Received: 6 September 2017 | Revised: 9 January 2018 | Accepted: 7 March 2018

DOI: 10.1111/jvim.15130

Journal of Veterinary Internal Medicine

STANDARD ARTICLE

Characteristics of hemostasis during experimental Ehrlichia canis

infection

Sarah Shropshire | Christine Olver | Michael Lappin

Department of Clinical Sciences, Colorado

State University, Fort Collins, Colorado, CO Background: Ehrlichia canis infection in dogs can cause thrombocytopenia and clinical evidence of
bleeding. It is unknown why some dogs show signs of bleeding whereas others do not despite clin-
Correspondence ically relevant thrombocytopenia.
Sarah Shropshire, 300 W. Drake Road, Fort
Collins, CO 80523. Hypothesis/Objectives: Activated platelets, decreased fibrinolysis or both mitigate bleeding tend-
Email: [email protected] ency. Assess standard hemostatic variables, platelet dynamics, and specialized coagulation testing
in dogs experimentally infected with E. canis to evaluate this clinical discrepancy.
Funding information
Center for Companion Animal Studies,
Animals: Four healthy laboratory beagles.
Colorado State University, Fort Collins, CO
Methods: Dogs were given blood infected with E. canis IV. Platelet indices of activation, platelet
aggregometry, antiplatelet antibodies (percent IgG), complete coagulation panel, and thromboelas-
tography (TEG) were measured before inoculation and on weeks 1-8. Dogs were treated with
doxycycline at approximately 5 mg/kg PO q12h between weeks 3 and 4 (day 24). For each vari-
able, 1-way repeated measures analysis (1-way ANOVA) with post-hoc analysis was performed
with statistical significance set at P < .05.

Results: Dogs had significantly lower platelet counts, evidence of activated platelets, and antiplate-
let antibodies during E. canis infection. Dogs also appeared hypercoagulable and hypofibrinolytic
using TEG as compared with baseline, changes that persisted for variable amounts of time after
doxycycline administration. No overt signs of bleeding were noted during the study.

Conclusions and Clinical Importance: Activated platelets and a hypercoagulable, hypofibrinolytic

state could explain the lack of a bleeding phenotype in some dogs despite clinically relevant throm-
bocytopenia. Findings from our pilot study indicate that additional studies are warranted.

KEYWORDS
aggregometry, platelet, thrombocytopenia, thromboelastography

Abbreviations: a, rate of clot formation; AA, arachidonic acid; ADP, adenosine diphosphate; APTT, activated partial thromboplastin time; AUC, area under the
curve; AUCAA, AUC for AA; AUCADP, AUC for ADP; AUCsaline, AUC for saline control; CL30, clot lysis 30 minutes after MA; CL60, clot lysis 60 minutes after MA;
HCT, hematocrit; K, clotting time; L, total lysis; LY30, percent clot lysis 30 minutes after MA; LY60, percent clot lysis 60 minutes after the MA; MA, maximum
amplitude; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; MPC, mean platelet component concentration; MPM, mean
platelet mass; MPV, mean platelet volume; MRTG, maximal rate of thrombus generation; OSPT, one-stage prothrombin time; PCDW, platelet component
distribution width; PCT, plateletcrit; PDW, platelet volume distribution width; percent IgG, percent of immunoglobulin associated platelets; TEG,
thromboelastography; TF, tissue factor; TF-TEG, TF-activated TEG; TG, total thrombus generated; TF 1 tPA-TEG, TF-activated TEG 1 tissue plasminogen activator;
TMRTG, time to maximum rate of thrombus generation; MRL, maximum rate of lysis; TMRL, time to maximal rate of lysis; R, reaction time
.......................................................................................................................................................................................
.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited and is not used for commercial purposes. Copyright V C 2018 The Authors. Journal of Veterinary Internal

Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

1334 wileyonlinelibrary.com/journal/jvim
J Vet Intern Med. 2018;1–9. | 1
J Vet Intern Med. 2018;32:1334–1342.
wileyonlinelibrary.com/journal/jvim
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SHROPSHIRE et al.of Veterinary Internal Medicine SHROPSHIRE 1335
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1 | INTRODUCTION inoculation, the dogs were observed daily for clinical abnormalities and
blood samples were scheduled to be collected on weeks 1–8. In addition
Ehrlichia canis infection in dogs can manifest with clinical signs related to testing for infection, other assays performed included CBC (Siemens,
to bleeding and also commonly causes thrombocytopenia, particularly ADVIA 120 Hematology System, Erlangen, Germany), plasma fibrinogen
1,2
in the acute phase of infection. The exact mechanisms of bleeding concentration (Tcoag AMAX Destiny Plus, Bray, Wicklow, Ireland), tis-
and thrombocytopenia are unknown but thought to be related to proc- sue factor (TF)-activated TEG (TEG 5000 Thromboelastograph Hemo-
esses such as vasculitis and immune and nonimmune processes affect- stasis Analyzer, Haemonetics Corporation, Braintree, Massachusetts;
3
ing platelets. For example, several studies have documented TF-TEG), TF-activated TEG (TEG 5000 Thromboelastograph Hemosta-
antiplatelet antibodies in dogs with ehrlichiosis.4–9 Platelet dysfunction sis Analyzer, Haemonetics Corporation) with tissue plasminogen activa-
in infected dogs with antiplatelet antibodies also has been identified tor (tPA) added (TF 1 tPA-TEG), whole blood impedance platelet
and 1 study proposed that these antibodies may interfere with primary aggregometry (Multiplate 5.0 Analyzer, Diapharma Group Inc, West
3,10
hemostasis thus contributing to bleeding events. Despite these Chester, Ohio), and direct flow cytometry detecting percent of immuno-
processes, not all dogs infected with E. canis show signs of bleeding.1 globulin-associated platelets (percent IgG) and are described below. The
Currently, it is not clear why some dogs show signs of bleeding dogs were fasted for 10 hours before each analysis and
whereas other dogs do not despite clinically relevant thrombocytope- approximately 12 mL of blood from the jugular vein was drawn with a
nia. We hypothesize that platelets become activated during infection, 20 gauge needle using a vacutainer (BD Vacutainer Single Use Needle
blood clots become resistant to fibrinolysis or both, factors that could Holder, Franklin Lakes, New Jersey). The blood for TEG and platelet
prevent a bleeding phenotype. A study in dogs naturally infected with aggregometry assays was drawn from the other jugular vein with a 20
Babesia rossi identified the presence of large activated platelets based gauge needle. For the TEG and aggregometry assays, each blood collec-
on hematologic platelet indices. This finding was theorized to contrib- tion event had to have a single penetration of the vein with no re-
ute to the lack of bleeding seen in dogs despite severe thrombocytope- direction. The appropriate volumes of blood were placed into the fol-
nia.11 Another study in dogs showed that systemic inflammation is lowing tubes: EDTA (BD Vacutainer K2 EDTA 3.6 mg 2.0 mL tubes,
associated with decreased fibrinolytic activity as determined by throm- Franklin Lakes, New Jersey), red top (Covidien Monoject Blood Collec-
boelastography (TEG).12 This situation could help prevent bleeding tion Tube No Additive 5.0 mL tubes, Minneapolis, Minnesota; serum),
events in dogs affected by an inflammatory disease such as ehrlichiosis. 3.2% sodium citrate buffered (BD Vacutainer 3.2% sodium citrate tubes,
Therefore, the purpose of our study was to assess platelet indices of Franklin Lakes, New Jersey), and a heparin tube (Sarstedt lithium heparin
activation, platelet function as assessed by whole blood impedance pla- micro tube, Numbrecht, Germany), respectively, and were gently
telet aggregometry, percentage of immunoglobulin associated platelets inverted 5 times to allow proper mixing.
(percent IgG), and TEG measurements including velocity curve (Vcurve) Dogs that developed findings suggesting clinical ehrlichiosis were
variables in dogs experimentally infected with E. canis. to be treated with doxycycline at 5 mg/kg PO q12h for 4 weeks and
supportive care as indicated.
2 | MATERIALS AND METHODS
2.2 | Complete blood cell count and standard
2.1 | Experimental E. canis infection coagulation tests
This prospective study was approved by the Institutional Animal Care
A CBC was performed on each sample collection day and the following
and Use Committee and used 4 healthy purpose-bred beagles and 1
variables were recorded: hematocrit (HCT), mean corpuscular volume
client-owned dog that was clinically normal, but positive for E. canis
(MCV), mean corpuscular hemoglobin concentration (MCHC), platelet
DNA in blood13 and Ehrlichia spp. antibodies in serum (SNAP 4Dx Plus,
count, mean platelet volume (MPV), plateletcrit (PCT), platelet volume
IDEXX Laboratories, Westbrook, Maine). The beagles were castrated
distribution width (PDW), mean platelet mass (MPM), mean platelet
males with a weight range of 13.8–15.7 kg and age range of 21–23
component concentration (MPC), and platelet component distribution
months at the start of the study. The beagles were housed under the
width (PCDW). Activated partial thromboplastin time (APTT; Tcoag
same conditions, were not receiving any medications, and did not have
AMAX Destiny Plus), one-stage prothrombin time (OSPT; Tcoag AMAX
a history of previous medication administration. Samples from all 4
Destiny Plus), and antithrombin activity (Tcoag AMAX Destiny Plus)
dogs were tested initially and at each week (week 1–8) for antibodies
were measured at week 0 and weeks 2–6 in all dogs. D-dimer concen-
against Anaplasma spp., Borrelia burgdorferi, and E. canis/E. ewingii, anti-
trations (Tcoag AMAX Destiny Plus) were measured at week 0 and
gens of Dirofilaria immitis, and DNA of Anaplasma spp., Babesia spp.,
weeks 2–8 in all dogs. Plasma fibrinogen concentration (Tcoag AMAX
Bartonella spp., Ehrlichia spp., the hemoplasmas, Neorickettsia spp., and
Destiny Plus) was measured at all time points (weeks 0–8).
Rickettsia spp. (SNAP 4Dx Plus, IDEXX Laboratories; Veterinary Diag-
nostic Laboratory, Colorado State University, Fort Collins, Colorado).13
2.3 | Thromboelastography
A total of 8 mL of anticoagulated blood was collected from the
client-owned E. canis-infected dog and all 4 of the E. canis-naïve dogs For TF-TEG, citrated blood samples were allowed to sit at room tem-
were each given 2 mL of this blood IV via the cephalic vein. After perature for 30 minutes before analysis. Briefly, the cups were warmed
SHROPSHIRE
1336 ET AL.
Journal of Veterinary Internal Medicine SHROPSHIRE et| al3.

