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Hemopoietic origin of factor XIII A subunits in

platelets, monocytes, and plasma. Evidence

from bone marrow transplantation studies.

M C Poon, … , B A Ruether, D I Hoar

J Clin Invest.

1989;

84(3)

:787-792.

https://doi.org/10.1172/JCI114237

.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic

contributions to FXIIIA in these components were studied in patients transplanted with

marrows from donors with different FXIIIA phenotypes. In three patients with successful

engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet

and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA

in platelets and monocytes is synthesized de novo and/or from their progenitor cells.

Plasma FXIIIA phenotype change after transplantation was more complex. Patient I

changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115

d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but

did show enrichment of the fastest band. Thus, while there is a definite contribution of donor

hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to

delay or prevent the phenotype change.

Research Article

(2)

Hemopoietic Origin of Factor Xil A Subunits in Platelets, Monocytes, and Plasma

Evidence from Bone Marrow Transplantation Studies

Man-Chiu Poon, JamesA.Russell, Sandra Low, Gary D. Sinclair, Allan R. Jones, WalterBlahey, Bernard A.Ruether, and David1.Hoar

DepartmentsofMedicine, Pediatrics, Medical Biochemistry, and Laboratory Medicine, UniversityofCalgary, FoothillsHospital and

theTomBaker Cancer Centre; Hemophilia Clinic andMolecularDiagnosticLaboratory, Alberta Children's Hospital; andthe

Canadian RedCross BloodTransfusion Service, Calgary, Alberta,Canada

Abstract

Factor XIII A subunit(FXIIIA) is found in plasma, platelets, and monocytes. Thehemopoietic contributions to FXIIIAin

thesecomponents werestudied inpatients transplanted with

marrows from donors with different FXIIIA phenotypes. In

threepatients with successful engraftment (byDNA genotyp-ing, red cell phenotypgenotyp-ing, and cytogenetic studies) plateletand monocyteFXIIIAchangedtodonorphenotypes with hemato-logic recovery. Thus, FXIIIA in platelets and monocytes is

synthesized denovoand/orfromtheirprogenitorcells. Plasma

FXIIIA phenotype change after transplantation was more

complex. Patient1 changedfrom phenotype 1-1(one

electro-phoretically fast band)to1-2(three bands)in115d; patients2

and 3 didnotchange completelyfromphenotype 1-2to1-1in

upto458d,but did show enrichment of the fastest band.Thus, while there isadefinite contribution of donorhemopoiesisto

plasma FXIIIA, anothersourceofrecipientFXIIIAappearsto

bepresenttodelayorprevent thephenotype change. Introduction

Activated FactorXIII(fibrin stabilizing factor)isa

transami-dase thatparticipatesin the final event of bloodcoagulation (1). It covalently crosslinks fibrinmonomersinto stablefibrin

clotby the formation ofe-(y-glutamyl) lysylbonds infibrin.

Patients with deficiency of this clotting factor may have as

manifestations excessive bleeding, defective wound healing, and fetalwastage (1). Plasma FactorXIII, with amolwt of

320,000, isatetramercomposedoftwoAandtwoBsubunits

(1-4). TheAsubunit, withamolwtof75,000,isthezymogen

thatpossessesthe FactorXIIIenzymatic activityafter

activa-tionby thrombininthepresenceof calcium ions. The B

sub-unit, withamolwtof80,000,functionsas acarrier for the A

subunit. Both platelets (2-8) and monocytes (9, 10) have

Portions of this workwerepresentedatthe 1987Annual Meeting of

the CentralSocietyfor ClinicalResearch, Chicago, IL,and the 1988 Annual Meeting of theAmerican Society of Hematology, San

An-tonio, TX, and were published in abstract form (1987. Clin. Res. 35:892A,and 1988. Blood.72[Suppl 1]:306a).

Address reprint requests to Dr. Man-Chiu Poon, University of Calgary,FoothillsHospital, 1403 29th StreetN.W., Calgary,Alberta, CanadaT2N2T9.

