Hemofilija Kod Pasa

7/30/2019 Hemofilija Kod Pasa

1/9

1993 81: 2644-2651

Griggs and MS ReadTC Nichols, DA Bellinger, RL Reddick, SV Smith, GG Koch, K Davis, J Sigman, KM Brinkhous, TR

Athrombosis: studies in canine von Willebrand disease and hemophiliaThe roles of von Willebrand factor and factor VIII in arterial

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests

Information about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/subscriptions/index.dtlInformation about subscriptions and ASH membership may be found online at:

.reservedHematology; all rightsCopyright 2010 by The American Society of

900, Washington DC 20036.weekly by the American Society of Hematology, 2021 L St, NW, SuiteBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published

For personal use only.by on December 14, 2010.www.bloodjournal.orgFrom
http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requestshttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requestshttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requestshttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintshttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintshttp://bloodjournal.hematologylibrary.org/subscriptions/index.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/index.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/index.dtlhttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintshttp://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests


2/9

T h e R o l e s of von W il lebrand Factor and Factor VI11 in Arteria l Th rom bosis:S tud ies in Canine von Wi l l ebrand D isea se and Hem ophi l ia ABy Timothy C.Nichols, Dwight A . Bellinger, Robert L. Reddick, Scott V. Smith, Gary G. Koch, Keir Davis, Jeff Sigman,

Kenneth M. Brinkhous, Thomas R. Griggs, and Marjorie S . ReadWe have studied the roles of von Willebrand factor (vWF)and factor Vlll in arterial thrombosis in four canine phe-notypes: normal (n =6). hemophilia A (n =1 ), von Wil-lebrand disease (vWD) (n = S), and hemophilia A/vWD (n=1).vWF activity was determined by botrocetin-inducedagglutination of fixed human platelets and vWF antigen(vWF:Ag) by Laurel1 electroimmunoassay and crossed im-munoelectrophoresis. Plasma from normal dogs and thosewith hemophiliaA had vWF activity, vWF:Ag, and a fullrange of vWF:Ag multimers on gel electrophoresis equiv-alent to normal canine plasmapool. Platelet cytosol con-tents were isolated by freezing and thawing, triton X-100solubilization, or sonication of washed platelets with andwithout protease inhibitors and inhibitors of platelet acti-vation. Washed platelets were also stimulatedwithcalciumionophore and MgCI2. There was no measurable vWF ac-tivity or vWF:Ag inplatelet lysates or releasates nany dogregardless of phenotype. All dogs were studied using a

ON WILLEBRAND FACTOR (vWF) s a large adhesiveV glycoprotein present in endothelial cells and suben-dothelium in humans,'-$ pigs,6 and vWF isalso presentin human plateletsg'" and porcine platelets.12,13 here areconflicting reports about whether or not canine platelets con-tain VWF.',~,'~,'~decrease in the degree of expression ofplasma or platelet vWF in humans can produce a bleedingdiathesis.'6-20 n addition, high molecular weight vWF mul-timers present in human endothelial cells and platelets maybe responsible for part of the biologic activity of vWF exVariations in both the compartmentalization andmultimeric distribution of vWF within these compartments,then, can alter the phenotype of a given species and may beimportant for the function(s) of the vWF protein.vWF has several complex roles in hemostasis and throm-bosis. A t sites of exposed subendothelium, vWF supports theinitial adhesion of platelets functioning as a ligand betweenplatelet membrane glycoproteins and the subendothelium.This adhesive property of vWF is particularly evident at highFrom the Departments of Medicine and Pathology and the Centerfor Thrombo sis and Hemostasis, School ofhfedic ine, the Departmentof Biostatistics, School of Public Health, and the Division of Labo-

ratory Animal Medicine, University of North Carolina at Chapel Hill,NC.Submitted August 10, 1992; accepted December 3 0, 1992.Supported in part by National Instilutes of Health Grants No.HL01648 and HL26309 and Navy Medical Research and Develop-ment C omm and, Naval Research 14-89-J-1712.Address reprint requesls to T imo thy C. Nichols, M D, Departmenlof Medicine. University of North C arolina School of Med icine, C B#7075, 349 Burnett- Wom ack Bldg, Chapel H ill, NC 27514.Th e publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. section 1734 solely toindicate lhis fact.0 I993 by The American Society of Hematology.0006-4971/93/8110-0017$3.00/0

standard arterial stenosis and injury procedure to inducearterial thrombosis. Thromboses were detected by cyclicreductions in Doppler blood flow velocity. Vessels were ex-amined by light and scanning electron microscopy. Throm-bosis developedinthe arteries of normal (9of 10) and he-mophiliaA dogs (16 of 16) but in none of the vWD dogs(0of 10). Infusion of canine vWF cryoprecipitate into vWDdogs markedly shortened bleeding time but did not supportthrombosis as seen in dogs with vWF inthe plasma andsubendothelium. Thrombosis, then, fails to occur when vWFis absent from the plasma and subendothelial compart-ments or present only in the plasma compartment. Thesedata are consistent with the hypothesis that vWF in theplasma and subendothelium supports thrombosis. Neitherplasma FVlll nor platelet vWF is essential for thrombosisin this model.0 1993by The American Society of Hematology.

