Platelets in bleeding and thrombosis

Transcription

1 Graduate Theses and Dissertations Iowa State University Capstones, Theses and Dissertations 2016 Platelets in bleeding and thrombosis Kelly M. Makielski Iowa State University Follow this and additional works at: Part of the Veterinary Medicine Commons Recommended Citation Makielski, Kelly M., "Platelets in bleeding and thrombosis" (2016). Graduate Theses and Dissertations This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact

2 Platelets'in'bleeding'and'thrombosis' ' by Kelly'Makielski' ' ' ' Athesissubmittedtothegraduatefaculty inpartialfulfillmentoftherequirementsforthedegreeof MASTEROFSCIENCE Major:VeterinaryClinicalSciences ProgramofStudyCommittee: DanaLeVine,MajorProfessor AlbertJergens AmandaFalesIWilliams IowaStateUniversity Ames,Iowa 2016

3 ii TABLEOFCONTENTS Page ACKNOWLEDGMENTS... iv CHAPTER1 INTRODUCTION... 1 Background... 1 Objectives... 2 ThesisOrganization... 2 CHAPTER2 DEVELOPMENTANDVALIDATIONOFANOVELCANINEIMMUNE THROMBOCYTOPENIABLEEDINGSCORE... 4 Abstract... 4 Introduction... 6 MaterialsandMethods... 7 Studydesign... 8 Statisticalanalysis... 9 Results Discussion Footnotes References Figures Tables CHAPTER3 COATEDPLATELETPOTENTIALINCANINECANCERPATIENTS Abstract Introduction MaterialsandMethods Animals Coatedplateletassay Statistics Results Discussion References Figures Tables... 40

4 CHAPTER4 GENERALCONCLUSIONS Briefsummary Futuredirections iii

5 iv ACKNOWLEDGMENTS Iwouldliketothankmycommitteechair,DanaLeVine,andmycommitteemembers, AlbertJergensandAmandaFalesIWilliams,fortheirpatience,guidance,andsupport throughoutthecourseofthisresearch.additionally,iwouldliketothanktheiowastate UniversityVeterinaryClinicalSciencesResearchIncentivegrantforfundingofthecoated plateletstudy,andtheamericankennelclubcaninehealthfoundationforthefundingofthe studyoncanineitpthatthedogibatstudywasapartof.finally,iwouldliketothankthe dogsandownerswhoparticipatedinthecoatedplateletstudyandtheitpstudybyproviding samples,andtheveterinarystudentvolunteerswhoparticipatedinthedogibatproject.

6 1 CHAPTER(1.(INTRODUCTION( Background( The(hemostatic(system(of(the(body(is(vital(and(has(major(implications(for(survival.(( Aberrations(in(hemostasis,(whether(an(increased(tendency(toward(clotting(or(an(increased( tendency(toward(bleeding,(can(have(significant(implications.((platelets(are(an(important( component(of(the(hemostatic(system.((traditionally,(platelets(have(been(the(main( determinant(of(primary(hemostasis,(or(formation(of(the(platelet(plug(at(the(site(of(vascular( injury.((secondary(hemostasis,(or(the(coagulation(cascade,(stabilizes(the(platelet(plug( through(a(crosslinked(fibrin(meshwork.((the(final(stage(involves(plasminogen(activation(to( plasmin,(which(leads(to(fibrinolysis(or(the(breakdown(of(the(fibrin(clot.((newer(evidence( suggests(that(this(traditional(viewpoint(of(hemostasis(with(platelets(involved(only(in(primary( hemostasis(is(overly(simplistic.((it(is(now(known(that(activated(platelets(have(a(vital(role(in( secondary(hemostasis,(as(the(surface(of(the(activated(platelet(provides(a(procoagulant( membrane(for(thrombin(generation,(leading(to(generation(of(coagulation(enzymes.(( Without(the(platelet(providing(this(procoagulant(surface,(clinical(hemorrhage(is(likely(to( occur(such(as(seen(in(canine(scott(syndrome((css).((this(uncommon(disorder(seen(in( German(shepherd(dogs(results(when(platelets(are(unable(to(generate(the(procoagulant( surface.(dogs(with(css(have(clinical(bleeding,(often(significant,(such(as(epistaxis,(bruising,( and(postpsurgical(hemorrhage.(perhaps(the(veterinary(disease(in(which(the(importance(of( platelets(is(most(appreciated(is(immune(thrombocytopenia((itp).((itp(is(caused(when( antibodies(bound(to(the(platelet(surface(lead(to(their(destruction(by(the(immune(system.((

7 2 Dogs(with(ITP(often(have(severely(decreased(platelet(counts,(and(often(have(very(significant( clinical(bleeding.(((despite(the(importance(of(platelets(in(preventing(significant(hemorrhage,( uncontrolled(clotting(can(have(potentially(significant(consequences(as(well.((numerous( conditions(in(veterinary(medicine(are(known(to(cause(hypercoagulability((an(increased( tendency(towards(abnormal(blood(clotting).(treatment(of(any(hemostatic(disorder(should( be(aimed(at(restoring(the(balance(between(prop(and(antipthrombotic(factors(that(exists(in( health.(( Objectives( The(aim(of(this(thesis(is(to(investigate(the(role(of(platelets(in(bleeding(and(in( thrombosis.((this(was(accomplished(using(two(projects.((the(first(is(the(development(and( validation(of(a(novel(canine(bleeding(score(to(be(used(in(canine(itp.((the(second(is(the( investigation(of(coated(platelet(potential(in(canine(cancer(patients.((( Thesis(Organization( The(format(of(this(thesis(is(based(on(the(use(of(journal(papers(as(chapters.(The( second(chapter(describes(the(development(and(validation(of(a(novel(bleeding(score(to(be( used(in(canine(itp,(the(dogibat.(this(study(was(presented(in(abstract(form(at(the(2016( American(College(of(Veterinary(Internal(Medicine(Forum,(and(is(being(prepared(for( submission(to(the(journal(of(veterinary(internal(medicine.(chapter(three(is(a(study(of( coated(platelet(potential(in(canine(cancer(patients.(this(has(been(submitted(for( presentation(at(the(veterinary(cancer(society(forum(and(platelets(international.((it(is(also( being(prepared(for(submission(to(the(both(the(second(and(third(chapters(will(be(submitted(

8 3 to(the(journal(of(veterinary(internal(medicine.(finally,(the(last(chapter(contains(the(thesis( conclusions(and(recommendations(for(future(research.(

9 4 CHAPTER(2.(DEVELOPMENT(AND(VALIDATION(OF(A(NOVEL(CANINE(IMMUNE( THROMBOCYTOPENIA(BLEEDING(SCORE( A(paper(to(be(submitted(to(the(Journal(of(Veterinary(Internal(Medicine( Kelly(M.(Makielski,(Marjory(B.(Brooks,(Chong(Wang,(Jonah(N.(Cullen,(Annette(M.(O Connor,( Dana(N.(LeVine( Abstract( Background:A(method(of(objective(quantification(of(clinical(bleeding(in(canine(immuneU mediated(thrombocytopenia((itp)(is(needed(because(itp(patients(have(variable(bleeding( tendencies(that(do(not(directly(relate(to(severity(of(thrombocytopenia.((a(consistent(description( of(their(bleeding(would(allow(more(direct(comparisons(of(this(bleeding(heterogeneity(in(multiu institution(studies.(((application(of(a(standardized(bleeding(assessment(tool(would(help(stratify( ITP(patients(in(clinical(trials(based(on(their(bleeding(severity(to(provide(an(objective(comparison( of(patient(outcomes(among(treatment(protocols.(( Objectives:In(this(study(we(aimed(to(develop(and(validate(a(daily(canine(ITP(bleeding( assessment(tool((dogibat)(and(to(evaluate(the(utility(of(a(training(course(for(improving( accurate(implementation(of(the(dogibat.(additionally,(we(aimed(to(use(the(dogibat(to(score( clientuowned(dogs(with(thrombocytopenia.( Methods:A(novel(bleeding(assessment(tool,(DOGiBAT,(was(developed(for(canine(ITP comprising(nine(different(anatomic(siteuspecific(bleeding(grades(ranging(from(0((none)(to(2( (severe).(a(computerized(training(course(was(developed(for(application(of(the(dogibat(to( score(clinical(cases.((a(caseubased(quiz(set(of(still(images(was(used(to(assess(the(trainees (ability( to(apply(the(dogibat(tool.(to(assess(the(efficacy(of(the(training(course,(70(veterinary(student(