to 378C and 20 mL of 0.2 M CaCl2, 10 mL of TF (Tcoag TriniCLOT PT diluent and incubated for 3 minutes as recommended by the manufac-
Excel, Bray, Wicklow, Ireland) at a final dilution of 1:1000, and 330 mL turer (Multiplate 5.0 Analyzer, Diapharma Group Inc). Adenosine
of citrated whole blood were added to the cup and analyzed. TF was diphosphate (ADP; Diapharma Group Inc, West Chester, Ohio) and ara-
prepared before each individual TEG analysis. The TEG tracings then chidonic acid (AA; Diapharma Group Inc) were used as platelet agonists
were generated for at least 60 minutes and the variables reaction time and area under the curve (AUC) for ADP (AUCADP) and for AA (AUCAA)
(R), clotting time (K), rate of clot formation (a), and maximum amplitude were recorded. Note that the AUC value is an arbitrary unit. As recom-
(MA) were recorded. mended by the manufacturer (Multiplate 5.0 Analyzer, Diapharma
For TF 1 tPA-TEG, citrated blood samples were allowed to sit at Group Inc), the final concentration of ADP was 6.5 mM and the final
room temperature for 30 minutes before analysis. Briefly, the cups concentration of AA was 0.5 mM. To serve as a control and to evaluate
were warmed to 378C and 20 mL of 0.2 M CaCl2 and 10 mL of TF for spontaneous platelet aggregation, an identical volume of saline was
(Tcoag TriniCLOT PT Excel) at a final dilution of 1:1000 were added to used in place of the agonists (AUCsaline). The reagents were reconsti-
the cup. To prepare tPA (Cathflo Activase [Alteplase], 2 mg Vial, Carroll, tuted and stored according to the manufacturer’s recommendations
Ohio), the vial was reconstituted with sterile water resulting in 1.08 (Diapharma Group Inc) in 60 mL aliquots.
million units/mL. Then, 4.1 mL of the reconstituted tPA was added to
996 mL of a phosphate-buffered solution (PBS) to make the stock tPA 2.5 | Direct flow cytometry assay for platelet-
solution. The stock solution (10 mL) was added to 400 mL of citrated associated immunoglobulin
whole blood, mixed gently, and 330 mL of this mixture was added to
the cup and analyzed. The TF was prepared before each individual TEG The protocol used for our study was modified from other protocols

analysis and the tPA solution was kept on ice between analyses but previously described in the literature.13,14 Blood (700 mL) anticoagu-

was discarded after each testing period. The TEG tracings then were lated with EDTA was mixed with an equal volume of sterile PBS. This

generated for at least 60 minutes and the variables R, K, a, MA, percent mixture was centrifuged at 200g for 1 minute 30 seconds at 208C to

of clot lysis 30 minutes after MA is reached (LY30), amount of clot lysis generate platelet-rich plasma (PRP). Platelet-rich plasma was removed

30 minutes after MA is reached (CL30), percent of clot lysis 60 minutes from the erythrocyte layer and placed into an Eppendorf tube (Light
after the MA is reached (LY60), and amount of clot lysis 60 minutes Labs SNAPLOCK Microcentrifuge Tubes, Dallas, Texas). Each PRP sam-
after MA is reached (CL60) were recorded. Additionally, velocity curve ple was adjusted to 2 3 106 cells/mL using a manual hemocytometer
variables were recorded, denoted as the maximal rate of thrombus gen- to provide a standard volume of PRP that then was pelleted by centrif-
eration (MRTG), time to maximum rate of thrombus generation ugation at 1000g for 5 minutes at 208C. The platelets were resus-
(TMRTG), total thrombus generated (TG), maximum rate of lysis (MRL), pended and washed 3 times at the same speed in a solution containing
time to maximal rate of lysis (TMRL), and total lysis (L). Controls (Level I 3 mM EDTA, 1% bovine serum albumin (BSA), and PBS. Each sample
[Levels I and II controls; Haemonetics Corporation, Braintree, Massa- was incubated at room temperature with 50 mL of a 1:200 dilution of
chusetts] and Level II [Levels I and II controls; Haemonetics Corpora- fluorescein isothiocyanate (FITC)-labeled rabbit anti-dog IgG (FITC-con-
tion]) were performed at each time point as recommended by the jugated AffiniPure rabbit anti-dog IgG (H 1 L) Jackson ImmunoResearch
manufacturer (TEG 5000 Thromboelastograph Hemostasis Analyzer, Labs, 304-095-003, West Grove, Pennsylvania) for 30 minutes. After
Haemonetics Corporation) before analysis of the samples. incubation, the platelets were washed 3 times with PBS-EDTA-BSA
Hypercoagulability, using TF-TEG, was defined as a statistically solution and resuspended in 200 mL of PBS-EDTA-BSA solution for
higher MA value as compared to baseline (week 0). Hypofibrinolysis, flow analysis. Gate settings used for our study were previously estab-
using TF 1 tPA-TEG was defined as an LY30 or LY60 statistically lower lished with PE-labeled mouse anti-human CD61 (Anti-human CD61
or a CL30 or CL60 statistically higher than the baseline (week 0) result. [Integrin beta 3] PE, VI-PL2, 12–0619-42, eBioscience, San Diego, Cali-
From the velocity curve variables, hypercoagulability was defined as an fornia) using healthy dog samples. Platelets from a healthy beagle
MRTG or TG statistically higher or a TMRTG statistically lower than served as a negative control at each time point. Samples were consid-
the baseline (week 0) result. From the velocity curve variables, hypofi- ered positive if the percent IgG was > 2 standard deviations above the
brinolysis was defined as an MRL statistically lower or a TMRL statisti- reference range determined from the negative control and the 4
cally higher than the baseline value. healthy beagles before inoculation with E. canis. All samples were ana-
lyzed using a Cyan ADP instrument (Cyan ADP instrument, Beckman
2.4 | Whole blood impedance platelet aggregometry Coulter, Miami, Florida) and the generated data was analyzed using
FlowJo software (FlowJo software, Tree Star, Ashland, Oregon).
For the multiple channel electrical impedance platelet aggregometry
(Multiplate 5.0 Analyzer, Diapharma Group Inc), heparinized blood sam-
2.6 | Statistical analysis
ples were kept at room temperature and analyzed within 40 minutes of
blood collection (range, 29–40 minutes) for 12 minutes. Before each For each response variable, a repeated measures analysis (1-way
analysis, the test cells were warmed to 378C within the aggregometer ANOVA) was performed using the lme4 and lmerTest packages in R (R
and an automated pipette was used to decrease preanalytical variabili- package, version 3.2.2). To account for repeated measures, dog was
ty. Heparinized blood samples (300 mL) were diluted with 300 mL of included as a random effect. Time points were compared with week 0
4 | Journal
SHROPSHIRE et al.of Veterinary Internal Medicine SHROPSHIRE 1337
ET AL.