shear rates.25Once platelets attach to the subendothelium,vWF supports platelet spreading in If the vessel isinjured at a stenotic site, vWF is required for the developmentof occlusive throm bo~ is. ~~" 'he relative contribution of vWFin plasma, platelets, endothelium, and subendothelial matrixin platelet attachment and spreading and arterial thrombosisis only partly understood and may differ between species.vWF serves as a carrier for factor VI11 (FVIII) in plasmain humans, pigs, and dogs. This carrier function may providefor the delivery of FVIII to sites of arterial injury. Such aseries of events could both localize FVIII activity where vWFattaches to exposed subendothelium and promote thrombosis.However, a monoclonal antibody raised to purified porcinevWF, BB3-BD5, both prevents and aborts thrombosis byneutralizing vWF when infused into normal pigs but doesnot affect FVIIl activity.29These data suggest that vWF sup-ports thrombosis independent of its function as a carrier forFVIII. Studies have not been done in vivo in the absence ofFVIII to confirm this finding.The purpose of this study was to examine the relative rolesof plasma and platelet vWF and FVIII in induction of arterialthrombosis in dogs. These studies were performed on theChapel Hill strains of dogs with normal, hemophilia A , vonWillebrand disease phenotypes, and a newly developed strainof bleeder dogs with combined defects, hemophilia A/vWD.~*No platelet vWF was detected in these dogs regardless ofphenotype. Thrombosis developed in 90% of stenosed andinjured arteries in normal dogs but in none of the vWD dogseven when they were infused with vWF concentrates. Re-placementof plasma vWF by infusion, then, does not supportthrombosis in vWD dogs as seen in dogs that have vWFwithinthe plasma and subendothelium. Thus, thrombosis fails tooccur when vWF is absent from the plasma and subendo-thelial matrix or present only in the plasma compartment.Moreover, thrombosis occurred in hemophilia A dogs asreadily as in normal dogs. vWF, then, supports thrombosisindependent of its association with FVIII and when it is absent

2644 Blood, Vol 81, No 10(May 15). 1993: pp 2644-2651

http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/


3/9

THROMBOSIS: CANINE vWF AND FVlll 2645from platelets. These data are consistent with the hypothesisthat vW F in the plasma and subendothelium supportsthrombosis. These data have been reported in part.33

MATERIALS AND METHODSNormal (n=2), hemophiliaA (n= I l),vWD(n=6), and hemophilia A/vWD (n= 1) dogs came from theclosed colony at the Francis Owen Blood Research Laboratory at theUniversity of North Carolina, Chapel Hill. Four additional normaldogs were purchased from the Division of Laboratory Animal Med-icine, University of North Carolina. All animals were treated accordingto the standards set in Guide for the Ca re and Use of LaboratoryAnimals (National Institutes of Health publication no. 85-23). Noanimal had received blood products within 2 weeks of these studies.Dogs with hemophilia A had lessthan 1%FVIII activity but normalvWF antigen and activity levels and normal saline bleeding times.34vWD dogs had less than 1% vWF activity and vWFAg, depressedlevels of FVIII activity, and bleeding times longer than 15 minutes.The dog with combined hemophilia A/vWD had no detectable FVIIIactivity, vWF activity nor vWFAg, and bleeding time of greater than

15 minutes. Dogs were classified as phenotypically normal if theyhad ( ) FVIII activity levels and plasma vW Factivity and vW Fantigen(vWAg) evels 260% ofthat found in normal canine pooled plasma,and (2) normal bleeding times.FVIII assays. FVIII coagulant activity was performed by a mod-ified one-stage method using a kaolin-activated MIIdeficient canineplasma substrate.35 Platelet-poor plasma (PPP) wasprepared by centrifugation of blood anticoagulated with 3.2% sodiumcitrate, pH 6.8 (1/9: vol/vol, 2,OOOg, 5 minutes). PPP was either testedimmediatelyor frozen at -70C until assayed. Platelet-rich plasma(PRP) was prepared by centrifugation of blood anticoagulated withacid-citrate-dextrose ACD: 100 mmol/L sodium citrate, 0.07 mol/L citric acid, 0.1 1 mol/L dextrose, pH 4.5, 7J42.5: vol/vol, 500g8 minutes). The platelets were washed three times by resuspensionin citrated saline (0.9% NaC1, 5 mmol/L sodium citrate, pH 6.5) andrepeat centrifugation. n some experiments, PRP was prepared fromblood anticoagulatedwith EDTA (20 mmol/L) containing leupeptin(10 pmol/L) and prostaglandin E-1 (PGE-I) (50 ng/mL). In theseexperiments, the platelets were washed in citrated saline containingEDTA, leupeptin, and PGE-I at the same concentrations as above.Platelet lysates were prepared by three different methods. First,washed platelets were frozen and thawed three times. Second washedplatelets were solubilized by the addition of 20% triton X-100.'5.36Third, washed platelets were disrupted by sonication (Branson SonifierCell Disrupter 200, Danbury, CT).With all three techniques, theplatelet debris was separated from the lysate by centrifugation (2,000g.10 minutes, 23C). Some lysates were prepared in the presence ofprotease inhibitors and inhibitors of platelet activation (ACD withaprotinin400KIU/mL and 300 mmol/L PGE- I or EDTA, leupeptin,and PGE-1 at the concentrations stated above). Normal pig and hu-man platelets were used as positive controls for these methods.In some experiments, washed canine platelets were stimulated toundergo release by the addition of calcium ionophore (A23187, 1rmol/L and 100 nmol/L) and MgCl, (0.02 mol/L). The stimulatedplatelets were removed by centrifugation (2,00Og, 10minutes, 23C)and the supernatant was assayed for vWF activity and antigen.Aliquots of the washed platelets were ked in glutaraldehyde beforeand just after producing platelet lysatesor releasates for examinationby transmission electron microscopy (TEM) to confirm platelet dis-ruption or degran~lation.~~Plasma, platelet lysates, and platelet releasates were assayed forvWF activity using the macroscopic tap tube procedure with botro-cetin and paraformaldehyde-fixed, lyophilized human platelets.374