10 5 volunteers(were(randomized(to(take(the(quiz(with((n=35)(or(without((n=35)(the(training.(all( students(scored(all(sites(from(all(cases(and(site(scores(were(considered(correct(if(they(agreed( with(the(investigators (preuspecified(classification.(the(frequency(of(correct(scores(was( compared(between(trained(and(untrained(students.(a(logistic(regression(model(was(used(to( assess(the(association(between(training(and(score,(while(adjusting(for(correlated(responses( from(sites(within(cases.( Results:Clinicians((n=13)(and(technicians((n=3)(taking(the(quiz(following(training(were( able(to(correctly(apply(the(dogibat,(scoring(100%(of(responses(correctly.(86.1%(of(responses( from(students(that(received(training(were(correctly(scored,(compared(to(only(78.2%(of( responses(from(students(that(did(not(receive(training.(the(odds(of(trained(students(giving( correct(answers(were(higher(than(untrained(students((p(<0.0001).((the(anatomic(site(for(which( training(was(most(important(for(correct(scoring(was(oral(bleeding((82.9%(with(training,(47.6%( without;(p(<0.0001).((similarly,(scoring(of(ocular((75.2%(with(training,(58.1%(without;(p=0.0097)( and(cutaneous(bleeding((84.8%(with(training,(66.7%(without;(p=0.003)(were(improved(by(the( training(course.((students(with(more(clinical(experience(were(better(able(to(apply(the(dogibat( correctly((fourth(years,(84.2%;(third(years,(80.1%;(p(<0.0149).((an(inverse(correlation(was(found( between(platelet(count(and(dogibat(score,(except(in(severely(thrombocytopenic(dogs(with( platelet(counts(under(30,000(μl.( Conclusions(and(Clinical(Importance:Our(study(suggests(that(the(DOGiBAT(is(simple( scoring(tool(for(classification(of(bleeding(severity(in(dogs(with(itp.(a(short(computerized( training(course(was(shown(to(improve(the(accuracy(and(consistency(of(dogibat(scoring.( Adoption(of(the(DOGiBAT(scoring(system(for(future(clinical(trials(of(canine(ITP(patients(will(

11 6 facilitate(more(rigorous(assessment(of(treatmentueffect(based(on(standardized(bleeding( outcomes.( Introduction( Immune(thrombocytopenia((ITP)(is(a(relatively(common(cause(of(significant( thrombocytopenia(in(dogs. 1 ((ITP(occurs(when(antiplatelet(antibodies(bound(to(the(platelet( surface(lead(to(their(premature(destruction(by(the(reticuloendothelial(system.(itp(is(considered( idiopathic(or(primary(when(diagnostic(evaluation(yields(no(apparent(underlying(cause(or( trigger,(and(secondary(when(an(underlying(cause(is(found.((mortality(rates(reported(with(itp( have(ranged(from(3(to(30%. 2,3,4 (((Glucocorticoids(administered(at(immunosuppressive(doses(are( the(mainstay(of(treatment.((many(adjunctive(treatments(have(been(suggested,(but(prospective( evaluation(of(their(efficacy(beyond(steroids(alone(is(limited.(additionally,(there(are(clear(risks( associated(with(overutreatment,(including(adverse(effects(of(medications,(immune(suppression,( and(potentially(hypercoagulability. 5 ((Some(of(the(difficulty(in(prospectively(evaluating( treatment(of(itp(can(be(attributed(to(the(lack(of(a(standardized(method(of(assessing(outcome.(( Several(studies(have(used(platelet(recovery(time(as(the(primary(outcome(measure,(typically( with(the(time(to(reach(a(platelet(count(greater(than(40,000(platelets(/(μl(as(the(primary( endpoint. 3,6,7 (Other(study(endpoints(evaluated(have(included(diseaseUrelated(mortality,(adverse( treatment(reactions,(cost,(hospitalization(time,((and(transfusion(requirements. 3,6,7( Platelet( count(has(been(shown(to(be(poorly(correlated(with(both(clinical(bleeding(and(with(survival(in( dogs. 2 (O Marra(et(al.(found(that(initial(platelet(count(and(lowest(platelet(count(recorded(during( hospitalization(were(not(associated(with(survival(to(discharge. 2 ((The(presence(of(melena(at( admission(was(associated(with(increased(transfusion(requirements(and(with(failure(to(survive(

12 7 to(discharge.((platelet(counts(were(similar(between(dogs(that(had(melena(and(those(without( melena. 2 (Humans(with(ITP(have(also(been(shown(to(have(heterogeneity(in(their(bleeding( phenotypes,(especially(when(platelet(count(is(under(30,000(platelets(/(μl. 8 (Due(to(the(variable( bleeding(in(canine(itp(and(its(inconsistent(correlation(with(platelet(count,(an(objective(method( of(bleeding(assessment(is(needed.(a(bleeding(score(metric(will(help(to(objectively(assess( treatment(efficacy(in(multicenter(clinical(trials,(stratify(patients(according(to(their(bleeding( severity,(and(individualize(treatment(based(on(a(patient s(bleeding(risk.(our(primary(objective( in(this(study(was(to(develop(and(validate(a(novel(canine(itp(bleeding(assessment(tool.((other( study(objectives(were(to(develop(a(training(deck(to(instruct(users(of(the(dogibat(on(its(correct( implementation,(and(to(apply(the(dogibat(to(clinical(cases(of(canine(thrombocytopenia. ( We(hypothesized(that(the(use(of(the(DOGiBAT(will(standardize(bleeding(severity(scoring,( allowing(for(consistency(among(clinicians,(and(that(reviewing(a(training(deck(will(improve( veterinary(students (ability(to(correctly(assign(bleeding(severity(scores(to(case(examples.(finally,( we(hypothesized(that(in(clinical(canine(cases(of(thrombocytopenia,(dogibat(score(and(platelet( count(would(be(inversely(correlated(except(in(severely(thrombocytopenic(dogs(with(counts(less( than(30,000(platelets(/(μl.( Materials(and(Methods(( A(novel(bleeding(assessment(tool,(the(DOGiBAT,(was(developed(for(canine(ITP((Table(1).(( This(tool(was(modelled(after(the(ITP(Bleeding(Scale((IBLS)(due(to(its(simplicity(and(validation(in( humans.((the(dogibat(comprised(nine(different(anatomic(sites((cutaneous,( catheter/venepuncture(bleeding,(oral(mucosa,(intraocular,(epistaxis,(gastrointestinal,(urinary,( pulmonary,(and(intracranial).((each(anatomic(site(was(given(a(siteuspecific(bleeding(grade(of(0(

13 8 (none),(1((mild),(or(2((severe),(with(the(exception(of(pulmonary(and(intracranial(that(were(given( only(grades(of(0(or(2.(( A(computerUbased(training(deck(was(developed(for(application(of(the(DOGiBAT(to(score( clinical(cases.(the(training(course(initially(introduces(the(participant(to(general(principles(of( scoring(within(the(dogibat(bleeding(tool,(and(then(provides(pictures(from(clinical(cases( demonstrating(to(the(participant(the(appearance(of(each(bleeding(grade(at(each(anatomic(site.(( A(caseUbased(quiz(set(of(still(images(was(then(developed,(using(images(from(clinical( cases.((a(caseubased(quiz(was(utilized(to(assess(the(trainees (ability(to(apply(the(dogibat(tool( rather(than(clinical(cases(for(consistency(across(a(multicenter(study.((the(quiz(provided(the(user( with(clinical(case(details(and(images(to(completely(score(three(cases(using(the(dogibat.(( Study(design( This(study(consisted(of(three(phases.((In(phase(1,(clinicians(and(technicians(were( provided(the(training(deck,(after(which(they(completed(a(caseubased(quiz(to(assess(their( implementation(of(the(dogibat.( In(phase(2,(third(and(fourth(year(veterinary(students(volunteers((n=70)(were( randomized(to(take(the(quiz(with((n=35)(or(without((n=35)(the(training(in(order(to(assess(the( effectiveness(of(the(training(deck.(the(study(protocol(was(considered(exempt(by(the(iowa(state( University(Institute(Review(Board.( In(phase(3,(the(clinicians(who(had(received(training(on(correct(implementation(of(the( DOGiBAT(in(phase(1(applied(the(DOGiBAT(bleeding(tool(to(clinical(cases(as(part(of(a(larger( multicenter(study(of(canine(thrombocytopenia.((canine(patients(presenting(to(iowa(state( University((ISU),(Cornell(University(Hospital(for(Animals((CUHA),(and(Cornell(University(

14 9 Veterinary(Specialists((CUVS)(with(a(platelet(count(of(less(than(50,000(platelets(/(μl(due(to(any( underlying(etiology(were(enrolled(with(client(consent.((cases(were(excluded(if(they(had( received(glucocorticoids(for(a(period(of(greater(than(48(hours(at(the(time(of(enrolment,(if(they( had(received(any(other(immunosuppressive(therapies,(or(if(body(weight(was(under(3(kg.( Enrolled(cases(were(separated(into(three(groups(based(on(underlying(etiology(of(the( thrombocytopenia,(which(were(primary(immune((pi),(secondary(immune((si),(and(nonuimmune( (NI).((Categorization(was(determined(through(discussion(between(three(of(the(authors((K.M.,( D.L.,(and(M.B.)(and(was(based(on(results(of(diagnostic(testing(performed(at(the(attending( clinician s(discretion.((treatment(decisions(for(the(clinical(cases(were(at(the(discretion(of(the( attending(clinician.((enrolled(cases(were(scored(daily(using(the(dogibat,(from(time(of( enrolment(until(discharge(from(the(hospital,(death(or(euthanasia,(or(until(a(maximum(of(7(days.( The(study(was(approved(by(the(Institutional(Animal(Care(and(Use(Committees(of(ISU(and(CUHA.( Statistical(Analysis( Sample(size(calculations(were(performed(using(online(resources a,b (to(determine(the( number(of(students(needed(per(group((students(with(access(to(the(training(deck(and(students( that(were(not(given(access(to(the(training(deck)(to(determine(a(significant(difference.(pilot(data( was(utilized(from(10(veterinary(students(who(scored(3(cases((cluster(size(30)(with(a(baseline( correct(response(rate(of(75%.(using(independent(samples,(an(alpha(=(0.05,(and(80%(power,(the( required(sample(size(to(detect(a(significant(difference((defined(as(a(20%(increase(in(number(of( correct(scores)(between(the(two(groups(was(calculated.((when(sample(sizes(were(adjusted(for( clustering(by(students((i.e.(each(student(answers(multiple(questions),(and(assuming(an(