T AB LE 1 Dunnett’s test results for the TF-activated TEG, TF 1 tPA TEG, and whole blood impedance platelet aggregometry variables in bea-
gles experimentally infected with E. canis

Measurement Time different from Week 0 Time different from Week 3

TF-activated R (min) Week 2, 3, 4, 5 Week 0, 8

TF-activated K (min) Week 1, 2, 3, 4, 5 Week 0, 2, 8

TF-activated a (degrees) Week 1, 2, 3, 4, 5 Week 0, 1, 2, 7, 8

TF-activated MA (mm) Week 1, 2, 3, 4, 5 Week 0, 1, 2, 6, 7, 8

TF 1 tPA R (min) None Week 1, 8

TF 1 tPA K (min) Week 3 None

TF 1 tPA a (degrees) Week 3, 4 Week 0, 1

TF 1 tPA MA (mm) Week 3, 4, 5 Week 0, 1, 2, 7, 8

TF 1 tPA LY30 (%) Week 1, 2, 3, 4, 5 Week 0

TF 1 tPA CL30 (%) Week 1, 2, 3, 4, 5 Week 0, 8

TF 1 tPA LY60 (%) Week 1, 2, 3, 4, 5 Week 0, 1, 2, 8

TF 1 tPA CL60 (%) Week 3 Week 0, 1

MRTG (dcs) Week 3, 4, 5 Week 0, 1, 2, 8

TMRTG (min) None None

TG (dcs) Week 3, 4, 5 Week 0, 1, 2, 5, 6, 7, 8

MRL (dcs) None None

TMRL (min) Week 2 Week 2

L (dcs) Week 3, 4 Week 0, 1, 2, 7, 8

AUCsaline None None

AUCADP Week 2, 3 Week 5, 6, 7, 8

AUCAA Week 2, 3 Week 0, 4, 5, 6, 7, 8

Abbreviations: a, rate of clot formation; AA, arachidonic acid; ADP, adenosine diphosphate; AUCAA, area under the curve for AA; AUCADP, area under
the curve for ADP; AUCsaline, area under the curve for saline; CL30, amount of clot lysis 30 minutes after MA is reached; CL60, amount of clot lysis 60
minutes after MA is reached; K, clotting time; L, total lysis; LY30, percent of clot lysis 30 minutes after MA is reached; LY60, percent of clot lysis 60
minutes after the MA is reached; MA, maximum amplitude; MRL, maximum rate of lysis; MRTG, maximal rate of thrombus generation; R, reaction time;
TG, total thrombus generated; TMRL, time to maximal rate of lysis; TMRTG, time to maximum rate of thrombus generation.

