Measurement of endogenous and exogenous alpha-granular platelet proteins in patients with immune and...

Measurement of endogenous and exogenous alpha-granular

platelet proteins in patients with immune and nonimmune

thrombocytopenia

MA RY HU G HE S, CATHERINE P. M. HAYWARD, PET ER HORSE WOOD,² THEODORE E. WARKENTIN AND JOHN G. KE LTON*²

Departments of Pathology and *Medicine, Faculty of Health Sciences, McMaster University Division,

Hamilton Health Sciences Corporation, and ²Canadian Red Cross Society, Hamilton, Ontario, Canada

Received 22 January 1999; accepted for publication 9 June 1999

Summary. Idiopathic thrombocytopenic purpura (ITP) iscaused by antiplatelet antibodies and is characterized byincreased platelet destruction and elevated levels of IgG(platelet-associated IgG, PAIgG). Nonimmune thrombocyto-penic patients also have elevated levels of PAIgG. In thisstudy we investigated two possible biological explanations forthe increased levels of PAIgG in these patients. The ®rsthypothesis suggests that a thrombocytopenic stress causesincreased thrombocytopoiesis with increased numbers andcontent of the platelet alpha granules. The second hypothesisis that for uncertain reasons (immunological or cytokine)there is increased absorption of plasma proteins by eithermegakaryocytes or by the platelets themselves. To addressthis issue, we compared the level of megakaryocytesynthesized alpha granular proteins [platelet factor 4 (PF4)and beta-thromboglobulin (b-TG)] to plasma-absorbed alpha

granular proteins (albumin, IgG and ®brinogen) in patientswith immune (n�39) and nonimmune (n�60) thrombo-cytopenias. Plasma-absorbed alpha-granular proteins wereelevated in both immune and nonimmune thrombocyto-penia with no increase in megakaryocyte synthesized alpha-granular proteins. These plasma-derived protein elevationswere not attributable to elevated mean platelet volumes orelevated plasma concentrations of the respective protein. Wehypothesize that the increased IgG in these platelets is notthe result of production of larger platelets, but re¯ects aselective increase in the endocytosis of plasma-absorbedalpha-granular proteins at the megakaryocyte and/orplatelet level.

Keywords: platelet-associated IgG, idiopathic thrombocyto-penic purpura, endocytosis, plasma proteins, megakaryocyte.

Idiopathic thrombocytopenic purpura (ITP) is a disordercharacterized by increased platelet destruction throughremoval of antibody-coated platelets by phagocytic cells ofthe reticuloendothelial (RE) system (McMillan, 1981). Thisdestructive process is mediated by antiplatelet autoantibodies(McMillan, 1981), directed against platelet surface glyco-proteins, with glycoproteins IIb/IIIa and Ib/IX being the mostcommon targets (van Leeuwen et al, 1982; Woods et al,1984a, b). It is not known what triggers the production ofantiplatelet autoantibodies, nor is it possible to predict whichpatients are at an increased risk for developing life-threatening haemorrhages (Lightsey et al, 1976).

For many years investigators have measured platelet-associated immunoglobulins (PAIgG) in patients with ITP.

These studies led to two generally accepted observations.Firstly, platelets from patients with ITP have elevated levels ofPAIgG (Kelton & Steeves, 1983; George, 1990; George et al,1985; George & Saucerman, 1988). Secondly, elevatedPAIgG is not speci®c for ITP and is observed in mostthrombocytopenic disorders, including nonimmune disor-ders, such as aplastic anaemia and leukaemia (George &Saucerman, 1988; Kelton et al, 1989; Heaton et al, 1988;George, 1989; Mueller-Eckhardt et al, 1980).

In this study we questioned the biological explanation forthe increased levels of PAIgG in both immune andnonimmune thrombocytopenic disorders. The currentexplanation for increased PAIgG levels in patients with ITPis that elevated PAIgG levels are a re¯ection of the increasednumbers of larger younger platelets circulating in thesepatients (George, 1990). Since larger platelets are thought tobe more dense and since alpha-granule content is theprimary determinant of platelet density, it would be expected

British Journal of Haematology, 1999, 106, 762±770

762 q 1999 Blackwell Science Ltd

Correspondence: Dr John G. Kelton, HSC 3W10, McMasterUniversity Medical Center, 1200 Main Street West, Hamilton,

Ontario, Canada L8N 3Z5.