15 10 intraclass(correlation(of(0.75,(the(required(sample(size(was(determined(to(be(35(students(in(the( trained(group(and(35(students(in(the(untrained(group.(( Quiz(site(scores(were(considered(correct(if(they(agreed(with(the(investigators (preu specified(classification.(the(frequency(of(correct(scores(was(compared(between(trained(and( untrained(students.(a(logistic(regression(model(was(used(to(assess(the(association(between( training(and(score,(while(adjusting(for(correlated(responses(from(sites(within(cases.(( Results( In(the(first(phase(of(this(project,(clinicians((n=13)(and(technicians((n=3)(taking(the(caseU based(quiz(following(training(were(able(to(correctly(apply(the(dogibat,(scoring(100%(of( responses(correctly.(( In(the(second(phase(of(this(project,(70(veterinary(students(were(administered(the(caseU based(quiz,(after(having(been(randomized(to(either(receive(the(training(deck((n=35)(or(no( training((n=35).((of(the(students(that(received(training,(17/35(were(third(year(veterinary( students(and(18/35(were(fourth(year(veterinary(students.(of(the(students(that(received(no( training,(18/35(were(third(year(veterinary(students(and(17/35(were(fourth(year(veterinary( students.(((students(that(received(training(correctly(scored(86.1%(of(responses,(compared(to( only(78.2%(of(responses(from(students(that(did(not(receive(training.(the(odds(of(trained( students(giving(correct(answers(were(higher(than(untrained(students((p(<0.0001).((the( anatomic(site(for(which(training(was(most(important(for(correct(scoring(was(oral(bleeding( (82.9%(with(training,(47.6%(without;(p(<0.0001).((Similarly,(correct(scoring(of(ocular((75.2%(with( training,(58.1%(without;(p=0.0097)(and(cutaneous(bleeding((84.8%(with(training,(66.7%( without;(p=0.003)(were(improved(by(access(to(the(training(course.((students(with(more(clinical(

16 11 experience(were(better(able(to(apply(the(dogibat(correctly((fourth(years,(84.2%;(third(years,( 80.1%;(p(<0.0149).( In(the(third(phase(of(this(project,(the(DOGiBAT(was(utilized(for(daily(assessment(of( bleeding(in(a(large(multicenter(study(of(canine(thrombocytopenia.(dogs((n=61)(with(platelet( counts(of(under(50,000(platelets(/(μl,(regardless(of(etiology,(were(enrolled.((these(cases(were( later(categorized(based(on(results(of(diagnostic(testing(as(primary(immuneumediated((pi;(34/61;( 56%),(secondary(immuneUmediated((SI;(17/61;(28%),(or(nonUimmuneUmediated((NI;(10/61;( 16%).((On(admission(to(the(hospital,(PI(dogs(had(higher(mean(total(DOGiBAT(scores((5)(than(SI( dogs((2.6)(or(ni(dogs((3).((figure(2).((platelet(counts(on(admission(were(also(lower(in(the(pi(dogs( (12,824(platelets(/(μl)(than(the(SI(dogs((22,200(platelets(/(μl)(or(NI(dogs((21,000(platelets(/(μl).(( The(most(common(anatomic(sites(of(bleeding(on(enrolment(in(the(study(were(cutaneous( bleeding((62%(of(enrolled(dogs),(followed(by(oral(mucosa((51%),(gastrointestinal((41%)(and( urinary((41%).(a(negative(correlation(existed(between(platelet(count(and(dogibat(score( (Figure(3A),(but(this(negative(correlation(was(less(strong(when(only(evaluating(bleeding(scores( on(days(platelet(count(was(under(30,000(platelets(/(μl.((figure(3b).( Discussion( There(are(several(bleeding(scores(used(in(the(human(literature.((The(World(Health( Organization(Bleeding(Scale(is(simple(and(easy(to(implement. 9 (However,(it(was(originally( designed(for(use(in(human(patients(undergoing(chemotherapy(and(must(be(extrapolated(for( use(in(other(patients.((additionally,(it(contains(somewhat(subjective(terms(such(as( mild (and( gross (that(are(not(defined(within(the(scoring(system.((in(contrast,(the(international(working( Group(on(ITP(in(humans(recently(established(a(bleeding(score,(the(ITPUBAT. 10 ((This(bleeding(

17 12 score(is(rigorous(and(thorough,(but(complicated(and(impractical(to(implement(in(clinical( practice,(taking(an(average(of(15(minutes(to(thoroughly(score(an(individual(patient.((a(third( bleeding(score,(the(itp(bleeding(scale((ibls)(is(simple,(yet(complete,(consisting(of(nine(anatomic( sites. 8 ( In(humans(with(ITP,(the(IBLS(correlates(well(with(platelet(count;(however,(this( correlation(does(not(persist(when(platelet(count(is(under(30,000(platelets(/(μl. 8 ((Due(to(the( simple(yet(thorough(nature(of(the(ibls(and(its(validation(with(human(itp,(the(dogibat(was( modelled(after(this(bleed(score.(( The(bleeding(score(developed(in(this(study,(the(DOGiBAT,(was(validated(with(clinicians( and(technicians(by(use(of(a(caseubased(quiz(utilizing(details(and(images(from(clinical(cases.((a( caseubased(quiz(was(utilized(for(the(validation(for(consistency(between(clinicians(and(between( institutions,(since(this(was(a(multicenter(project.((( A(training(module(was(utilized(to(instruct(the(user(of(the(DOGiBAT(on(its(correct( implementation.(to(demonstrate(the(effectiveness(of(the(training(deck,(third(and(fourth(year( veterinary(student(volunteers(were(randomized(to(take(the(caseubased(quiz(having(either( received(training(or(with(no(training.((veterinary(students(were(utilized(as(a(large(population(of( individuals(that(were(less(experienced(than(clinicians,(and(therefore(more(likely(to(benefit(from( additional(training.((we(found(that(students(who(received(training(scored(more(correct( responses(than(those(who(had(received(no(training.((certain(anatomic(sites((oral(cavity,(ocular,( and(cutaneous)(were(most(affected(by(having(access(to(training.(it(is(not(unexpected(that( certain(sites(may(be(more(affected(by(access(to(the(training(deck,(as(some(anatomic(site( bleeding(grades(are(inherently(easier(to(understand(without(training((e.g.(presence(or(absence(

18 13 of(epistaxis)(than(others((e.g.(funduscopic(bleeding(or(hyphema(with(ocular(bleeding).((as(the( training(deck(provides(the(user(a(written(definition(and(pictorial(example(of(each(bleeding( grade(at(each(anatomic(site,(it(may(help(clarify(grading(sites(that(are(less(clear.( In(this(study,(we(found(that(clinical(experience(was(beneficial(for(the(correct( implementation(of(the(dogibat.((clinicians(and(technicians,(following(review(of(the(provided( training(deck,(were(able(to(score(100%(of(responses(correctly,(likely(due(to(the(clinical( experience(of(these(individuals.(we(also(determined(that(fourth(year(veterinary(students( scored(more(responses(correctly(compared(with(third(year(veterinary(students,(independent(of( their(access(to(the(training(deck.(this(is(suspected(to(be(due(to(the(clinical(experience(that( fourth(year(veterinary(students(gain(from(their(clinical(rotations(during(their(senior(year.( The(DOGiBAT(was(easy(to(implement(in(clinical(practice(when(scoring(patients(with(ITP( or(other(significant(causes(of(thrombocytopenia.(similar(to(what(is(seen(in(human(itp(patients, 8 ( we(found(a(negative(correlation(between(bleed(score(and(platelet(count((in(other(words,(a( lower(platelet(count(was(associated(with(a(higher(dogibat(score).((however,(this(correlation( was(less(apparent(when(only(bleed(scores(from(days(with(platelet(counts(of(less(than(30,000( platelets(/(μl.( The(exact(mechanisms(for(the(heterogeneity(in(bleeding(phenotypes(between(patients( with(similar(platelet(counts(have(yet(to(be(determined.(several(reasons(have(been(proposed,( including(differences(in(platelet(activation, 11 (interference(of(antibodies(with(platelet( function 12,13,(the(effects(of(anemia(on(platelet(function,(abnormalities(with(the(vasculature, 14 ( presence(of(procoagulant(microparticles, 15,16 (and(abnormalities(of(other(components(of( hemostasis.((

