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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1533-1540
PLENARY PAPER
From the Cancer Research and Treatment Center and the Department of
Pathology, the University of New Mexico Health Sciences Center,
Albuquerque, NM; the Department of Microbiology, School of Medicine,
Korea University, Seoul, Korea; and the Departments of Pathology and
Laboratory Medicine and Pediatrics, University of Pennsylvania School
of Medicine, Philadelphia, PA.
Antibodies to PF4/heparin can be demonstrated in almost all patients
with heparin-induced thrombocytopenia/thrombosis (HIT/HITT) and in some
persons exposed to heparin who do not have clinical manifestations. The
role of anti-PF4/heparin antibodies in the pathogenesis of HIT/HITT has
been difficult to establish because the antibodies found in serum are
generally polyclonal and polyspecific. To circumvent this problem, we
developed a murine monoclonal antibody (mAb) to human (h) PF4/heparin
complexes. A monoclonal IgG2b
Heparin-induced thrombocytopenia/thrombosis (HIT/HITT)
is a life-threatening complication that develops in 1% to 5% of
patients exposed to intravenous heparin.1 Autoantibodies to
PF4/heparin complexes can be identified in the plasma of more than 90%
of patients in whom the clinical syndrome develops.2
However, anti-PF4/heparin antibodies can also be demonstrated in a
significant proportion (15% to 70%) of asymptomatic patients
repetitively exposed to heparin.3-6 Why symptomatic disease
develops in only a subset of immunized patients is
unknown.1,7,8
Heterogeneity in disease expression may, in part, reflect differences
in comorbid factors that predispose to thrombosis, such as
atherosclerosis, surgery, and vascular trauma.9,10 Others have implicated differences in antibody titer,11
affinity,12 isotype,13
subclass,11,14 and platelet Fc receptor polymorphism (Fc An additional explanation may lie in the heterogeneity of
anti-heparin/PF4 antibodies themselves. Anti-PF4/heparin antibodies found in serum differ in their antigen
specificities,11,18-20 though the
responsible determinants have not been clearly delineated. Certain
PF4/heparin antibodies may affect the capacity of PF4 to modulate
heparin-dependent antithrombin,21 protein C
co-factor,22 or other procoagulant and anticoagulant
activities. However, the polyclonal nature of the naturally occurring
immune response complicates any attempt to determine whether a subset
of anti-PF4 antibodies is responsible for thrombosis. To begin to
address this possibility, we have developed a murine monoclonal
antibody (mAb) to hPF4 and hPF4/heparin complexes. In this article, we
describe the antigenic specificity of one such hPF4/heparin mAb,
designated KKO, and its relationship to naturally occurring human
antibodies from patients with HIT/HITT.
Materials
Patient samples
Development of monoclonal antibodies to hPF4/heparin and hPF4 Four 6- to 8-week-old female Balb/c mice were injected intraperitoneally on day 0 with a 50 µL sterile solution composed of 25 µL phosphate-buffered saline (PBS) containing recombinant hPF4 (50 µg), heparin (2 U), and 25 µL Freund's complete adjuvant. Subsequent injections containing 50 µg hPF4 and 2 U heparin in PBS were administered intraperitoneally or through the tail vein on days 12, 30, 41 and 48, and 62. Mice were given an intravenous boost of heparin and hPF4 on day 66, 3 days before they were sacrificed. Titers of anti-hPF4/heparin were monitored by ELISA (see below). The 2 mice expressing the highest serum titers (more than 1:100,000) of anti-hPF4/heparin antibodies were sacrificed, and their spleens were removed for fusion. Fusion and hybridoma selection were optimized using standard methodology.23 Hybridomas were cultured for 7 days, and their supernatants were screened for antibodies to hPF4/heparin and hPF4 by ELISA. Wells considered positive (A405 > 0.8) were weaned from HAT supplement over 7 to 10 days, subcloned by limiting dilution, and grown in pristane-primed mice to generate ascites. Monoclonal antibodies to hPF4/heparin (KKO) and hPF4 alone (RTO) were isolated from ascitic fluid using the Hi Trap affinity columns according to the manufacturer's instructions. Isotyping was performed using the ImmunoPure Monoclonal Antibody Isotyping kit according to the manufacturer's instructions.Preparation of human and murine PF4, hPF4 mutants, NAP-2, and IL-8 Recombinant wild-type human and mouse PF4, mutant hPF4, hNAP-2, and hIL-8 were expressed in E coli as described.24 Briefly, cDNA constructs for each chemokine (see below) were inserted in a pT7-7 vector, introduced into E coli BL21(DE3) pLysS, and grown in Luria broth containing 100 µg/mL ampicillin. Bacteria were grown to an A600 of 1.0, followed by 3 hours of induction at 37°C with 1 mmol/L IPTG. Bacteria were lysed and sonicated, and the chemokine was purified at room temperature by affinity chromatography using heparin-agarose equilibrated with 50 mmol/L Tris HCl and 1 mmol/L EDTA, pH 8, and was eluted using a 0.2 to 2.0 mol/L NaCl gradient. Eluted proteins were further purified by reverse-phase chromatography using a ProRPC FPLC column.