Experimental animals.

Plasma and platelet v WF assays.

Fixed human platelets are the standard platelet reagent for vWF assayin our laboratory. We have obtained comparable results with fixedhuman or fixed canine platelets in this assay." Appropriate dilutionswere done to exclude an inhibitor of vWF activity. vWFAg wasassayed by Laurel1 electroimmunoassay?' The vWFAg multimeranalysis was performed by sodium dodecyl sulfate (SDS) gel electro-phoresis as described." The dry gels were scannedby laser densitom-etry for the distribution of vW F multimers with the LKB UltroscanXL (Pharmacia LKB, Piscataway, NJ). Crossed immunoelectropho-resis (CIE) of plasma and platelet lysates was performed on 1%agarosegels with and without denaturing agent^.'^^^^Rabbit anti-caninev WF antibody. A rabbit anti-canine polyclonalantibody was raisedagainst canine vWF by the method of Lamb etal." Canine cryoprecipitate from the plasma of hemophilia A dogswas chromatographedon 4% agarose (Biogel A- 15m, 200-400 mesh;Bio-Rad Laboratories, Richmond, CA). The void volume fractionswere pooled and used as antigen for immunizations. The antiserumwas made monospecific for vWF by adsorption of 1 vol of antiserumwith cryoprecipitate from 2 vol of plasma from a dog homozygousfor vWD. This monospecific antibody did not inhibit canine FVIIIactivity (data not shown) and was used to detect vWFAg in all assaysand to stain gels. Canine fibronectin wasmeasured in platelet lysates, platelet releasates, and PPP by enzyme-linked immunosorbent assay (ELISA) with a polyclonal rabbit anti-canine antibody.43Fibronectin in the platelet lysates and releasateswas used as a control for recovery of platelet a-granule contents.Bleeding time . Saline ear bleeding times were performed on all

Arterial stenosis and injury procedure: Animal preparation, his-tologic studies, and vessel morphometry. The stenosis and injuryprocedure was performed under general anesthesia and with physi-ologic monitoring as This model was modified fromthat reported by Folts and U ~ h i d a . 4 ~ ~ ~ ~riefly, general anesthesiawa smaintained with halothane (1% o 3%) from a vaporizer (FluomaticVaporizer, Foregger, Smithtown,NY). he vaporizer was periodicallychecked for calibration by a Type 18 Riken Gas Analyzer (A.M.Bickford, Wale City, NY).Stenosisand injury were then applied to one or both ofthe carotidarteries and/or a coronary artery (left circumflex or left anterior de-scending) with a Goldblatt clamp. Thrombosis was detected by cyclicflow reductions in arterial blood flow velocity (Doppler, 20 MHz)following stenosis and injury. After each dog was killed with an over-dose of pentobarbital, the heart was pressure perfused and placed infixatives with the carotid arteries. The degree of arterial stenosis andinjury was determined by computer-assisted planimetry of light mi-croscope arterial cross sections (Ziess Videoplan). Coronary and ca-rotid arterial injury was assessed by the amount of smooth musclecell damage and disruption of the internal elastic Thepresence or absence of thrombosis was also determined from ex-amination of arterial sections with light microscopy.Cryoprecipitate containingvWFbut lackingFVIII (FVIIIdeficient cryoprecipitate) was prepared fromPPP of hemophilia A dogs?7 The cryoprecipitate was stored at -70Cand dissolved in a minimal volume of 0.9% NaCl just before infusion.Fresh normal canine PPP was prepared for infusion into one vWDdog. v W F activity, antigen concentration,and multimeric distributionwere determined for each infusate. One unit of vWF activity is definedas that equal to the amount present in 1 mL of normal canine pooledplasma.The products were infused over approximately 60 minutes afterthe surgical preparation had been completed and all baseline sampleshad been obtained but before the stenosis and injury procedure. Fol-lowing vWF product infusion, the stenosis and injury procedure was

Canine plasma and plateletfibronectin.

Isolation and infusion of v WF.