Receivedfor publication 18November 1988andinrevisedform 7

April1989.

been found to contain Factor XIII in their cytoplasm, but

only the 75,000 mol wt Asubunits are present. The Factor

XIII A subunits (FXIIIA)' in platelets, monocytes, and

plasmaarebiochemically,functionally,andimmunologically similar(2-10).

It hasbeenestimatedthat only abouthalf ofthe total cir-culating FXIIIA is present in plasma (6, 1 1); the other half is contained in the bloodcells.Hemopoiesis has been suggested as a sourceof FXIIIA in these components, but the extent of this contribution, especiallytoplasma, has not been conclu-sivelydemonstrated. Polymorphism of FXIIIA has been ob-served (12). Using an agarose gel electrophoresis system, three commonphenotypes have been described. Phenotype 1-1 consists of asinglefast-moving band; 2-2 consists of a single slow-moving band; and1-2consists ofboth the fast-and slow-moving bands, plus an intermediate moving band. In this study we used the FXIIIA polymorphism to follow the contri-bution ofhemopoiesis toplasmaand cellular FXIIIA in pa-tients after bone marrowtransplantation.

Methods

Patients. Patients with aplastic anemia or hematologic malignancy acceptedtothe bonemarrowtransplantation programatthe

Univer-sityofCalgarywerestudied.FXIIIAphenotypesweredeterminedon

thesepatients and their HLA A, B, andDRidenticalsibling donors. When theFXIIIAphenotypes in the recipient and donor pair were

different,the plasma and cellularFXIIIAphenotypes of therecipient

patientsweredeterminedatintervals aftertransplantation. The trans-plantation protocol included conditioning therapy before infusion with 1.8-3X108donormarrownucleated cells perkilogram recipient bodyweight. The conditioningtherapy wasintravenous cyclophos-phamide(120mg/kg) plus various combinations of total body

irradia-tion,intravenouscytosine arabinoside,and oralbusulfan accordingto

theunderlyingdisorders.Acombination of intravenous methotrexate andcyclosporinAwasusedfor prophylaxis ofgraftvs.hostdisease.

Plasma and cell specimens. Venous bloodwascollected in

vacu-tainertubescontainingK3EDTA (10.5 mg/7 ml blood; Becton

Dick-inson, Mississauga,ON). Plasma and platelets washed twice with saline

wereprepared from the EDTA anticoagulated blood according to the methodsdescribed by Board et al. (12). The separated platelets con-tained< 103total whitecells/106 platelets. Monocytes were separated bydensity gradientcentrifugationincolloidalsilica supplied

commer-cially (Sepracell-MN; Sepratech Corp., Oklahoma City, OK), and

washedtwice in PBS (0.009Mphosphate, 0.003 MKCI, 0.14 MNaCl,

pH 7.4) containing 0.1% BSA according to the manufacturer's in-structions. The recovery of monocytes was 88±9.7%, with

lympho-cytesasthemajorcontaminantsand noappreciable contamination with platelets (<102platelets/106 monocytes) and PMN.Allplasma

aliquotsand pellets of plateletsandmonocytes were frozenat-70°C.

Frozen cells werethawed andusedonly once.

1.Abbreviationsused in this paper: FXIIIA, Factor XIII A subunit.

J.Clin.Invest.

© TheAmericanSociety for Clinical Investigation, Inc. 0021-9738/89/09/0787/06 $2.00

(3)

Factor XIII studies. FXIIIA phenotype determination was per-formed accordingtothemethodof Grahametal.(13).The FXIIIA

variants were separated by agarose gel (Seakem ME; FMC Corpora-tion, Rockland, ME)electrophoresisand visualized andphotographed

with ultraviolet light illumination after incorporation of monodansyl cadaverinetocaseinbythe thrombin- andCa++-dependent transglu-taminase activity of FXIIIA. 5-Ml sampleswereapplied for electropho-resis. Plasma samples were applied after dilutions with TEB buffer (0.18MTris, 0.04 M Na2EDTA, 0.1Mboricacid, pH 8.6). Except for thetitration study (see below), plasmawasroutinely studiedat1/4-1/8 dilution.Atlowerplasma dilution the bands tendtosmudge. Frozen platelet or monocyte pellets separated from7mlblood were preincu-batedwith 50 Ml 1% TritonX-100 inTEBbufferat roomtemperature for 30 min, and 5 Mlappliedwithout further dilution.

