WO1994013307A1

WO1994013307A1 - Platelet activation and function

Info

Publication number: WO1994013307A1
Application number: PCT/US1993/012016
Authority: WO
Inventors: Peter G. Carroll; Douglas C. Stafford; John A. Kink
Original assignee: Steritech, Inc.
Priority date: 1992-12-11
Filing date: 1993-12-10
Publication date: 1994-06-23
Also published as: AU5870994A

Abstract

Screening both human and non-human platelets for the expression of Granule Membrane Glycoprotein-140 (GMP-140) using a polyclonal antibody in a simple, convenient, nonradioactive assay. An interspecies cross-reactive, anti-GMP-140 antibody, i.e. antibody that reacts with GMP-140 on the platelets of more than one species.

Description

PLATELET ACTIVATION AND FUNCTION

FIELD OF THE INVENTION

The invention generally relates to methods of measuring platelet activation. In particular, the invention relates to methods for screening both human and non- human platelets for the expression of Granule Membrane Glycoprotein-140 (GMP-

140), an indicator of platelet activation.

BACKGROUND

With the recent and rapid expansion in the field of transfusion medicine, the establishment of an excellent quality control program becomes of paramount importance. In particular, it is necessary to monitor the quality of platelets used in transfusions to ensure that they are not activated. The transfusion of platelet concentrates (PC) is an essential supportive therapy in patients with a variety of medical and surgical problems. M.B. Simpson, In: Platelets, D.M. Smith and S.H. Summers (eds.) (American Association of Blood Banks 1988) (pp. 129-165). Current AABB standards require that platelets from whole blood contain at least

5.5 x 10¹⁰ platelets in at least 75% of the units tested, whereas platelets collected by an apheresis procedure should contain a minimum of 3 x 10" platelets in at least 75% of the units tested.

Post-transfusion monitoring involves measuring the increased increment in the platelet count (if any) and the impact on clinical bleeding. L.A. Harker and

S.L. Slichter, New Eng. J. Med. 287:155 (1972). Normal function and survival of stored platelets following transfusion are essential for therapeutic benefit. S. Murphy and F.H. Gardner, New Eng.J.Med. 280:1094 (1969). G. Rock et al- Transfusion 24:147 (1984). Traditional in vitro tests for platelet quality have been less than adequate.

Tests such as platelet aggregation, pH, ATP secretion, cell count and morphology have been employed to predict platelet survival and function. But these tests do not always indicate when platelets are activated and are only weakly correlated with platelet survival in vivo. Further, some require expensive machinery or are quite time consuming. Recently, focus has turned to more direct measurements of platelet activation. Upon activation, platelets release several substances that mediate platelet responses, resulting in irreversible aggregation and activation of the coagulation system. This is called "secretion" or the "platelet release reaction". E.L. Snyder et al., Vox Sang. 41:172 (1981). Release of the granule contents of platelets is initiated by a number of agonists binding to their receptor. Platelets contain three different types of secretory granules: dense granules, alpha-granules and lysosomal granules. The granules differ in respect to the stimulus necessary to induce secretion. Secretion of the dense- and alpha-granules contents is observed after stimulation with all platelet agonists. Degranulation occurs by movement of the secretory granules to the platelet periphery and fusion of the granule membranes directly with the plasma membrane, resulting in secretion of the granule contents. Granule membrane proteins are expressed on the plasma membrane once fusion occurs. GMP-140 (or "P-Selectin") is present in the alpha-granule membrane. P.

Stenberg et al., J. Cell. Biol. 101:880 (1985). It is expressed on the plasma membrane after fusion. GMP-140 expression is therefore reflective of alpha- granule secretion and platelet activation. J.N. George, J. Clin. Invest. 78:340 (1986). GMP-140 appears to function as an adhesive platelet ligand; thus, it is possible that platelets expressing this molecule would be preferentially cleared from the circulation. Indeed, there is experimental evidence to suggest that activated human platelets that express GMP-140 are preferentially cleared from the circulation after transfusion. M. Rinder, H. M., et al. Transfusion 31:409 (1991).

SUMMARY OF THE INVENTION

The invention relates to methods for screening both human and non-human platelets for the expression of Granule Membrane Glycoprotein-140 (GMP-140), an indicator of platelet activation. The present invention contemplates an anti-GMP- 140, interspecies cross-reactive, polyclonal antibody as well as a method using an anti-GMP-140, interspecies cross-reactive, polyclonal antibody to measure platelet activation. In one embodiment, the polyclonal antibody reacts with activated human and rodent platelets. Importantly, the antibody is substantially unreactive with unactivated, human and rodent plalelets. By "substantially unreactive" it is meant that the specific antibody is approximately as reactive on unactivated platelets as a non-specific antibody.

In one embodiment, the polyclonal antibody is an avian antibody, i.e. immunization with GMP-140 was performed in an avian species and antibody was thereafter recovered. Where an avian antibody is contemplated, it is preferably a chicken antibody. In such a case, the antibody is conveniently obtained from the egg yolk of a chicken egg and is, therefore, IgY antibody. For specificity, the antibody is affinity purified against immobilized GMP-140 or a portion of GMP- 140.

