The Elusive Hypercoagulable Patient

The Elusive Hypercoagulable Patient
Benjamin Brainard VMD, Dipl ACVAA, ACVECC
Critical Care, University of Georgia, Athens, GA ([email protected])
Many diseases in veterinary medicine are complicated by the occurrence
of micro or macro-thrombosis, which can magnify the morbidity and mortality of
the condition. In diseases where hypercoagulability is suspected, anti-platelet or
anticoagulant drugs are frequently recommended, without clear evidence of the
effect on outcome or on the incidence of coagulopathy, consumptive or
otherwise. Frequently, the choice for either anti-platelet or anti-coagulant drug is
made due to convenience and owner compliance rather than an extensive
knowledge of the origin of the hypercoagulability.
The consequence of the presence of macrothrombi is significant disruption
of blood flow to large areas of the body (e.g. lungs, hind limbs), while the upshot
of microthrombi is more insidious organ dysfunction caused by thrombi lodged in
small capillaries that gradually cause widespread organ hypoperfusion.
Thrombotic disease has been reported in dogs suffering from immune-mediated
hemolytic anemia (IMHA), sepsis, pancreatitis, heartworm disease, parvoviral
enteritis, and neoplasia, among other diseases.1-3 In cats, pancreatitis, sepsis,
and altered blood flow due to cardiomyopathy are associated with thrombus
formation.4-6 Horses with colic, especially those with strangulating intestinal
lesions or colitis, may have a presdisposition for venous thrombosis.7 Given the
heterogeneity of diseases associated with thrombosis, the diagnosis of
hypercoagulabity in a patient prior to the onset of coagulopathy is critical.
Identification of the hypercoagulable animal will allow delivery of targeted therapy
to the animals most at risk, but the best technique to achieve an antemortem,
pre-thrombosis diagnosis of hypercoagulability is elusive, and may vary
depending on thrombotic triggers in the different disease states.
Hypercoagulability is generally suspected in animals with diseases that
have a strong inflammatory component, because systemic inflammation can
result in endothelial cell (EC) and monocyte activation and exposure of tissue
factor (TF), shedding of TF-expressing microparticles from ECs and monocytes,
increased circulating fibrinogen concentration, and generation of thrombin.
Activation of platelets by inflammatory stimuli can also play a role, resulting in
exposure of membrane P-selectin (CD62P) and phosphatidylserine (PS) on the
platelet membrane that can interact with circulating mononuclear cells and
coagulation factors, respectively.
In addition to systemic inflammation, conditions that result in abnormal
blood flow or blood stasis such as neoplasia, arterio-venous fistulae, or cardiac
disease and arrhythmias (eg., atrial fibrillation8) may also presdispose to clot
formation. The presence or absence of endogenous and exogenous substances
(eg., prednisone therapy,9 low levels of antithrombin [AT]10) may drive animals
towards a hypercoagulable state. The contributions of endothelial damage,
blood stasis, and a hypercoagulable state towards thrombus formation was first
described in the 1850’s by Rudolph Virchow, and are referred to today as
Virchow’s triad.
Identification of a hypercoagulable state: plasma testing
Traditional coagulation tests, such as platelet count, activated partial
thromboplastin time (aPTT), and prothrombin time (PT), are most useful for the
demonstration of hypocoagulability; they do not reliably identify
hypercoagulability. Prolongations of coagulation times in concert with a
decreased platelet count may appear in patients with a consumptive
coagulopathy. In clinical practice, a drop in circulating platelet count
accompanied by a prolongation of at least 20% in baseline aPTT in an at-risk
patient is generally the first indication of a consumptive coagulopathy.11
Another option for identification of a hypercoagulable state focuses on the
measurement of specific procoagulant or anticoagulant molecules in circulation.
These molecules can include fibrinogen concentration (when elevated
contributing to a procoagulant state) or AT activity (decreased in hypercoagulable
states). Other procoagulant molecules, such as coagulation factor V or VIII
activity, or levels of anticoagulant proteins C and S, may be used to discover the
overall balance of hemostasis in an individual.