19 14 There(were(some(limitations(to(this(study.((First,(the(validation(of(the(DOGiBAT(used(a( caseubased(quiz(rather(than(actual(clinical(cases.((however,(this(allowed(for(a(consistent( method(of(assessing(correct(implementation(of(the(dogibat(across(multiple(institutions.(( Additionally,(data(from(the(larger(clinical(study(of(canine(thrombocytopenia(suggests(validation( with(clinical(cases.((second,(the(dogibat(total(score(is(not(intended(to(be(directly(correlated( with(clinical(severity,(as(the(severity(of(bleeding(at(each(site(is(not(necessarily(equivalent.(a( truly(summative(score(would(be(ideal,(but(would(require(assigning(a( severity(factor (to(each( site.((in(the(absence(of(large(studies(with(extensive(followuup(data,(this(would(be(arbitrary.( Our(study(suggests(that(the(DOGiBAT(is(simple(scoring(tool(for(classification(of(bleeding( severity(in(dogs(with(itp.(a(short(computerized(training(course(was(shown(to(improve(the( accuracy(and(consistency(of(dogibat(scoring.(in(clinical(cases(of(thrombocytopenia,(dogibat( score(and(platelet(count(were(inversely(correlated(except(in(severely(thrombocytopenic(dogs( (platelet(count(less(than(30,000(platelets(/(μl),(similar(to(the(association(between(ibls(score( and(platelet(count(in(humans(with(itp. 8 (Adoption(of(the(DOGiBAT(scoring(system(for(future( clinical(trials(of(canine(itp(patients(will(facilitate(more(rigorous(assessment(of(treatmentueffect( based(on(standardized(bleeding(outcomes.( (

20 15 Footnotes( a (OpenEpi,(Atlanta,(GA( b ( ( (

21 16 References( 1.( ( Botsch( V,( Kuchenhoff( H,( Hartmann( K,( et( al.( Retrospective( study( of( 871( dogs( with( thrombocytopenia.(vet(rec(2009;164:647u651.( 2.((O'Marra(SK,(Delaforcade(AM,(Shaw(SP.(Treatment(and(predictors(of(outcome(in(dogs(with( immuneumediated(thrombocytopenia.(j(am(vet(med(assoc(2011;238:346u352.( 3.((Balog(K,(Huang(AA,(Sum(SO,(et(al.(A(prospective(randomized(clinical(trial(of(vincristine(versus( human(intravenous(immunoglobulin(for(acute(adjunctive(management(of(presumptive(primary( immuneumediated(thrombocytopenia(in(dogs.(j(vet(intern(med(2013;27:536u541.( 4.((Putsche(JC,(Kohn(B.(Primary(immuneUmediated(thrombocytopenia(in(30(dogs((1997U2003).(J( Am(Anim(Hosp(Assoc(2008;44:250U257.( 5.((O'Marra(SK,(Shaw(SP,(deLaforcade(AM.(Investigating(hypercoagulability(during(treatment(for( immuneumediated( thrombocytopenia:( a( pilot( study.( J( Vet( Emerg( Crit( Care( (San( Antonio)( 2012;22:126U130.( 6.((Bianco(D,(Armstrong(PJ,(Washabau(RJ.(A(prospective,(randomized,(doubleUblinded,(placeboU controlled( study( of( human( intravenous( immunoglobulin( for( the( acute( management( of( presumptive( primary( immuneumediated( thrombocytopenia( in( dogs.( J( Vet( Intern( Med( 2009;23:1071U1078.( 7.((Rozanski(EA,(Callan(MB,(Hughes(D,(et(al.(Comparison(of(platelet(count(recovery(with(use(of( vincristine( and( prednisone( or( prednisone( alone( for( treatment( for( severe( immuneumediated( thrombocytopenia(in(dogs.(j(am(vet(med(assoc(2002;220:477u481.( 8.( ( Page( LK,( Psaila( B,( Provan( D,( et( al.( The( immune( thrombocytopenic( purpura( (ITP)( bleeding( score:(assessment(of(bleeding(in(patients(with(itp.(br(j(haematol(2007;138:245u248.(

22 17 9.((Webert(KE,(Arnold(DM,(Lui(Y,(et(al.(A(new(tool(to(assess(bleeding(severity(in(patients(with( chemotherapyuinduced(thrombocytopenia.(transfusion(2012;52:2466u2474;(quiz(2465.( 10.((Rodeghiero(F,(Michel(M,(Gernsheimer(T,(et(al.(Standardization(of(bleeding(assessment(in( immune( thrombocytopenia:( report( from( the( International( Working( Group.( Blood( 2013;121:2596U2606.( 11.(( Panzer( S,( Rieger( M,( Vormittag( R,( et( al.( Platelet( function( to( estimate( the( bleeding( risk( in( autoimmune(thrombocytopenia.(eur(j(clin(invest(2007;37:814u819.( 12.((Chu(XX,(Hou(M,(Peng(J,(et(al.(Effects(of(IgG(and(its(F(ab')2(fragments(of(some(patients(with( idiopathic(thrombocytopenic(purpura(on(platelet(aggregation.(eur(j(haematol(2006;76:153u159.( 13.( ( Olsson( A,( Andersson( PO,( Tengborn( L,( et( al.( Serum( from( patients( with( chronic( idiopathic( thrombocytopenic(purpura(frequently(affect(the(platelet(function.(thromb(res(2002;107:135u 139.( 14.( ( Goerge( T,( HoUTinUNoe( B,( Carbo( C,( et( al.( Inflammation( induces( hemorrhage( in( thrombocytopenia.(blood(2008;111:4958u4964.( 15.( ( Tantawy( AA,( Matter( RM,( Hamed( AA,( et( al.( Platelet( microparticles( in( immune( thrombocytopenic(purpura(in(pediatrics.(pediatr(hematol(oncol(2010;27:283u296.( 16.( ( Jy( W,( Horstman( LL,( Arce( M,( et( al.( Clinical( significance( of( platelet( microparticles( in( autoimmune(thrombocytopenias.(j(lab(clin(med(1992;119:334u345.(

23 18 Figures( Fig$1:$Mean(DOGiBAT(scores(on(enrolment(into(the(study.(PI( (primary(immune?mediated;(si( (secondary(immune? mediated;(ni( (non?immune?mediated.( ( ( ( $ $ $$$$$$$$$$$$$$(A)( ( ( ( ( (((((((((((((((B)( $ Fig$2:$$Daily(DOGiBAT(score(versus(platelet(count((platelets/μL)(when(all(scores(and(platelet(counts(included((A)( and(when(only(dogibat(scores(on(days(where(platelet(count(<(30,000/μl(included((b).(

24 19 Tables( Table&1.Thecanineimmunethrombocytopeniadailybleedingassessmenttool(DOGiBAT). Site( Skin$$ Catheter/venipuncture/ other$cutaneous$bleed$ Bleeding$grade( ( 0$ 1$ 2$ No( No( Petechiae(/( ecchymoses( Single(site( Self?limiting(/(( <(5(min( Petechiae/( ecchymoses(( >(1(anatomic(site( >(5(min(and/or( intervention(to( control( Oral$mucosa$$ No( Petechiae(( Frank(hemorrhage( Intraocular$$ No( Funduscopic(( Hyphema( Epistaxis$$ Gastrointestinal$$ Urinary$$ Pulmonary$ (suspected/observed)$ Intracranial$ hemorrhage$ (suspected/observed)$ No( Occult(blood?( (Hema?chek ( Guaiac(?)( No( Unilateral(and(( <(5(min( Occult(blood+((Hema? chek ( Guaiac(+)( Microscopic( (dipstick)( Bilateral(or(( >(5(min( Hematemesis,( hematochezia,(( melena( Macroscopic( No( N/A( Yes( No( N/A( Yes(

25 20 CHAPTER3.COATEDPLATELETPOTENTIALINCANINECANCERPATIENTS KellyM.Makielski,UnityJeffery,LeslieE.Fox,ChadM.Johannes,AshleyJ.Graef,JongH.Kim, BrianD.Husbands,JaimeF.Modiano,DanaN.LeVine ApapertobesubmittedtotheJournalofVeterinaryInternalMedicine Abstract Introduction:Neoplasiaisassociatedwithanincreasedriskofthrombosis.Coated plateletsareaplateletsubpopulationformedbystimulationwiththrombinandaglycoprotein (GP)VIagonist,suchasconvulxin.TheydisplayincreasedproTcoagulantsurfaceproteinsand enhancedthrombingeneration,andincreasedcoatedplateletsareassociatedwiththrombosis inhumans.wehypothesizedthatdogswithneoplasiaproducemorecoatedplateletsthan healthycontroldogs. Materials&Methods:ClientTowneddogsdiagnosedwithlymphoma/leukemia(n=20)or solidtumors(n=14)werecomparedwithhealthycontrols(n=14).plateletswerestimulatedex# vivowiththrombinandconvulxin.flowcytometrywasusedtoquantifythepercentageof coatedplateletsbasedonhighlevelsofsurfacefibrinogen.ananovafollowedbyan uncorrectedfisher slsdtestwasusedtocomparecoatedplateletpercentagesbetweenthe groups. Results:Healthycontroldogs(mean30.81%;range %)produced significantlylowercoatedplateletsthandogswithsolidtumors(mean41.7%;range %;p=0.0328),butnotdogswithlymphoma/leukemia(mean31.4%;range %;

