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 acid sequence and reactivity to agonists such as ristocetin [7] M.V.Ragni,R.C.Jankowitz,H.L.Chapmanetal.,“Aphase and botrocetin. 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