In one embodiment, the method of measuring platelet activation, comprises: a) providing platelets and an anti-GMP-140, interspecies cross-reactive, polyclonal antibody; b) reacting said polyclonal antibody with said platelets; and c) measuring the amount of said antibody bound to said platelets.

DESCRIPTION OF THE FIGURES

Figure 1 schematically shows the GMP-140 transcript and four constructs for protein expression. Figure 2 shows the purification of the pET expressed GMP-140 fusion product by gel electrophoresis.

DESCRIPTION OF THE INVENTION

The present invention provides an interspecies cross-reactive, anti-GMP-140 antibody, i.e. antibody that reacts with GMP-140 on the platelets of more than one species. Such an antibody is necessary to conclusively correlate platelet activation

(as measured by GMP-140 expression) with platelet survival and function, since controlled studies are not really possible in humans. That is to say, fresh human platelets from a blood bank are from random (outbred) donors and are processed by the particular method of platelet preparation for that institution, even if that method is inherently damaging to the platelets. The survival data obtained with such human platelets is therefore complicated by other factors.

To overcome these variables, the present invention provides an interspecies cross-reactive, polyclonal antibody. This antibody reacts with activated rodent platelets (e.g. mouse platelets) and, thus, allows for the controlled studies to show that platelet activation (as measured by GMP-140 expression) correlates with in vivo survival.

The present invention also provides a simple, convenient method for measuring platelet activation with the antibody. This method does not have the costs and complexities of the current GMP-140 assay, i.e. flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an interspecies cross-reactive, polyclonal antibody raised against a human GMP-140 peptide fragment. The fragment used was a fragment of the GMP-140 protein (as encoded by the E4 cDNA of Johnston, et al., Cell 56:1033-1044, 1989). Expression of the protein in an expression vector is much more efficient than extracting GMP-140 from platelets by biochemical techniques. Furthermore, it provides a renewable resource.

It is not intended that the present invention be limited by the construct or the expression system employed. While an amino terminal fragment can be conveniently be expressed in E. coli, other constructs can be used and expression can be in other systems, including systems where the protein is glycosylated.

The present invention contemplates raising antibody to GMP-140 (or a portion thereof) in a non-mammal, such as an avian host. While murine monoclonal antibodies have been raised to human GMP-140, these antibodies do not react with rodent platelets. Consequently, they are not suited to correlating

GMP-140 expression with platelet in vivo survival using simple animal model systems. Indeed, these reagents have not been useful in a conclusive correlation of GMP-140 expression in vitro and platelet survival in vivo.

The present invention contemplates generation of an interspecies cross- reactive, polyclonal antibody to measure platelet activation in animal model systems. Furthermore, once the correlation of GMP-140 expression with survival in animals is established with the antibody of the present invention, its similar reactivity with human platelets will allow for diagnostic applications.

The present invention also contemplates a simple assay for measuring antibody binding to GMP-140. In one embodiment, the present invention contemplates an agglutination assay employing anti-GMP-140 antibody bound to a solid surface (e.g. beads, or other microparticles). Such an assay allows for a quick and semi-quantitative detection method for GMP-140 expression in vitro. Other assay formats to determine surface GMP-140 on platelets are contemplated. A hemagglutination type assay is amenable to multi-sample testing using 96-well round bottomed microtiter plates. Serial dilutions of patient platelets with a constant number of anti-GMP-140 particles can be mixed in the wells and observed for agglutination at the bottom of the wells.

In another embodiment, a competitive inhibition ELISA assay using either free anti-GMP-140 or anti-GMP-140 coated particles mixed with platelets and added to GMP-140 coated microtiter wells. A reduction in signal after using an anti-chicken enzyme conjugate would indicate the presence of surface GMP-140.

Alternatively, an ELISA capture assay can be adapted to recognize platelet surface

GMP-140.

Anti-GMP-140 antibody coated plates would "capture" platelets expressing surface GMP-140 followed by a anti GMP-140 enzyme conjugate. Production of signal after substrate addition would indicate presence of platelet surface GMP-140.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); °C (degrees

Centigrade); HPLC (High Pressure Liquid Chromatography); OD (optical density); SDS (sodium dodecylsulfate); CPDA (Citrate-Phosphate-Dextrose-Adenine); CEPT (Citrate-EDTA-Prostaglandin El-Theophylline); EDTA (ethylenediaminetetracetic acid).

In some of the examples below, mouse platelet concentrates are used. These are prepared by the following method. First, an anticoagulant was prepared, either CPDA or CEPT. Citrate, EDTA, Prostaglandin El (PGEl), Theophylline anticoagulant (CEPT) was prepared as follows:

A solution of 130 mM Na₃*Citrate*2H₂O, 10 mM Disodium EDTA*2H₂O, and 100 mM theophylline was prepared and deionized water was added with heat until the theophylline was dissolved. 5 ml aliquots of the solution were stored at -

20 C. Just prior to use, the tube was thawed and 1 ul of 5 mg/ml PGEl in ethanol was added.

Swiss Webster mice at 30 grams of body weight and 9 weeks old were used for the study. Whole blood was collected by heart puncture from mice into a 3cc syringe with a 23 Gl needle containing 85 ul of working anti-coagulant solution.