763Measurement of Alpha-granular Platelet Protein in ITP

q 1999 Blackwell Science Ltd, British Journal of Haematology 106: 762±770

that platelets in ITP patients would contain more alpha-granule proteins such as IgG and albumin (George, 1990).

In this study we investigated this hypothesis directly bymeasuring several different alpha-granule proteins inpatients with ITP and nonimmune thrombocytopenias.The strategy we employed was to compare the amount ofmegakaryocyte-synthesized alpha-granule proteins (endo-genous platelet proteins; b-thromboglobulin and plateletfactor 4) to the amount of plasma-derived alpha-granuleproteins (exogenous platelet proteins; IgG, albumin and®brinogen). We postulated that by measuring both endo-genous and exogenous platelet proteins, and relating theseresults to platelet size (mean platelet volume), we coulddifferentiate whether the increased PAIgG in thrombocyto-penic patients was due to the production of larger platelets,a general overall increase in alpha-granular content, or aselective alteration in the pool of plasma-derived alpha-granular proteins.

MATERIALS AND METHODS

Patients and controls. The study was approved by aUniversity and Hospital Ethics Review Committee andsamples were collected with informed consent. We studied39 patients with ITP; 29 had active ITP and 10 patients werein remission. Patients were considered to have ITP if they metthe following criteria: thrombocytopenia (<150 ´ 109/l),a nonpalpable spleen, normal or increased numbers of

megakaryocytes in the bone marrow (if performed), and theabsence of secondary causes of thrombocytopenia (McMil-lan, 1981; Kelton & Gibbons, 1982). ITP patients weresubdivided into two groups, those with active disease(thrombocytopenia present) and those in remission. Patientswith a past history of ITP and normal platelet counts(>150 ´ 109/l), who were not on any immunosuppressivetherapy at the time of investigation, were classi®ed as havingITP in remission. The control patients (n�60) with otherhaematological disorders included patients with multiplemyeloma, aplastic anaemia, thrombotic thrombocytopenicpurpura, systemic lupus erythematosus, iron de®ciencyanaemia, myeloproliferative disorder, hypersplenism, spleno-megaly, leukaemia, lymphoma, alcoholic liver cirrhosis andincidental thrombocytopenia of pregnancy. These patientswere subdivided according to their platelet counts (throm-bocytopenic, n�26; normal platelet count, n�34). Thenormal controls (n�13) were a group of nonthrombocyto-penic, aspirin-free, healthy male and female laboratorypersonnel.

Platelet collection and preparation. Whole blood wascollected into acid citrate dextrose (ACD, pH 4´5, 6:1,vol:vol). Platelets were isolated by differential centrifugationand washed ®ve times in phosphate-buffered saline (PBS, pH6´5) with 10 mM ethylenediaminetetra-acetic acid disodium(EDTA). Washed platelets were resuspended to 1 ´ 108

platelets/ml in buffer (25 mM Tris, 100 mM NaCl, 0´02%bovine serum albumin (BSA), containing the protease

Fig 1. Measurement of PAIgG in immune and nonimmune patients and healthy controls. Each symbol represents a single patient or healthy

control. The mean (B) and standard deviation for each patient or healthy control group is indicated.

inhibitors leupeptin (50 mg/ml), 10 mM EDTA and phenyl-methylsulphonyl ¯uoride (0´25 mM). Platelets were lysed bythe addition of 0´5% Triton X-100 (Pierce, Rockford, Ill.).Following lysis (30 min at 228C), platelet lysates werecentrifuged (14 000 rpm for 20 min), and the supernatantswere used for all protein measurements.

Measurement of platelet-associated IgG (PAIgG). Platelet-associated IgG in lysed platelets was measured using animmunoradiometric assay. Anti-human IgG monoclonalantibody, CAG-2 (Warkentin et al, 1994), raised in ourlaboratory, was coated onto Immulon II (Dynatech, Chan-tilly, Va.) microtitre strip wells in 0´2 mol/l carbonate buffer(pH 9´5) overnight at 48C. This antibody recognized allsubclasses of IgG (Warkentin et al, 1994). The wells werewashed once with wash buffer (0´9% NaCl, 0´05% Tween-20) and blocked with 2% BSA/PBS for 2 h at roomtemperature. Platelet lysates were diluted (6´25±1´25 ´ 106 platelets/ml) in 2% BSA/PBS and duplicate100 ml samples were added to the wells. Plates wereincubated for 3 h at room temperature and then washed®ve times with wash buffer.

