets were from the same species. Platelets mixed with EC42 cells and incubated at 37 °C were used as negative controls. To determine the effect of Abs on rosette formation, platelets were preincubated with anti-factor H or anti-Crry F(ab')2 Abs at 10 µg/ml for 15 min at room temperature before the addition of EC423b.

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)-- Total RNA was isolated from rat platelets, kidney, and liver using TriZol reagent (Life Technologies, Inc., Manassas, VA). cDNA was produced from 5 µg of total RNA by RT using oligo-dT primers (Life Technologies, Inc.). Subsequent PCR was performed with 20 mM Tris-HCl, 2 mM MgCl2, 100 µM of each deoxynucleotide triphosphate, 0.1 µM of each primer, and 2.5 units of Taq polymerase. The primers were designed from the known sequence of mouse factor H, spanning bases 2556-3028 (GenBankTM accession number M12660) (20): sense, 5'-TGATTGAAACCACCGTGAAA-3' and antisense 5'-AATAAATGCTGGTTCCATC-3'. Thirty cycles of 1 min denaturation at 94 °C, 1 min annealing at 56 °C and 1 min extension at 72 °C were performed. PCR products formed were electrophoresed through a 2% agarose gel and stained with ethidium bromide.

3'-Rapid Amplification of cDNA Ends (RACE)-- The nucleotide sequence at the 3'-end of factor H mRNA from rat platelets was analyzed using a 3' RACE kit (Life Technologies, Inc.). The first strand of cDNA synthesis was initiated at the poly(A)+ tail of rat platelet mRNA using an oligo-dT adapter primer. Amplification was done using a factor H specific primer: 5'-CCACCAACATGCTTACATGC-3' spanning bases 3641-3660 of mouse factor H sequence and an antisense abridged universal amplification primer that had a complementary region to the adapter primer used in the first strand cDNA synthesis.

Sequence Analysis-- PCR products obtained by RT-PCR and 3'-RACE were extracted from the gels with a commercially obtained kit (Qiagen, San Diego, CA). The purified PCR products were inserted into the TA vector (Invitrogen, Carlsbad, CA). Positive clones were sequenced on an ABI 373A DNA Sequencer using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and M13 reverse primers. Sequence comparisons were made with Genetics Computer Group software (Madison, WI).

Northern Analysis-- 20 µg of total RNA was electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde, transferred by capillary action to a nylon membrane, and cross-linked by ultraviolet irradiation. Membranes were prehybridized and hybridized at 42 °C in buffer containing 5× SSPE, 5× Denhardt's solution, 200 µg/ml of denatured salmon sperm DNA, 0.1% SDS, and 50% formamide. The PCR product obtained from platelets, used as probe, was random prime-labeled with [alpha -32P]dCTP to activities of ~109 cpm/µg (Roche Molecular Biochemicals) (31). After hybridization with the probe, the membranes were washed with 2× SSC, 0.1% SDS and then at 65 °C with 0.1× SSC, 0.1% SDS. Autoradiography was subsequently performed at -80 °C with an intensifying screen. The blots were stripped and complete probe removal verified by autoradiography. The blots were then probed for GAPDH as a housekeeping gene (32) and subjected to autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CR1 Is Not the Mouse IAR-- To confirm that CR1 is not the IAR in mice, platelets were isolated from CR1-/- mice. Platelets from both CR1-/- and wild-type mice formed rosettes with EC423b clearly evident by light microscopy (Fig. 1). Rosettes were not formed with platelets from either strain and EC42 cells (not shown). Thus, CR1 does not function as the IAR of mouse platelets


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  		Fig. 1.   EC423b rosette with platelets from CR1-/- (A) and wild-type (B) mice. EC423b, prepared as described under "Experimental Procedures," were incubated with platelets from CR1-/- (A) or wild-type (B) mice for 20 min at 37° C, and the appearance of rosettes containing EC423b bound to IAR-containing platelets was visualized by light microscopy. Magnification, × 200.