4/9

2646 NICHOLS ET ALTable 1. Effect of Canine Phenotype, FVIII, and P latelet and P lasma vWF Ac tivity and Antigen on the P roduction ofArterial Thrombosis

Thrombosislatelet. PlasmaB ed ng PlateletPhenotype Time FVlll Count vWF Act iv ity vWF:Ag vWF Act iv ity vWF:Ag per No . of

(n) lnfusate (min) (%) (mm3x I@) (%) (%I (%I (%) AneriestNormal(6) None 2.3 94 297.5 41 < 1 99 85 1011 tvWD (6) None >15 15.5 265 < 1 < 1 < 1 < l 0/10t

(1.5.2.6) (66, 22) (135, 65) (61, 58) (68, 24)(7, 0) (225, 75)

(1 1) (1.3.4.4) (230, 60) (87, 60) (76, 52)cryoprecipitate (295, 00) (44, 62) (36, 4)

Hemophilia A None 2.7 < 1 270 < l < 1 135 110 16/16tvWD (2) FV III-deficient canine 4.1 17 298 < 1 < 1 103 41.5 014vWD (1 ) Normal canine plasma 12 22 305 < 1 < 1 54 67 012Hemophilia FV111-deficient canine >15


5/9

THROMBOSIS: CANINE vW F AND FVlll 2647

or leupeptin) a nd a platelet m embrane stabilizer (PGE- 1).None of these procedures yielded detectable canine plateletvW F antigen or activity (Table l ), nor were any vW F mul-timers detected (Fig I ) . Dilution studies failed to detect a ninhibitor of vW F activity in canine platelets. In addition, noprecipitin arc was seen on CIE of the platelet lysates of anycanine phenotype (data not show n). This finding was in con-trast to the precipitin arc detected on CIE of plasmas fromnormal dogs and hemophilia A dogs that was comparable tothat found in norm al canine pooled plasma (data not show n).Platelet lysates prepared by these methods from normal pigand huma n platelets contained normal am ounts of vW F ac-tivity and antigen and a normal distribution of multimers( da ta n ot ~h o w n ) .~ ' .~ 'Com plete disruption of platelets by each method (ie, freez-ing and thawing, Triton X-100, and sonication) was con-firmed by transmission electron micrograph (TEM ). Plateletdegranulation following stimulation with MgC12 and twoconcentrations of A23 187 was confirmed by TEM taken be-fore and after stimulation (data not shown). Finally, therewas no detectable vW F functionally or antigenically in anyof the platelet washes before stimulation or disruption.The platelet lysates and releasates from all proceduresyielded detectable fibronectin with an average of 5.2 k 1.3pg/mL X 10" platelets. These data suggest that platelet a-granule co ntents w ere successfully released and recovered bythese physical and chemical methods. Th e incidence ofthrombosis in the four canine phenotypes is listed on T able1.Ten of 1 1 arteries (6 coronary, 5 ca rotid) tested in 6 norm aldogs developed cyclic flow reductions (CFR) indicatingthrombosis was induced by stenosis and injury. In addition,CFR occurred in all 16 arteries (5 coronary, 11 carotid) testedin 1 1 hemophilia A dogs (Fig 2). In contrast, no CFRs weredetected in any of 10 arteries (2 coronary, 8 carotid) in sixvWD dogs (Fig 2). We have previously found that the inci-dence of thrombosis in these two arteries relative to the pres-ence or absence of vWF is eq~ iva len t,~ 'nd all dogs in thispresent study with assessment for both carotid and coronaryarteries had the same outcome. This finding has allowed thegrouping of the two circulations for statistical analysis. Thedifference between the incid ence of thrombosis betw een phe-notypes in the present study is significant ( P


6/9

2648 NICHOLS ET AL

Fig 2. Photomicrographs ofstenosed and injured carotidarteries from dogs with hemo-philia A and vWD. (A)Throm-bosis ina carotid artery of a he-mophilia A dog that developedat the site of stenosis and pinchinjury. There are regions withinthe thrombus where plateletsare predominant and otherswhere erythrocytes are pre-dominant. (B) Carotid arteryfrom adogwithvWD taken fromthe stenosis and pinch-injurysite. There is no detectablethrombus. Hematoxylin-eosin.Bar= 1mm.

Subjects with hemophilia A lack FVIII and have seriousimpairment of thrombin generation. Because human arterialthrombi are rich in fibrin in addition to platelets, arterialthrombosis occumng in the presence of vWF without FVIIImay seem paradoxical. However, it has been observed thathuman patients with severe hemophilia A are not protectedfrom myocardial infarction or thrombosis complicating aorticatheroscler~sis.~~n both humans and dogs, then, vWF sup-ports arterial thrombosis independent of its association withFVIII.

The mechanism by which vWF supports arterial throm-bosis in dogs may involve vWF in two compartments: plasmavWF and exposed subendothelial matrix vWF. This hypoth-

esis is supported by the finding that infused plasma vWF failsto support thrombosis in the vWD dogs with vWF-deficientsubendothelial matrix (Table 1) . Infusion of FVIII-deficientcryoprecipitate into vWD dogs provided plasma vWF witha full range of multimers that was readily detectable func-tionally and antigenically but would not have replaced sub-endothelial vWF. In dogs, then, vWF in the subendothelialmatrix may be needed to support thrombosis in addition toplasma vWF. Studies in vWD pigs have also shown thatplasma vWF alone is insufficient to support arterial throm-bosis, whereas the combination of plasma and platelet vWFdoes support thrombosis.**.29. n vWD pigs, then, plateletvWF may interact with plasma vWF in the formation of