Blood was pooled from 7 mice for each tube and centrifuged at 1800 rpm (17 cm average radius) for 3 minutes to obtain platelet rich plasma (PRP). PRP was transferred into clean polystyrene tubes, the volume was restored with buffered saline containing glucose and citrate (BSGC), the samples were centrifuged, and the supernatant was added to the PRP. PRP was then pooled and centrifuged at

2600 rpm for 10 minutes.

In some of the Examples below, flow cytometry was employed to measure platelet survival. In flow cytometry, antibodies bound to the surface of platelets are detected by the addition of fluorescence labeled secondary antibodies. The instrumentation measures the fluorescent intensity of each platelet as it passes through a small orifice.

To measure GMP- 140 in vitro by flow cytometry, a small aliquot of platelet rich plasma was placed in HEPES buffer containing a GMP- 140 binding antibody or control antibody. After incubation, a secondary reagent (e.g. Goat Anti-avian antibody conjugated to FTTC) was then added to the tube in saturating amounts.

Finally, the cells were diluted in isotonic saline, fixed with paraformaldehyde and analyzed on a FACSCAN (Becton Dickinson, Mountian View, CA). Activated platelets were made by adding Phorbol Myristate Acetate (PMA) to the test system at a final concentration of 2 x 10^"7 M (Sigma, St. Louis, MO; working concentration was 10 ug/ml and was stored at -40°C and dissolved in DMSO). 1% Paraformaldehyde (PFA) (Sigma, St. Louis, MO) was prepared by adding 10 grams paraformaldehyde to 1000 ml deionized water. This was heated to 70°C, whereupon 16.2 mM of dibasic sodium phosphate was added until the solution was clear. The solution was cooled and the pH was adjusted to 7.4 with 3.8 mM of monobasic sodium phosphate. This was filtered and stored in the refrigerator. Mouse platelets were harvested in the manner described above and tested with the anti-GMP-140 mouse monoclonal antibody CD62 (Becton Dickinson, Mountian View, CA) conjugated to phycoerythrin. When evaluated by flow cytometry in the manner outlined above, no staining was observed above background (even for PMA activated mouse platelets), confirming the fact that such antibodies are not useful for small animal studies (data not shown).

To measure platelet recovery in. vivo by flow cytometry, platelet concentrate was first labeled with a PKH2-GL fluorescent cell linker kit (composed of PKH2 dye stock and diluent A; Zynaxis Cell Science, Inc., Malvern, Pa) according to the method described by Horan, P.K., et al.,in Flow Cytometry. Darzynkiewicz, Z. and Crissman, H. (eds.) pg. 480 - 483. The method used in the following examples differs from that method described in Flow Cytometry in that instead of using a column purification step, the present examples used centrifugation to wash away ecess dye. Each ml of platelet concentrate was treated with 1 ml of Diluent A containing 4 ul PKH2-GL probe from Zynaxis. The reaction was carried out without light for 10 minutes. Add 1:1 BSGC (a buffered Saline-Glucose-Citrate-

PGE1 solution containing: 13.6 mM Na₃Citrate*2H₂O, 11.1 mM Glucose, 8.0 mM Na₂HPO₄*7H₂O, 1.6 mM KH₂PO₄, 0.9 mM Na₂EDTA*2H₂O, pH adjusted with 1M NaOH) to dilute dye solution. The labelling solution was centrifuged at 3000 g for 10 minutes. Supernatant was removed and platelet pellets were resuspended in BSGC to a concentration of 1 x 10⁶ platelets/ul. The platelets were then ready to inject into the mice. A capillary tube treated with EDTA and Heparin was used for eye bleeding. Blood was drawn at various time points after injection. 30 ul blood from each sample was added to 1 ml of saline. The blood was again diluted by adding 200 ul to another 1 ml of saline. The dilute blood was then ready for analysis on the FASCAN.

EXAMPLE 1 This example describes the expression of a GMP- 140 fragment by E. coli.

The expression rational was as follows (see Figure 1 for diagrams and cloning details). The GMP- 140 transcript encodes a secreted protein of an estimated MW of 86,244 (the much larger in vivo size of 140,000 MW is presumed to be due to extensive glycosylation of the protein). Following a predicted 41 aa leader peptide, the translated sequence predicts a 789 aa protein, containing five different structural domains. These include a "Lectin" domain, "EGF" domain, nine tandem "C3b-C4b Regulatory Protein" repeats, a putative transmembrane domain and a presumed cytoplasmic segment This organization is strongly conserved between GMP-140 and at least 2 other (closely linked) genes; this gene family is referred to as "selectins" (Bevilacqua, et al., Cell 67: 233, 1991). Within this family, the lectin and EGF domains show the strongest protein conservation.