26 21 p=0.8974).whendogsinremissionwereexcluded,therewasnosignificantdifferencein percentageofcoatedplateletsbetweenhealthycontrols(mean30.81%)anddogswithsolid tumors(n=12;mean41.39%;p=0.0542)orlymphoma/leukemia(n=13;mean28.4%;p= 0.646). Conclusions:Thisdatasuggeststhatcoatedplateletpotentialmaybehigherindogs withsolidtumorscomparedtohealthycontrols.furtherstudiesinvestigatingcoatedplatelets inspecificsubsetsofneoplasiaandinvestigatingadditionalmechanismsofhypercoagulabilityin dogswithneoplasiaarewarranted. Introduction Dogsandhumanswithneoplasiahaveanincreasedriskofvenousthromboembolism (VTE),whichhasthepotentialtosignificantlyimpactmorbidityandmortality.Humancancer patientshavea4tto7tfoldincreasedrelativeriskofvtecomparedtohealthyhumans. 1T6 While VTEeventscanbeasymptomaticanddiscoveredincidentallyduringcancerstaging, 1,7T10 they canalsocontributesignificantlytopatientmorbidityandmortality.mortalityinhumancancer patientswithahistoryofvtewas26%,comparedto4%inthosewithoutdocumentedvte. 11 VTEhasbeenidentifiedasoneoftheleadingcausesofmortalityinhumancancerpatients. 11,12 Inaprospectiveobservationalstudyofcausesofmortalityinhumancancerpatientsreceiving chemotherapy,thrombosiswasthesecondmostcommoncauseofmortalitybehind progressionofcancer. 12 Neoplasiaisthemostcommonunderlyingdiseaseindogswith documentedthromboticevents,bothvenousandarterial. 13T18 Theexactmechanismsofthe hypercoagulabilityassociatedwithneoplasiaareincompletelyunderstood,andarelikely multifactorial.

27 22 Coatedplateletsareasubpopulationofplateletsthatareformedex#vivoafterdual stimulationwiththrombinandaglycoprotein(gp)viagonist.thesurfaceofcoatedplateletsis coatedwithprotcoagulantglycoproteinsandalphagranulecontents,bridgedbyserotonin. 19 Coatedplateletshaveanenhancedabilitytogeneratethrombinproduction,makingthem highlyactivated.thisamplifiedthrombinproductionmayplayaroleinthepropagationofvte. Increasedcoatedplateletpotentialisassociatedwithanincreasedriskofthromboembolic disease.humanswithischemicstrokehistoryproduceagreaterpercentageofcoatedplatelets whencomparedwithhealthycontrols. 20T22 Conversely,decreasedcoatedplateletpotentialis associatedwithincreasedclinicalbleeding.humanswithhemorrhagicstrokehistoryform decreasedcoatedplateletscomparedwithhealthycontrols. 23 DogswithcanineScottsyndrome haveadecreasedabilitytoformcoatedplatelets,whichisassociatedwithincreasedclinical bleeding. 24 Additionally,humansonserotoninTspecificreuptakeinhibitors(SSRIs)havea tendencytoformfewercoatedplatelets. 25 HumansonSSRIsalsohaveamildincreasein bleedingtendencyandamildlyreducedriskofvte,likelyduetotheroleofserotonininthe formationofcoatedplatelets. TheroleofcoatedplateletsinVTEindogswithneoplasiaisnotknown.Ifcoatedplateletsarefoundtoplayarole invteindogswithneoplasia,theycouldrepresentapotentialtherapeutictarget. Theprimaryobjectiveofthisstudywastoevaluatecoatedplateletpotentialindogs withneoplasiacomparedwithhealthycontroldogs.itwashypothesizedthatdogswith neoplasiawouldproduceagreaterpercentageofcoatedplateletswhencomparedwithhealthy controldogs.

28 23 MaterialsandMethods Animals# ClientTowneddogspresentingtoaveterinaryteachinghospitalwithacytologicor histopathologicdiagnosisoflymphoma/leukemiaorsolidtumors(e.g.carcinoma,sarcoma, etc.)wererecruitedforinclusioninthestudy.stafftandstudenttowneddogsthatwerefreeof diseaseandnotonanymedicationsotherthanstandardpreventativetreatmentswere recruitedashealthycontroldogs.dogswereexcludedfromenrollmentinanygroupifbody weightwaslessthan5kgoriftheyhadreceivedanyanticoagulantorantiplateletmedications (e.g.nontsteroidalantitinflammatorymedications)withinthe96hoursprecedingsampling.all diagnosticandtherapeuticplanswereatthediscretionoftheattendingclinician.dogswere includedregardlessofdiseasestatus,butwereseparatedintogroupsbasedontheirclinical remissionstatusfordataanalysis(eitherremissionornontremission).dogsinthelymphoma groupwereclassifiedwithregardstoclinicalstageandsubstageaccordingtotheworldhealth Organization(WHO)clinicalstagingsystemforlymphomaindomesticanimals(Table1).Stage wasdeterminedbasedonphysicalexamandstandarddiagnostictesting.manydogsdidnot havecompletestagingperformed,andwerecategorizedaccordingtoavailableinformation. Substagewasdeterminedbytheattendingclinician.Dogswereincludedindependentof whethertheywerereceivingchemotherapytreatment.allprocedureswereapprovedbythe InstitutionalAnimalCareandUseCommitteesofIowaStateUniversityandtheUniversityof Minnesota. # #

29 24 Coated#platelet#assay## AsampleofvenousbloodwascollectedintoasyringecontainingacidTcitratedextrose (ACD).Samplesofserumandcitratedplasmawerealsoobtainedandbankedforlateruse.To generateplateletrichplasma(prp)forflowcytometryreactions,theacdbloodwascombined withanequalvolumeofbufferedsalineglucosecitrate(bsgc;129mmnacl,14mmna 3 citrate, 11mMglucose,10mMNaH 2 PO 4,pH7.3)andcentrifugedat170Gfor8minutesatroom temperature(21 C).ThesupernatantPRPwasharvestedandflowcytometryreactionswere performedwithin4hours. Allactivationexperimentswereperformedina100μl(total)assayvolumecontaining1 μlprpandthefollowingreagents(finalconcentrations):2.5mmcacl 2,1.25mMMgCl 2,1μg/ml biotintfibrinogen(giftofgeorgedale,universityofoklahoma),2.5μg/mlrattlesnaketoxin(gift ofgeorgedale,universityofoklahoma),0.5u/mlbovinethrombin,0.1mmglytprotargtprot NH 2,150mMNaCl,and10mMHEPES,pH7.5.Thereactiontubeswereincubatedat37 Cfor 10minutes,andindividuallylabeled,aspreviouslydescribed, 24,26T29 withthefollowingspecific markersofcoatedplatelets:phycoerythrintstreptavidin(petsa)andfluoresceinisothiocyanatet abciximab(fitctabx)tolabelsurfaceboundbiotinylatedfibrinogenandidentifyplatelets. FlowcytometrywasthenperformedonaBDFACSCcan(BDBiosciences,Spark,MD)or BDLSRII/Fortessa(BDBiosciences,Spark,MD)flowcytometer.Plateletswerefirstidentifiedby theforwardandsidescatterproperties(figure1a)andthenbasedonfitctabxpositivity (Figure1B).Withintheplateletpopulation,coatedplateletswereidentifiedandgatedbasedon theirpetsapositivity(figure1c).allflowcytometrydataanalysiswasperformedwithflowjo (TreeStarInc.,Ashland,OR).

30 25 Statistics# Samplesizeswerecalculatedpriortostudyinitiation.Samplesizeswerecalculatedto detecta10%differencebetweencasesandcontrols,adifferenceapproximatetothat previouslydetectedbetweennormalpeopleandthosewithischemicstroke, 20T22 with80% powerandanalphaof0.05.calculationswereperformedusingthestandarddeviationofa publishedcoatedplateletassay. 30 Basedonthesecalculations,itwasdeterminedthateachof thethreegroups(healthycontroldogs,dogswithlymphomaorleukemia,anddogswithsolid tumors)neededtocontain13dogstodetectasignificantdifference. Threeofthereactiontubescontainedidenticalreagents,andthereforewereconsidered technicalreplicatesandaveraged.resultswerediscardedifoneofthetechnicalreplicateswas consideredanoutliercomparedtotheothertwo(definedasadifferenceof>10%coated platelets)orifassayfailureoccurred. AD AgostinoandPearsontestfailedtorejectnormality.AnANOVAfollowedbyan uncorrectedfisher slsdtestwasusedtocomparethepercentageofcoatedplateletsbetween thethreegroups.ptvaluesetat0.05. AMannTWhitneytestwasusedtocompareplateletcountsbetweenlymphomadogs anddogswithsolidtumors,usingaptvalueof0.05. Results Intotal,54dogswerescreenedforenrollmentinthestudyand48wereenrolled.The 48dogsincludedinthestudyconsistedofhealthycontrols(n=14),dogswithadiagnosisof lymphoma/leukemia(n=20),anddogswithsolidtumors(n=14).clinicalcharacteristicsofthe healthycontroldogsarelistedintable2.thehealthycontroldogshadameanageof5.0years