using Dunnett’s method with the lsmeans package in R (R package, ver- within 4 days of doxycycline administration and all dogs became PCR
sion 3.2.2). To investigate the effects of doxycycline on the measured (Veterinary Diagnostic Laboratory, Colorado State University) negative
variables, an additional analysis was performed where time points were for E. canis at week 5 and remained PCR negative for the duration of
compared with week 3 using Dunnett’s method with the lsmeans pack- the study. None of the dogs developed positive test results for any
age in R (R package, version 3.2.2). For all tests, a P < .05 was consid- other tested infectious agent during the course of the study.
ered significant. The repeated measures 1-way ANOVA for the TF-TEG, TF 1 tPA-
TEG, and whole blood impedance platelet aggregometry identified sta-
3 | RESULTS tistical differences for all variables with the exception of the fibrinolysis
value MRL and the platelet aggregometry value, AUCsaline. Note that
No complications were observed after the inoculations. All dogs inocu- TEG in dogs can be performed using an activator (eg, TF, kaolin) or
lated from the client-owned donor dog became persistently positive without an activator (native); however, it is recommended to use an
for E. canis/E. ewingii antibodies in serum (SNAP 4Dx Plus, IDEXX Lab- activator such as TF or kaolin.15 Tissue factor was chosen as the activa-
oratories Inc) and were positive by PCR (Veterinary Diagnostic Labora- tor for our study because the reference ranges used at our institution
tory, Colorado State University) for E. canis by week 2. At week 3 after are based on TF-activated TEG analyses.
inoculation, all 4 of the dogs had lost weight, were hyporexic and sub- Several statistically significant differences from baseline were iden-
jectively lethargic. Thus, all dogs were treated with doxycycline at tified in all dogs for TEG and platelet aggregometry at multiple time
approximately 5 mg/kg, PO, q12h for 4 weeks starting between weeks points (Table 1) with the exception of only 4 variables: TF 1 tPA-TEG
3 and 4 (day 24) of the study. All 4 dogs became clinically normal variables R, TMRTG, and MRL and platelet aggregometry variable
SHROPSHIRE
1338 ET AL.
Journal of Veterinary Internal Medicine SHROPSHIRE et| al5.

F I G U R E 2 Mean MRTG and mean TMRL from TF 1 tPA TEG over

FIGURE 1 Mean MA values and mean percent clot lysis 30
time in beagles experimentally infected with E. canis. *The vertical
minutes after MA (LY30) from TF 1 tPA TEG over time in beagles
solid line represents when all dogs were administered 5 mg/kg PO,
experimentally infected with E. canis. *The vertical solid line
twice daily of doxycycline
represents when all dogs were administered 5 mg/kg PO, twice
daily of doxycycline
with the exception of PDW. Multiple statistically significant differences
from baseline in all dogs for the hematology and flow cytometry varia-
AUCsaline. Similarly, Dunnett’s method identified multiple statistically
bles at multiple time points (Table 2), with the exception of only 4 vari-
significant differences from week 3 in all dogs at multiple time points
ables: OSPT, PDW, MPC, and PCDW, were observed. Similarly,
(Table 1) with the exception of 4 variables: TF 1 tPA-TEG variables K,
Dunnett’s method identified multiple statistically significant differences
TMRTG, and MRL and platelet aggregometry variable AUCsaline. For
from week 3 in all dogs at multiple time points (Table 2) with the
TF-TEG, dogs had shorter R times, smaller K values, larger a values,
exception of PDW. Overall, dogs had lower antithrombin concentra-
and higher MA values as compared with baseline at the respective time
tions, platelet counts, PCT, HCT, and MCHC as compared to baseline
points (Table 1). For TF 1 tPA-TEG, dogs had smaller values for K,
at the respective time points (Table 2). In contrast, dogs had higher D-
LY30, LY60, and higher values for a, MA, CL30, CL60, MRTG, TG,
dimers, OSPT, plasma fibrinogen concentration, MPV, MPM, MCV, and
TMRL, and L as compared with baseline at the respective time points
percent IgG as compared to baseline at the respective time points
(Table 1). After doxycycline administration on day 24, several statistical
(Table 2). The average plasma fibrinogen concentrations and average
differences were noted as compared to week 3 (Table 1). Overall, for
MPM values over time in relationship to doxycycline administration are
TF-TEG, at week 3, dogs had shorter R times, smaller K values, larger a
presented in Figure 3. After doxycycline administration on day 24, sev-
values, and larger MA values as compared to other time points. Overall,
eral statistical differences were noted as compared with week 3. Over-
for TF 1 tPA-TEG, at week 3, dogs had smaller values for R, K, LY30,
all, at week 3, dogs had lower antithrombin concentrations, platelet
LY60, and TMRL and higher values for a, MA, CL30, CL60, MRTG, TG,
counts, PCT, MPC, HCT, and MCHC as compared with other time
and L as compared with the respective time points. The average MA
points (Table 2). In contrast, at week 3, dogs had higher D-dimers,
values and average LY30 values over time in relationship to doxycy-
OSPT, APTT, plasma fibrinogen concentrations, MPV, MPM, PCDW,
cline administration are presented in Figure 1. The average MRTG and
MCV, and percent IgG as compared with the respective time points
average TMRL values over time in relationship to doxycycline adminis-
(Table 2). The average platelet counts and average percent IgG over
tration are presented in Figure 2.
time in relationship to doxycycline administration are presented in
For platelet aggregometry, no significant differences were identi-
Figure 4.
fied in the values for AUCsaline as compared with baseline or week 3.
However, the AUCADP and AUCAA were significantly lower than base-
line at the respective time points (Table 1). To further evaluate the 4 | DISCUSSION
effect of doxycycline administration, for AUCADP, at week 3, dogs had
significantly lower values as compared to other time points (Table 1). Ehrlichia canis infects dogs worldwide and clinical manifestations can
Specifically, the AUCADP was statistically higher at weeks 5 (difference, include bleeding but it is unknown why some dogs do not exhibit signs
106.50; P-value, .042), 6 (104; 0.049), 7 (166.75; 0.001), and 8 (149.00; of hemorrhage despite clinically relevant thrombocytopenia.1 Overall,
0.002) as compared with week 3. Similarly, for AUCAA, at week 3, dogs we found using coagulation testing that the dogs became hypercoagu-
also had significantly lower values as compared to other time points lable and hypofibrinolytic based on TEG after becoming infected with
(Table 1). Specifically, the AUCAA was statistically higher at weeks 0 E. canis. Additionally, certain measures of platelet activation increase
(difference, 98.00; P-value, .009), 4 (86.00; 0.026), 5 (100.25; 0.007), 6 during infection, including during the thrombocytopenic phase.
(115.25; 0.002), 7 (150.00; <0.001), and 8 (149.25; <0.001) as com- The results from TF-TEG and TF 1 tPA-TEG showed that experi-
pared with week 3. mentally inoculated dogs appeared hypercoagulable as compared with
Results from the repeated measures 1-way ANOVA for the hema- baseline as defined by a higher MA value and hypofibrinolytic as
tology and flow cytometry were significantly different for all variables defined by a lower LY30 and LY60 and a higher CL30 and CL60.16
6 | Journal
SHROPSHIRE et al.of Veterinary Internal Medicine SHROPSHIRE 1339
ET AL.