PF4/heparin ELISA Binding of antibody to either PF4, PF4 variants, PF4 bound to various GAGs, or other chemokines complexed to heparin was measured using an ELISA-based method as previously described.2 Briefly, to screen hybridoma culture supernatants, 96-well microtiter plates were coated overnight at room temperature with 50 µL/well PBS containing PF4 (final concentration, 10 µg/mL) in the presence or absence of heparin (0.2 U/mL). The plates were then washed 3 times with Tris-buffered saline/0.01% Tween-20, blocked with 10% fetal calf serum (FCS) (200 µL/well) in PBS for 2 hours at room temperature, and washed once more. Culture supernatant was added (50 µL/well) for 1 hour at room temperature. Unbound antibody was removed by washing, and 50 µL/well of alkaline-phosphatase-conjugated goat antimouse IgG, diluted 1:1000 in 10% FCS/PBS, was added for 1 hour at room temperature. After washing, 50 µL/well Sigma Fast p-nitrophenyl phosphate substrate was added, and the absorbance at 405 nm was measured. Binding of KKO to PF4 and related chemokines, or PF4 bound to various GAGs, was measured in the same manner except that an incubation volume of 100 µL/well was used.Competition assays by ELISA The capacity of KKO to bind hPF4/heparin-coated wells in the presence of HIT sera was assessed using a concentration of KKO (diluted in 10% FCS/PBS) that produced 75% of maximal binding. The diluted KKO was added to wells in the presence of varying concentrations of HIT/HITT sera, and the binding of KKO to hPF4/heparin was measured by ELISA as described above.Cell-associated ELISA Cultured human umbilical vein endothelial cells (HUVECs) were prepared as described.26 Cells were grown to confluence in complete media containing M199 supplemented with 20% FCS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL amphotericin B, endothelial cell growth supplement, and heparin (100 µg/mL) in 75 cm2 flasks. The cells were then seeded onto 96-well microtiter plates26 at a density of 32,000 cells/well in the absence of heparin. After 48 hours in heparin-free medium, the cells were fixed with 0.05% glutaraldehyde, and binding of KKO or RTO was measured by ELISA as described above. In other experiments, binding of KKO to Chinese hamster ovary (CHO) cells lacking xylosyl transferase (provided by C. Esko, University of California and K. Williams, Thomas Jefferson University) was conducted in essentially the same manner.Platelet activation by KKO Platelet activation by KKO in the presence of PF4 and heparin was assayed by the release of 14C-serotonin,2 with modification for a microtiter-well format. Briefly, citrated platelet-rich plasma obtained from aspirin-free healthy donors was labeled with 14C-serotonin (22.5 nCi/mL PRP) for 30 minutes at 37°C, after which platelet uptake of 14C-serotonin was blocked by the addition of excess imipramine (1 µmol/L final concentration). KKO or isotype control (30-320 µg/mL) in modified Tyrode's buffer (137 mmol/L NaCl, 3 mmol/L KCl, 0.4 mmol/L NaH2PO4, 12 mmol/L NaHCO3, and 1 mmol/L MgCl2·6H2O, pH 7.0) was preincubated with either hPF4 (10 µg/mL) alone, heparin alone (0.2 U/mL), or hPF4 (10 µg/mL) plus heparin (0-100 U/mL). Labeled platelets (75 µL) were added in triplicate to wells containing antigen/antibody-containing mixture (20 µL) with 5 µL heparin or buffer to yield a 100 µL final volume. After a 1-hour incubation at room temperature, the reaction was terminated by the addition of 100 µL of 0.5% Na EDTA (pH 8.5), platelets were pelleted, and the release of 14C-serotonin was measured by liquid scintillation. In other experiments, 14C-labeled platelets were incubated with the Fc RIIA blocking antibody mAb IV.3 (7.2 or 72 µg/mL)27 for 1 hour before the addition of KKO (250 µg/mL), PF4, and heparin. Controls for the assay included plasma from
patients with HIT (SRA+, positive control), normal plasma,
mIgG2b (isotype control), RTO, and calcium ionophore A23187.