31 26 (range1 9years),andconsistedof7spayedfemales(50%)and7castratedmales(50%). Breedsrepresentedinthehealthycontrolgroupweremixedbreed(n=3),Australianshepherd (n=2),pitbullterrier(n=2),labradorretriever(n=2),and1eachofgermanwirehairedpointer, roughtcoatedcollie,goldenretriever,greyhound,andbordercollie. Clinicalcharacteristicsofthelymphoma/leukemiadogsarelistedinTable3.Ofthedogs inthelymphoma/leukemiagroup,19/20hadlymphomaand1/20hadleukemia.ofthedogs withadiagnosisoflymphoma/leukemia,13wereconsideredtohaveclinicaldiseaseatthetime ofenrollmentand7wereconsideredinclinicalremission.dogsinthelymphomagrouphada meanageof9.2years(range3 15years),andconsistedof9spayedfemales(45%),9 castratedmales(45%),1intactfemale(5%),and1intactmale(5%).breedsrepresentedinthe lymphomagroupincludedbeagle(n=2),goldenretriever(n=2),and1eachofvizsla,bernese mountaindog,mixedbreed,doberman,jackrussellterrier,bordercollie,boxer, Newfoundland,standardpoodle,Cairnterrier,Germanshorthairpointer,ChowChow,Cocker spaniel,germanshepherd,bassethound,andirishsetter.diagnosisoflymphomawasmost commonlybasedonacombinationofcytologyandclonalitytesting(n=13),followedby histopathology(n=5),thencytologyalone(n=2).themostcommonwhostageatthetimeof diagnosisoflymphomawasstageiii(n=9),followedbystagev(n=5),stageiv(n=2),andstage II(n=2).Atthetimeofdiagnosis,10dogswereconsideredsubstageb,and9dogswere substagea.onedoghadchroniclymphocyticleukemia(cll),andthereforethewhoclinical StagingSystemwasnotapplicable.ImmunophenotypingwasconsistentwithBTcellneoplasiain 10casesandTTcellneoplasiain5cases.Immunophenotypewasnotavailablein4dogswith lymphoma.theonedogwithadiagnosisofcllhadanimmunophenotypeconsistentwithtt

32 27 cellleukemia.twelveofthelymphomadogswerereceivingchemotherapyatthetimeof enrollment,including5ofthe13dogsthatwerenotinclinicalremissionandallofthe7dogs thatwereinclinicalremission.themostcommonchemotherapyprotocolwasthechop protocol(n=7).otherchemotherapyusedincludedamodifiedmoppprotocol,chlorambucil, vincristineandcyclophosphamide,doxorubicinandccnu,anddoxorubicinalone,eachusedin onedog.elevenofthedogsweretreatedwithsystemicglucocorticoids(prednisone(n=10)and prednisolone(n=1))aspartofthechopprotocol(n=7),moppprotocol(n=1),concurrentlywith chlorambucil(n=1),concurrentlywithdoxorubicin(n=1),andastheonlytreatment(n=1)atthe timeofenrollment. Ofthedogswithsolidtumors,12wereconsideredtohaveresidualdiseaseatthetime ofenrollmentand2wereconsideredinclinicalremission.clinicalcharacteristicsofthedogsin thesolidtumorgrouparelistedintable4.dogsinthesolidtumorgrouphadameanageof9.4 years(range5 13years),andconsistedof9castratedmales(64%),4spayedfemales(29%), and1intactmale(7%).breedsrepresentedinthesolidtumorgroupincludedmixedbreed (n=4),englishspringerspaniel(n=2),goldenretriever(n=2),and1eachofboxer,bostonterrier, Englishsetter,Germanshepherd,Labradorretriever,andAustralianshepherd.Diagnosisof neoplasiaindogswithsolidtumorswasbasedonhistopathology(n=7),cytology(n=2),orboth (n=2).onedogwasenrolledthathadapresumptivediagnosisofaninsulinomabasedon clinicalsigns,documentedhypoglycemiawithconcurrentelevatedbloodinsulinlevels,and evidenceofapancreaticmassoncomputedtomography. Dogswithsolidtumorshadavarietyofcytologicorhistopathologictumortypes, includinganalglandadenocarcinoma(n=3),insulinoma(n=2;1/2presumptiveand1/2

33 28 confirmedbasedonhistopathology),osteosarcoma(n=2),chrondrosarcoma(n=2),and1each ofmastcelltumor,nasaladenocarcinoma,hemangiosarcoma,oralmelanoma,oligodendroglial ganglioglioma,andclearcelladnexalcarcinoma.onedoghadanoralmelanomathathadbeen surgicallyexcisedwithincompletemarginsandananalglandadenocarcinomadiagnosedthe dayofstudyenrollmentandthereforehadnotbeentreated.twodogswerereceiving chemotherapeutictreatmentatthetimeofenrollment,includingonethathadpreviously receivedafullcourseoffractionatedradiationtherapythatwasreceivingcarboplatinforanal glandadenocarcinoma,andonedogwithhemangiosarcomathatwasreceivingdoxorubicinand animmunotherapeutictreatmentaspartofanongoingclinicaltrial.thedogthatwasreceiving carboplatinwasalsoreceivingmetoclopramideandmetronidazoleforchemotherapytrelated sideeffects.threedogswerereceivingsystemicglucocorticoids(prednisone(n=2)and prednisolone(n=1)). Treatmentdataforthedogsinthelymphomagroupandthedogsinthesolidtumor group,includingconcurrentmedicalconditionsandtreatmentsunrelatedtotheircancer,are locatedintables5(lymphoma/leukemiadogs)and6(solidtumordogs). Whenalldogswereincluded,thepercentageofcoatedplateletsproducedbydogswith solidtumors(mean41.7%;range %)wassignificantlyhigherthanthepercentage ofcoatedplateletsproducedbyhealthycontroldogs(mean30.81%;range %;p= ).However,therewasnosignificantdifferencebetweenthepercentageofcoated plateletsproducedbyhealthydogsanddogswithlymphoma/leukemia(mean31.4%;range %;p=0.8974)(Figure2A).

34 29 Whendogsinclinicalremissionwereexcluded,therewasnosignificantdifferencein percentageofcoatedplateletsbetweenhealthycontroldogs(mean30.81%)anddogswith solidtumors(n=12;mean41.39%;p=0.0542)orlymphoma/leukemia(n=13;mean28.4%;p= 0.646)(Figure2B). Theaverageplateletcountofthecasesincludedinthisstudywas333x10 3 /μl (lymphoma323x10 3 /μl;solidtumor349x10 3 /μl).therewasnosignificantdifferencein plateletcountbetweendogswithlymphomaanddogswithsolidtumors(p=0.215).ofthe lymphomadogs,3/20(15%)hadplateletcountsoutsideofthereferencerange(200t500x10 3 / μl);1/3wasbelowthereferencerangeand2/3wereabovethereferencerange.ofthesolid tumordogs,1/13(7.7%)hadaplateletcountabovethereferencerange;noneofthesolid tumordogshadaplateletcountbelowthereferencerange.onedoginthesolidtumorgroup didnothaveaplateletcountperformedonthedayofenrollment,buthistoricallyhadplatelet countswithinthenormalreferencerange.noneofthedogsincludedinthisstudyhadevidence ofthromboticeventsatthetimeofenrollment,historically,orafterenrollmentpriortothe completionofthismanuscript.withtheexceptionofthedogwithhematuria,whichwas presumedsecondarytoeitheraurinarytractinfectionorsterilehemorrhagiccystitissecondary tocyclophosphamide,nodogincludedinthisstudyhadevidenceofclinicalbleeding. Discussion Theprimaryobjectiveofthisstudywastoevaluatecoatedplateletpotentialindogs withneoplasiacomparedtohealthycontrols.thedatafromthisstudysuggeststhatcoated plateletpotentialindogswithsolidtumorsmaybehigherwhencomparedwithhealthy

35 30 controls.however,whendogsthatwereinclinicalremissionwereexcludedfromanalysis, therewasnosignificantdifferencebetweenthethreegroups. Hypercoagulabilityhasbeenwelldocumentedinbothhumanandcaninecancer patients.neoplasiawasfoundtobethemostcommonunderlyingdiseasefoundindogswith documentedpulmonarythromboembolism, 14,15 portalveinthrombosis, 16,18 andsplenicvein thrombosis. 31 Thoughthedatafromthisstudydidnotdocumentadefinitiveroleforcoated plateletsinallcanineneoplasia,thereisstillmuchtobelearnedregardingtheunderlying causesofthehypercoagulabilitythatisseenincancerpatients.intheveterinaryliterature, severalstudieshavedocumentedatendencytowardhypercoagulabilityincaninecancer patients.andreasenet#al.showedthathypercoagulabilitywasthemostcommonhemostatic abnormalitydocumentedincaninecancerpatients,with66%havingparametersconsistent withhypercoagulability.carcinomapatients,withtheexceptionofmammarycarcinomas,had increasedclotstrengthonthromboelastography(teg)andincreasedfibrinogen.dogswith distantmetastaseshadsignificantlyelevatedfibrinogenanddtdimerlevelscomparedtothose withonlylocaldisease. 32 AnotherstudyevaluatingTEGindogswithneoplasiafoundsimilar results,with50%ofdogswithmalignantdiseasehavingtegparametersconsistentwith hypercoagulability. 33 Adenocarcinomaswerethemostcommonneoplasiaassociatedwith hypercoagulabilityinthatstudy.kolet#al.#showedthat88%ofdogswithlymphomahaveteg parametersconsistentwithhypercoagulability,andthetendencytowardhypercoagulability persistedthroughouttreatmentandforuptoamonthafterclinicalremissionwasattained. Acceleratedclotformationattheendofthechemotherapyregimenwasassociatedwith decreasedsurvival. 34