Monoclonal anti-human IgG, HB43 (American TypeCulture Collection, Rockville, Md.), was labelled with37 MBq Na125I per ml using the chloramine-T method andhad an average speci®c activity of 6´99 GBq/mg protein. The

125I-HB43 was diluted (1/1000) in 2% BSA/PBS and 100 mlwas added to each well. Following an incubation of 2 h atroom temperature, the wells were washed ®ve times withwash buffer and each well was transferred into 75 ´ 12 mmplastic tubes (Sarstedt, Newton, N.C.). An LKB 1275minigamma counter (Fisher Scienti®c, Nepean, Ont.) wasused to determine counts per minute per tube. Puri®edhuman IgG (Sigma-Aldrich, Oakville, Ont.) was diluted in2% BSA/PBS with 0´5% Triton X-100, and used to generatestandard curves. The results were expressed as mg IgG/109

platelets, which is equivalent to fg IgG/platelet.Measurement of platelet-associated albumin. Platelet-

associated albumin was measured using a similar method.ELISA plate wells were coated with monoclonal anti-humanalbumin (Clone HSA1/25.1.3; Cedarlane Laboratories,Hornby, Ont.) in 0´2 mol/l carbonate buffer (pH 9´5)overnight at 48C. Wells were washed once with washbuffer and blocked with 2% BSA/PBS for 2 h at roomtemperature. Platelet lysates were diluted (1´5±6´25 ´ 106

platelets/ml) in 2% BSA/PBS and duplicate 100 ml sampleswere added to plate wells. Plates were incubated for 3 h atroom temperature and then washed ®ve times with washbuffer. A second monoclonal anti-human albumin (Clone943127; Cortex Laboratories, San Leandro, Calif.) labelledwith 37 MBq Na125I per ml (average speci®c activity


764 Mary Hughes et al

Fig 2. Measurement of platelet albumin in immune and nonimmune patients and healthy controls. Each symbol represents a single patient or

healthy control. The mean (B) and standard deviation for each patient or healthy control group is indicated.



18´69 GBq/mg protein) was diluted (1/1000) in 2% BSA/PBS and 100 ml was added to each well. Following anincubation of 2 h at room temperature, plate wells werewashed ®ve times with wash buffer. Each well was placedinto 12 ´ 75 mm plastic tubes and counted in a gammacounter. The two monoclonal anti-human albumin anti-bodies used in this assay speci®cally recognized humanalbumin and did not recognize bovine serum albumin.Puri®ed human albumin (Sigma-Aldrich, Oakville, Ont.)diluted in 2% BSA/PBS, with 0´5% Triton X-100, was usedto generate standard curves. The results were expressed asmg/109 platelets.

Measurement of beta-thromboglobulin and platelet factor 4.Beta-thromboglobulin (b-TG) and platelet factor 4 (PF4)were measured on washed lysed platelets using commercialEIA kits obtained from Asserachrom (Murex Diagnostics,Guelph, Ont.), according to the manufacturer's speci®ca-tions. b-TG and PF4 concentrations were expressed as mg/109 platelets.

Measurement of platelet-associated ®brinogen. Platelet-asso-ciated ®brinogen was measured using an enzyme-linkedimmunosorbent assay described previously (Hayward et al,1996). Puri®ed human ®brinogen (Alexis Biochemicals, SanDiego, Calif.), diluted in 2% BSA/PBS (with 0´5% Triton X-100) was used as the standard. The platelet ®brinogenresults were expressed as mg/109 platelets.

Mean platelet volume. Blood samples were collected intoEDTA anticoagulated tubes (Becton Dickinson, Mississauga,Ont.) and analysed on a Coulter S-Plus Counter (CoulterElectronics, Hialeah, Fla.), for determination of mean plateletvolumes (¯).

Serum IgG and serum albumin measurements. Serum IgGand albumin was measured using a Kallestad QM 300Protein Analysis System (Sano® Diagnostics Pasteur,Chaska, Min.).