The procedure couldidentifyFXIIIAphenotypesataplasma

sam-pleconcentrationof0.035U/ml for phenotype 1-1 and 0.050 U/ml for phenotype 1-2. The minimal numbers of platelets and monocytes re-quired for phenotype identificationwere1 xI05 and 1 X 103per5Ml, respectively.Attheseconcentrations the bands of all phenotypeswere

clearly visible and could be photographed. For each individual studied, theFXIIIAphenotype of plasma, platelets, and monocytes is identical. Preliminary experiments showed that lymphocytes andPMNdidnot

containFXIIIAusing this system, andareconsistent with the observa-tions of Henriksson(9), Dvilansky(11), and their colleagues.

Plasma FXIIIA activitywasquantitated by itsabilitytoincorporate

fluorescent monodansyl cadaverineto casein in the presence of cal-cium and thrombinaccordingto themethod ofSheltawyet al. (14).

EDTAanticoagulated plasmawasused,andthe standardcontrolwas a

pool of normal EDTAanticoagulated plasmaobtained from 20

nor-malindividuals.

DNAstudies.DNAwasextracted fromperipheralbloodormarrow

aspirateusing standardtechniques (15).DNA(2Mg)wasdigestedwith Pst I(hMF-l)orSinI(3HVR), separatedon 1% agarose gels, alkaline blotted, and probed with the hMF-l or 3HVRprobe aspreviously

described(16, 17).The hMF-l sequence from theD1Z2locusof chro-mosome 1 recognizes complex allelicpatterns thatcan differentiate

mostunrelatedindividuals. Withinfamilies,on theaverageoneinfour will shareallelesand beidentical. The 3HVR probe isonchromosome 16 andrecognizesatwo-allelepatternathigh stringency.Wehave used thisatlowstringencytoidentifymorecomplexpatterns derived from

multiplechromosomes useful forDNAgenotyping.

Cytogeneticstudies. Cytogeneticstudieswereperformed by

stan-dard methods(18, 19).

Redcellphenotype studies.Redcellantigen phenotypingwas stud-ied by standardtechniques (20).In oneRh(D) negative patient

trans-planted withanRh(D)positivedonor, theproportionof Rh(D)

posi-tive cells aftertransplantationwasestimatedbythedifferential

aggluti-nation methoddescribedbyMollison(20).

Results

26patient-donor pairswerestudied,and6werefoundtohave different FXIIIA phenotypes. Five ofthese

patients

were

transplanted, with four followed for 13-22mo.

FXIIIA

phenotype

ofplasma

mixtures

Mixtures of different

proportions

of

phenotype

1-1 and 1-2 plasmas ofknown FXIIIA

activity

were

electrophoresed

at

sample

dilutions of

1/4.

In three

experiments

the mixture would assume a 1-2 phenotype when the

proportion (by

FXIIIAactivity) of1-2inthe mixturewas0.26±0.03

(1 SD)

or

greater, and would assumethe 1-1 phenotype whenthe

pro-portion

of1-1 inthemixturewas0.83±0.04(1 SD)orgreater. The resultssuggest that if donor

hemopoietic

cellscontribute to plasmaFXIIIA after transplantation, it would befasterand

easiertochange from plasma phenotype 1-1 to1-2,thanfrom plasma phenotype 1-2 to 1-1.

Marrow

transplantation

with

donors ofdifferent

FXIIIA

phenotype

Patient 1. Fig. 1 A shows the FXIIIA phenotype studies on patient 1, a 32-yr-old womantransplanted foracute

promy-elocytic

leukemia in second

remission.