While low AT levels may represent true pathology (eg., in patients with
protein-losing nephropathy), they can also be indicative of utilization, in scenarios
where large amounts of thrombin have been formed (eg., during growth of a
thrombus) and it can sometimes be difficult to distinguish the chicken from the
egg in terms of AT levels and hypercoagulability in patients with thrombi.12 A
recent study of dogs with septic peritonitis described decreased AT and protein C
activity in dogs who did not survive, although almost all of the dogs had AT lower
than the reference interval.13 When AT combines with thrombin, a tertiary
compound is created, the thrombin-antithrombin (TAT) complex. Elevated levels
of circulating TAT complexes indicate that thrombin has been formed in the
patient, and are a strong clue for an underlying hypercoagulable state. TAT is
assayed using an ELISA (Dade/Siemens) that is relatively expensive and difficult
to have available for use on individual clinical patients. TAT has been evaluated
in dogs with hyperadrenocorticism,14 carcinoma,15 Babseiosis,16 and congestive
heart failure17 and appears to be elevated in conditions associated with
coagulation or inflammation.
Other markers of ongoing thrombin generation include prothrombin
activation fragments (F1+2), which are generated by the activity of factor Xa on
factor II, and fibrinopeptides A and B. These have been rarely evaluated in dogs,
and poor cross-reactivity to the reagents in the human-based assay has been
Whole blood viscoelastic testing:
Coagulation testing that assesses more than one aspect of the
coagulation cascade at the same time may be a useful adjunct in determining
hypercoagulable states. Commonly available viscoelastic coagulation devices
include thromboelastography (TEG) or rotational thromboelastometry (ROTEM),
both of which can monitor coagulation kinetics in whole blood or plasma
samples. Whole blood coagulation analysis has a theoretical benefit over
plasma-based coagulation testing because the interaction of all cellular
components of blood (minus the endothelium) contribute to the result.
Viscoelastic testing evaluates the time to initial fibrin cross-linking, the rate of clot
formation, and the strength and viscoelastic characteristics of the clot that is
formed,19 Hypercoagulable samples start to clot more quickly, with a faster rate
of clot formation, and greater clot strength.
TEG has been used in an attempt to identify animals and people with
hypercoagulability, with mixed results, partially due to the nature of the test. The
TEG is sensitive to the RBC concentration in the sample; as hematocrit drops,
the TEG tracing appears progressively hypercoagulable, even in normal
animals.20,21 This complicates the TEG-based diagnosis of hypercoagulability in
cases such as IMHA, where anemia is an important aspect of the disease itself.
TEG has also been evaluated in veterinary medicine using a number of different
protocols for rest time, rest temperatures, and activators. Most studies will use
citrated blood that is rested for 30 minutes, either at room temperature or at 37°
C (rest temperature does not significantly affect the results22). Some published
studies have used TF as an activator (in at least 3 different concentrations) and
others kaolin, while other studies have used no activator other than
recalcification. As the blood sits during the rest period, the sample itself will
become hypercoagulable through activation of factor XII and the contactactivation pathway; the use of a strong activator overcomes the slight changes
that this may cause in the tracing. While activators (or not) affect the duration of
time prior to initial fibrin formation (ie., the reaction, or R time), they do not
substantially affect the maximum clot strength (“maximum amplitude”, MA), which
is dependent primarily on the platelets and fibrinogen concentration in the
sample. MA is sometimes transformed to the elastic shear modulus (“G”) which
is calculated as: (5000*MA)/(100-MA) and reported in dynes/sec. The
coagulation index (CI) is a unitless number calculated by an algorithm that
integrates the main TEG parameters to arrive at a single number (0.1227R +
0.0092K + 0.1655MA – 0.0241(angle) – 5.0220, derived for dogs using citrated
whole blood),19 and those patients with a CI higher than the normal range are
hypercoagulable, and those below the range are hypocoagulable. In general, the
reference interval for CI is -3 – 3.