Identification and Characterization of C3b-binding Proteins from Rat Platelet Membranes-- To isolate C3b-binding proteins from platelets, solubilized platelet membranes were subjected to C3b affinity chromatography. As shown in Fig. 2, a predominant 150-kDa band was recovered. Under reducing conditions, this protein migrated at 175-kDa, consistent with the presence of intrachain disulfide bonds. In addition, a 115-kDa band was also recovered by C3b affinity chromatography.


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  		Fig. 2.   Isolation of rat platelet C3b-binding proteins. Solubilized rat platelet membranes were subjected to C3b affinity chromatography. Proteins bound to the C3b column were eluted and subjected to non-reducing SDS-PAGE followed by staining with Coomassie Blue.

Tryptic peptides from the 150-kDa C3b-binding protein were analyzed by µLC/MS/MS and found to be identical to the sequence of mouse factor H (20). Direct comparisons were made between the platelet 150-kDa C3b-binding protein and factor H purified from rat plasma. The amino acid sequences of peptides from both platelet and plasma proteins were identical (Table I). The peptides corresponded to residues 39-200 and 719-842 of the known 1216 amino acid mouse factor H sequence, and encompassed SCRs 1-4 and 12-14 of this 20 SCR-containing protein (20). Identical peptides were obtained from the 115-kDa platelet C3b-binding protein, consistent with its identity to factor H through residue 842. 

                              
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Table I
Amino acid sequences obtained from tryptic peptides derived from the platelet 150-kDa C3b-binding protein and rat plasma factor H

As an independent verification that the platelet C3b-binding proteins were comparable to factor H, immunoblotting was performed. The 150-kDa and 115-kDa C3b-binding proteins isolated from rat platelets were recognized by goat anti-human factor H by Western blotting (Fig. 3, lane 1). As a positive control, plasma factor H was run in parallel (lane 2). Therefore, rat platelets have a 150-kDa C3b-binding protein with biochemical and immunochemical relatedness to factor H. 


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  		Fig. 3.   Rat platelet C3b-binding proteins are recognized by anti-factor H Abs. Rat platelet C3b-binding proteins were electrophoresed under non-reducing conditions and subjected to Western blotting with anti-human factor H Abs (lane 1). As a positive control, rat plasma factor H was treated identically (lane 2).

Factor H Is Present on the Surface of Rodent Platelets-- By IF microscopy, platelets from rats (Fig. 4A) and mice (not shown) were positively stained with anti-factor H Abs. Staining for the complement inhibitory protein, Crry, present on the surface of platelets (Fig. 4B) (18), and with polyclonal anti-platelet Abs (Fig. 4C) were used as positive controls. The surface expression of factor H on rat platelets was further substantiated by flow cytometry (Fig. 5B), with Crry again serving as the positive control (Fig. 5C). Rat factor E subjected to flow cytometry were not stained with anti-factor H Abs (not shown), indicating factor H was not passively adsorbed to circulating blood cells. As these studies were performed without plasma membrane solubilization, this indicates factor H is a membrane-bound protein on the rat and mouse platelet surface.


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  		Fig. 4.   Rat platelets bear factor H by IF microscopy. Formaldehyde fixed rat platelets were stained with anti-factor H F(ab')2 (A), with anti-Crry (B), and anti-thrombocyte (C) Abs as positive controls. Specifically bound primary Abs were detected with FITC-conjugated secondary Abs. Platelets stained with nonimmune F(ab')2 were not visible and, as such, are not shown. Magnification, × 400.


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  		Fig. 5.   Flow cytometric analysis of factor H on rat platelets. Rat platelets were incubated with nonimmune (A), anti-factor H (B), or anti-Crry F(ab')2 (C) Abs. In further studies, platelets were pretreated prior to analysis. The black line represents the results from platelets exposed to PIPLC (D and E), or neuraminidase (F), whereas the color-filled areas indicate platelets exposed to appropriate buffer control alone. Platelets were incubated in PIPLC followed by anti-factor H (D) or anti-DAF (E), or treated with neuraminidase and then anti-factor H Abs (F). In all instances, bound primary Abs were detected with FITC-labeled secondary Abs and analyzed by FACScan. The increased fluorescence of platelets in neuraminidase buffer alone stained with anti-factor H (F, filled red area) was a consistent finding in all experiments.

