Coagulopathies�SOTAL

Urs Giger United States

State of the Art Lecture

Urs Giger

PD, Dr. Med.Vet., MS, FVH, Dipl. ACVIM & ECVIM

Urs Giger received his veterinary degree from the University of Z�rich, Switzerland, where he also pursued his initial clinical training in small animal medicine and surgery and a doctoral thesis on the orthopedic correction of canine hip dysplasia. In 1981, he moved as a postdoctoral fellow to the United States where he subsequently completed a residency in small animal medicine at the University of Florida.

He then joined the faculty of the School of Veterinary Medicine at the University of Pennsylvania in Philadelphia and has been a clinician in Medicine, Oncology and Pediatrics/Genetics. He is now the Charlotte Newton Sheppard Professor of Medicine and Chief of the Section of Medical Genetics and has a secondary appointment at the University of Zurich. He is a diplomate of the American and European College of the Veterinary Internal Medicine and is heading the Pediatrics and Genetics Clinic, the Josephine Deubler Genetic Disease Testing Laboratory and the Transfusion Medicine Program at the University of Pennsylvania. His clinical and research expertise and interests are in hereditary and hematologic disorders of small animals.

Bleeding is a common clinical presentation in small animal practice. Bleeding disorders occur more commonly in dogs than in cats. Clinical manifestations of bleeding disorders may range from mild and self-limited to life-threatening hemorrhage requiring immediate medical attention. Animals may bleed due to vascular injury caused by any trauma, surgery, ulcer, and tumor. In case of hemostatic disturbances, the hemorrhagic tendency is exaggerated as exemplified by spontaneous, multifocal, and unexpected severe bleeding.

Hemostasis is the complex physiologic response to bleeding and can be divided into primary and secondary hemostasis. Vascular endothelium, platelets, and vonWillebrand factor (vWF) are required for primary hemostasis or the formation of the unstable platelet plug, which is sufficient to stop capillary bleeding. Von Willebrand factor, a large plasma protein synthesized by endothelium, facilitates adhesion between platelets and subendothelium. With larger vessel injury, coagulation factors are also needed to form a stable fibrin clot, known as secondary hemostasis. Hemostatic disorders can be conveniently classified into vasculopathies, thrombocytopenias, thrombopathias, vonWillebrand disease (vWD), and coagulopathies, realizing that some disease processes cause combined hemostatic defects (e.g., disseminated intravascular coagulopathy [DIC]). Hemorrhagic presentations may suggest certain hemostatic disorders and practical tests are available to characterize the bleeding tendency. An accurate diagnosis of the cause of bleeding will influence the treatment and outcome of a patient.

Coagulation Cascade

The coagulation cascade is a series of enzymatic reactions involving coagulation factors, denoted by Roman numerals. This complex biochemical pathway plays three pivotal roles in the formation of fibrin from fibrinogen:

1.   Acceleration of fibrin generation > 10 million-fold.

2.   Regulation of fibrin plug size appropriate for injury.

3.   Localization of fibrin clot formation to site of injury.

The coagulation cascade can be divided into an extrinsic and intrinsic system that merges into the common pathway. When blood vessel walls are injured, the extrinsic coagulation system is initially activated by tissue factor (thromboplastin produced in the subendothelium), which combines with factor VII. The small amount of thrombin produced through factor VIIa appears sufficient to activate factor XI to factor XIa, and to activate other cofactors (factors V, VIII, XIII). Through the action of factor XIa on factor IX, thrombin formation is maintained. The other contact phase coagulation factors include factor XII, prekallikrein and High Molecular Weight Kininogen and are in vivo of lesser importance in the activation of the intrinsic system. The above described coagulation process explains why hemophilic (factor VIII and IX deficient) or factor VII deficient animals bleed, whereas factor XII deficient animals do not.

Coagulation factors are enzymes (serine proteases), cofactors, or substrates of particular reactions of the coagulation cascade. Most factors are given roman numerals from I-XIII, but the numbering is not sequential and there is no factor VI. Calcium (factor IV) is required for most reactions and is the reason why chelators, e.g., citrate and EDTA, are used for blood collection and processing where plasma or blood cells are analyzed. All glycoproteins of the coagulation cascade are synthesized in the liver and circulate as inactive precursors in the plasma. They need to be activated at the site of vessel injury (depicted with letter a following their Roman numeral). Vitamin K is needed for the functional synthesis of the coagulation factor II, VII, IX, and X. The half-lives of the coagulation factors varies from hours (factor VII) to a few days (Fibrinogen). It should be noted that in vitro coagulation factor interaction, known as clotting, proceeds somewhat differently from the above described in vivo coagulation cascade, but is helpful in identification of specific coagulopathies. Following a fibrin plug formation, plasminogen will be activated and plasmin, an unspecific protease will commence breaking down fibrinogen as well as fibrin which results in fibrinogen lysis and fibrin(-ogen) split product (FSP) as well as D-dimer formation.