T AB LE 2 Dunnett’s test results for hematology and flow cytometry variables in beagles experimentally infected with E. canis

Measurement Time different from Week 0 Time different from Week 3

Antithrombin activity (%) Week 3, 4 Week 0, 2, 4, 5, 6

D-dimers (ng/mL) Week 3 Week 0, 2, 4, 5, 6, 7, 8

APTT (s) Week 3 Week 0, 2, 4, 5, 6

OSPT (s) None Week 2, 4

Fibrinogen (mg/dL) Week 1, 2, 3, 4, 5 Week 0, 4, 5, 6, 7, 8

3
Platelet count (x 10 cells/mL) Week 1, 2, 3 Week 0, 5, 6, 7, 8

MPV (fL) Week 2, 3, 4 Week 0, 1, 5, 6, 7, 8

PCT (%) Week 1, 2, 3 Week 0, 4, 5, 6, 8

PDW (%) None None

MPM (pg) Week 1, 2, 3, 4 Week 0, 5, 6, 7, 8

MPC (g/dL) None Week 1, 8

PCDW (g/dL) None Week 1

Hct (%) Week 2, 3, 4, 5, 6, 7, 8 Week 0, 1, 2, 6, 7, 8

MCV (fL) Week 4, 5, 6 Week 2, 4

MCHC (g/dL) Week 3, 4, 5, 6 Week 0, 2

Percent IgG (%) Week 3 Week 0, 1, 4, 5, 6, 7, 8

Abbreviations: APTT, activated partial thromboplastin time; HCT, hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpus-
cular volume; MPC, mean platelet component concentration; MPM, mean platelet mass; MPV, mean platelet volume; OSPT, one-stage prothrombin
time; PCT, plateletcrit; PCDW, platelet component distribution width; PDW, platelet volume distribution width; percent IgG, percent of immunoglobulin
associated platelets.

Additional analyses were performed to compare week 3 results to statistically different from week 3 but did not return to baseline until
those from subsequent time points, because it was unknown if doxycy- week 8 with the exception of CL60. Thus, the results suggest that the
cline administration would affect TEG parameters. The time point week dogs were hypofibrinolytic until week 8 (Figure 1) approximately 5
3 was chosen for comparison because the dogs were most clinically weeks after doxycycline administration. The velocity curve variables
affected at this time and because doxycycline administration was are generated from the TEG tracing and represent the first derivative
started on day 24 of the study. After doxycycline administration, TF- of the associated waveform. These variables provide information
TEG MA and TF 1 tPA-TEG MA did not statistically differ from week 3 regarding clot formation and clot breakdown.16 The MRTG reflects the
until week 6 or week 7, respectively. After doxycycline administration, maximal velocity of increase in clot strength and observed clot growth
it took 2 weeks (week 6) for TF-TEG MA and 3 weeks (week 7) for whereas TMRTG reflects the time interval required to reach maximal
TF 1 tPA-TEG MA to return to baseline. Before doxycycline adminis- rate of clot formation.17,18 Total thrombus generated reflects amount
tration, the fibrinolysis variables LY30, CL30, LY30, and CL60 all were of clot strength produced and represents total area under the velocity

F I G U R E 3 Mean fibrinogen and MPM over time in beagles F I G U R E 4 Mean platelet counts and mean percent IgG over time
experimentally infected with E. canis. *The vertical solid line in beagles experimentally infected with E. canis. *The vertical solid
represents when all dogs were administered 5 mg/kg PO, twice line represents when all dogs were administered 5 mg/kg PO,
daily of doxycycline twice daily of doxycycline
SHROPSHIRE
1340 ET AL.
Journal of Veterinary Internal Medicine SHROPSHIRE et| al7.