Intuitively, for the purpose of generating an interspecies cross-reactive polyclonal antibody, an expression vector was constructed which included (at least) both these regions. The first (larger) fragment included this region and all nine regulatory protein repeats (a total of 661 aa). This fragment terminates at Hinc II restriction site immediately 5' to the transmembrane domain; this site was chosen, since membrane spanning domains are often toxic in E. coli. The second fragment has the same 5' end, but contains only 4 regulatory repeats (a total of 365 aa; a fusion of a similar fragment of GMP- 140 has been made to the hinge domain of human IgGl, and is properly expressed in transfected COS cells; Aruffo, et al,

Cell 67:35-44. 1991). Expression constructs with this shorter derivative were constructed as a backup if the larger construct was toxic in E. coli.

Two different E. coli expression systems were employed. The first was the expression vector pET3c; this allows a protein of interest to be expressed as a fusion (with a 13 aa leader) under the control of a strong, inducible promoter (T7

RNA polymerase; see Novagen product catalogue). The advantages of this system is that the gene of interest is expressed at high levels and has very little fusion protein sequences present The expression proteins are often insoluble, facilitating easy purification via inclusion body preparation as described by S. Carroll, U.S. Patent Appl. Ser. No. 07/946,927, hereby incorporated by reference. The second expression system utilized was the Glutathione S-transferase

(GST) gene fusion system (product information available from Pharmacia); insert open reading frames are expressed as carboxyl terminal fusions with the GST protein, under the control of an inducible tac promoter. Although protein induction is not as high in this system, it is still an excellent complement to the pET system for several reasons. First, unfused proteins are often unstable in E. coli; however, fusion to the GST protein may stabilize fusion proteins, thus allowing purification. As well, if the expressed protein is soluble in both systems, it is simple to purify soluble GST fusion proteins on Glutathione Sepharose 4B columns. The GST system is also well designed for protein-protein and protein-DNA biochemical analysis. If a protein is expressed intact and insoluble in both systems, generally the pET system is used for large scale purification due to the higher levels of induction driven by the T7 promoter.

In frame fusions of both described fragments were constructed in each system; the orientation and integrity of each construct was confirmed via restriction analysis. A T7 promoter primer (Novagen #69348-1) was utilized in sequencing reactions from both pET constructs; the resulting sequence confirmed that both constructs the pET leader was correctly fused to GMP- 140 sequences as verified by a sequencing gel. Poor protein expression was observed from the larger expression construct in either vector. The accumulation of low levels of inducible protein was observed, but the MW was much smaller than predicted; it is likely protein degradation occurred with this construct, possibly due to the presence of nine consecutive repeats in the expression protein. Since this degradation was observed in protease deficient E. coli hosts (Ion-), it appeared unlikely that the large construct would be useful for preparative protein purification. However, the smaller construct expressed high levels of inducible protein of approximately the predicted molecular weight in either the pET and GST systems. The fusion band is clearly a pET leader-GMP-140 fusion, since Western analysis utilizing an antibody specific to the T7 leader peptide (Novagen #69455-1) demonstrated the presence of the pET leader peptide in the expressed protein (combined with the sequencing gel result, this indicates that the fusion protein must contain the product of the GMP- 140 reading frame downstream of the pET leader). The pET expression construct was selected for large scale purification, since it induced higher levels of completely insoluble protein. (Deposited with American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville MD 20852 USA, accession number ATCC 69511, deposited on December 8, 1993. The E. coli host deposited is identified by the following characteristics: BL21 (DES) lys S strains containing pET GMP140 (53-418) expression plasmid.)

Figure 2 illustrates the purification of the pET expressed GMP-140 fusion. The arrow indicates the induced protein, which is not present in a soluble protein preparation, but was highly pure in isolated inclusion bodies. The yield was approximately 10 - 20 mg of fusion protein from 500 ml of liquid culture.

EXAMPLE 2

This example describes immunization in an avian species using the GMP- 140 fragment of Example 1. This was followed by antibody collection and liter determihation.

Immunization. One white leghorn hen obtained from a local breeder was immunized with 1 mg of GMP 140 fusion protein [1 mg/ml in PBS plus 0.01%

SDS emulsified with an equal volume (1 ml) of complete Freund's adjuvant (GIBCO)]. The hen was boosted at day 14 and day 21 with 500 ug GMP 140 fusion protein in incomplete Freund adjuvant (GIBCO).

Antibody Collection. Chicken immunoglobulin (IgY) was extracted according to a modification of the methods of A. Poison, et al., Immunol. Comm. 9:493 (1980).

Yolks were separated from the egg whites and yolks were blended with four volumes of egg extraction buffer [0.01M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 0.005% thimerosal and PEG 8000 (Amresco, Solon, OH)]. The polyethylene glycol was added slowly to the mixture to a final concentration of 3.5%. After all the PEG dissolved, the protein precipitate that formed was pelleted by centrifugation at 9000 x g for 10 minutes. The supernatant was decanted and filtered through cheesecloth to remove the lipid layer, and PEG was again added to a final concentration of 12%. After centrifugation at 9000 x g for 10 minutes, the supernatant was discarded and the pellet containing the IgY was dissolved in the original yolk volume in phosphate buffered saline (PBS) (0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5) with 0.005% thimerosal.