7/9

THROMBOSIS: CANINE vWF AND FVlll 2649

thrombosis at sites of arterial stenosis and injury in the ab-sence of subendothelial matrix vWF. Plasma vWF is thecommon denominator in a two-compartment model of vWF-dependent arterial thrombosis in these two vWF-deficientspecies: plasma and subendothelial matrix vW F in vWD dogsand plasma and platelet vW F in vWD pigs. C onsidered to-gether, these data support the hypothesis that plasma vWFmay activate, be activated by, or function as a cofactor tovWF in another compartment to support arterial thrombosis.Bleeding time shortening following vWF infusion appearsto differ among vW F-deficient species. Infusion of vWF co n-centrates largely corrected bleeding times in vWD dogs inthis study but has failed to do so in vWD pig^.*^,^' This dif-ference in bleeding time response to vW F infusion betweenvWD pigs and dogs was observed even with plasma vWFactivity levels g reater than 100% n both species. In hu ma nswith vW D, a divergence also has been noted between bleedingtime and vW F activity following vW F infusion.54 t appearsin humans with type I11 (severe homozygous) vWD that re-placement of plasma and platelet vW F are required to nor-malize the bleeding time, w hereas plasma v W F alone is in-~ u f f i c i e n t . ~ " ~ ~ur c urrent study in dogs considered with thoseperformed in pigs and hu ma ns suggests there may be speciesdifferences in the bleeding time shortening in vWF -deficientsubjects.Several possibilities could e xplain these species differencesin bleeding time shortening. Clearly, the variability in vWFcompartmentalization among dogs, pigs, and hu mans couldaffect this test. It also is possible that plasma vWF in dogscorrects prolonged bleeding time by a mechanism tha t differsfrom that by which it supports thrombosis. This hypothesisis supported by the observation that infusion of vWF sup portsvWF-dependent bleeding time correction without supportingvWF-dependent arterial thrombosis. A lternatively, cryopre-cipitation may concentrate other plasma constituents thatcould shorten the bleeding time in dogs independent of vWF.In addition, there may be changes in the molecular confor-mation of vWF induced by plasma harvesting or cryoprecip-itation that would shorten bleeding time but not sup port ar-terial thrombosis. Th is hypothesis is supported by a notherreport from o ur laboratory that docum ented a difference inthe half-life of FVIII complexed with endogenous and ex-ogenous vW F.'~Regardless of the m echanism, bleeding timeshortening by plasma v W F in the absence of platelet or sub-endothelial matrix vW F in vW D dogs suggests that plasmavWF is the main determinant of the bleeding time in thisspecies.Other investigators have noted that occasionally mongreldogs fail to develop thrombosis in this stenosis and injuryThree such dogs had a vW F antigen of 92% k 11%;however, the administration of deamino-8-D-argenine va-sopressin (DDA VP), produced a rise in vWF antigen to 133%f 12% that was accompanied by cyclic flow reductions. Noother data on vW F were given in th at abstract.Other studies that have exam ined canine platelets for vW Fantigen have yielded variable results, both the presence andabsen ce o f p la te le t V W F . ~ , ~ , ' ~ , ' ~latelet v W F activity was notreported in these studies. In the present stu dy, we used severalphysical and chem ical procedures to isolate platelet vWF from

PRP in several anticoagulant regimens. These procedureswere successfully used to isolate porcine a nd hum an plateletvWF. For this study, we developed a rabbit anti-canine vW Fantibody that was raised against v W F rom a dog with he-mophilia A and rendered monospecific by adsorption withcanine vW D cryoprecipitate. This antibody readily detectedvW F antigen in can ine plasma bu t not platelet lysates. Thesefindings are in agreement with those of Potter et al, who usedrabbit anti-canine v W F antibod ies7 There are two reportsthat identified small amou nts of vWF antigen in the plateletsof mongrel dogs. In the first study, anti-human and anti-canine antibodies were used th at had been rendered m ono-specific by adsorption with hu ma n but not canine vW Dp 1 a ~ m a . I ~his anti-human vW F antibody was shown to bemonospecific for hum an bu t not canine v W F by a rocketinhibition method against commercial anti-huma n v W F an-tisera.60 n the second study, an anti-human vW F antibodyfrom a commercial source was used.I5 No antibody charac-terization data were given in that study. It is possible thatthese anti-human vWF antibodies that were not renderedmonospecific by adsorption with canine plasma or cryopre-cipitate may have recognized epitopes other than canine vW Fin canine platelets. It may also be that some breeds of dogsfail to express vW F in their platelets, whereas others expresssmall amounts. We used dogs primarily from t he closed col-ony of inbred dogs, thus providing homogeneous groups ofnormal, hemophilia A, and vW D. Th e source of normal ca-nine platelets was mongrel dogs in th e studies that detectedplatelet vW F antigen. Dogs, like human s, may have discor-dance in the expression of platelet and plasma vW F . '~ , '~Halothane was used by itself for anesthesia in this study.This agent has been successfully used in studies of vW F ina rte ria l t h r o m b o ~ i s . * ~ - ~ ~ther investigators have shown th athalothane in combination with thiamyl interrupted cyclicthrombo sis in this model.'' It may be tha t the antiplateleteffect of barbiturates on platelets accounts for the apparentdiscrepancy between our report and that of Bertha et al.6'Our findings corroborate those of Barr et al., who found thathalothane did not induce an antiplatelet effect in dogs.62Our study has shown that when vWF is absent, arterialthrombosis does not occur in this model. An interesting al-ternative hypothesis for the protection from thromb osis af-forded by vW D in dogs could be a concomitant lack of othe rendothelial cell products suc h as issue plasminogen activator.Such a combination of defects has been documented in hu-m a n ~ . ~ ~o confirm such a possibility would require char-acterization of the plasminogen activator response in ourvW D dogs. This possibility will require further inves tigation.vWF supports platelet adhesion, platelet spreading, andarterial thrombosis in several species. The contribution ofvW F within plasma, endothelial cells, subendothelial matrix,and platelets to each of these processes is complex. Th e dif-ference in expression of vW F by canine and porcine endo-thelium and platelets suggests that these two an ima l speciescan be used to explore differences in the function of vW F inits various compartments. Also, agents designed to preventor treat thrombosis or hemorrhage by modifying vW F inplasma or platelets could be tested for differential results inthese two species. Additional comparative studies in dogs,