Plasma ®brinogen measurements. Plasma ®brinogen levelswere measured using the Clauss ®brinogen assay and anMLA Electra 1600C Automatic Coagulation Analyzer(Medical Laboratory Automation, Montreal, Quebec).

Statistical analysis. Statistical analyses were performedusing the Corel Quattro Pro. (Version 7) data analysisprogram. Inferences about the corresponding measurementsof endogenous and exogenous alpha granular plateletproteins, total platelet protein and mean platelet volume,for each patient group, was made by calculating the meanand standard deviation for each data set. The quantitativeupper limit of normal for each platelet protein measured wasbased on the data set of the healthy control group (mean 62standard deviations). The Pearson product moment coef®-cient of correlation, r, was calculated to determine whetherdata sets were correlated. In addition, the calculated meanfor each data set was compared by using an analysis of

Fig 3. Measurement of platelet ®brinogen in immune and nonimmune patients and healthy controls. Each symbol represents a single patient or

healthy control. The mean (B) and standard deviation for each patient or healthy control group is indicated.

variance (ANOVA, one-way) to determine whether differencesamong the population means were signi®cant (P<0´05).

RESULTS

Plasma-derived platelet proteins were measured in healthycontrols, patients with active ITP (thrombocytopenic), andpatients with ITP in remission (normal platelet counts)(Figs 1±3). These assays were also performed in healthycontrols and patients with nonimmune thrombocytopeniaand nonimmune patients with normal platelet counts(Figs 1±3). The mean values and standard deviations forPAIgG, platelet albumin and platelet ®brinogen for thecontrol group and each patient group are summarized inTable I.

The mean values of the plasma-derived alpha-granularproteins, IgG, albumin and ®brinogen, were signi®cantlyhigher in platelets from thrombocytopenic patients, com-pared to the nonthrombocytopenic controls (P <0´05).These elevations were consistent and independent of themechanism responsible for the thrombocytopenia. With theexception of ®brinogen, these proteins were not signi®cantlyelevated in platelets from patients with normal plateletcounts (P >0´05). The one exception was observed innonthrombocytopenic patients with nonimmune haemato-logical disorders. The mean platelet ®brinogen for thispatient group was signi®cantly elevated, compared tonormal controls (P�0´0004). Patients with elevatedPAIgG, platelet-associated albumin, and/or platelet ®brino-gen, had normal levels of serum IgG, serum albumin, andplasma ®brinogen.

Megakaryocyte synthesized platelet proteins were mea-sured in healthy controls, patients with active ITP (throm-bocytopenic), and patients with ITP in remission (normalplatelet counts) (Fig 4). These assays were also performed inhealthy controls and patients with nonimmune thrombo-cytopenia and nonimmune patients with normal plateletcounts (Fig 4). The mean values and standard deviations forthe alpha-granular proteins, PF4 and b-TG, are summarizedin Table I. Whereas thrombocytopenic patients consistentlydemonstrated elevated plasma-derived platelet proteins, aparallel increase in megakaryocyte synthesized proteins wasnot observed in any patient. The mean values for theseproteins (b-TG and PF4) were not signi®cantly elevated inthrombocytopenic patients or any of the patient groups,compared to healthy controls (P >0´05).

To determine whether elevated plasma-derived plateletproteins re¯ected production of larger platelets in thrombo-cytopenic patients, we studied a parameter of platelet size;the mean platelet volume (MPV). The MPV for boththrombocytopenic and nonthrombocytopenic patientgroups were within the established normal range (Fig 5).Furthermore, there was no correlation between MPVelevation and the levels of PAIgG in the thrombocytopenicpatients with ITP (r�ÿ0´23), or in the thrombocytopenicpatients with nonimmune haematological disorders(r�ÿ0´26). Similarly, there was no correlation betweenMPV and elevated platelet albumin in these two patientgroups (r<0´15).



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DISCUSSION

Platelet populations from ITP patients have been shown tohave a greater size heterogeneity as re¯ected by an increasedpercentage of megathrombocytes (Karpatkin, 1985; Khanet al, 1975; Zucker-Franklin & Karpatkin, 1977; Garg et al,1972). It has been hypothesized that this altered largerplatelet population may be responsible for the elevated PAIgGlevels observed in ITP patients (Shulman et al, 1982; Pfuelleret al, 1981, 1986). Indeed, Corash et al (1977) demonstratedthat large-heavier platelets contained more alpha granules.Based on these observations, the purpose of our study was todetermine if platelets from ITP patients, who presumablyhave `larger and heavier' platelets containing more alphagranules, do in fact have an increased alpha-granule proteincontent.