Anti-GMP-140 Titer. The IgY antibody of eggs harvested on day 32 and day 33 were assayed by ELISA. To prepare for the ELISA, a 96-well Falcon Probind Microtiter plate (Becton Dickinson, Lincoln Park, NJ) was coated overnight at 4°C with a 100 ul/well of GMP- 140 fusion protein (see Example 1) at 2.5 mg/ml in PBS with 0.005% thimerosal. The wells were blocked for nonspecific binding with PBS containing 1% bovine serum albumin (BSA) for 1 hour at 37°C.

The immune IgY or preimmune IgY (IgY from an unimmunized hen) were diluted at various concentrations in PBS (containing 1% BSA and 0.05% Tween

20) and added to the wells, and the plate was incubated for 1 hour at 37°C. The plates were then washed three times with PBS with 0.05% Tween 20 and three times with only PBS. Alkaline phosphate conjugated rabbit anti-chicken IgG (Fisher Biotech, Pittsburgh, PA) was diluted 1:750 in PBS (containing 1% BSA and 0.05% Tween 20) and added to the plate and incubated for 1 hour at 37°C.

The plates were then washed as before and p-nitrophenyl phosphate substrate (Sigma, St. Louis, MO) was added at 1 mg/ml in 0.05 M NajCO_j pH 9.5, with 10 mM MgCl₂. The plate was then read at 410 nm using a Dynatech MR300 Micro ELISA reader at about 10 minutes after substrate addition. The results are shown in Table 1. TABLE 1

Results of ELISA

O.D. reading of preimmune IgY was subtracted from Immune IgY reading.

The results clearly show that high titer antibody to the fusion protein was made. Good reactivity to a dilution of 1:18750 or greater was apparent with the unpurified immune antibody to GMP- 140.

EXAMPLE 3 This example describes the affinity purification of the IgY from the eggs of the birds immunized in Example 2. This involved coupling GMP- 140 fusion protein to a resin and purifying the anti-GMP-140 antibody over the resin.

Antigen Coupling. Five (5) milligrams of GMP- 140 fusion protein (5 mis) from Example 1 (above) was coupled to 6 grams of ACTIGEL A (Sterogene Biochemicals, San Rafael, California) according to manufacturer's instructions.

Briefly, the ACTIGEL resin was washed with 10 volumes of PBS before mixing with GMP-140, followed by the addition of 1.1 ml of 1 M NaCNBH₃ coupling solution. The mixture was mixed overnight at room temperature and then the resin was washed with 100 mis of PBS. The coupled resin was then washed with 1 volume of 4 M Guanidine-HCl (pH 8.0) to remove any nonspecifically bound GMP-140, then rewashed with PBS.

The coupling efficiently was determined by O.D. reading at 215 nm. The standard 280 O.D. reading for protein quantitation cannot be used due to the nucleic acid contamination of the GMP-140 protein preparation, whereas 215 nm is more specific for quantitating only protein, i.e. the peptide bond absorbing range. From this, the GMP- 140 coupling efficiency to ACTIGEL was determined to be 76%.

Antibody Purification. The GMP- 140 affinity resin was packed into a 10 ml column, equilibrated with PBS containing 0.005% thimerosal with a flow rate at 2 ml per minute. Twenty (20) mis of the PEG purified anti GMP-140 IgY (see Example 2) was loaded and the flow through was collected. The GMP- 140 affinity matrix was washed successively with several bed volumes of BSS Tween (O.l M boric acid, 0.025 M sodium borate, 1M NaCl, 0.1% v/v Tween 20 pH 8.3), then the specific anti GMP- 140 IgY was eluted with 8 mis of ACTISEP (Sterogene

Biochemicals, San Rafael, California) according to manufacturer's instructions, followed by 10 mis of PBS with 0.005% thimerosal. The ACTISEP, PBS eluate was dialyzed against Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.2) with 0.005% thimerosal at 4°C. The A^_n protein determination was made on all the washes and eluates and the affinity purified anti-GMP-140 had a protein concentrate of about 100 mg/ml or about 1% of the total protein of the starting PEG-IgY preparation (more booster immunizations of the hens can increase the specific IgY to up to 10% of the total protein). The starting PEG preparation was compared to the affinity purified anti- GMP antibody by ELISA and it was determined that it was about 100-fold increase in purity (data not shown). EXAMPLE 4

This example involves coupling the antibody to polystyrene particles, reacting the particles with antibody, and reacting the particles with GMP-140 protein.