Evaluation of Factor H Attachment to the Platelet Surface-- To determine the mechanism by which factor H is bound to the platelet surface, platelets were subjected to flow cytometry after the following treatments: 1) high salt washing; 2) pretreatment with PIPLC, and 3) pretreatment with neuraminidase. High salt washing did not remove factor H from the surface of platelets, nor, as predicted, did it remove the type I membrane protein, Crry (not shown). PIPLC treatment did not eliminate Crry (not shown) or factor H (Fig. 5D) from the platelet surface, whereas DAF, attached to the platelet surface by a GPI anchor (33, 34) was removed (Fig. 5E). In contrast, neuraminidase treatment reduced the binding of anti-factor H (Fig. 5F) but not anti-Crry (not shown) to the platelet surface demonstrating that factor H is bound to platelets through sialic acid residues.

Platelets Can Synthesize Factor H-- To determine whether rat platelets express factor H mRNA transcripts, RT-PCR analysis was performed with primers designed from the known sequence of mouse factor H. As shown in Fig. 6, a PCR product of the predicted size of 472 base pairs was amplified from rat platelets (lane 1) and liver (lane 2). By sequence analysis, the rat platelet and liver sequences were identical, and 90% similar to the known sequence of mouse factor H encompassing amino acids 802-958 (20); the predicted translated protein product was 85% identical to mouse factor H as shown in Table II. Using this PCR product as probe in Northern analyses, RNA from platelets, kidney, and liver had identically sized RNA transcripts of 5.5, 3.4, and 1.8 kilobases (Fig. 7).


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  		Fig. 6.   Platelets bear mRNA for factor H. RT-PCR analysis using primers spanning bases 2556-3028 of mouse factor H, showing that rat platelets (lane 1) contain RNA of the predicted size and comparable with that identified in rat liver (lane 2).

                              
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Table II
Predicted amino acid sequence of rat platelet factor H compared to that of mouse liver factor H
Shown is the amino acid sequence of rat platelet factor H predicted from the nucleotide sequence obtained from RT-PCR. This is directly compared to the sequence of SCR's 14-16 of mouse liver factor H. Where there are amino acid differences, the mouse sequence is provided below the rat platelet sequence. The conserved cysteine residues are underlined. In bold type is a peptide sequence obtained from rat platelet C3b-binding proteins by µLC/MS/MS.


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  		Fig. 7.   Rat platelets contain factor H mRNA. Northern analyses show that rat platelets (lane 1) have factor H mRNA of comparable size to that identified in rat kidney (lane 2) and liver (lane 3). The positions of ribosomal RNA in the gel are indicated with arrows.

By 3'-RACE of RNA isolated from rat platelets, a product of the predicted size of 699 base pairs was amplified. The nucleotide sequence of this product was identical to the known sequence of mouse liver factor H, except for one base at residue 4169 in the 3'-untranslated region. These results indicate rat platelets have the inherent capacity to produce factor H protein, which is predicted to be a soluble protein. Thus, any alterations accounting for its capacity to bind to the platelet membrane are likely to occur after protein translation.

Factor H Functions as the Rodent IAR-- Platelets from normal rats and mice formed rosettes with EC423b cells (Fig. 8, A and C). Preincubation with anti-factor H F(ab')2 Abs inhibited rosette formation in platelets of both species (Fig. 8, B and E). As a negative control, antibodies to Crry, a complement regulator on the rodent platelet surface, were used. Platelets pretreated with anti-Crry F(ab')2 clearly rosetted with EC423b (Fig. 8D). Rosette formation was eliminated when platelets were preincubated with neuraminidase (Fig. 8F). Thus, factor H on the rodent platelet surface is the functional IAR.