For this report we measured two types of alpha-granuleprotein based on their origin of synthesis. These proteinsincluded several megakaryocyte synthesized (endogenousalpha-granular platelet proteins; bTG and PF4) as well asseveral plasma-derived alpha-granular proteins (exogenousalpha-granular platelet proteins; IgG, albumin and ®brino-gen) in platelets from patients with a variety of thrombocyto-penic and nonthromboctyopenic disorders. We postulatedthat if the explanation for elevated PAIgG was megakaryo-cytic stimulated production of larger heavier platelets thenthere would be an increase in mean platelet volume, with anincrease in all components of the alpha granule. In thismodel, levels of plasma-derived alpha-granular proteins maybe normal or increased. However, if the stimulus occurred atthe circulating platelet level, then one would expect plateletswith normal levels of endogenous alpha-granular proteins,but elevated levels of plasma-derived alpha-granularproteins.

Previously, we have postulated that elevated levels of PAIgGmight suggest that immune mechanisms could contribute tothe thrombocytopenia in patients with historically classi®ed`nonimmune' thrombocytopenic disorders (Sinha & Kelton,

1990). Indeed, Hymes et al (1979) reported that some patientswith thrombocytopenia in disorders other than ITP havecirculating anti-platelet antibody. In our study we attempted toexclude `nonimmune' thrombocytopenic patients who testedpositive for the most commonly detected anti-platelet anti-bodies in ITP (anti-glycoprotein IIb/IIIa and anti-glycoproteinIb/IX), by screening platelet lysate samples from each patientin an antigen capture assay according to Warner et al (1999).Based on the results of this assay (not shown), all patientscategorized with ITP demonstrated detectable anti-glycopro-tein IIb/IIIa or anti-glycoprotein Ib/IX antibody, whereas allpatients categorized with nonimmune thrombocytopeniafailed to demonstrate anti-platelet antibodies in this assay.Although this screening approach does not rule out thepossibility that patients categorized with `nonimmune' throm-bocytopenia may still have an anti-platelet immune compo-nent with speci®cities other than glycoprotein IIb/IIIa orglycoprotein Ib/IX, it is clear that the `immune mechanisms'responsible for the thrombocytopenia in ITP versus `non-immune' thrombocytopenic disorders, are distinct.

Our de®ned quantitative upper limit of normal for eachplatelet protein measured was based on values obtained froma group of healthy controls (n�13). The results we obtainedwere similar to those obtained by other investigators usingdifferent techniques. Together these studies indicate that thenormal amounts of protein from solubilized platelets as<5 mg PAIgG/109 platelets (Kelton & Steeves, 1983; Keltonet al, 1989), <15 mg albumin/109 platelets (Kelton & Steeves,1983), 80±140 mg ®brinogen/109 platelets (Colman et al,1994; McKeown et al, 1993), 30±80 mg b-TG/109 platelets(Colman et al, 1994; McKeown et al, 1993; Bellon et al,1993; Kerry & Curtis, 1985) and <5±14 mg PF4/109

platelets (Colman et al, 1994; McKeown et al, 1993; Bellonet al, 1993; Kerry & Curtis, 1985).

When platelets from groups of immune and nonimmunethrombocytopenic patients were studied we observed aconsistent increase in plasma-derived platelet proteins,with no parallel increase in megakaryocyte synthesized



Fig 5. Measurement of mean platelet volume (MPV) in immune and nonimmune patients and healthy controls. Each symbol represents a singlepatient or healthy control. The mean (B) and standard deviation for each patient or healthy control group is indicated.



proteins (Figs 1±4). In this study not all individual patientswith immune and nonimmune thrombocytopenia demon-strated increased PAIgG levels. These ®ndings suggest thatthere may be two subsets of thrombocytopenic patients:those with increased PAIgG levels and those with noincrease. In our study we focused on whether there was adifference in the overall average level of measured alpha-granule proteins between different patient groups, ratherthan individual patients. Although it remains debatable as towhich assay measures the true amount of IgG on washedplatelets, it is clear that the overall mean PAIgG value fromthe group of patients with ITP and the group of patients withnonimmune thrombocytopenia both have a signi®cantincrease in PAIgG compared to normal controls whenassayed by the same technique. We suggest that thesedifferences are not a re¯ection of the platelet preparation orassay techniques, but rather a re¯ection of the patientpopulation as a whole.

