Porcine and Canine von Willebrand Factor and von Willebrand Disease: Hemostasis, Thrombosis ...

12 Pages·2014·1.43 MB·English

HindawiPublishingCorporation
Thrombosis
Volume2010,ArticleID461238,11pages
doi:10.1155/2010/461238
Review Article
Porcine and Canine von Willebrand Factor and von Willebrand
Disease: Hemostasis, Thrombosis, and Atherosclerosis Studies
Timothy C. Nichols,1,2 Dwight A. Bellinger,2,3 Elizabeth P. Merricks,2 Robin A. Raymer,2
MarkT.Kloos,2NatalieDeFriess,2MargaretV.Ragni,4,5andThomasR.Griggs1,2
1DepartmentofMedicine,FrancisOwenBloodResearchLaboratory,UniversityofNorthCarolina,ChapelHill,NC27516,USA
2PathologyandLaboratoryMedicine,FrancisOwenBloodResearchLaboratory,UniversityofNorthCarolina,ChapelHill,
NC27516,USA
3DivisionofLaboratoryAnimalMedicine,FrancisOwenBloodResearchLaboratory,UniversityofNorthCarolina,ChapelHill,
NC27516,USA
4Medicine/Hematology/Oncology,UniversityofPittsburghMedicalCenter,Pittsburgh,PA15261,USA
5HemophiliaCenterofWesternPA,Pittsburgh,PA15213,USA
CorrespondenceshouldbeaddressedtoTimothyC.Nichols,[email protected]
Received27October2010;Accepted29December2010
AcademicEditor:DavidH.Farrell
Copyright©2010TimothyC.Nicholsetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Use of animal models of inherited and induced von Willebrand factor (VWF) deficiency continues to advance the knowledge
of VWF-related diseases: von Willebrand disease (VWD), thrombotic thrombocytopenic purpura (TTP), and coronary artery
thrombosis.First,inhumans,pigs,anddogs,VWFisessentialfornormalhemostasis;withoutVWFbleedingeventsaresevereand
canbefatal.Second,theADAMTS13cleavagesiteispreservedinallthreespeciessuggestingallusethismechanismfornormal
VWFmultimerprocessingandthatallaresusceptibletoTTPwhenADAMTS13functionisreduced.Third,whiletheroleofVWF
inatherogenesisisdebated,arterialthrombosiscomplicatingatherosclerosisappearstobeVWF-dependent.Thedifferencesinthe
VWFgeneandproteinbetweenhumans,pigs,anddogsarerelativelyfewbutimportanttoconsiderinthedesignofVWF-focused
experiments.Thesehomologiesanddifferencesarereviewedindetailandtheirimplicationsforresearchprojectsarediscussed.
ThecurrentstatusofporcineandcanineVWDarealsoreviewedaswellastheirpotentialroleinfuturestudiesofVWF-related
disordersofhemostasisandthrombosis.
1.Introduction aseveremucosalandcutaneousbleedingdiathesisthatmost
often involves nasal, oral, gastrointestinal, and genitouri-
AnimalmodelsofvonWillebrandfactor(VWF)deficiency, nary mucosa. Without prompt treatment, bleeding can be
bothinheritedandinduced,haveprovidedkeyinsightsinto crippling or fatal in all three species. In addition, humans
VWF-related diseases. This paper focuses on the current andanimalswithVWDareoftenrecognizedwhenexcessive
status of knowledge of porcine and canine VWF and von bleedingoccursinassociationwithrelativelyminortrauma
Willebrand disease (VWD) and how investigations with or surgery. Affected pigs and dogs have undetectable VWF
theseanimalshaveadvancedtheunderstandingoftheseem- antigen, activity, and multimers (Figure1, Table1). The
inglyparadoxicalrolesofVWFinhemostasisandthrombosis inheritance pattern across species occurs in an autosomal
aswellasatherogenesis[1]. fashion[2–6].Understandingthemolecularandbiochemical
In humans, pigs, and dogs, VWF is a large, adhesive similarities and differences between human, porcine, and
glycoproteinthatsupportshemostasisbymediatingplatelet canine VWF and VWD has proven seminal in characteriz-
adhesion to injured vessel surfaces. Porcine and canine ing critical protein-protein interactions involved in VWF-
VWDmirrortype3humanVWD,phenotypicallyexhibiting mediatedhemostasisandthrombosis.