8/9

2650 NICHOLS ET AL

pigs, and h um ans should con t inue to clarify th e relative con-tribution of v W F in its different comp artme nts to th rom bos i sand hemostas is .

ACKNOWLEDGMENTThe author s thank Ro bin Raymer, M anager of the Francis Owen

Blood Research Laboratory, and her staff for excellent care and man-agement of the animals used in this study.REFERENCES

I . Bloom AL, Gidd ings JC, Wilks CJ: Factor VI11 on th e vascularintima: Possible importance in haemostasis and thrombosis. Nature241:217, 19732. Hoy er L, de 10s Santos R, Hoyer J: Antihemophilic factor an-tigen, localization in endothelial cells by immunofluorescent mi-croscopy. J Clin Invest 52:2737, 19733. Jaffe E, Hoyer LW, Nachma n RL: Synthesis of antihemophilicfactor antigen by cultured human endothelial cells. J Clin In vest 52:2757, 19734. Sakariassen KS, Bolhuis PA, Sixma JJ: Human blood plateletadhesion to artery subendothelium is mediated by factor VIII/vonWillebrand factor bound to the subendothelium. Nature 279:636,1979

5. Spom L, Marder V, Wagner D: Inducible secretion of large,biologically potent von Willebrand factor multimers. Cell 46: 185,19866. Bahnak BR, Wu QY , Coulombel L, Assouline A, K erbiriou D,Pietu G, Drouet L, Caen JP, Meyer D: Expression of von W illebrandfactor in porcin e vessels: Heterogene ity at the level of von W illebrandfactor mRNA. J Cell Physiol 138:305, 19897. Potter EV, Shau ghnessy MA, Green D Some immunofluores-cent observations on factor VIII/von Willebrand factor in dogs.Thromb Haemost 4456 , 19808. Meyers KM , Wardrop K J, Helmick CM, White F P Immu-

nohistochemical detection o f von Willebrand factor in vascular en-dothelium bu t not platelets from plasma VI1R:AG deficient and co n-trol dogs and species differences in platelet VI1I:RAG. Fed Proc 46:424, 19879. Koutts J, Walsh PN, Plow EF, Fenton JW, Bouma BN, Zim-merman TS: Active release of human platelet factor VIII-relatedantigen by adenosine diphosphate, collagen, and thrombin. J ClinInvest 62:1255, 197810. Zucker M, Broekman M, Kaplan K: Factor VIII-related an-tigen in hum an blood platelets: Localization and release by throm binand collagen. J Lab Clin M ed 94:675, 19791 . Sporn LA, Chavin SI, Marder V, Wagner D: Biosynthesis ofvon Willebrand protein by hum an megakaryocytes. J Clin Invest 76:1102, 198512. Cramer EM, Caen JP, Drouet L, Breton-Conus J: Absence

of tubular structures and immunolabeling for von Willebrand factorin the platelet a-gr anules from po rcine von W illebrand disease. Blood86:774, 198613. Bowie EJW, Solberg LA, Fass DN, Johnson CM, KnutsonGJ, Stewart ML, Zoecklein LJ: Transplantation of normal bonemarrow into a pig with severe von Willebrand's disease. J Clin Invest78:26, 198614. McCarroll DR, Waters D, Steidley KR, Clift R, McDonaldT P Canine platelet von W illebrand factor: Quantification and mul-timeric analysis. Exp Hematol 16:929, 198815. Parker MT, Turrentine MA, Johnson GS: Von Willebrandfactor in lysates of washed canine platelets. Am J V et Res 52:119,199116. Mannucci PM, Lombardi R, Bader R, Vianello L, FedericiAB, Solinas S, Mazzucconi MG, Mariani G: Heterogeneity of type