The results of our study failed to demonstrate an increasein the level of megakaryocyte synthesized proteins in eitherimmune or nonimmune thrombocytopenic patients. These®ndings support earlier studies where no difference wasfound in platelet density between normal controls, ITPpatients and patients with nonimmune thrombocytopenicdisorders (Illes et al, 1987). Furthermore, studies by Pfuelleret al (1986), in contrast to Corash et al (1977), found thatPAIgG of both controls and patients with immune andnonimmune thrombocytopenia is preferentially located inthe lightest platelets rather than in heavier platelets. Wesuggest that elevated levels of PAIgG observed amongthrombocytopenic patients are not due to the presence of agreater percentage of megathrombocytes found among thesepatients, but is a re¯ection of an altered process of IgGaccumulation by platelets and/or megakaryocytes.

It is well accepted that the mean platelet volume inthrombocytopenic patients is generally elevated in compar-ison to patients with normal platelet counts (Garg et al,1971; Branehog et al, 1975). However, in our study we failedto observe an increase in overall mean platelet volumebetween patient groups in comparison to normal controls.Although some individual thrombocytopenic patients hadincreased mean platelet volumes, because of the greatvariation within the patient groups, the differences did notreach statistical signi®cance for the group as a whole. In ourstudy we focused on whether there was a difference in theoverall mean platelet volume between different patientgroups, rather than individual patients. Our results agreewith earlier studies by Kelton et al (1979) and Holme et al(1988) who also found no relationship between increasedlevels of platelet associated IgG and any particular size classof platelets.

The observation that IgG is elevated in parallel with otherplasma proteins (albumin and ®brinogen) in thrombocyto-penic patients provides further information about PAIgG.Firstly, not only are measurements of PAIgG of no diagnosticusefulness, but measurements of platelet albumin, or anyother plasma-derived alpha-granule protein, will yield thesame information as IgG measurements. Secondly, anisolated increase in the exogenous alpha-granule protein

content in platelets from thrombocytopenic patients suggeststhat the mechanism of internalization of IgG and otherplasma proteins is altered in platelets and/or megakaryo-cytes in these patients. Thirdly, the observation that elevatedPAIgG measurements are observed in patients with diverseclinical aetiologies (both immune and nonimmune), sug-gests that increased PAIgG measurements may be unrelatedto platelet clearance.

There are few precedents for cells acquiring proteinscontained within regulated secretory granules by endocy-tosis. In platelets, speci®c receptors such as GP IIb/IIIa andFcgRII may be responsible for the internalization of®brinogen and IgG in a receptor-speci®c manner (George1990; George et al, 1985; George & Saucerman, 1988;Harrison et al, 1989). However, a speci®c receptor has notbeen identi®ed for albumin. Presently, there are no reportedstudies on the quantitation of platelet receptors in ITPpatients versus normal controls. However, the observationthat increased levels of PAIgG are not con®ned to anyparticular size class of platelets (Kelton et al, 1979; Holmeet al, 1988) suggests that there may not be a quantitativedifference in the number of IgG platelet receptors in ITPpatients, but rather a difference in the presence of astimulating factor for IgG internalization.

Based on the results of our study, we hypothesize thatstimuli associated with thrombocytopenia may be respon-sible for the altered uptake of plasma-derived alpha-granularproteins in thrombocytopenic patients. Our data suggest thatthis stimulus may be independent of the mechanism ofthrombocytopenia. We hypothesize that stimuli in thrombo-cytopenic patients leads to increased endocytosis by mega-karyocytes and/or platelets, resulting in platelets withelevated levels of plasma proteins.

ACKNOWLEDGMENTS

This work was supported by a grant from the MedicalResearch Council of Canada and the Heart and StrokeFoundation of Ontario to J. G. Kelton and a Bayer/CanadianRed Cross Society Research Studentship to M. Hughes. C. P.M. Hayward is a Research Scholar of the Heart and StrokeFoundation of Ontario.

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Measurement of endogenous and exogenous alpha-granular platelet proteins in patients with immune and...

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