2 Thrombosis
Human Dog Pig charged region flanking the amino terminal side of the A1
domain disulfide loop that is involved in interaction with
NL VWD NL VWD NL VWD
the platelet-binding agonist, ristocetin. This region is also
critical to the regulation of VWF binding to platelet GP1b
under physiologic conditions and maintenance of the A1
loop in an unreactive configuration. Porcine plasma VWF
spontaneously binds human platelets in the absence of an
agonist such as ristocetin or botrocetin, and the deletion
of these four residues in this regulatory area of human
VWF-GP1b binding may help explain this observation
[2, 4, 17, 21–23]. Interestingly, botrocetin supports pig
VWF and pig platelet agglutination but ristocetin does
Figure1:MultimerdistributionofvonWillebrandfactorantigen not [4, 21–23]. Botrocetin also supports agglutination of
innormal(NL)andvonWillebranddisease(VWD)human,dog, pig platelets by human VWF [22]. In humans, a D1472H
and pig plasma samples. Human plasmas were obtained from polymorphismisassociatedwithdecreasedristocetinactiv-
GeorgeKingBio-Medical,Inc.,OverlandPark,Kansas.Thehuman ity, and pigs have a leucine at the homologous position
VWDplasmawasfromapatientwithseveretype3VWDplasma (Figure2) [24]. The pig platelet Gp1b sequence is con-
with VWF:RCo 15IU/dl and VWF:Ag 1IU/dl. The dog and
served (not shown) suggesting the D1472L substitution in
pig plasmas were prepared at the Francis Owen Blood Research
pig VWF may, in part, account for the relative lack of
LaboratoryattheUniversityofNorthCarolinaatChapelHillusing
ristocetin reactivity. The RGD sequence is conserved and
normalandsevereVWDanimalsthathadnodetectibleactivityor
likely mediates platelet integrin αIIbβ3 binding (Figure2).
antigen in either species. None of the subjects had recently been
Recombinant porcine VWF trafficked to storage granules,
transfused with VWF-containing products. Anti-VWF antibodies
forimmunostainingwerepurchasedfromDako(A082,Carpintera, is efficiently multimerized and secreted, and spontaneously
CA)(1.5%agarosegel)[7–9].Theidentityoftheverybottomband agglutinateshumanplatelets[18].
seeninalllanesisunknownbutisaconsistentfindinginmultiple
laboratoriesandisalsoseeninmurineVWDplasma[7–11].Itis
possible that this band simply represents nonspecific binding of 3.CanineVWFandVWD
theantibodytotheleadingedgeoftheproteinsattheendofthe
electrophoresisprocedure.
The canine VWF gene has been localized to canine chro-
mosome 27 (http://www.ncbi.nlm.nih.gov/gene/399544) in
aregionthatissyntenicwithhumanchromosome12p13.2.
2.PorcineVWFandVWD Like human VWD, canine VWD appears to be the most
prevalent inherited bleeding disorder in various strains of
The porcine VWF gene is on pig chromosome 5q21, a dogs,and,thus,thereissignificantinterestindetectionand
regionthatissyntenicwithhumanchromosome12p13.2,the characterizationofthediseaseandfindingnewtreatmentsin
locationofthehumangene[12,13].ThehumanVWFgene all species [3, 31–34]. The Chapel Hill, canine VWD strain
islocatednearthetelomere,perhapsinpartaccountingfor wasderivedfromashow-dogstrainofScottishTerriersand
thehighfrequencyofmutationsresultinginvarioustypesof thiscolonywasestablishedin1978fromasingleheterozygote
VWD,whilethepigVWFgeneismorecentromeric.Porcine [4]. This causative mutation in the Chapel Hill strain of
VWD appears to be among the oldest recognized animal canine type 3 VWD is due to a single nucleotide deletion
modelsofaninheritedbleedingdisorder[14].Themolecular in the canine VWD sequence at base pair 255 that causes a
defect in porcine VWD has been linked to the VWF locus, frameshiftandaprematurestopcodoninexon4[35].These
but the precise molecular mechanism has remained elusive VWDdogsmakenodetectableVWFmRNAorprotein.