I von Willebrand disease: Evidence for a subgroup with abnormalvon Willebrand factor. Blood 66:796, 19 8517. Gralnick H R, Rick ME, M cKeown LP, W illiams SB, ParkerRI, Maisonneuve P, Jenneau C, Sultan Y: Platelet von Willebrandfactor: An imp ortant determ inant of the bleeding time in type I vo nWillebrand's disease. Blood 68:58, 198618. Ruggeri ZM, Zim merm an TS: The complex multimeric com-position of factor VIII/von W illebrand factor. Blood 57:1140, 198119. Rodeghiero F, Castaman G, Ruggeri M, Tosetto A: Thebleeding time in normal subjects is mainly determined by plateletvon Willebrand factor and is dependent on blood group. ThrombRes 65:605, 199220. Eller Th, Brauer P, Albert J, Keller F Significance and quan-titative analysisof von W illebrand factor in h uman platelets. Throm bRes 65:631, 199221. Ruggeri 2,Zimmerman T: Variant von Willebrand's diseasecharacterization of two subtypes by analysis of multimeric compo-sition of factor VIII/von Willebrand factor in plasma and platelets.J Clin Invest 65:1318, 198022. Meyer D, Obert B, Pieta B, Lavergne T, Zimm erman T: Mul-timeric structure of factor VIII/von Willebrand factor in von Wil-lebrand's disease. J Lab Clin Med 95 :590, 19 8023. Moake JL, Turner NA, Stathopoulos NA, Nolasco LH, Hel-lums JD: Involvement of large plasma von Willebrand factor mul-timers and unusually large vWF forms derived from endothelial cellsin shear stress-induced platelet aggregation. J Clin Invest 78: 1456,198624. Spom L, Marder V, Wagner D von W illebrand facto r releasedfrom W eibel-Palade bodies binds more avidly to extracellular matrixthan that secreted constitutively. Blood 69:1531, 198725. Weiss HJ: von Willebrand factor and platelet function. AnnNY Acad Sci 614:125, 199126. Reddick R, GriggsT, Lamb M, Brinkhous K: Platelet adhesionto damaged coronary arteries: Comparison of normal and von Wil-lebrand diseased swine. Proc Natl Acad Sci USA 79 507 6, 198227. Reddick RL, Read MS, Brinkhous KM , Bellinger DA, Nicho lsTC , Griggs TR : Coronary atherosclerosis in the pig: Induced-plaqueinjury and p latelet response. Arteriosclerosis 105 4 , 199028. Nichols TC, Bellinger DA, Johnson TA, Lamb MA, GriggsTR: von Willebrand's disease prevents occlusive thrombosis in ste-nosed and injured porcine coronary arteries. Circ Res 59:15, 198629. Bellinger DA, Nichols TC, Read MS, Reddick RL, Lamb MA,Brinkhous KM , Evatt BL, Griggs TR: Prevention of occlusive cor-onary artery thrombosis by a murine mo noclonal antibody to porcinevon W illebrand factor. Proc Natl Acad Sci USA 84:8100, 198730. Nichols TC, Bellinger DA, Tate DA , Reddick RL, Read MS,Koch GG, Brinkhous KM, Griggs TR: V on W illebrand factor andocclusive arterial thrombosis: A study in normal and von Willebrand'sdisease pigs with diet-induced hypercholesterolemia and atheroscle-

rosis. Arteriosclerosis 10:449, 19903 . Bellinger DA, Nichols TC, Reddick RL, Read MS , Koch GG,Brinkhous KM. GriggsT: Role of plasma and platelet von W illebrandfactor in a porcine arterial thrombosis model. Circulation 82:III-595a,199032. Smith SV, Reddick RL, Brinkhous KM, Read MS: Immu-nohistological evidence for a blood vessel wall protein analogous tobotrocetin. Blood 78:397a, 199133. Nichols TC, Bellinger DA, Read MS, Reddick RL, K och GG ,Brinkhous KM, Griggs T von Willebrand factor, factor VIII, andarterial thrombosis: Studies in canine von Willebrand disease andhemophilia A. Thromb Haemost 65:855a, 199134. Graham JB, Buckwalter JA, Hartley LJ, Brinkhous KM: Ca-nine hemophilia: Observations on the course, the clotting animaly,and th e effects of blood transfusion. J E xp Med 90:97, 1949



9/9

THROMBOSIS: CANINE vWF AND FVll l 2651

35. Langdell RD, Wagner R H, Brinkh ous KM : Effect of antihe-mophilic factor on one-stage clotting tests. J Lab Clin Med 41:637,195336. Rodeghiero F, Castaman G, Tos etto A, Lattuada A, M annucciP: Platelet von Willebrand factor assay: Results using two m ethodsfor platelet lysis. Th rom b Res 59:259, 1990

37. Read MS, Shermer RW , Brinkhous KM : Venom coagglutinin:An activator of platelet aggregation depend ent on von W illebrandfactor. Proc Natl Acad Sci USA 75:45 14, 197838. A llain J, Cooper H, Wagner R, Brinkhous K Platelets fixedwith paraformaldehyde: A new reagent for assay of von W illebrandfactor and platelet aggregating factor. J Lab C lin Med 85:318, 197 539. Read MS, Smith SV, Lam b MA, Brinkhous KM : Role ofbot-rocetin in platelet agglutination: Formation of an activated complexof botrocetin an d von W illebrand factor. Blood 74: 1031, 198940. Read MS, Potter JY, Brinkhous KM : Venom coagglutinin fordetection of von W illebrand factor activity in anim al plasma. J L abClin Med 101:74, 198341. Laurel1 CB: Quantitative estimation of proteins by electro-phoresis in agarose gels containing antibodies. A nal Biochem 15:45,1966

42. Lamb MA, Reisner HA, Cooper HA, Wagner RH: The mul-timeric distribution of factor VIII-related antigen studied by an im -proved crossed-immunoelectrophoresis technique. J Lab Clin Med98:751, 198143. ON eill SL, Feldman BF, Boothby JT: Measurem ent of canineplasma fibronectin by rocket immuno electrophoresis. Am J Vet Res46:1175, 198544. Mertz E T The anomaly of a normal Duke's and very pro-longed saline bleeding time in swine suffering from an inheritedbleeding disease. Am J P hysiol 136:360, 194245. Uchida Y, oshimoto N, Murao S: Cyclic fluctuations in cor-onary blood pressure and flow induced by caronary artery constriction.