Coupling Antibody. Polystyrene particles (0.65 microns) from Spherotech

(Libertyville, 111.) at 5% w/v, 100 ul each, were washed 3 times with TBS (50mM Tris, 150 mM NaCl, pH 7.2) with 0.005% thimerosal, by centrifugation using a microcentrifuge. The particles were then passively coated with different concentrations (100 ug, 50 ug or 25 ug) of affinity purified anti-GMP-140 antibody (see Example 3) in a final volume of 1 ml of TBS. This was done overnight on a rotator at room temperature.

The coated particles were washed with TBS, PBS containing 0.05% Tween 20, then TBS again. The final washed pellet was resuspended in 200 ul (2.5% w/v particles) in TBS. To determine whether the chicken IgY was passively bound to the particles,

10 ul of particles (as sample from each of the concentrations) was mixed on a slide with 20 ul of rabbit anti-chicken Ig (Sigma, St Louis, MO) diluted 1:100 in antibody dilution solution (PBS with 0.005% thimerosal, 0.05% Tween-20 and 1% BSA). Washed uncoated particles were used as a control. Visible agglutination occurred in all of the coated particles after several minutes, while no agglutination occurred in the uncoated control particles (data not shown). Agglutination of the particles coated with 100 ug/ml of anti-GMP-140 antibody occurred at the fastest rate and was the only coated particle used subsequently.

To determine whether the coated antibody was immunologically active on the particles, 10 ul of undiluted GMP- 140 protein (1 mg/ml) or diluted protein (10,

100 or 1000 fold in antibody dilution solution) was mixed on a slide with 10 ul of the 100 ug/ml antibody-coated particles. Uncoated particles were again used as a control.

After 3 minutes of gentle rocking, GMP- 140 dilutions of 1:10 or 1:100 resulted in the agglutination of particles (data not shown). This indicated that the antibodies were active on the microparticle surface and the passive coating was successful.

EXAMPLE 5 This example describes a rapid, inexpensive agglutination assay having a single use assay format for the determination of surface GMP- 140 on human platelets. The 100 μg/ml anti-GMP-140 coated particles from Example 4 were used to evaluate 3 to 4 day-old human platelets (American Red Cross, Madison, WI). Uncoated particles were initially tested by mixing them with equal volumes of platelets on a slide and were found to non-specifically agglutinate platelets. To reduce this non-specific binding, both uncoated an coated particles were pretreated for 2 hours at room temperature with different dilutions of (1:5, 1:10, 1:100) of PBS with 1% bovine serum albumin (Sigma, St. Louis, MO). This served to overcoat the particles and prevent nonspecific agglutination of the uncoated particles with platelets.

Ten μl of overcoated particles were mixed with equal volume of human platelets on a slide and gently rocked for 2-3 minutes. Clear agglutination occurred using the anti GMP- 140 coated particles; uncoated particles (without anti GMP- 140) did not agglutinate either platelet sample (data not shown). Pretreating the particle with an overcoat at a 1:5 dilution resulted in the best differentiation between coated and uncoated particles (i.e. best reduction of nonspecific agglutination in the uncoated particles with platelets without affecting specific anti GMP- 140 reactivity to the platelets).

EXAMPLE 6

This example, using flow cytometry, evaluates the optimum incubation time for the Goat anti-Chicken FITC labeled antibody to bind to the affinity purified Chicken anti-human GMP- 140 antibody of Example 3. The Goat anti-Chicken antibody was used as a label to determine the quantity of Chicken anti-human

GMP-140 antibody that has bound to platelets. Detecting the labelled Goat anti- Chicken antibody by analysis on FASCAN allows for the measurement of the amount of bound Chicken anti-human GMP- 140 antibody, which in turn indicates the level of GMP- 140 expression. For the analysis, 24 tubes were prepared, 8 each for the test, control and positive control samples. Each tube contained 10 ul platelets prepared from mice (see above) at a concentration of 200,000 platelets per microliter. The positive control and test tubes contained 200 ng Chicken anti-human GMP- 140 antibody in 40 ul Hepes buffer. Positive control tubes also contained Hepes buffer and PMA, a platelet activator, at 4 x 10^"7 M. The negative control tubes contained 200 ng

Non-specific Chicken antibody in 40 ul Hepes buffer. The tubes were incubated for 45'. Then 10 ul Goat anti-Chicken FITC (at a 1:10 dilution) was added to each tube. The tubes were incubated for 10', 20', 25.', 30', 35', 40', 45', or 60'. The results are shown in Table 2.

TABLE 2

GMP-140 Expression on Mouse Platelets

The results indicate that binding of the secondary Goat antibody is optimal after 45'. The reaction between GMP- 140 and the Chicken antibody to GMP- 140 on mouse platelets starts to plateau after 30'.

EXAMPLE 7

This example evaluates a method of fixing platelets with PFA prior to evaluating them for expression of GMP- 140. Mouse platelet concentrates drawn and harvested as described above, were resuspended in 15% plasma / 85% protein- free media and prepared for an in vitro test of GMP- 140 expression, measured by binding of Chicken anti-human GMP- 140 polyclonal antibody, as follows. The platelets were first activated with 4 x 10^"7M PMA or incubated in Hepes solution. 1% PFA was then added to each tube and incubated at room temperature for 1 hour. Platelets were washed with 1% BSA-Hepes and resuspended in 250 ng Chicken anti-human GMP- 140 antibody and incubated for 40 minutes. The washing and resuspending was repeated. Then the platelets were washed and resuspended in Hemaline II. The results are in TABLE 3.