[15]. The complete cDNA sequence of canine VWF predicts
The complete cDNA sequence of porcine VWF pre- a protein of 2,813 amino acids and thus is identical in
dicts a protein of 2,807 amino acids, six residues shorter the number of amino acids to human VWF (Figure2) [36,
than human VWF (Figure2) [17–19]. Porcine and human 37]. The full-length human and canine VWF are 87.1%
VWF share 84.3% identity, with additional 7% conser- identical at the nucleotide level and 86.2% at the protein
vative substitutions. The propeptide and mature portions level,withanadditional6%conservativesubstitutions.The
of porcine VWF are equally homologous to the human propeptideandmatureportionsofcanineVWFare87.3and
sequence. Both human and porcine pre-pro-VWF contain 92.8% homologous, respectively, to the human sequence.
234 cysteines conserved in perfect register. Vicinal cysteine Like porcine VWF, canine pre-pro-VWF contains 234 cys-
motifs(CXXC)thoughttobeinvolvedindisulfideisomerase teines conserved in perfect register with the homologous
activity[20]arealsoconservedinD1,D2,andD3domains portion of human VWF. Vicinal cysteine motifs (CXXC)
of each of these species. Using the human numbering are also conserved in D1, D2, and D3 domains of each of
systemforreference,thefollowingresiduesaredeletedfrom these species. Canine VWF does not react with ristocetin;
porcine VWF: G215 (mid-D1 domain), GGLV1241–1244 botrocetin however, is a reliable canine VWF activator and
(D3/A1 domain boundary), and 1494S (A2 domain). The supportstheagglutinationofcanineplateletsbycanineand
deleted residues 1241–1244 are located within a negatively humanVWF[4,21,22].Thereasonforthelackofristocetin