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  		Fig. 8.   Factor H is the functional IAR of rodent platelets. Rosetting of rat (A, B) and mouse (C-F) platelets with EC423b was determined following pretreatment with preimmune F(ab')2 (A, C), anti-Crry F(ab')2 (D), anti-factor H F(ab')2 (B, E), or neuraminidase (F). Rosetting with EC423b (A, C) was inhibited with anti-factor H Abs (B, E) or neuraminidase pretreatment (F), but not with anti-Crry Abs (D). Magnification: A-B, × 400; C-E, × 200.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The IA reaction was defined by Nelson (2, 3) as the interaction between cells and complement-coated particles. Immune complexes bearing the activated complement component, C3b, bind to cells containing IAR, which then transport them to the mononuclear phagocyte system for their disposal. In primates, IA function clearly is served by CR1 on erythrocytes. In contrast, the immune complex clearance mechanism in non-primates, including rodents, is carried out by platelets rather than erythrocytes (13, 35). The mouse complement receptors CR1 and CR2 were identified on the basis of similarity to their human counterparts. However, notable differences are that: 1) they are derived from alternatively spliced transcripts of the same gene, Cr2 (27, 36), 2) they have similar but not identical ligand binding characteristics compared with humans (37), and, 3) neither of the proteins are present on platelets, erythrocytes, or unstimulated neutrophils (16), confirming that CR1 does not function as the rodent IAR. We showed that rosetting occurred, as an in vitro assay for the IA reaction, with platelets isolated from CR1-/- mice. Therefore, the IAR in rodents has gone unidentified.

In this study, we purified a 150-kDa single chain protein from platelets by C3b affinity chromatography. Its characteristics by SDS-PAGE are similar to the C3b-binding protein isolated from rabbit platelets by the Atkinson laboratory (17). The 150-kDa C3b-binding platelet protein and factor H isolated from rat plasma were identical in the regions where the peptide sequences were obtained, which were localized to two areas spanning 23.3% of the length of the protein and encompassing SCRs 1-4 and 12-14. The 115-kDa C3b-binding platelet protein also was related to factor H, both at an amino acid sequence level and by immunoblotting with anti-human factor H Abs. The factor H family continues to expand as more factor H-related proteins are identified (38). However, to date, none are larger than 62 kDa nor do they include the combination of SCRs 1-3 and 14 as identified by mass spectrometry in the 115-kDa protein (39). It is more likely that this 115-kDa protein is either a breakdown product in the preparation of solubilized platelet membranes or a spliced gene product, which occurs frequently in the regulators of complement activation gene family (36, 40).

Factor H displays several similarities with the human IAR, CR1. Both are large glycoproteins possessing only N-linked carbohydrate moieties (41, 42). Both belong to the regulators of complement activation family, which also includes CR2, DAF, and membrane cofactor protein (11, 12). Genes of this family are encoded on the long arm of chromosome 1q32 in humans (43) and a conserved linkage group in mouse chromosome 1 (44). These proteins share a tandemly repeated SCR motif of ~60 amino acids, containing a conserved amino acid framework around four invariant cysteines, which form two disulfide bonds. Like CR1, factor H is a versatile protein with several different functions. It inhibits the formation and accelerates the decay of C3 convertases, serves as a cofactor for factor I (45), displays chemotactic activity for monocytes (46), and possibly participates in interactions with extracellular matrix and leukocytes (47).

Any candidate for the rodent IAR must be present on the surface of platelets. By flow cytometry and IF, factor H was present on the platelet membrane. Neither technique detected factor H on rat erythrocytes. This is an important control, as erythrocytes do not participate in IA in non-primates (6, 13), and it also indicates not all circulating blood cells contain factor H. Flow cytometry further indicated that when rodent platelets were washed with high salt, factor H was not removed, providing evidence that factor H did not just passively adsorb from plasma onto the platelet membranes. Neuraminidase treatment of platelets reduced factor H, suggesting that factor H is bound to the platelet membrane by sialic acid residues, whereas Crry, a type I membrane protein, remained unaltered. PIPLC treatment caused a substantial decrease in DAF, a GPI-linked protein on the platelets but no change in factor H or Crry, indicating that factor H is not GPI-linked to the platelets.