TABLE 3

The results show that fixing the cells with PFA prior to introducing the antibodies resulted in poor binding of the antibody to GMP- 140. This confirms the protein nature of the binding site.

EXAMPLE 8

In previous examples, blood was drawn from mice using CPDA anticoagulant. This example evaluates the expression of GMP- 140 in mouse platelets after storage with CEPT. A simultaneous test of treated platelets' survival in vivo serves to compare the methods of the present invention with actual platelet survival.

In this experiment, platelet concentrates drawn and harvested as described above, were resuspended in 15% plasma / 85% protein-free, buffered media or were left as platelet rich plasma (PRP). The platelets were stored for 24 or 42 hours. Next the samples were split. One fraction of each sample was prepared for an in vivo test of survival by injecting Zynaxis PKH2-G1 labeled platelets from each sample into 3 mice. Blood was then drawn at time points, diluted and processed on the FASCAN instrument, as previously described, to determine the percent of injected platelets which remain in circulation. The zero time point value used in the calculations of the in. vivo test, was the average of the labeled platelet count at 2 and 4 hours.

The other fraction of each sample was prepared for an in. vitro test of GMP- 140 expression, measured by binding of Chicken anti-human GMP- 140 polyclonal antibody, as follows. Each experimental tube was prepared with 1000 ng Chicken anti-human GMP- 140 antibody in 50 ul. The following samples were set up in duplicate. The positive controls contained PMA at a concentration of 4 x 10^"7 M. 10 ul of a platelet solution (200,000 platelets/ul) was added to each tube. Samples were incubated for 45'. Then 10 ul of a 1:10 dilution of goat anti-Chicken FITC antibody was added to each sample. The samples were incubated for an additional 45'.

The results are shown in Table 4. Clearly the best survival in vivo is seen with either fresh PRP (Sample 5) or platelets stored for only 24 hours in 15% plasma / 85% protein-free media (sample 4). Platelets stored for 24 hours and 42 hours in platelet rich plasma (Sample 3 and Sample 1, respectively) or stored for 42 hours in 15% plasma / 85% protein-free media (Sample 2) showed poor survival.

The survival numbers correlated well with the antibody staining. The highest GMP-140 Antibody binding was with the platelets showing the poorest survival (Sample 1). Interestingly, these same cells did not PMA activate to a high level, suggesting that, indeed, the granules were released during storage. Shape change suggested complete activation.

TABLE 4

The lowest level of GMP-140 expression (Sample 5) showed good survival. These same cells also PMA activated to the highest level.

EXAMPLE 9

This example evaluates the GMP- 140 expression of stored mouse platelets in a manner similar to EXAMPLE 8. Binding of the chicken antibody to human

GMP-140 was compared with a simultaneous test of treated platelets' survival in vivo.

Platelet concentrates drawn and harvested as described above, were resuspended in 15% plasma / 85% protein-free media. Then platelets were tested either fresh (Sample 3) or after 24 and 48 hours of storage (Sample 2 and 1, respectively). Next the samples were split. One fraction of each sample was prepared for an in vivo test of survival by injecting labeled platelets from each sample into 3 mice. Blood was then drawn at time points, diluted and processed on the FASCAN instrument, as previously described, to determine the percent of injected platelets which remain in circulation. The zero time point value used in the calculations of the in vivo test, was the the labeled platelet count at 6 hours.

The other fraction of each sample was prepared for an in vitro test of GMP- 140 expression, measured by binding of Chicken anti-human GMP-140 polyclonal antibody, as follows. Each experimental tube was prepared with 929 ng Chicken anti-human GMP- 140 antibody in 50 ul. The following samples were set up in duplicate. The positive controls contained PMA at a concentration of 2 x 10^"7 M. 10 ul of a platelet solution (200,000 platelets/ul) was added to each tube. Samples were incubated for 45'. Then 10 ul of a 1:10 dilution of goat anti-Chicken FITC antibody was added to each sample. The samples were incubated for an additional 45'.

The results are shown in Table 5. Clearly the best survival in vivo is seen with fresh platelets (Sample 3). Platelets stored for only 24 hours in 15% plasma / 85% protein-free media show lower survival (Sample 2) and platelets stored for 48 hours (Sample 1) showed poor survival. TABLE 5

The survival numbers correlated well with the antibody staining. The highest GMP- 140 Antibody binding was with the platelets showing the poorest survival (Sample 1). The lowest level of GMP- 140 expression (Sample 3) showed good survival.