Thrombosis 3
Human 824 LERCPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKY 883 D3
d
Porcine 825 LQRCPCFHQGREYAPGETVKVDCNTCVCRDRKWSCTDHVCDASCSALGLAHYLTFDGLKY 883 o
m
Canine 825 LERCPCFHQGQEYAPGETVKIDCNTCVCRDRKWNCTDHVCDATCSAIGMAHYLTFDGLKY 883 ain
* :******** :*********: .********** * .******** :** : :* :***********
Human 1244 VVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDM 1303
Porcine 1240 -VPPTEGPVSPTTPYEEDTPEPPLHDFFCSKLLDLVFLLDGSDKLSEADFEALKVFVVGM 1299
Canine 1244 VVPPTEGPIGSTTSYVEDTPEPPLHDFHCSRLLDLVFLLDGSSKLSEDEFEVLKVFVVGM 1303
**** : .* : . .** * ** .******* .** :*********** . :*** :* * .** .*** .*
Human 1304 MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKY 1363
Porcine 1300 MEHLHISQKHIRVAVVEYHDGPHAYISLQDRKRPSELRRIASQVKYAGSEVASISEVLKY 1359
Canine 1304 MEHLHISQKRIRVAVVEYHDGSHAYIELKDRKRPSELRRITSQVKYAGSEVASTSEVLKY 1363
A
** :* :**** :********** .**** * :*********** :******** : *** ***** * 1d
o
m
a
Human 1364 TLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLK 1423 in
Porcine 1360 TLFQIFGRVDRPEASRIALLLMASQEPRRLAQNLARYLQGLKKKKVTVIPVGIGPHVSLK 1419
Canine 1364 TLFQIFGKIDRPEASRIALLLMASQEPSRLARNLVRYVQGLKKKKVIVIPVGIGPHASLK 1423
****** . : : ****************** * : : :* : .** :******** ** ******* . .* *
Human 1424 QIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLL 1483
Porcine 1420 QIRLIEKQAPENKAFVVSGVDELEQRKNEIISYLCDLAPEVPAPTRRPLVAQVTVAPELP 1479
Canine 1424 QIHLIEKQAPENKAFVFSGVDELEQRRDEIINYLCDLAPEAPAPTQHPPMAQVTVGSELL 1483
** :****** ******* .* .****** : : :** : .******** .* .** * : ***** . . *
Human 1574 NRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPN 1633 A2
d
Porcine 1569 NRTNTGLALQYLSEHSFSASQGDREEAPNLVYMVTGNPASDEIKRMPGDIQVVPIGVGPD 1628 o
m
Canine 1574 NRTNTGLALQYLSEHSFSVSQGDREQVPNLVYMVTGNPASDEIKRMPGDIQVVPIGVGPH 1633 a
in
********* :*** :*** .****** : .****************** :************* .
Human 1664 LVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANI 1723
Porcine 1659 LVLQRCCSGEGPHLPTQAPVPDCSQPLGVVLLLDGSSSLPASYFDEMKSFTKAFISKANI 1718
Canine 1664 LVLQRCCSGEGLQIPTLSPTPDCSQPLDVVLLLDGSSSIPASYFDEMKSFTKAFISRANI 1723
A
3
*********** : :** :* .******* .* :******** :*********** :*****: *** d
o
m
a
Human 1724 GPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTS 1783 ni
Porcine 1719 GPQLTQVSVLQYGSITTIDLPWNMPLEKAHLRGLVDLMQREGGPSQIGDALGFAVRYVMS 1778
Canine 1724 GPRLTQVSVLQYGSITTIDVPWNVAYEKVHLLSLVDLMQQEGGPSQIGDALSFAVRYVTS 1783
** :**************** :*** : ** .** .*** : ** :*********** .***** : *
Human 2504 GSPRGDSQSS 2513 C
1
Porcine 2499 GSFRGDSVSY 2508 do
m
Canine 2504 GSPRGDAQSH 2513 a
in
** *** : *
Figure 2: Alignments of Human, Porcine, and Canine VWF. (a) Region of D(cid:2)/D3 domain highlighting the F.VIII:C binding region
(underlined).(b)RegionofA1domainshowingtheconserved1272–1458disulfidebonds(C1272andC1458)inredandGP1bbinding
sitesinblackunderlined.TheD1472Hhumanpolymorphismsiteishighlightedingreen.(c)RegionofA2domainshowingtheADAMTS13
cleavagesite(underlined).(d)RegionofA3domainhighlightingCollagenbindingsite.(e)RegioninC1domainindicatingtheRGDbinding
siteofintegrinαIIb/βIII.TheVWFaminoacidsequenceswereanalyzedbyClustalWmultiplesequencealignmentprogram[16]andderived
fromNCBIAccessionNP 000543(Human),AF052036andAY004876(Porcine),andNP 001002932(Canine).
4 Thrombosis
Table1:DifferentialcorrectionofphenotypeofVWDswinebytransplantationofliver,kidney,bonemarrow,andlung.
VWF
Transplant Cell(s)synthesizing
Plasma Platelet F.VIII Bleedingtime[25] Thrombosis References
procedure VWF
None None None None Low Prolonged Notdone [14,25]
Normalliverto Hepatic Nearnormalto Nearnormal Nearnormalto
Notdone Notdone [26]
VWD endothelium normal tonormal normal
Normalkidneyto Presumed
None Notdone Low Prolonged Notdone [26]
VWD endothelium
Normalbone
Megakaryocyte Littletonone Present Low Prolonged No [9,27,28]
marrowtoVWD
VWDbonemarrow Nearnormalto
Endothelium Normal Absent Normal Yes [9,28]
tonormal normal
NormalLungto Pulmonary
∼5% Absent Prolonged Notdone [29]
VWD endothelium
AdaptedwithpermissionfromBrinkhousKM,ReddickRL,ReadMS,NicholsTC,BellingerDA,GriggsTR.vonWillebrandfactor,andanimalmodels:
contributionstogenetherapy,thromboticthrombocytopenicpurpura,andcoronaryarterythrombosis.MayoClinProc.1991;66:733–742[30].