She hadaFXIIIA phe-notypeof1-1 (plasma, platelets, and monocytes), and her fe-male donorhadaphenotypeof1-2.Aftertransplantation, the first study was performed on day 31 when the patient was hematologicallynormalwithaplateletcountof171 X 109/liter and awhitecountof5.4X

109/liter

with6% monocytes,and when she had not received anybloodcomponents for 12 d. The patient had received plateletsbut no additional plasma after the

transplantation.

At thistime both the platelets and monocytes hadassumed the donor phenotype of 1-2, and re-mained phenotype 1-2uponmultiple

follow-up

studiesover a 22-moperiod. Theplasma phenotypestudiedatintervals from days 31 to 93 continuedto be that of therecipient 1-1, but changedto 1-2 when studiedon day 115, andremained 1-2 thereafter. The plasma FXIIIA

activity

of the patient after transplantation did not vary

significantly

from the pretrans-plant level(1.09 U/ml) duringtheinitial 100dof follow-up.

Thepatienthad acontinuingnormal peripheral blood and

marrow

picture, withno blood componenttransfusionsince day 19

posttransplantation.

Her red cell phenotype also changed from pretransplantgroup A to donor group 0 with-outresidualgroup A cells. Peripheral blood cellDNAstudies using hMF- 1 (Fig. 2 A) and 3'HVR (Fig. 3 A) on day 295 posttransplantation showedachangeofphenotype to one that hadclear donor-specific fragments confirming

marrow

trans-plantation.However, there was apersistence of recipient-spe-cific fragmentsin the peripheral blood cellDNA, indicating thattherestill existedapopulation of recipient elements. Anal-ysis with 3'HVR suggested the chimerismwasapproximately equally

representing

donorand

recipient

(Fig. 3 A).

Patient 2.Fig. 1 B shows the FXIIIA phenotypestudieson

patient

2,a39-yr-oldmantransplanted foracute

granulocytic

leukemia in first remission. He had a FXIIIA phenotypeof 1-2, and his male donor hadaphenotype of

1-1.

After

trans-plantation,

thefirst studywasperformedatday23 when the

patient

hadnotreceivedany blood componentsfor 11 d,and whenhis plateletcount was68X

109/liter

andhiswhitecount was 2.3 X

109/liter

with 51% monocytes. This patient had received platelets but no additional plasma after the trans-plantation.Atthis timetheplateletand monocyte FXIIIAhad assumedthe donorphenotype of1-1 and remained soupon multiple follow-up studiesover a 15-mo period. The plasma FXIIIA phenotype remained 1-2 and had notchanged when studied on day 233

posttransplantation.

The plasmaonday 288posttransplant clearly showedadecreasedintensity of the two slow-moving bands compared with the

fastest-moving

band. Theintensity ofthe two

slow-moving

bandscontinued todecreasegradually,buttheywerestillfaintly visible

(with

an

enriched fast-moving band)when the plasma from day 458 posttransplantwaslaststudied.TheplasmaFXIIIA

activity

of the

patient

after

transplantation

dropped slightly,from 0.87to

0.78U/ml by day 23,and thenincreasedgraduallyto1.3 U/ml

by day

77. The

activity

then stabilized at 0.95

U/ml

from day87after

transplantation.

(4)

DONOR

RECIPIENT

PRE

POST

PA

M

P

PA M P

PA M

P-1

P-2

D R p D R P Figure 2. DNA

geno-and 2 (B) using the

410 ,! hMF-Iprobe. DNA

iso-*;- . _- 4* latedfrom peripheral

3<;; t : _ X 10 bloodwasdigestedwith

PstI. Triangles,

frag-t

*"

~4

46 * mentsshared

by

donor

(lane D)and

posttrans-4.6 metshrdbdor plant recipient

(lane

); diamonds, recipient-specificfragments(lane

2.6

11!

23.0

R)

remaining

after

25 transplantation. Num-A B bers refer to

approxi-matesizes ofDNA

frag-mentsinkilobases.Lane Pin panelBis fromashortexposureof the

sameautoradiograph used in lanes D and R.