There are numerous examples of factor H binding to the surface of a variety of cells, including nucleated cells (48, 49), viruses (50), bacteria (30, 51-53), and parasites (54). In the majority of cases, this is mediated via interactions of several sites on factor H with sialic acid residues on the host cell (30, 55, 56), although protein-protein interactions have also been described (57). For the most part, the binding of factor H has conferred a resistance to complement activation, presumably through its capacity to decay C3 convertases as well as act as a factor I cofactor (39). Factor H mediates these actions through its affinity for C3b (58, 59), although specific binding to C3b is not a demonstrated feature of most of these interactions. As with other molecules with low affinity interactions, clustering of molecules can considerably increase binding avidity (60).

The PCR product obtained from rat platelet RNA differed from mouse liver factor H by only 15% at the protein sequence level. Furthermore, by 3'-RACE, nucleotide sequences corresponding to SCRs 19-20 and into the 3'-untranslated region were identical to that from mouse liver factor H. Northern analysis recognized factor H transcripts of similar lengths in rat liver, kidney, and platelets. Such differently sized transcripts have also been observed for human factor H mRNA, which is compatible with alternative mRNA splicing (61). In humans, the smaller 1.8 kilobase factor H mRNA transcript gives rise to the 45-kDa factor H-like protein 1 (39, 61). Despite the presence of a similarly sized transcript in platelets, we did not observe a 45-kDa C3b-binding protein from platelets in this study. Native factor H has three potential binding sites for both C3 and sialic acid, whereas factor H-like protein 1 has only one of each (39). As such, factor H would have superior binding capabilities for both sialic acid residues on the platelet surface and C3b in immune complexes.

The PCR and Northern analysis data clearly demonstrate the intrinsic ability of platelets to synthesize factor H. Factor H has been known to be associated with human platelets (62). This platelet-associated factor H resides in alpha  granules, is functionally active, and is released upon complement activation or other stimuli (63, 64). Our data show that rodent platelets are different from their human counterparts as they contain factor H on their surface. In this regard, they are similar to U937 monocytic cells, which synthesize and bear factor H on their plasma membrane while not secreting it as a soluble protein (65).

In summary, we have isolated a 150-kDa C3b-binding protein from rat platelets. By biochemical and immunochemical criteria, the protein has characteristics of factor H and resides on the surface of platelets, which also have the inherent capacity to produce this protein. When the function of factor H is blocked on rodent platelets with neutralizing Abs, rosetting with EC423b is prevented, establishing factor H as the rodent IAR. Further studies of platelet-associated factor H will help elucidate mechanisms of immune complex clearance and inflammatory cell accumulation in various rodent models of immune complex-mediated diseases. These animal models are particularly useful in this era where genetic manipulations of individual genes are possible. The generation of factor H-deficient mice will allow dissection of the role of the IAR in vivo.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01DK48173 and R01DK55357 and a chapter grant from the Arthritis Foundation, Greater Chicago Chapter.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Training Grant T32DK07510.

§ To whom correspondence should be addressed: The University of Chicago, Section of Nephrology, 5841 S. Maryland Ave., MC5100, Chicago, IL 60637. Tel.: 773-702-4796; Fax: 773-702-5818; E-mail: jalexand@medicine.uchicago.edu.

Published, JBC Papers in Press, June 13, 2001, DOI 10.1074/jbc.M101299200

    ABBREVIATIONS

The abbreviations used are: IA, immune adherence; Ab, antibody; C, complement; Crry, CR1-related gene y; DAF, decay accelerating factor; FITC, fluorescein isothiocyanate; GPI, glycosylphosphatidyl inositol; IF, immunofluorescence; PBS, phosphate-buffered saline; PIPLC, phosphatidylinositol-specific phospholipase C; SCR, short consensus repeat; TBST, Tris-buffered saline with 0.5% Tween 20; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; µLC/MS/MS, microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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