reactivity may in part be explained by the D1472P amino to clear human and species-specific VWF in a comparable
acid substitution in canine VWF similar to that in porcine fashion.TheresultsofpreclinicaltestingofrhVWFhavebeen
VWF as described above (Figure2) [24]. The dog platelet predictive of the half-life found in early testing in human
Gp1b sequence has an M239T substitution that does not subjects[46].
appeartohavea“gainoffunction”substitutionasreported
fortheM239VmutationinhumanplateletGP1b[24].Itis
4.2. VWF Secretion with DDAVP and VWF mRNA Upreg-
alsopossiblethatbotrocetinmorespecificallyactivatesVWF
ulation by rhIL-11. Recombinant human IL-11 (rhIL-11,
than does ristocetin [38]. The RGD sequence is conserved
Neumega), a glycoprotein 130- (gp130-) signaling cytokine
and most likely mediates platelet integrin αIIbβ3 binding
that is approved for treatment of thrombocytopenia, has
in humans, pigs, and dogs. Recombinant canine VWF
been shown to induce elevations in VWF and F.VIII in
established VWF-dependent Weibel-Palade body formation
humansandmice[47].Intheseinitialstudies,itwasunclear
inculturedprimaryVWDendothelialcells[35].
if the mechanism for VWF elevation was mediated by
increasedsecretionorincreasedproductionbyupregulation
4.ResearchUtilizingVWDPigsandDogs of VWF mRNA. If the latter mechanisms were shown to
be correct, then rhIL-11 could be used as an additional or
4.1. Replacement Products. Pigs and dogs with VWD have alternativetherapyto1-desamino-8-D-argininevasopressin
beenusedtodeterminesafety,efficacy,pharmacodynamics, (DDAVP), a standard therapy for increasing VWF levels in
and pharmacokinetics in the preclinical testing of plasma- humansthroughtheWeibel-Paladebodysecretorypathway
derived and recombinant human VWF (rhVWF) [10, 39– [34, 48, 49]. To compare DDAVP and rhIL-11, dog models
42]. Importantly, rhVWF reduces the saline bleeding time were informative since canine but not murine species
[25]from>15minutesto∼5minutes[10].Thecorrectionof respond to DDAVP. rhIL-11 produces a gradual, sustained
thesalinebleedingtimemayportendsupportofhemostasis elevationofVWFandF.VIIIlevelsinbothnormalandhet-
whenrhVWFisgiventoVWF-deficienthumans. erozygous VWD (VWF +/−) dogs, while DDAVP produces
Infusion of plasma-derived and recombinant human a rapid, nonsustained increase. rhIL-11 treatment produces
VWF into VWD pigs and rhVWF into VWD dogs is a2.5to11-foldincreaseinVWFmRNAinnormaldogsbut
accompaniedbyadelayedriseinfactorVIIIactivity(F.VIII). notinhomozygous(VWF−/−)VWDdogs,thusidentifying
Conversely, when human F.VIII is infused into VWD dogs, amechanismforelevationofplasmaVWFinvivo.Moreover,
thehalf-lifeoftheinfusedF.VIIIismarkedlyreducedwhen dogs pretreated with rhIL-11 retain a DDAVP-releasable
compared to infusing the same amount of human F.VIII pool of VWF and F.VIII, suggesting that rhIL-11 does not
intohemophiliaAdogswithnormalVWFlevels[43].Taken significantly alter trafficking of these proteins to or from
together,thesedataareconsistentwithhumanVWFbinding storage pools. The half-life of infused VWF is unchanged
toandstabilizationofF.VIIIinbothspeciesinvivo. by rhIL-11. These results strongly suggest that rhIL-11 and
InVWDdogs,thehalf-lifeofrhVWFisbetween10.2and DDAVP raise plasma VWF levels by different mechanisms,
13.0 hours [10] and the half-life of plasma-derived canine and that rhIL-11, like DDAVP, could be an alternative to
VWF is between 12 and 18 hours [44]. In VWD pigs, the plasma-derived products for some VWD or hemophilia
half-lifeofrhVWFis∼10to16hours[11,39],andthehalf A patients who are unresponsive to DDAVP or in whom
life of plasma derived porcine VWF is between 10 and 18 DDAVP is contraindicated [50]. Likewise, rhIL-11 therapy
hours[11,45].Thus,bothpigsanddogswithVWDappear could also reduce use of plasma products in some patients.