DONOR

RECIPIENT

PRE

POST

I'.

B

PA M

P

PA

M

P

PA

M

P-1 P-2

DONOR

RECIPIENT

PRE

C

POST

1.1-S

PA

M

P

PA

M

P

PA M

P-1

P-2

Figure 1. FXIIIA phenotypesoftransplant donor (DONOR) and

re-cipient (RECIPIENT), pretransplant (PRE)and posttransplant

(POST) platelets(PA),monocytes(M),and plasmas (P).Triangles,

thepointsofsample applicationatthe cathodal end. (A) Studieson

patient 1. The posttransplant samplesweretakenasfollows:platelet,

day31 aftertransplantation;monocytes,day 31; plasma sampleP-I, day 31;andplasma sample P-2, day 115. The number of lysed cells

in5-id samples appliedtotheposttransplantation laneswere as

fol-lows:platelets, 1.6X 107;monocytes, 1X 106. (B) Studiesonpatient

2. Theposttransplant samplesweretakenasfollows:platelets, day

23;monocytes,day 23; plasma sample P-1, day 23;and plasma

sam-ple P-2, day 288. The number of lysed cells in 5-iu samples applied

platelet counts by day 32 and had not required any blood

componenttransfusionsinceday 12. That he hadasuccessful marrowtransplantation wasshown by normal follow-up

pe-ripheral blood andmarrowstudies,acomplete change of the peripheral blood cell DNA genotype to the donorgenotype

(Fig. 2 B), andachangetodonor redcellphenotypes. Patient3. Fig. 1 Cshows the FXIIIAphenotype studieson

patient 3, a 49-yr-old mantransplanted for chronic

granulo-cytic leukemia in the accelerated phase. He had a FXIIIA phenotype of 1-2, and his male donor hadaphenotype of 1-1.

Thepatternof cellular andplasma FXIIIA phenotype change aftertransplantation wassimilartothatdescribed forpatient 2.Theplatelet andmonocyte FXIIIAwaspersistently ofdonor phenotype 1-1 since first studiedatday 60 posttransplantation.

Thelast platelet transfusionwas onday 16 after transplanta-tion. PlasmaFXIIIAphenotype remainedrecipient pretrans-plant phenotype 1-2 (three bands), but began toshow a

de-creasedintensity ofthe two slower-moving bands compared with thefastest band by day 250posttransplant. The slower-moving bands continued tobe presentbut in decreasing

in-tensity compared with the fastestbandwhen theplasmawas

last studiedat day 399 posttransplant. After transplantation the patient's spleen became smaller but remained palpable, andalthough therewasnormalmarrowrecovery, the periph-eralwhitecellandplateletcountdidnotreach normal values

untilday 100 aftertransplantation.Successfultransplantation inthis patientwasindicated by the disappearance ofthe

Phila-delphia chromosome abnormality,achange of theperipheral

and marrow cellular DNA genotype to the donor genotype

(Fig. 3 B), andachange of red cellstodonorphenotypes. Patient 4. Patient 4was a45-yr-oldmantransplanted for

myeloproliferative disorder in the chronic phase. He had a

FXIIIAphenotype of 1-2, and his male donor hadaphenotype

totheposttransplantation laneswere asfollows:platelets, 1.5 X 107;

monocytes,3X 105.Thetwoslower-moving bands in lane P-2were

faintanddidnotreproduce well. (C) Studiesonpatient 3. The post-transplant samplesweretakenasfollows: platelets, day 60;

mono-cytes,day 60; plasma sample P- 1, day 60; and plasma sample P-2, day294.The numberoflysed cells in5 Mlappliedtothe

posttrans-plantation laneswere asfollows: platelets, 4.9X 106;monocytes, 1.4

x I05. Thetwoslower-moving bands inlane P-2werefaintanddid

notreproduce well.