Jpn Heart J 16:454, 197546. Folts JD, Crowell EB Jr, Rowe GG : Platelet aggregation inpartially obstructed vessels and its elimination by aspirin. Circulation54:365, 197647. GriggsTR , Potter J , McC lanahan SB, Webster WP, B rinkhousKM : Macrom olecular factor VI11 complex: Functional and structuralheterogeneity observed in von Willebrand swine with transfusion.Proc Natl Acad Sci USA 74:759, 197748. Thomas KB, Firkin BG, Howard MA: Analysis of plateletvon W illebrand factor antigen. Haemostasis 17:217, 1987

49. Lopez-Fernandez MF, Batlle J, Ruggeri ZM, Z immerm an TS:Secretion of von Willebrand factor from platelets. Methods Enzym ol169:244, 198950. Solymoss S, Golden EA, Bovill EG: Measurement of plateletvon W illebrand factor is dependent on method of platelet isolation.Am J Clin Pathol 93:400, 1990

5 1. Bellinger DA, Nichols TC, Samama CM , Reddick RL, Bon-neau M, Read MS, Bailliart 0, Koch GG, Vaiman M, BrinkhousKM, Pignaud GA, Sigman JL, Gnggs TR, Drouet L Altered phe-notypes of normal a nd vo n Willebrand disease pigs by bone m arrowtransplantation: Effects on thrombosis and hemostasis. Blood 78:179a, 1991 (abstr, suppl 1)52. B ertha BG, Folts JD, Nugent M, Rusy B F Halothane, butnot isoflurane or enflurane, protects against spontaneous and epi-nephrine-exacerbated acute thrombus formation in stenosed dogcoronary arteries. Anesthesiology 71:96, 198953. Brinkhous KM , Reddick RL, Griggs T R Arterial thrombosis,atherosclerosis, and th e factor VIII/von W illebrand factor complex,in Zim merman TS, Ruggeri ZM (eds): Coagulation an d BleedingDisorders: Th e Role of Factor VI11 and von W illebrand Factor, V ol9. New York, NY, Marcel Dekker, 1989, p 28354. Blatt PM, Brinkhous KM , Culp HR , Krauss JS, Roberts H RAntihemoph ilic factor concentrate therapy in von Willebrand disease:Dissociation of bleeding-time factor and ristocetin co-factor activities.JAMA 236:2770, 197655. Mannucci P, M oia M, Rebulla P, Altieri D, Monteagudo J,Castillo R: Correction of the bleeding time in treated patients with

severe von Willebrand d isease is not solely depend ent on the no rmalmultimeric structure of plasma von Willebrand factor. Am J Hematol2555, 198756. Smiley R, Titley P, Rock G: Studies on the prolonged bleedingtime in von Willebrand's disease. Thro mb Res 53:417, 198957. Castillo R, Mateagudo J, Escolar G, Ordinas A, Magallon M,Villar JM: Hem ostatic effect of normal platelet transfusion in severevon W illebrand disease patients. B lood 77: 1901, 199158. Brinkhous KM, Sandberg H, Garris JB, Mattsson C, Palm M,G r ig s T, Read MS: Purified human factor VI11 procoagulant protein:Comp arative hemostatic response after infusions into hem ophilic andvon W illebrand disease dogs. Proc Natl Acad Sci USA 82:8752 , 198 559. Folts JD, Coller BS: Role of von W illebrand factor in acuteplatelet thrombus formation in stenosed dog coronary arteries. Blood7040 2a, 1987 (abstr, suppl 1)60. McCarroll DR, W aters DC, Steidley KR , Clift R, McDonaldT P Canine yon W illebrand factor expresses a m ultimeric compositionsimilar to hum an von Willebrand factor. Exp Hematol 16:1060, 198761. O'Rourke ST, Folts JD, Albrecht RM: Inhibition of canineplatelet aggregation by barbiturates. J Lab C lin Med 108:206, 198662. Barr SC, Ludders JW, Loon ey AL, Gleed RD, Erb H N: Plateletaggregation in dogs after sedation with aceprom azine an d atropineand during general anesthesia and surgery. Am J Vet Res 53:2067,199263. Ludlam CA, Peake IR, Davies BL, Furlong RA, B loom AL:Factor VI11 and fibrinolytic response to deamino-8-D-argenine va-sopressin in normal subjects and dissociate response in some patientswith haemophilia and von Willebrands disease. Br J Haem atol45 :499, 1980


Hemofilija Kod Pasa

Documents

Transcript of Hemofilija Kod Pasa