curve during clot formation.18 As a result, an increased MRTG, 2, 3, and 4 (increased MPM) as compared with baseline. After doxycy-
decreased TMRTG, and increased TG could represent a hypercoagu- cline administration, MPV and MPM returned to baseline and were stat-
lable state.17,19 With regards to clot breakdown, MRL, TMRL, and L can istically different when results from week 3 and week 5 were compared.
be evaluated. The MRL reflects maximal velocity of clot breakdown, The dogs were noted to have significant decreases from their baseline
TMRL reflects the time interval required to reach maximal rate of clot platelet counts during weeks 1, 2, and 3 and had platelet counts that
lysis, and L represents total amount of clot lysis. Therefore, an ranged from 20 to 100 3 103/mL. Therefore, there was evidence of pla-
increased MRL, decreased TMRL, and increased L could represent a telet activation during the periods of most severe thrombocytopenia,
hyperfibrinolytic state and vice versa for a hypofibrinolytic state.20 but it is also plausible that these changes could have been related to
Overall, velocity curve results aligned with traditional TEG results in increased platelet release from the bone marrow as a response to
that the dogs appeared hypercoagulable and hypofibrinolytic. When thrombocytopenia. The differentiation between these 2 possibilities
evaluating the results from the dogs as compared with baseline, the was not possible in our study. Because there was evidence of potential
dogs appeared hypercoagulable based on significantly larger MRTG platelet activation during these time periods, such a situation could have
and TG values (at weeks 3, 4, and 5) and also demonstrated hypofibri- contributed to why no bleeding events were observed despite the dogs
nolysis based on a longer TMRL value (at week 2). It also was observed having significant thrombocytopenia.
that the dogs appeared hypercoagulable by week 3 (increased MRTG) Possible causes for thrombocytopenia and subsequent bleeding in
and week 5 (increased TG) and these changes persisted and did not E. canis infected dogs include destruction of platelets by platelet-
return to baseline until week 8. The cumulative findings from both TEG directed antibodies, increased clearance of platelets from circulation by
analyses and from velocity curve results indicated that dogs were the spleen and sequestration as a result of vasculitis.4–7,9,23 The per-
hypercoagulable and hypofibrinolytic at certain time points over the cent IgG was statistically higher than baseline at week 3 but statistically
course of infection and that these changes persisted for variable significant thrombocytopenia also was identified at weeks 1, 2, and 3.
amounts of time after doxycycline administration.16 A hypercoagulable This finding is in agreement with other studies in which the thrombocy-
and hypofibrinolytic state could result in a hemostatic phenotype topenia observed in E. canis infections likely was caused by several
rather than a bleeding phenotype, which could help explain why bleed- mechanisms and not solely platelet-directed antibodies. Interestingly,
ing may not be observed in dogs infected with E. canis despite severe the percent IgG decreased after administration of doxycycline in all
thrombocytopenia. dogs and they all were considered negative by week 5 (Figure 2). This
The overall function of platelets also could explain why bleeding is finding was surprising because the half-life of most of the immunoglob-
or is not observed in E. canis infection. If platelets are in an activated ulin G (IgG) subclasses in dogs is estimated to be similar to that of
state, they will function in primary hemostasis to prevent bleeding.21 humans, which is approximately 20–21 days. Four subclasses of IgG
However, if a thrombocytopathy is present, bleeding may be observed occur in both humans and dogs, but they are categorized differently.24
regardless of the platelet count, but particularly if the platelet count is A previous study reported that natural and experimental E. canis infec-
low. We investigated platelet function using both whole blood imped- tions in dogs are associated with a predominance of IgG2 rather than
ance aggregometry and parameters measured on the Advia 120 hema- IgG1.25 Immunoglobulin G2 is the functional human analog to the
tology analyzer. The platelet aggregometry was normal except for time canine IgG subclass A which is not thought to be involved in antibody-
periods when the dogs were thrombocytopenic. Significant decreases in dependent cell-mediated cytotoxicity or complement-dependent cyto-
platelet count were observed by week 1 and remained statistically lower toxicity, but research is ongoing to better characterize the canine IgG
than baseline until week 4 and similarly, AUCADP and AUCAA were stat- subclasses.24 The rapid decrease in percent IgG after doxycycline may
istically lower than baseline at weeks 2 and 3. Additionally, the initiation be related to other nonantimicrobial effects of this drug. For example,
of doxycycline administration between weeks 3 and 4 likely affected it has been previously documented that dogs infected with E. canis and
the platelet count and resultant AUC readings.22 The results show that healthy dogs had an increase in platelet counts suggesting that doxycy-
platelet count returned to baseline by week 5 (Figure 2) and AUC cline may result in platelet proliferation.22 Additionally, doxycycline has
returned to baseline by weeks 4 and 5, approximately 1–2 weeks after been shown to decrease antibody production and inhibit proliferating
doxycycline administration. Therefore, no evidence of platelet dysfunc- lymphocytes.22,26 It also has been shown to decrease the expression of
tion was detected before thrombocytopenia was documented or once IgG on granulocytes, but it is unknown if this occurs with platelets.27
the platelet count started to rebound and normalize. Additionally, plate- Therefore, if the platelets have decreased survival in circulation
let activation parameters were increased. Therefore, another reason because of antibody binding but doxycycline causes proliferation of
why dogs infected with E. canis may not show evidence of bleeding platelets and decreased antibody production, this could explain why
despite severe thrombocytopenia is if platelets are in an activated state. the percent IgG decreased so rapidly. It is currently unknown if doxycy-
Variables indicating increased platelet activity include increased MPV, cline decreases IgG expression on platelets similar to granulocytes or if
11
MPM, and PCDW and decreased MPC. These changes occur in the it interferes with antibody-antigen binding on the platelet surface. Fur-
platelet because of release of intracellular components such as dense ther studies are needed to determine the relationship between doxycy-
granules in addition to morphology changes in their integral shape.11 cline and the percent IgG response in thrombocytopenic dogs.
Based on our results and aforementioned indices, there was evidence of Another notable finding observed during our study was that all of
platelet activation at weeks 2, 3, and 4 (increased MPV) and at weeks 1, the dogs were strongly positive on serology (SNAP 4Dx Plus, IDEXX
8 | Journal
SHROPSHIRE et al.of Veterinary Internal Medicine SHROPSHIRE 1341
ET AL.