(5)

R D P R D P

Figure

3. DNA

geno-typing on patients 1 (A) and 3 (B)using the

23 23

3'HVR

probe. DNA

10 10 isolated fromperipheral

4_O>-*__ bloodwasdigestedwith Sin

I,

andhybridization

4i-5 _6. 5 was performed at 55°C.

Washeswereat

50°C

in

25 0.2Xstandardsaline

ci-25

trate (0.15 MNaCl, 0.015MNacitrate).

-w 9 Closed diamonds,

frag-4X * ments shared by donor

A B (lane D) and

posttrans-plant recipient (lane P); opendiamonds, recipient-specific fragments (lane R) remaining after transplantation. The posttransplant samplefrom patient 1 (A) was obtained on day 295, and from patient 3 (B) on day 64 after trans-plantation. Numbers refer to approximate sizes of DNA fragments in kilobases.

of 1-1. After transplantation, although his peripheral blood andmarrowpicture became normal quantitativelyand quali-tatively by microscopy,FXIIIAphenotypeofplatelets,

mono-cytes,and plasma continuedtobe thatof therecipient and did notchangeuponmultiple studiesover a 19-mo period(data

notshown).Inthis

patient

themultiple marrowchromosomal abnormalities persisted after transplantation and the periph-eral blood DNAstudies withhMF-1 and 3HVRon day281 after

transplantation

alsoshowednochange ingenotype(data

notshown),suggesting thatatleastthemyeloid and lymphoid cellseries didnot

engraft.

Redcellphenotype study, however, didsuggestsome

erythroid engraftment.

Thepatientwasblood group Rh(D) negative and the donor was Rh(D)

positive.

Studyatday 281 after

transplantation using

the red cell differ-ential

agglutination

method showedthat - 5%of his

circulat-ing redcellswereRh(D)positive.

Marrow

transplantation

with donors

of

identical

FXIIIA

phenotype

Twopatients with similar recipientand donor FXIIIA pheno-types, one pair with phenotype 1-1, another with 1-2, were

restudied after successful marrow

transplantation

foracute

granulocytic leukemiainremission.Nochange inFXIIIA phe-notypesontheplatelets,monocytes, andplasmawasobserved,

suggesting

thatthe FXIIIA

phenotypic changes

in

patients

1,

2,

and 3 after

transplantation

were notinduced

by

theprocessof

transplantation

itself,

but ratherbythe

engraftment

of hemo-poieticcellsof different phenotype.

Discussion

Patients 1-3 appearto have hadasuccessful transplantation, althoughDNAstudies

identify patient

1 as abloodchimera. Herposttransplant

peripheral

blood DNAshowed bothdonor and

recipient pretransplant-specific

bands

(Figs.

2Aand 3

A).

Patient4appears to have hadanengraftment ofonlyasmall

population

oferythroid elements, sincenoevidenceforDNA genotypechangescould beobtainedon

peripheral

blood DNA where 10% engraftmentwould bereadilydetected.

Theplateletand monocyteFXIIIAphenotypesof

patients

1-3 assumed those of the donors after marrow

transplanta-tions, suggesting that FXIIIA in these two cell lines was synthe-sized de novo and/or by their progenitor hemopoietic cells. Similar observations on platelets were obtained by Wolpl et al. (21), but they did not study the monocytes. These conclusions areanticipated byanumber ofprevious studies,but the mar-row transplantation model provides further definitiveproof. In addition to platelets, FXIIIA has been demonstrated in megakaryocytes (22). Recently, FXIIIA was identified in the cytoplasm (9, 10) and on the surface (23) of monocytes and macrophages. That the monocyte FXIIIA is synthesized de novo was also suggested by several lines of evidence. Func-tional andimmunologically identifiable FXIIIA has been re-covered from themedia(serum and platelet free) of monocyte cultures (23). The monocyte surface expression of FXIIIA could be augmented by such soluble monocyte activators as phorbolmyrisacetate,LPS, and

y-IFN.

Finally, usingacloned FXIIIA gene,Weisbergetal. (24, 25) were able to demonstrate mRNAby Northernblotanalysis from preparationsof mono-cytesand the U937monocytic cellline.