Diagnostic Approach

Signalment and Family History

Although hereditary coagulopathies may occur in any breed, each coagulopathy has thus far only been reported in certain breeds. Hemophilia A and B occur in many different breeds and are X-chromosome recessively inherited; thus, only males are generally affected and females are asymptomatic carriers. Signs of bleeding typically occur at an early age and are often recurring, but may not be recognized until adulthood.

History

Bleeding may be induced (trauma, surgery) or appear spontaneously. Careful history taking may reveal exposure to toxins (rodenticides, mushrooms) and drugs (warfarin, heparin). It is important to identify the specific product, as, for example, different rodenticides have quite varied potency. Any evidence of other diseases, for example, hepatopathies and cancer, may be responsible for the hemorrhage.

Physical Examination

Careful clinical evaluation may differentiate between primary and secondary hemostatic defects. Surface bleeding is typically seen with primary hemostatic disorders. Petechia and ecchymosis are hallmark features of thrombocytopenias and thrombopathias. In Von Willebrand disease, bleeding occurs at sites of injury (trauma, dental disease, estrus, gastrointestinal), rather than petechia or ecchymosis. Coagulopathies may be associated with single or multiple sites of bleeding characterized by cavity bleeding such as hematoma, hemarthrosis, hemomediastinum, and hemoperitoneum and �thorax, but gastrointestinal hemorrhage and bruising may also occur. Signs of other underlying diseases may be recognized.

Hemostatic Tests

Hemostatic tests are indicated:

1.   Whenever an animal is bleeding excessively.

2.   Prior to surgery when an increased bleeding tendency is suspected.

3.   To monitor therapeutic interventions.

4.   For genetic screening in certain breeds or families with a known bleeding disorder.

Hemostatic abnormalities should be assessed prior to instituting therapy whenever possible or at least appropriate blood samples should be collected pretreatment. Excellent venipuncture with discarding of first few drops of blood (to avoid platelet activation and tissue factor) and extended compression over jugular, saphenous or femoral vein is required. The cuticle bleeding time crudely assesses overall hemostasis but is not standardized and is painful, and therefore, not recommended. A minimal database includes a packed cell volume and total protein evaluation. Evaluation of a blood smear can provide a platelet estimate and identify platelet size and clumping as well as schistocytes. The results can provide some measure of the extent of blood loss and red blood cell transfusion requirement.

Primary Hemostasis: Platelets and vWF

Since 8-15 platelets are normally found per high power oil emersion microscopic field, an absence or low number of platelets suggests a severe thrombocytopenia. Hemorrhage is generally not observed unless the platelet count is < 40,000/µl (normal 150-500,000/µl). Detection of platelet-associated antibodies further supports an immune-mediated thrombocytopenia, but this test is rarely available. Serum titers for Ehrlichia canis and E. platy are indicated in certain states and countries. The presence of schistocytes along with thrombocytopenia, suggests intravascular disseminated coagulation, where intravascular fibrin strands fragment erythrocytes. Because von Willebrand disease is such a common mild primary hemostatic defect in dogs, plasma vWF measurements by ELISA are indicated. Alternatively, DNA testing is available in some canine breeds for specific vWD-causing mutations and is useful for detection of carriers. Finally, in light of normal platelet count and plasma vWF values, a prolonged buccal mucosal bleeding time (BMBT) indicates a thrombopathia. Disposable devices are available that facilitate making one to two standard 1 mm deep mucosal incisions. The platelet function analyzer (PFA100) is a simple tool to functionally assess primary hemostasis. Electron microscopic and platelet aggregation and nucleotide studies allow further characterization of platelet dysfunctions in specialized laboratories.

Secondary Hemostatic Tests: Coagulation Tests

Whereas the whole blood clotting time test is insensitive and inaccurate, there are several standardized coagulation screening tests useful for defining coagulopathies in clinical practice. The screening tests assess coagulation in vitro, which is helpful, but is now known not to be identical with the in vivo coagulation process. Nearly all coagulation tests assess the function of certain parts of the coagulation system in using fresh whole blood or fresh (frozen) plasma to generate fibrin in a fibrometer. Recalcified citrated plasma is used and many tests compare a patient�s sample directly with a simultaneously obtained control or pool plasma (plasma from 10 animals). In general, coagulation times (the time to clotting and fibrin formation) are much shorter in small animals than in humans, thus tests need to be validated for the particular animal species.

