A variety of tests for platelet function are available. These tests assess all aspects of platelet activation, including adhesion, release reaction (P selection expression, ADP release), membrane flipping (phosphatidylserine exposure) and microvesiculation, activation of the fibrinogen receptor (GPIIb/IIIa), and aggregation. Genetic tests are available for specific defects, including CALDAG-GEF1 defect in Basset Hound thrombopathia and GPIIb/IIIa defects in Glanzmann’s thrombasthenia in various dog breeds, including Otterhounds and Great Pyrenees. There are also global assays of primary hemostasis that evaluate different aspects simultaneously including the buccal mucosal bleeding time (BMBT) and platelet function analyzers. Virtually all of these (with the exception of the BMBT), are only available at specialized veterinary laboratories. Furthermore, fresh platelets (and the production of platelet-rich plasma) are required; hence samples cannot be sent through the mail for platelet function testing. Therefore, if a thrombopathia (abnormal platelet function) is suspected, the patient should be referred to the laboratory or veterinary school performing the testing. Only more commonly used tests will be discussed here.
The most common tests performed for evaluation of platelet function are platelet aggregation tests. In these tests, platelet aggregation is induced in vitro when one or more agonists are introduced and bind to specific receptors in the platelet membrane. Typical agonists used for stimulation of aggregation are collagen, ADP, arachidonic acid, epinephrine, and thrombin. These agonists induce platelet aggregation by fibrinogen binding to glycoprotein IIb/IIIa. The majority of dogs with inherited disorders of platelet have abnormal platelet aggregation to one or more of these agonists. A defective response to ADP only as an agonist led to the identification of a defect in the ADP receptor as a cause of thrombopathia in a dog. Other agonists induce platelet aggregation with vWf, not fibrinogen. These include ristocetin (which binds to glycoprotein Ib-IX) and botrocetin (which binds to glycoprotein IIb/IIIa). These agonists were used for the diagnosis of von Willebrand disease (in human patients in particular) rather than diagnosis of thrombopathias, but are now mostly obsolete.
There are two types of aggregometers, optical and impedance. Platelet aggregation produces a change in light transmission (for the optical aggregometer) or in electrical resistance across two electrodes (impedance aggregometer), which can be measured by the instrument. Both the lag time between introduction of the agonist and platelet aggregation, as well as the rate of aggregation (determined from the slope of the aggregation curve) are measured by the instrument. The impedance method can measure platelet aggregation in whole blood as well as platelet-rich plasma (whole blood is considered to be a more physiologic medium) whereas the optical method only uses platelet-rich plasma. Special optical aggregometers, lumi-aggregometers, measure ATP release simultaneously with aggregation (thus evaluating the release reaction and platelet aggregation).
Platelet function analyzer
With advances in technology, new instruments that assess platelet function, such as the PFA-100 platelet analyzer, have been developed. With the PFA-100, citrate anticoagulated blood is passed through an aperture cut in a collagen-coated membrane, which is impregnated with ADP or epinephrine, which act as agonists for the platelets. As the platelets encounter the agonists, they aggregate and seal off the membrane, resulting in decreased blood flow that is detected by the analyzer. The time taken to seal off the membrane is called the closure time, which is dependent on vWf and normal platelet function in human patients. Testing in dogs has shown that, with the ADP channel, the closure time is abnormally long in dogs with vWD, Glanzmann’s thrombasthenia and Bassett Hound thrombopathia, but normal in dogs with anticoagulant rodenticide toxicosis (which specifically affects primary hemostasis. The epinephrine channel does not appear to work in the dog. This can be used as a screening tool for thrombopathia in a bleeding animal with normal platelet counts and screening coagulation assays, instead of the BMBT (it is far more standardized than the latter test and less subject to inter-observer variability), however the specific instrumentation is required.
The ADP/collagen channel should be used because very prolonged times (>300 seconds) are seen with the epinephrine/collagen channel in healthy dogs. Short closure times have not been associated with enhanced platelet or vWf function, so only prolonged times are considered relevant. Unfortunately, studies have shown that the analyzer is insensitive to drug-induced thrombopathia (due to aspirin and non-steroidal anti-inflammatory agents). Long closure times can be seen in the following conditions:
- Thrombocytopenia: Moderate thrombocytopenia (<100 x thousand/ul) in dogs may prolong the times, however there is weak to no correlation between platelet counts and collagen/ADP closure time (dogs with severe thrombocytopenia can have only mildly prolonged closure times).
- vWD: This will only detect moderate to severe reductions in vWf (<25% vWf:Ag) and cannot be used for carrier detection.
- Inherited thrombopathia: Times are likely to be prolonged in dogs with Glanzmann’s thrombasthenia or Bassett Hound thrombopathia. However, this has not yet been studied.
Closure times are also affected by hematocrit. Animals with anemia may have long closure times, whereas animals with high hematocrits (e.g. healthy Greyhounds) could have short closure times.