Theoriginofplasma FXIIIA appears to be more complex. Ourstudysuggests that thereisadefinite and major contribu-tion toplasmaFXIIIAbythetransplantedhemopoietic cells. This is clearly demonstrated by the complete change-over from recipient 1-1 to donor 1-2 phenotypein patient 1, and by the increasing intensity ofthe fastest-moving band (due to enrichment of donor phenotype 1-1) inpatients2and 3. The hemopoietic cells' contributionto plasma FXIIIA is probably viathe platelets and monocytes and/or their precursors, but we cannotconclude whether one or both ofthese cell lines con-tribute. Previous studies have shown that sickle cell disease patients with thrombocytosis may have anincreased plasma concentration ofFXIIIA (26),whilesomepatients with idio-pathic thrombocytopeniapurpura(22) and cytopenias second-arytoacuteleukemia (27)had lowerplasmaFXIIIAlevels.In

experimental

studies ratsrenderedcytopenic by cytotoxicor other agentshad decreasedFXIIIAlevels(28). However, these studies also showed thatinlong-standing severely thrombocy-topenic patients the reduction of plasmaFXIIIA waslessthan expected (22), suggesting that plateletswere notthe sole con-tributor. HowplateletFXIIIAreaches theplasma is unknown as FXIIIA isnotreleasedduring platelet release reactionand clot retraction (29). How monocytes would contribute to plasma levels ofFXIIIAhasyet tobedetermined, although in vitro culture studies suggestthat FXIIIA is secreted intothe culturemedia (23).

The long time period required to change the plasma FXIIIAphenotype of patient1(between 93 and115d) and the inability oftheplasmaFXIIIAofpatients2 and 3 tochange completely from

recipient

1-2 to donor 1-1 phenotypeby 15 and 13mo,

respectively,

after

transplantation

also suggestthat FXIIIA sources other thanthe donor

hemopoietic

cellsmay also be present. When amixture of1-1 and 1-2 plasmawas

(6)

cell and platelet counts were observed by day 32 in patients 1 and 2, and by day 100 in patient 3.

Although blood chimerism demonstrated by DNA geno-typing in patient 1 makes interpretation of delayed change-over of plasma FXIII phenotype impossible, chimerism is un-likely inpatients 2 and 3. In patient 3 complete donor marrow engraftment was suggested by peripheral blood and marrow DNAgenotyping studies,red cell

phenotyping,

and the cytoge-netic demonstration of the disappearance of the original dis-ease marker Philadelphia chromosome. Successful donor marrowengraftmentin patient 2 was demonstrated by red cell phenotypeand peripheralblood DNA genotyping studies. Al-though marrow DNA was not studied in patient 2, an unde-tected significant megakaryocyte/platelet chimerism is un-likely. In Fig. 1 B the posttransplant platelet FXIIIA was clearly a single band (donor phenotype 1-1). The

concentra-tion of this sample ( 150X I05platelets/5 pA1) was 150-fold the concentration required for phenotype identification and shouldhave shown the extra twoslower-moving bandsifthe recipient pretransplant platelets (with phenotype 1-2) were present at > I1/150 (0.67%)of the sample.

Onepossiblerecipient source of FXIIIA that would delay theplasma FXIIIA phenotype change aftertransplantationis residual recipient tissue (hepatic, splenic, and pulmonary al-veolar) macrophages. These tissue cells, derived from trans-formed monocytes (30-32), can apparently proliferate inde-pendently ofthe marrow precursors(30), and have a longer disappearance time than circulating monocytes (31). After marrowtransplantation residentrecipient pulmonary alveolar macrophagescould berecovered in decreasingnumbers until approximatelyday 81 (31). This lifespan figure of81 d after marrowtransplantation isprobably applicable to other tissue macrophages, as recipient macrophages could no longer be identified intheliver ofapatientwhodied 104 d after trans-plantation (32). While these tissue macrophage sources will certainlycause adelayinplasmaFXIIIAphenotype change in our patients, their lifespan isnot sufficientlylong toexplain totallythelong delayseen inpatients2and3, suggestingthat extrahemopoieticsources may be present.