36 31 Coatedplateletshavenotpreviouslybeenevaluatedincaninecancerpatients,and wereinvestigatedasapotentialmechanismforthedocumentedhypercoagulability.the currentstudyshowedthatdogswithsolidtumorsformedagreaterpercentageofcoated plateletscomparedtohealthycontrolsanddogswithlymphoma.however,whendogsthat wereinclinicalremissionwereexcluded,therewasnosignificantdifferencedetectedbetween thethreegroups.thereareseveralpotentialreasonsforthis.first,thedatausedforsample sizecalculationswasfromadifferentstudyutilizingaslightlydifferentassayforcoated platelets 30 withlowervariabilitycomparedtothisstudy. 30 Thiscouldhaveresultedinan underestimationofthesamplesizeneededtodetermineasignificantdifference,and subsequentunderpoweringofthisstudy.alternatively,thesignificantdifferencedetected couldbespurious,andthereisnotruedifferencebetweenthethreegroups,regardlessof remissionstatus. Therewasnosignificantdifferencebetweendogswithlymphomaandhealthydogswith regardstocoatedplateletpotential.thisissomewhatsurprising,giventhedocumented hypercoagulabilitythathasbeenshownindogswithlymphoma.reasonsforthiscouldinclude potentialunderpoweringofthestudyduetothehighassayvariabilityasmentionedprevious, oritcouldbethatthehypercoagulabilityseenwithlymphomaisduetoanothercomponentof thecoagulationsystem.furtherstudiesinvestigatingcoatedplateletsinmorehomogeneous populationsofdogswithneoplasiaarewarranted.ifcoatedplateletsaretrulyfoundtobe increasedindogswithneoplasia,theycouldrepresentanexcitingnewtherapeutictarget. Potentialmedicaltherapiestargetingcoatedplateletscouldincludeglycoprotein(GP)VI antagonists,toblockcoatedplateletformation,andssris.ssrisareparticularlyintriguingasa

37 32 potentialtherapy,astheyarereadilyavailableandarerelativelywellttoleratedbymostcanines withminimalsideeffects.therapiestargetingcoatedplateletsarelikelytohaveless hemorrhagicsideeffects,astheyaretargetingthisveryspecificsubsetofactivatedplatelets ratherthaninterferingwiththecoagulationcascade(suchaswithwarfarintbasedproducts). Thecasesenrolledwereveryheterogeneouswithregardstotreatmentstatus,with somedogshavinghadsurgicaltreatmentsoftheirneoplasia,dogsthatwerereceiving chemotherapy,dogsthatwerereceivingglucocorticoids,anddogsthatwerereceivingother medications.theeffectsofeachofthesetreatmentsoncoatedplateletsisnotcurrently known.itwasdecidedtoincludedogsthatwerereceivingchemotherapyandglucocorticoids becauseregardlessofwhetherunderlyingneoplasiaortreatmenteffectsaltercoatedplatelet levelsindogswithcancer,thepotentialimplicationforthromboticoutcomesremains. Endogenousandexogenousglucocorticoidshaveadocumentedassociationwith hypercoagulabilityinbothdogsandhumans. 35T38 Theeffectofglucocorticoidsoncoated plateletformationiscurrentlynotknown.humancancerpatientsreceivingchemotherapyhave anincreasedriskofthromboticdiseasecomparedtohumancancerpatientsthatarenot receivingchemotherapy. 3,12,39,40 Similarly,thrombosisisalsoaknownpotentialcomplication aftercancersurgeryinhumans. 41 Therewereseverallimitationstothisstudy.Thecasesincludedinthisstudywerevery heterogeneous.thedogsinthesolidtumorgrouphadavarietyofdifferenttumorsubtypes, anddogsinthelymphomagrouphadavarietyofdifferentanatomicsitesandclinical presentations.thesamplesizeforeachgroupwasrelativelysmall,especiallyconsideringthe heterogeneousnatureoftheenrolledcases.samplesizecalculationswereperformedpriorto

38 33 enrollment;however,thedatausedforsamplesizecalculationswasfromadifferentstudy utilizingaslightlydifferentassayforcoatedplatelets. 30 Thedatafromthatstudyhadlower variabilitycomparedwiththevariabilityincoatedplateletpercentageinthedogsenrolledin thisstudy. 30 Thesamplesizeswerealsocalculatedtodetectadifferenceof10%incoated plateletproduction.thispercentagewasconsideredclinicallysignificant,asthisisthe differenceincoatedplateletpercentagebetweenhealthyhumansubjectsandhumanswith historyofischemicstroke. 21,22 However,thedifferenceincoatedplateletpercentagerequired forclinicalsignificanceindogsiscurrentlyunknown.anotherlimitationofthisstudyisthatthe casesenrolledwereveryheterogeneouswithregardstodiseaseburdeninbothcancergroups, andwithregardstotumortypeinthesolidtumorgroup.futurestudiescomparingcoated plateletsinamorehomogeneouspopulationofdogswithneoplasiaarewarranted.thoughour exclusioncriteriaexcludeddogsthathadreceivedanticoagulantorantiplateletmedicationsin the96hoursprecedingsampling,wedidnotexcludedogsonmedicationsknowntoeffect coatedplateletformationinhumans(e.g.ssris).however,noneofthepatientsincludedin thisstudywereonmedicationsknowntoeffectserotonin.asmallnumberofdogsincludedin thisstudyhadconcurrentdiseases.thesedogswerenotexcludedastheconcurrentdiseases wereallconsideredtobechronicconditionsthatwerestableonmedicalmanagementas necessary.asneoplasiatendstobediagnosedinmiddletagedtogeriatricpatientsmoreoften, chronicconcurrentconditionsarerelativelycommonatthetimeofneoplasiadiagnosis. Insummary,thisdatasuggeststhatcoatedplateletpotentialmaybehigherindogswith solidtumorscomparedtohealthycontroldogs.furtherstudiesinvestigatingcoatedplatelet productioninmorespecificsubsetsofneoplasiaandinvestigatingadditionalmechanismsof

39 34 hypercoagulabilityindogswithneoplasiaarewarranted.itmayalsobewarrantedto investigatepotentialtherapiestomitigatetheformationofcoatedplateletsindogsatriskof thrombosis,suchasserotonintspecificreuptakeinhibitors.

40 35 References 1.##Connolly#GC,#Menapace#L,#Safadjou#S,#et#al.#Prevalence#and#clinical#significance#of#incidental#and#clinically# suspected#venous#thromboembolism#in#lung#cancer#patients.#clin#lung#cancer#2013;14:713g718.# 2.##CroninGFenton#DP,#Sondergaard#F,#Pedersen#LA,#et#al.#Hospitalisation#for#venous#thromboembolism#in# cancer#patients#and#the#general#population:#a#populationgbased#cohort#study#in#denmark,#1997g2006.#br#j# Cancer#2010;103:947G953.# 3.##Falanga#A.#The#incidence#and#risk#of#venous#thromboembolism#associated#with#cancer#and#nonsurgical# cancer#treatment.#cancer#invest#2009;27:105g115.# 4.##Falanga#A,#Russo#L.#Epidemiology,#risk#and#outcomes#of#venous#thromboembolism#in#cancer.# Hamostaseologie#2012;32:115G125.# 5.##Khorana#AA,#Connolly#GC.#Assessing#risk#of#venous#thromboembolism#in#the#patient#with#cancer.#J#Clin# Oncol#2009;27:4839G4847.# 6.##Sud#R,#Khorana#AA.#CancerGassociated#thrombosis:#risk#factors,#candidate#biomarkers#and#a#risk#model.# Thromb#Res#2009;123#Suppl#4:S18G21.# 7.##Di#Nisio#M,#Lee#AY,#Carrier#M,#et#al.#Diagnosis#and#treatment#of#incidental#venous#thromboembolism#in# cancer#patients:#guidance#from#the#ssc#of#the#isth.#j#thromb#haemost#2015;13:880g883.# 8.##Douma#RA,#Kok#MG,#Verberne#LM,#et#al.#Incidental#venous#thromboembolism#in#cancer#patients:# prevalence#and#consequence.#thromb#res#2010;125:e306g309.# 9.##Khorana#AA,#O'Connell#C,#Agnelli#G,#et#al.#Incidental#venous#thromboembolism#in#oncology#patients.#J# Thromb#Haemost#2012;10:2602G2604.# 10.##Menapace#LA,#Peterson#DR,#Berry#A,#et#al.#Symptomatic#and#incidental#thromboembolism#are#both# associated#with#mortality#in#pancreatic#cancer.#thromb#haemost#2011;106:371g378.# 11.##Monreal#M,#Falga#C,#Valdes#M,#et#al.#Fatal#pulmonary#embolism#and#fatal#bleeding#in#cancer#patients#with# venous#thromboembolism:#findings#from#the#riete#registry.#j#thromb#haemost#2006;4:1950g1956.# 12.##Khorana#AA,#Francis#CW,#Culakova#E,#et#al.#Thromboembolism#is#a#leading#cause#of#death#in#cancer# patients#receiving#outpatient#chemotherapy.#j#thromb#haemost#2007;5:632g634.#