In terms of actual prediction of thrombotic tendencies, very little outcomerelated data exists in the human or veterinary TEG literature. Some studies have
related the TEG variable MA to the occurrence of myocardial infaction (MI) in
humans undergoing general surgery and following trauma,23,24 and others have
related this value to the likelihood of coronary stent thrombosis following PCI. 25
Other studies in various populations of people have not found this correlation.26
An early study of 9 dogs with parvoviral enteritis described hypercoagulability in
all dogs (based on increased MA), and 4 of these dogs developed jugular venous
In dogs and humans with trauma, the presence of hypocoagulability on the
TEG is correlated with morbidity and mortality, but hypercoagulability on the TEG
was not associated with risk for venous thromboembolism. In dogs with septic
peritonitis, the preoperative MA was hypercoagulable and higher in those dogs
who survived hospitalization, which may also represent a more compensated
coagulation state (vs. those who were relatively hypocoagulable due to
consumption). A recent necropsy-based study failed to find a association
between the presence of hypercoagulability on the TEG and thrombosis in 26
dogs, but the criteria had the TEG performed within 7 days of necropsy, so may
not have reflected the underlying state at the time of thrombus formation.28 With
the relatively low incidence of definitively-diagnosed thrombotic events in
veterinary medicine, a standardized multi-center study may be necessary to
determine whether TEG, alone or in concert with other testing, can identify those
animals at high risk for thromboembolic disease, and to see beyond the inherent
heterogeneity of the underlying diseases.
Calibrated automated thrombin generation (CAT) has been less studied in
dogs, but shows promise as a test that can demonstrate the kinetics of thrombin
formation in a sample of citrated plasma or PRP. Through the use of a
fluorogenic substrate, thrombin production is tracked and graphed as a thrombin
generation curve. The area under the curve indicates the total capacity for
thrombin generation in the sample and is called the endogenous thrombin
potential (ETP). The slope of the curve gives the rate of thrombin generation and
it is possible to calculate the total amount of thrombin formed as well. CAT has
been investigated in veterinary medicine in the context of therapeutic monitoring
for anticoagulant treatment,29,30 but has been used in human medicine to identify
hypercoagulability in certain patient populations.
Flow cytometric determination of hypercoagulability
Assays for markers of platelet activation such as P-selectin expression or
platelet-neutrophil aggregates may indicate a propensity towards platelet
activation in specific patients.31 Flow cytometric techniques also can also be used
to document the presence of procoagulant MPs and PS externalization on
platelet membranes. Standardization of techniques is necessary to assay for
MPs because of their small size (less than 1.5 microns). Increased expression
of platelet P-selectin has been reported in patients with IMHA,32 and has been
studied as a marker of therapeutic effect in patients given platelet inhibiting
drugs.33 In humans and pigs, high levels of soluble P-selectin have been
associated with inflammation and venous thrombosis.
The Advia 120 hemostasis analyzer (Bayer Healthcare, Shawnee Mission,
KS) reports a parameter called mean platelet component (MPC), which is related
to the granularity of the circulating platelets. After activation, the granularity of
platelets decreases, and thus a decreased MPC may represent activated
platelets, although a recent study did not find an association between MPC and
SIRS criteria or general disease type (inflammatory vs. non-inflammatory).34
Another indirect measure of hypercoagulability is fibrinolysis; decreased
fibrinolysis can contribute to the persistence of thrombi, and the morbidity may be
indistinguishable from patients with hypercoagulability. Hypofibrinolysis is also
common in patients with systemic inflammation, who demonstrate increased
levels of α2-antiplasmin, plasminogen activator inhibitor-1, and thrombinactivatable fibrinolysis inhibitor (TAFI). In a necropsy study of horses with severe
gastrointestinal disease, persistent fibrin deposition was described in multiple
organs.35 The evaluation of fibrin(ogen) degradation products (FDP) and ddimers (produced by breakdown of cross-linked fibrin) do not reliably support or
undermine the presence of a thrombus, at least in the context of pulmonary
thromboembolic disease (PTE),36 although other studies of patients with
presumed hypercoagulable conditions (e.g. septic peritonitis) have demonstrated
persistent elevations in d-dimers, although these levels were not predictive of
TEG can also be used to assess fibrinolysis in dogs, but requires added
tPA to force the fibrinolysis to occur within a reasonable time. This technique has
been used to assess the presence of fibrinolysis in dogs that have an acute
coagulopathy secondary to hemoperitoneum.38
There are many tools and assays that con contribute to a global
assessment of the coagulation status of a patient. Used in together, and used for
serial assessments, these parameters, in addition to a good physical
examination, may give a leg up on the ante-thrombosis diagnosis of
hypercoagulability. As with any disease, the clinician must weigh the pros and
cons of anticoagulation, with a view towards the expected duration and resolution
of the illness. If a rapid resolution is not in sight, thromboprophylaxis may be
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