Laboratories Inc) testing for Ehrlichia spp. at week 1 but were PCR neg- caught R. sanguineus are used to initiate infections. A final limitation is
ative. At week 2, all dogs exhibited a weak positive result by serology the small number of dogs that were used. Thus, whether these findings
(SNAP 4Dx Plus, IDEXX Laboratories Inc) testing but all were PCR posi- are clinically relevant is unknown and additional studies are warranted.
tive. Then, at week 3, 2 of the dogs were negative and 2 dogs were Although the mechanisms of thrombocytopenia and bleeding are
positive on serology testing. Not until week 6 were all dogs noted to not completely understood in dogs infected with E. canis, our results
be serology positive again. Experimentally infected dogs can become showed that activated platelets and a hypercoagulable, hypofibrinolytic
seropositive to E. canis as early as 7 days postinoculation, but seroposi- state may explain the lack of a bleeding phenotype in some dogs
tivity may not be seen for 28 days postinoculation.1 We suspect that despite substantial thrombocytopenia. Additionally, further studies are
the strong positive results initially seen in all 4 dogs were actually anti- needed to investigate the relationship between doxycycline administra-
bodies transferred passively from the E. canis positive donor blood. tion and platelet dynamics in thrombocytopenic dogs.
This would also explain why the subsequent weeks showed a weak
positive result, which transitioned into a negative result in 2 of the
ACKNOWLE DGME NTS
dogs. Because we suspect the dogs were not only inoculated with E.
The study was performed at Colorado State University (CSU) Veterinary
canis positive blood but also with antibodies against E. canis from the
Teaching Hospital. The study was funded through the Center for Com-
donor dog, this also could explain the relatively delayed conversion to
panion Animal Studies at CSU. Partial findings were presented as an
seropositivity in some of the dogs.
abstract at the 2017 ACVIM Forum, National Harbor, MD. The authors
Dogs infected with E. canis have been shown previously to have
thank Dr. Ann Hess for her guidance for the statistical analysis, the staff
decreased platelet function based on several platelet aggregation stud-
3,10
ies, but to our knowledge, no studies have been performed using in Clinical Pathology at CSU for processing our samples, and Dr. Cody

multiple channel electrical impedance platelet aggregometry. However, Minor for helping to collect the blood samples.

when using impedance platelet aggregometry, decreased platelet

counts and a high HCT can cause in vitro effects on platelet aggregom- CONFLI CT OF INT ER ES T DE C LAR ATION
28–30
etry resulting in lower AUC readings. As a result, AUC readings Authors declare no conflict of interest.
may appear erroneously low because of thrombocytopenia rather than
reflecting true platelet dysfunction. To address this, an alternative tech-
OFF- LAB EL ANT IMIC ROBI AL DE CLAR AT ION
nique is to standardize the platelet count in all samples before perform-
Authors declare no off-label use of antimicrobials.
ing aggregometry. Doing so requires manipulation of the platelets
(centrifugation) and the test is not performed in whole blood which
may not reflect the true changes occurring in vivo (in whole blood).3,10 INST IT UT IONAL ANIMAL C AR E AND U SE COMMIT TE E

Our testing was performed using whole blood, and the platelet count (IACU C) OR OT HE R AP PR OVAL DE CLAR AT ION
was not adjusted. As a result, the observed AUC readings likely were Authors declare no IACUC or other approval was needed.
affected by the significant changes in platelet count and HCT observed
at certain time points, which is a limitation of the study. ORC ID
There were several other limitations to this study not previously
Sarah Shropshire http://orcid.org/0000-0002-6472-5452
mentioned. The primary limitation was use of blood from a naturally
Michael Lappin http://orcid.org/0000-0003-1605-9667
infected dog to initiate E. canis infection rather than tick infestation. It
currently is unknown whether factors imparted by Rhipicephalus sangui-
RE FE RE NC ES
neus could result in different findings to those obtained by inoculation
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How to
How to cite
cite this
this article: Shropshire S,
article: Shropshire S, Olver
Olver C,
C, Lappin
Lappin M.
M.
[18] Nielsen VG, Audu P, Cankovic L, et al. Qualitative thrombelasto-
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Characteristics hemostasis during
during experimental
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2007;104:59–64. canis infection.
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1111/jvim.15130
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