Thrombosis 5
It is important to bear in mind that DDAVP causes an in the Folts stenosis and injury model [8, 64, 68]. In addi-
immediatereleaseofVWF,andtachyphylaxislimitsrepeated tion, neutralizing VWF activity interrupts VWF-dependent
dosing in the short term. In contrast, rhIL-11 induces a arterial thrombosis in nonatherosclerotic pigs with normal
sustainedincreaseofVWFthat,onceestablishedoverafew VWF expression [69, 70]. Several molecules that inhibit
days,appearstopersistuntiladministrationisdiscontinued. VWF activity have been developed as potential therapeutic
Thus, the two drugs may well be complementary in many agents, and this work has recently been reviewed [71]. One
patientsifshowntobesafeinclinicaltrials. of the most promising novel approaches is the aptamer
Recently, rhIL-11 was administered subcutaneously to ARC1779 [72–74]. Aptamers are synthetic nucleotides that
nine subjects with mild or type 1 VWD [51] (5F, 4M, age bindwithhighaffinitytoatargetproteinandneutralizeits
21–49yr) in an FDA-approved phase 2 open label, dose function. The current status of the VWF-binding aptamer
escalation study [7]. The drug was well tolerated and these ARC1779 suggests that it will be a safe and effective new
data confirm that rhIL-11 increases VWF in humans with therapeuticthataddressesunmetneedsfortreatingpatients
mild or type 1 VWD by means other than the DDAVP witharterialthromboticdiseases,particularlythosethatare
releasable pool of VWF, and possibly by increasing VWF VWFmediated[75].
mRNA.Toourknowledge,ifthismechanismwasconfirmed VWF serves as a carrier for plasma F.VIII in humans,
inadditionalstudies,rhIL-11wouldbethefirstmedication pigs,anddogsandtherebyprotectsF.VIIIfromdegradation
for treating an inherited bleeding disorder that actually by activated protein C [76]. This carrier function may
targets the relevant gene in situ. These encouraging results also deliver F.VIII to sites of arterial injury and localize
haveprovidedthefoundationforobtainingFDApermission F.VIII activity to sites of VWF and platelet attachment on
to perform two phase 2 efficacy studies in mild or type 1 exposed subendothelium and promote thrombosis [77]. In
VWD patients, the first in patients with menorrhagia and addition, hemophilia A dogs (i.e., no detectable F.VIII but
the second in patients undergoing elective surgery. These normal VWF) form occlusive arterial thrombosis as readily
ongoing studies will determine if the results with rhIL-11 as normal dogs using the same experimental model [8]. In
therapy found in the VWD dogs and the phase 2 dose pigs, neutralizing VWF activity while leaving F.VIII activity
escalationstudycanbetranslatedintoasafe,newtreatment intact prevents the development of occlusive thrombosis
forVWF-deficienthumans. [69]. Human subjects with hemophilia A that lack F.VIII
have serious impairment of thrombin generation but are
not protected from myocardial infarction or thrombosis
4.3. Atherosclerosis. The role of VWF in atherosclerosis has
complicatingaorticatherosclerosis[78–80].Moreover,VWF
been a subject of debate. A mechanism linking VWF to
levelinhumansalsocorrelatesdirectlywiththrombosisrisk
atherosclerosis is based on the fact that VWF mediates
andinverselywithbleedingrisk[1].Theseresultssupportthe
platelet adhesion to injured arterial walls, thus delivering
hypothesisthatVWFhasanintrinsicpropertythatsupports
platelet contents, including platelet-derived growth factor
arterialthrombosisindependentofitsassociationwithF.VIII
(PDGF),tothearterialwall.Thehypothesisisthatrelatively
inhumans,pigs,anddogs.
highconcentrationsofPDGFpromoteatherogenesisatfocal
points of vascular injury [52–54]. The corollary is that the
absenceofVWFreducesatherogenesis.