Liverhas been suggested as a possible extrahemopoietic source ofplasmaFXIIIAbysome, but notall,studies.Patients with liver disease may have a decreased plasma FXIIIA level (27), butit isnotknownif thisis secondary to a decrease in the carrierprotein Factor XIII B subunits synthesized by the he-patocytes (21, 33, 34). Immunological and in vitro culture studies of hepatocytesandhepatoma cell lineshavegiven con-flicting results. Synthesiswas shown by some studies (33, 34) butnotby others (35, 36). Recently, FXIIIAmRNAhas been found in liverbiopsy preparations by Northern blot analysis witha cloned FXIIIA gene (24). However, a contribution of FXIIIA mRNAfrommacrophages in the preparation has not beendefinitelyexcluded, and asimilarstudy by Grundmann etal.(37) failedto identify FXIIIA mRNA in the liver.

Ourstudy, therefore,allows us toidentifythe transplanted hemopoietic

cells

as a source of plasma FXIIIA; however, the data do not allow usto conclude whether the contribution is from platelets, monocytes, or both. We also conclude that there are additional sources ofplasma FXIIIA. One source could beresidualrecipient tissue macrophages, also of hemo-poietic origin, but additional extrahemopoietic sources are distinct possibilities. Identification ofthe exact tissue site(s) will

require

further study. A marrow transplantation model

was also used by Wolpl et al. (21) to study plasma FXIIIA origin, but the studywas incomplete for interpretation. The study demonstrated that the plasmaFXIIIA of two patients with phenotype 1-1 and one patient with phenotype 2-2 all changed to the donor phenotype 1-2 in 11, 8, and 6 d, respec-tively, after the last platelettransfusions. The relationship of this time periodtothedayoftransplantationwas notstated,so it is not

possible

to assess

whether

othersources of plasma FXIIIA were present.Furthermore, they didnot study a phe-notype 1-2 (three bands) recipient transplanted with marrow from a donor with phenotype 1-1 or 2-2 (both with one band). Assuggested inourstudies,

transplantation

ofmarrowfroma donor with theFXIIIAphenotype consisting ofoneband(i.e., 1-1 or2-2)to arecipient with the phenotype consisting of three bands(i.e., 1-2) requires higher proportionsof donor FXIIIA (oragreaterdegree ofrecipientFXIIIAdepletion) for expres-sion ofthe complete change. The latter studieswehave per-formed should, therefore,be more sensitive for detecting low levels ofFXIIIAcontributionbyrecipientsources.

Althoughthegenesforboth FXIIIA andtheHLAantigens are located on chromosome6 (12, 25, 38-40), ourabilityto findHLAidentical siblings with

different

FXIIIAphenotypes confirms the observations of Board etal. (38), Olaisenetal. (39), and

Zoghbi

etal. (40), that HLAandFXIIIA genes are not closely linked. Platelet and monocyte FXIIIA polymor-phism can,

therefore,

be used as a marker for bone marrow

engraftment

inthe

appropriate

patients. Thismarker hasthe advantagethatitcan beusedasearlyasthefourth week after

transplantation during

early hemopoieticrecovery. Ourstudy also

provides

anexample of howDNA

genotyping

canbe used tofollowmarrow

engraftment

after

transplantation.

This tech-nique has recently been applied for evaluation of long-term

engraftment

after marrow

transplantation

in patients with

aplastic

anemia (41).

Acknowledgments

Wearegrateful for the nursing, secretarial, and technical assistance of Ms. LoisDumenko, Ms. CarolynKelly,Ms.Rita Peterson, Ms. Linda

Berreth,andMr.EricChing.

This workwassupported inpartby research funds from the Alberta Medical Services Inc. Foundation (No. 438), Canadian Red Cross BloodTransfusion Services (No.CA83-01), andthe Alberta

Heredi-taryDisease Program.

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