41 36 13.##Boswood#A,#Lamb#CR,#White#RN.#Aortic#and#iliac#thrombosis#in#six#dogs.#J#Small#Anim#Pract#2000;41:109G 114.# 14.##LaRue#MJ,#Murtaugh#RJ.#Pulmonary#thromboembolism#in#dogs:#47#cases#(1986G1987).#J#Am#Vet#Med# Assoc#1990;197:1368G1372.# 15.##Johnson#LR,#Lappin#MR,#Baker#DC.#Pulmonary#thromboembolism#in#29#dogs:#1985G1995.#J#Vet#Intern#Med# 1999;13:338G345.# 16.##Respess#M,#O'Toole#TE,#Taeymans#O,#et#al.#Portal#vein#thrombosis#in#33#dogs:#1998G2011.#J#Vet#Intern#Med# 2012;26:230G237.# 17.##Palmer#KG,#King#LG,#Van#Winkle#TJ.#Clinical#manifestations#and#associated#disease#syndromes#in#dogs# with#cranial#vena#cava#thrombosis:#17#cases#(1989g1996).#j#am#vet#med#assoc#1998;213:220g224.# 18.##Van#Winkle#TJ,#Bruce#E.#Thrombosis#of#the#portal#vein#in#eleven#dogs.#Vet#Pathol#1993;30:28G35.# 19.##Dale#GL.#CoatedGplatelets:#an#emerging#component#of#the#procoagulant#response.#J#Thromb#Haemost# 2005;3:2185G2192.# 20.##Prodan#CI,#Dale#GL.#CoatedGplatelets#in#ischemic#stroke#G#potential#insight#into#the#etiology#of#stroke# subtypes.#int#j#stroke#2008;3:249g250.# 21.##Prodan#CI,#Joseph#PM,#Vincent#AS,#et#al.#CoatedGplatelets#in#ischemic#stroke:#differences#between#lacunar# and#cortical#stroke.#j#thromb#haemost#2008;6:609g614.# 22.##Prodan#CI,#Vincent#AS,#Dale#GL.#CoatedGplatelet#levels#are#elevated#in#patients#with#transient#ischemic# attack.#transl#res#2011;158:71g75.# 23.##Prodan#CI,#Stoner#JA,#Cowan#LD,#et#al.#Lower#coatedGplatelet#levels#are#associated#with#early#hemorrhagic# transformation#in#patients#with#nonglacunar#brain#infarction.#j#thromb#haemost#2010;8:1185g1190.# 24.##Brooks#MB,#Catalfamo#JL,#Friese#P,#et#al.#Scott#syndrome#dogs#have#impaired#coatedGplatelet#formation# and#calceingrelease#but#normal#mitochondrial#depolarization.#j#thromb#haemost#2007;5:1972g1974.# 25.##Prodan#CI,#Joseph#PM,#Vincent#AS,#et#al.#CoatedGplatelet#levels#are#influenced#by#smoking,#aspirin,#and# selective#serotonin#reuptake#inhibitors.#j#thromb#haemost#2007;5:2149g2151.# 26.##Alberio#L,#Safa#O,#Clemetson#KJ,#et#al.#Surface#expression#and#functional#characterization#of#alphaGgranule# factor#v#in#human#platelets:#effects#of#ionophore#a23187,#thrombin,#collagen,#and#convulxin.#blood# 2000;95:1694G1702.#

42 37 27.##Brooks#MB,#Catalfamo#JL,#Brown#HA,#et#al.#A#hereditary#bleeding#disorder#of#dogs#caused#by#a#lack#of# platelet#procoagulant#activity.#blood#2002;99:2434g2441.# 28.##Dale#GL,#Friese#P,#Batar#P,#et#al.#Stimulated#platelets#use#serotonin#to#enhance#their#retention#of# procoagulant#proteins#on#the#cell#surface.#nature#2002;415:175g179.# 29.##Remenyi#G,#Szasz#R,#Friese#P,#et#al.#Role#of#mitochondrial#permeability#transition#pore#in#coatedGplatelet# formation.#arterioscler#thromb#vasc#biol#2005;25:467g471.# 30.##Knudsen#T,#Kjalke#M,#Tranholm#M,#et#al.#Development#of#a#flow#cytometric#assay#for#detection#of#coated# platelets#in#dogs#and#evaluation#of#binding#of#coated#platelets#to#recombinant#human#coagulation#factor#viia.# Am#J#Vet#Res#2011;72:1007G1014.# 31.##Laurenson#MP,#Hopper#K,#Herrera#MA,#et#al.#Concurrent#diseases#and#conditions#in#dogs#with#splenic#vein# thrombosis.#j#vet#intern#med#2010;24:1298g1304.# 32.##Andreasen#EB,#Tranholm#M,#Wiinberg#B,#et#al.#Haemostatic#alterations#in#a#group#of#canine#cancer# patients#are#associated#with#cancer#type#and#disease#progression.#acta#vet#scand#2012;54:3.# 33.##Kristensen#AT,#Wiinberg#B,#Jessen#LR,#et#al.#Evaluation#of#human#recombinant#tissue#factorGactivated# thromboelastography#in#49#dogs#with#neoplasia.#j#vet#intern#med#2008;22:140g147.# 34.##Kol#A,#Marks#SL,#Skorupski#KA,#et#al.#Serial#haemostatic#monitoring#of#dogs#with#multicentric#lymphoma.# Vet#Comp#Oncol#2015;13:255G266.# 35.##Van#Zaane#B,#Nur#E,#Squizzato#A,#et#al.#Hypercoagulable#state#in#Cushing's#syndrome:#a#systematic#review.# J#Clin#Endocrinol#Metab#2009;94:2743G2750.# 36.##Klose#TC,#Creevy#KE,#Brainard#BM.#Evaluation#of#coagulation#status#in#dogs#with#naturally#occurring# canine#hyperadrenocorticism.#j#vet#emerg#crit#care#(san#antonio)#2011;21:625g632.# 37.##Kol#A,#Nelson#RW,#Gosselin#RC,#et#al.#Characterization#of#thrombelastography#over#time#in#dogs#with# hyperadrenocorticism.#vet#j#2013;197:675g681.# 38.##Park#FM,#Blois#SL,#AbramsGOgg#AC,#et#al.#Hypercoagulability#and#ACTHGdependent#hyperadrenocorticism# in#dogs.#j#vet#intern#med#2013;27:1136g1142.# 39.##Khorana#AA,#Kuderer#NM,#Culakova#E,#et#al.#Development#and#validation#of#a#predictive#model#for# chemotherapygassociated#thrombosis.#blood#2008;111:4902g4907.#

43 38 40.##Scappaticci#FA,#Skillings#JR,#Holden#SN,#et#al.#Arterial#thromboembolic#events#in#patients#with#metastatic# carcinoma#treated#with#chemotherapy#and#bevacizumab.#j#natl#cancer#inst#2007;99:1232g1239.# 41.##Trinh#VQ,#Karakiewicz#PI,#Sammon#J,#et#al.#Venous#thromboembolism#after#major#cancer#surgery:# temporal#trends#and#patterns#of#care.#jama#surg#2014;149:43g49.#

44 39 Figures( ( ( ( (A)( ( ( (((((((((((B)(( ( (((((((((C)( Figure'1.'Flow'cytometric'analyses'of'platelet'surface'fibrinogen'expression.(Dot(plots(show(platelet(rich(plasma( dual(stimulated(with(thrombin((0.5(u/ml)(and(rattlesnake(toxin((2.5(μg/ml),(and(double(labeled(to(detect(platelet( membrane(gp(ii b III a ((fluorescein(isothiocyanatejabciximab((fitcjabx))(and(biotinylated(fibrinogen((phycoerythrinj streptavidin((pejsa)).(((a(gate(is(constructed(around(the(characteristic(forward(and(side(scatter(of(platelets((a).(the( platelet(population((plt1)(is(then(further(determined(based(on(fitcjabx(labeling((b).((the(platelet(population((plt2)( is(then(further(separated(based(on(pejsa(labeling((c).((in(this(example,(30.5%(of(platelets(are(expressing( biotinylated(fibrinogen(on(their(surface(following(dual(stimulation(with(thrombin(and(rattlesnake(toxin((coats).( ( A * B % coats 40 % coats Healthy Lymphoma/leukemia Solid tumor 0 Healthy Lymphoma/leukemia Solid tumor ( Figure'2.'(Coated(platelet(percentages(in(healthy(dogs(compared(to(those(with(lymphoma/leukemia(and(solid( tumors(including(dogs(in(clinical(remission((a)(and(when(only(dogs(with(active(disease(were(included((b).( *p=0.0328( ( (