EXAMPLE 10

This example evaluates CEPT andand CPDA for their effects on GMP 140 expression. A simultaneous test of treated platelets' survival in vivo serves to compare the methods of the present invention with actual platelet survival. Mouse platelet concentrates were drawn and harvested as described previously, and were resuspended in 15% plasma / 85% protein-free media for storage (24 hours or 48 hours) in the presence of CEPT or CPDA. Fresh platelets were used as controls. PRP contained 60% plasma.

Next the samples were split. One fraction of each sample was prepared for an in vivo test of survival by injecting Zynaxis PKH2-GL labeled platelets from each sample into 3 mice. Blood was then drawn at time points, diluted and processed on the FASCAN instrument, as previously described, to determine the percent of injected platelets which remain in circulation. The zero time point value used in the calculations of the in vivo test, was the labeled platelet count at 6 hours.

The other fraction of each sample was prepared for an in vitro test of GMP- 140 expression, measured by binding of Chicken anti-human GMP- 140 polyclonal antibody, as follows. Each experimental tube was prepared with 1000 ng Chicken anti-human GMP- 140 antibody in 50 ul. The samples were set up in duplicate. The positive controls contained PMA at a concentration of 2 x 10^"7 M. 10 ul of a platelet solution (200,000 platelets/ul) was added to each tube. Samples were incubated for 45'. Then 10 ul of a 1:10 dilution of goat anti-Chicken FITC antibody was added to each sample. The samples were incubated for an additional 45'. The results are shown in Table 6. Clearly the best survival in vivo is seen with fresh platelets, whether in CEPT (Sample 5) or CPDA (Sample 6). Platelets stored for only 24 hours in 15% plasma / 85% protein-free media show lower survival, whether stored in CEPT (Sample 3) or CPDA (Sample 4). Platelets stored for 48 hours showed poor survival; platelets stored in CEPT (Sample 1) appeared to be better than those in CPDA (Sample 2). TABLE 6

The survival numbers correlated well with the antibody staining. The highest GMP- 140 Antibody binding was with the platelets showing the poorest survival (Sample 2). The platelets with the lowest level of GMP-140 expression (Samples 5 and 6) showed good survival.

EXAMPLE 11

This example, using flow cytometry, evaluates the reactivity of the anti- GMP-140 antibody of the present invention with canine platelets.

For the analysis, control and positive control samples were prepared. Each tube contained 10 ul platelets prepared from dogs at a concentration of 200,000 platelets/ul. The positive control and test tubes contained 200 ng Chicken anti- human GMP- 140 antibody in 40 ul of Hepes buffer.. Positive control tubes also contained Hepes buffer and PMA, a platelet activator, at 2 x 10^"7 M. The negative control tubes contained 200 ng Non-specific Chicken antibody in 40 ul Hepes. The tubes were incubated for 45'. Then 10 ul Goat anti-Chicken FITC (at a 1:10 dilution) was added to each tube.

The results are shown in Table 7. The reactivity of the anti-GMP-140 antibody of the present invention with canine platelets is excellent. No reactivity is seen with the preimmune chicken control antibody.

TABLE 7

From the above it is evident that the present invention provides an interspecies cross-reactive, anti-GMP-140 antibody, i.e. antibody that reacts with GMP-140 on the platelets of more than one species. Furthermore, the antibody of the present invention allows platelet activation (as measured by GMP- 140 expression) to be correlated with platelet survival and function, without the accompanying difficiencies of prior art methods. All patent publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

Claims

~27 PATENTCLAIMS

1. An anti-GMP-140, interspecies cross-reactive, polyclonal antibody.

2. The polyclonal antibody of Claim 1, wherein said polyclonal antibody reacts with activated human and rodent platelets.

3. The polyclonal antibody of Claim 2, wherein said antibody is substantially unreactive with unactivated human and rodent plalelets.

4. The polyclonal antibody of Claim 1, wherein said antibody is an avian antibody.

5. The avian antibody of Claim 4, wherein said antibody is a chicken antibody.

6. The chicken antibody of Claim 5, wherein said antibody is IgY antibody.

7. The IgY antibody of Claim 6, wherein said antibody is affinity purified.

8. A method of measuring platelet activation, comprising: a) providing platelets and an anti-GMP-140, interspecies cross- reactive, polyclonal antibody; b) reacting said polyclonal antibody with said platelets; and c) measuring the amount of said polyclonal antibody bound to said platelets.

9. The method of Claim 8, wherein said polyclonal antibody is an avian antibody.

10. The avian antibody of Claim 9, wherein said antibody is a chicken antibody.

11. The chicken antibody of Claim 10, wherein said antibody is IgY.

12. The IgY antibody of Claim 11, wherein said antibody is affinity purified.

13. A method of measuring platelet activation, comprising: a) providing platelets and microparticles coated with an anti- GMP- 140, interspecies cross-reactive, polyclonal antibody; b) reacting said polyclonal antibody with said platelets; and c) measuring agglutination of said platelets.

14. The method of Claim 13, wherein said microparticles are polystyrene beads.

15. The method of Claim 14, wherein said polystyrene beads are coated with a substance that prevents non-specific sticking of said polystyrene beads to said platelets.