The intrinsic and common pathways are assessed by either the activated coagulation time (ACT) or activated partial thromboplastin time (PTT). Factor XII of the intrinsic cascade is activated by diatomaceous earth (celite) in the ACT test and by kaolin or other contact phase substrate in the PTT test.

The extrinsic and common pathways can be assessed by either the prothrombin time (PT) or the protein induced by vitamin K antagonism or absence (PIVKA) test. Different tissue factors (thromboplastins) are activating factor VII that in turn will lead to fibrin formation. It should be noted that the PIVKA test is not specific for the detection of anticoagulant rodenticide poisoning, but detects any coagulation factor deficiency of the extrinsic and common pathway and is therefore not that helpful.

Until recently the ACT tube test and PIVKA tests were the only point of care tests available for clinical practice, whereas PTT and PT tests were performed in reference laboratories. There is now a new point of care coagulation instrument (SCA2000) that is capable of rapidly determining the PTT and PT on small amounts (50 µl) of citrated whole blood, thereby making the chilling, rapid separation of citrated plasma and shipment of frozen plasma on dry ice to the laboratory for initial coagulation screening unnecessary.

In fact, a reasonable and simple approach for a bleeding animal to be screened for a coagulopathy would be to measure the ACT or PTT first as either test detects all coagulopathies (except for hereditary factor VII deficiency in Beagles). If the PTT (or ACT) is prolonged, a PT test would be indicated to differentiate between an intrinsic and common pathway defect or a combined coagulopathy involving several coagulation factors.

Although hereditary coagulopathies can be suspected based upon the pattern of coagulation test abnormalities, specific factor analyses are needed to confirm a diagnosis. A bleeding male animal with a prolonged PTT (or ACT) and normal PT likely has hemophilia A or B (factor VIII or IX deficiency), an X-chromosomal recessive disorder. However, factor XI deficiency is associated with the same test abnormalities and is inherited by an autosomal recessive trait (e.g. Kerry Blue Terriers). Finally, factor XII deficiency, particularly common in domestic shorthair cats, and prekallikrein deficiency causes marked PTT (ACT) prolongations but no excessive bleeding tendency.

Rodenticide poisoned animals that are bleeding or are at risk for bleeding will have prolongations in all of the above coagulation tests, but would have a normal thrombin time (TT). The thrombin time is independent of vitamin K-dependent coagulation factors and is a functional assay for fibrinogen to form fibrin. A toxicological investigation (product identification, blood toxicology analysis) may confirm the rodenticide poisoning. Moderate thrombocytopenia may be associated with warfarin type-rodenticide poisoning.

All liver diseases may result in varied coagulopathies due to impaired coagulation factor synthesis and vitamin K malabsorption. Similarly, disseminated intravascular coagulopathies (DIC), due to many different disorders is associated with variably prolonged coagulation times. More helpful to the diagnosis of DIC are the recognition of schistocytes, thrombocytopenia, low antithrombin III levels, and increased D-dimers and fibrin split (degradation) products. Recently a simple kit for canine D-dimers has become available.

Therapy and Monitoring

The above coagulation tests are also useful to monitor therapeutic interventions. Heparin drastically prolongs the PTT/ACT and to a much lesser extent the PT. In contrast, warfarin causes PT and PTT prolongations like the anticoagulant rodenticides. Thus, heparin and warfarin anticoagulant therapy are monitored by PTT (ACT) or PT, respectively. Similarly, vitamin K therapy (< 5 mg/kg/d PO or SC) should correct vitamin K deficiency states and can be assessed by either PT or PTT measurements.

The effect of transfusion support in bleeding animals with coagulopathies is also evaluated by coagulation screening tests and PCV. Fresh frozen plasma (10 ml/kg every six hours) is appropriate for all coagulopathies, whereas cryoprecipitate (2�5 ml/kg) is useful for control of bleeding in hemophilia A and von Willebrand disease. In addition, packed red cells or fresh whole blood alone could be used to correct the associated anemia.

In order to best assess the patient�s changes in the ability to coagulate, it is important to use the same technique and ideally same operator/laboratory. These above described simple guidelines should assure the correct diagnosis and successful management of animals with hemorrhage.

^� A search for an underlying disorder should also be pursued.

* Use veterinary laboratory�s established reference range.

SCA 2000 instrument using fresh citrated whole blood.