4.5.VWDandBleedingTimeProlongation. Inhumans,pigs,
Since dogs tend not to get atherosclerosis, pigs are a
and dogs, VWF is synthesized in endothelial cells that
more appropriate animal model in which to test these
store and secrete the protein into the subendothelium and
hypotheses[55].Todate,variationsinthedegreeofcoronary
plasma(Table1)[81,82].VWFisalsosynthesizedinhuman
and abdominal aortic atherosclerosis have been reported
and porcine megakaryocytes and is present in human and
for normal and VWD pigs [56–64]. However, these results
porcine platelet alpha granules [27, 83–85]. VWF does not
seemtobeimpactedbycholesterollevels.Inaretrospective
appear to be synthesized in canine megakaryocytes nor is
study,polymorphismsintheapolipoproteinB100genotype
it present in platelet alpha granules [8, 35]. Studies in the
were found to significantly influence the severity of high-
threespecieshaveshownthatbleedingtimeprolongationis
fat diet-induced atherosclerotic plaque formation in VWD
variablyaffectedbydecreasedplateletVWFandplasmaVWF
and normal swine without regards to the VWD genotype
[9, 27, 86, 87]. In porcine VWD and human type 3 VWD,
[65]. Humans with various types of VWD including type
replacementofbothplasmaandplateletVWFbytransfusion
3 are not protected from developing coronary and aortic
is required to normalize the bleeding time, whereas plasma
atherosclerosis but may develop fewer occlusive thrombi
VWF alone is insufficient [9, 27, 88–90]. Dogs that have
with ensuing organ infarction [66, 67]. Thus, VWF may
no platelet VWF but normal plasma and endothelial VWF
mediate thrombotic complications of atherosclerosis rather
have normal bleeding times [8]. In contrast to humans
thanatherogenesisperseasdiscussednext.
and pigs with VWD, infusion of canine VWF into VWD
dogs nearly normalizes bleeding time [8]. Thus, bleeding
4.4. VWD and Occlusive Arterial Thrombosis. The potential time, as a measure of hemostasis, appears to have species-
roleofVWFinthedevelopmentofarterialthrombosisnoted specificity related to VWF distribution in plasma, platelets,
in VWD humans with atherosclerosis has been studied in andendothelium.
pigs and dogs. VWD pigs with or without atherosclerosis OrgantransplantationcantransfertheentireVWFgene
andVWDdogsdonotdevelopocclusivearterialthrombosis in situ and provides a model to study VWF function
6 Thrombosis
in various compartments (Table1). For example, bone xenograft dysfunction and is a potential drug target for
marrowtransplantationinporcineVWDallowsforseparate improvingxenograftsurvival.
expressionofVWFintheplateletorplasma,endothelial,and
subendothelial compartments [9, 27, 28]. In these studies,
4.8. Thrombotic Thrombocytopenia Purpura (TTP). Dis-
plasma VWF alone was sufficient to support bleeding time
covery of the roles of VWF and its cleavage enzyme
and the development of occlusive arterial thrombosis in
ADAMTS13 in TTP has revolutionized the understanding
the Folts arterial stenosis and injury model [9]. Likewise,
of the underlying mechanisms that mediate this previously
transplantation of normal liver and lungs into VWD pigs
poorly understood disorder [101]. In studies performed
hasprovidedcirculatingVWFwithoutplateletVWF[29,91].
prior to this discovery, normal pigs and dogs injected with
Thetransplantednormalliverprovidessufficientamountsof
botrocetin developed thrombocytopenia, microthrombi in
plasmaVWFtocorrectthebleedingtimefrom>15minutes
lungs and spleen but not kidney or brain, and initial
to≤5minutes[26].Thetransplantednormallung,however,
depletion of VWF multimers followed by the appearance
provides∼5%ofnormalVWFbutisinsufficienttocorrect
of ultra large VWF multimers during recovery. VWD pigs
the prolonged bleeding time. Thrombosis studies have not anddogswerenotaffectedbybotrocetininfusionsuggesting
beenperformedintheliverandlungtransplantationexper-
that plasma VWF mediates this botrocetin-induced throm-
iments.Thus,bleedingtimeprolongationinpigsappearsto
botic thrombocytopenia in the absence of platelet VWF
occurwhenVWFismarkedlydecreasedorabsentfromthe
[102]. Since ADAMTS13 had not been discovered when
plasma and/or subendothelial compartments. The precise
these studies were performed, it was not assayed in these
roleofplateletVWFinthisscenarioisunknown.