Sequence analysis of KKO and RTO Total RNA was prepared from approximately 4 × 106 hybridoma cells for clones KKO and RTO (TRIzol reagent) followed by cDNA synthesis primed with oligo dT following the manufacturer's instructions. Heavy- and light-chain immunoglobulin variable regions were amplified using the PCR as previously described28 according to the following framework 1 and constant region primers for murine and  chains: heavy-chain forward 5'-GAGGTGAAGCTGGTGGAG(T/A)C(T/A)GG-3',
heavy-chain reverse 5'-GGGGCCAGTGGATAGAC-3',
light-chain forward 5'-CCAGTTCCGAGCTCCAGATGACCCAGACTCCA-3', light-chain reverse 5'-GTTGGTGCAGCATCAGC-3'. The PCR
products (350-400 bp) were gel purified by electroelution and directly sequenced using the above oligonucleotides and automated fluorescence sequencing at the Nucleic Acid/Protein Research Core Facility of The
Children's Hospital of Philadelphia. Use of these "universal" variable region framework 1 primers provided heavy- and light-chain sequences that began at the 8th and 9th amino acid residues,
respectively. To determine the authentic amino acid N-terminal residues
for KKO chains, putative leader sequences were determined by searching Genbank for other murine monoclonal antibodies with close homology to
KKO. For the heavy and light chains of KKO, sequence accession numbers
AF025443 and M20830 provided candidate leader sequences. Sets of
5' PCR primers beginning at the 5' end of the leaders were
synthesized (5'-ATGGGATGGAGCTATATCATCC-3' for heavy chain and 5'-ATGATGAGTCCTGCCCAGTTCC-3' for light chain). PCR
amplifications of KKO heavy- and light-chain cDNA were performed using
these primers paired with the original set of constant region reverse primers. PCR products of the appropriate size (400-450 bp) were obtained and served as templates to provide full-length variable region
KKO sequences. Immunoglobulin gene family assignments for heavy and
light chains were determined using the Kabat29 and Genbank
databases. Alignments of the predicted amino acid sequences were
performed using the MacVector software package.
Isolation and screening of murine mAb Seven days after fusion, 128 of 1152 wells contained antibodies to either hPF4/heparin or hPF4 by ELISA using A405 0.8 as an arbitrary cut-off value. Cells were subcultured when their supernatants generated A405 ratios to hPF4/heparin versus
hPF4 > 1.5. Cell populations showing the greatest relative
specificity for hPF4/heparin versus hPF4, and those with high
reactivity to hPF4 alone, underwent 3 additional rounds of subcloning.
Two monoclonal antibodies, an IgG2b
hPF4/heparin-dependent antibody designated KKO and an
IgG2b anti-hPF4 designated RTO, were ultimately isolated and subjected to further characterization. KKO exhibited an
A405 ratio of >30 to hPF4/heparin versus hPF4,
whereas RTO demonstrated an A405 ratio of  1. Monoclonal
antibodies purified from ascites were used for all subsequent studies.
Specificity of KKO and RTO for PF4/heparin complexes KKO bound to hPF4/heparin complexes in a dose-dependent manner (Figure 1A; half-maximal binding 0.036 µg/mL). The relative specificity of KKO was determined by adding various dilutions of KKO to wells coated with hPF4/heparin or hPF4 alone. Specificity for the complex was evident at all antibody concentrations tested (0.007-36 µg/mL; Figure 1A). At concentrations less than 0.14 µg/mL, the ratio of binding of KKO to PF4/heparin versus PF4 determined by A405 was more than 400. No binding of KKO to PF4 alone was evident at concentrations less than or equal to 0.072 µg/mL. KKO did not bind to immobilized heparin at any concentration tested (0.007-36 µg/mL). Although KKO demonstrated heparin-dependent binding to hPF4, binding of RTO to hPF4 was unaffected by the presence of heparin at all concentrations of antibody tested (Figure 1B).
Binding of KKO to human PF4 in complex with GAGs Another feature of naturally occurring HIT antibodies is their cross-reactivity with complexes composed of PF4 and other sulfated GAGs.31,32 Similarly, KKO bound to complexes formed between hPF4 (10 µg/mL) and 0-500 µg/mL chondroitin sulfate A, chondroitin sulfate B, or dermatan sulfate, chondroitin sulfate C, heparan sulfate, and dextran sulfate (Mr 8000) following a pattern similar to that reported previously for HIT antibodies33 (Figure 2).
Cell-reactivity of KKO Similar to HIT-IgG,34 KKO bound to cultured endothelial cells (Figure 3) and CHO cells (not shown) in the presence of exogenous hPF4 but not to CHO cells lacking heparan sulfate- or chondroitin sulfate-containing proteoglycans under the same conditions (data not shown). Binding of KKO to HUVECs was inhibited by heparin at concentrations (0.2 U/mL) known to dissociate PF4 from the cell surface (Figure 3). In comparison to KKO, equimolar concentrations of RTO bound one-third as well to HUVEC in the presence of either PF4 or PF4 and heparin (data not shown), consistent with ELISA data shown in Figures 1A and 1B. Whereas binding of KKO to PF4 was enhanced in the presence of heparin (Figure 1A), binding of RTO was diminished (Figure 1B), suggesting that RTO recognized an epitope on PF4 that was masked or altered by heparin or heparinlike molecules.
Platelet activation by KKO HIT antibodies27 and immune complexes containing murine IgG2 antibodies activate human platelets through a process that requires FcRIIA.35,36 To
determine whether KKO activates platelets through a similar pathway,
14C-serotonin-labeled platelets were incubated with KKO
(30-320 µg/mL) in the presence of either hPF4, heparin, or
hPF4/heparin. KKO (80 and 160 µg/mL) stimulated
14C-serotonin release in a heparin-dependent manner (Figure
4) when preincubated with hPF4 (10 µg/mL). Somewhat higher concentrations of antibody (KKO > 180
µg/mL) were required to initiate serotonin release when the antibody
was preincubated with hPF4 (10 µg/mL) complexed to heparin (1 U/mL)
(data not shown). Neither KKO nor isotype control activated platelets
in the presence of buffer alone or heparin alone (data not shown).
14C-serotonin release induced by KKO was almost completely
inhibited by the FcRIIA-specific mAb IV.3 (less than 5% release at
0.5, 1, and 5 U/mL heparin) (data not shown). RTO did not effect
serotonin release in the presence or absence of hPF4 or hPF4/heparin at all concentrations tested (31-250 µg/mL).
Epitope specificity of KKO We previously reported that a subset of HIT/HITT antibodies require an epitope in the third domain of hPF4 to bind in the presence of heparin. This region was defined using chimeras between hPF4 and the structurally related chemokine, NAP-2, which is not recognized by HIT antibodies.18 We now have investigated both the specificity of this subset of HIT antibodies in greater detail and the involvement of this domain in the binding of KKO. To do so, single amino acid substitutions were introduced into the third domain of PF4, between Cys36 and Cys52. Each PF4 variant was incubated with an optimal concentration of heparin18 and the binding of 23 HIT sera and KKO was measured.
Sequence analysis of KKO and RTO
We generated an mAb, KKO, that shares important serologic and
functional properties with naturally occurring anti-PF4/heparin antibodies found in patients with heparin-induced
thrombocytopenia/thrombosis. KKO recognizes PF4 in complex with heparin
over a narrow range of molar ratios approaching 1:1, similar to the
behavior of naturally occurring HIT antibodies.37 KKO also
recognizes complexes between PF4 and other GAGs, but not heparin itself
or heparin complexed with either mPF4, NAP-2, or IL-8, all features
shared with more than 95% of HIT antibodies. KKO binds to cells that
express GAGs, such as endothelial cells, only when PF4 is provided
exogenously,17,34,38 but it does not bind to cells that
lack GAGs and that are therefore unable to form the requisite antigenic
complex. KKO also activates human platelets through a heparin- and
PF4-dependent mechanism that is mediated through
Fc We are indebted to Joyce Lehner and Charles Hart of ZymoGenetics, Inc,
Seattle, WA, for their technical advice on monoclonal antibody
preparations. We thank Tara Hendry-Hofer for her excellent technical assistance.
Submitted June 24, 1999; accepted October 28, 1999.
Supported in part by NIH research grants HL54749 (MP, DBC), HL35246
(WK), HL61844 (DLS), P20RR11830 (GMA), KO8 HL04009
(GMA), and by the University of New Mexico Research
Allocation Committee Award (GMA).
Reprints: G. M. Arepally, Department of Medicine,
Hematology/Oncology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131; arepally{at}unm.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.
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Top
Abstract
Introduction
antibody (designated KKO)
was identified that bound specifically to hPF4/heparin complexes.
Maximal binding of KKO to hPF4/heparin complexes occurred at similar
molar ratios of PF4:heparin observed for HIT/HITT antibodies. KKO also
bound to hPF4 in association with other glycosaminoglycans. Platelet
activation by KKO required heparin and was abrogated by blockade of
Fc
FIX 129SV library from
Stratagene (LaJolla, CA), TRIzol reagent and Superscript RT-PCR kit
from Gibco/BRL, and AmpliTaq from Perkin-Elmer (Branchburg, NJ).
MacVector (v. 6.0) software package was purchased from Oxford Molecular
Group (Oxford, UK). All other chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO).
) or
hPF4/heparin (
) expressed as absorbance at 405 nm. (B) Binding of
RTO to wells coated with hPF4 (
) or hPF4/heparin (
). (C) Heparin
dependence of KKO was measured by ELISA using wells coated with a fixed
concentration of hPF4 and varying concentrations of heparin. The data
shown represent the mean ± 1 SD of duplicate wells and are
representative of 3 independent measurements.