animals.Recently,infusionofshigatoxinhasbeenshownto
support the development of a thrombotic microangiopathy
4.6. Gene Transfer. An inherited bleeding disorder such as inADAMTS13-deficientmice,buttheeffectoftheinfusing
VWD is an attractive target for gene transfer using viral botrocetinonADAMTS13orintheabsenceofADAMTS13,
vectors for several reasons. First, there is a single gene ifany,isunknown[103–106].TheADAMTS13cleavagesite
defect. Second, the protein can be expressed from many is preserved in both porcine and canine VWF (Figure2). It
target organs that will secrete the transgene product into ispossible,then,thatexpressionofporcineorcanineVWF
plasma. Third, successful gene transfer would reduce the in VWD pigs or dogs with mutations in the ADAMTS13
requirement for blood products and the associated invasive cleavage site, respectively, could result in a TTP syndrome.
procedures required for administration which could very Application of such information to the human condition
realistically be expected to improve patient comfort and wouldneedtoconsiderthepossibilitythatcharacterization
well-being. Fourth, expression of VWF at relatively low of ADAMTS13 is incomplete in many species and that
levels may provide some degree of phenotypic correction important functional differences may exist between them
frombleeding;overexpression,however,potentiallycouldbe [107].
associated with thrombotic side effects [1]. Contemporary Importantly, a novel approach to the treatment of
challengesforgenetherapyarewellrecognizedandVWFis TTP in humans has been to neutralize VWF activity with
no exception [92–94]. Nonetheless, human VWF has been the aptamer ARC1779 [108, 109]. Administration of this
expressedinbloodoutgrowthendothelialcellsisolatedfrom aptamer was well tolerated and appeared to be associated
VWD dogs [95], and progress has been made with murine with a significant increase in platelet counts in a case of
models [96, 97]. The availability of porcine and canine refractory TTP. Currently, ARC1779 is being tested as an
VWF cDNA and newer gene transfer vectors provides an “add-on”therapytoplasmaexchangeinpatientswithTTP.
opportunitytotransferthisworktolargeanimalmodelsthat
havearecognizedstrongpreclinicalpredictivevalue[98]. 4.9. Endocarditis Susceptibility. VWD swine appear to be
resistant to experimentally induced group C streptococ-
cal endocarditis, whereas normal pigs readily develop the
4.7. Lung Xenotransplantation and VWD. Separate from
infection [110]. These data suggest that normal VWF-
the role of VWF in hemostasis and thrombosis, lung
mediated platelet function is necessary for establishing this
transplantation studies have focused on the binding of
particular infection. Understanding of platelet function in
porcine VWF and primate xenoreactive antibodies. Swine
inflammationandinfectionisevolvingrapidly,andpigsand
pulmonary xenografts, as opposed to cardiac and renal
dogswithVWDlikelywillhavearoleinthesestudies[111–
xenografts, release large amounts of porcine VWF when
113].
transplanted into nonhuman primates. Human and non-
human primates have xenoreactive antibodies that bind
to carbohydrate side chains on porcine VWF [99]. Also, 5.SummaryandConclusions
porcine VWF spontaneously agglutinates human platelets
[2]. The absence of porcine VWF in VWD donor lungs PigsanddogswithVWDhaveprovidedfaithfulphenotypic
results in longer survival of the transplant in nonhuman models for the study of basic aspects of human type
primates possibly due to mechanisms mediated by reduced 3VWD,aswellaspowerfultoolsforrevealingpathogenesis,
orabsentVWF-mediatedxenoantibodybindingandplatelet developing new therapies, and testing their safety and
agglutination [100]. These findings strongly support the efficacy. This conclusion is remarkable, considering the
hypothesis that porcine VWF is a mediator of pulmonary important differences in porcine and canine VWF amino
Thrombosis 7
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