Chapter 6 Hemostasis

 

 

 

                        

6.1 Required reading assignment:

Schalm's Veterinary Hematology -- Jain:

• 1. Coagulation and its Disorders -- pp 388-425.

Veterinary Clinical Pathology - Coles:

• 1. Hemastasis and Coagulation of Blood -- pp 98-113

 6.2 Objectives and/or study questions:

Questions: 1. Compare the difference in the type of bleeding observed in patients with platelet disorders compared to coagulation factor disorders. 2. What pathways of coagulation are measured by the ACT, APTT, PT and TT? Which test could be performed easily in a clinical practice setting? What abnormality could prolong the ACT in spite of the existence of normal coagulation factor activities? Are the APTT and PT tests capable of detecting carrier states? Why or why not? 3. What is the anticoagulant of choice for coagulation testing? How important is the ratio of anticoagulant to blood? Is EDTA an acceptable alternative? Why or why not? 4. If you have a patient with a history of excessive bleeding and test results indicate a prolongation in the APTT but a normal PT, what specific coagulation factor(s) are possibly absent or diminished in activity? Can this patient have warfarin/ indanedione toxicity? 5. Is it possible for a patient to have a prolonged APTT in the absence of clinical bleeding? Name an example. Is it possible for a patient to have a prolonged PT in the absence of clinical bleeding? Name an example. 6. What is the function of antithrombin III? What compound enhances its activity? Name 3 mechanisms by which antithrombin III deficiency may occur. Patients with antithrombin III deficiency are at what risk? 7. What are the inheritance patterns for factor VIII and factor IX deficiencies? Is it possible for a male to be a carrier for Hemophilia A or B? Do carrier females have problems with excessive bleeding? Name two genetic mechanisms by which a female may be affected with Hemophilia A or B. 8. What are the vitamin K-dependent coagulation factors? What are 2 other proteins that are vitamin K-dependent but function to prevent thrombosis? Name 6 causes of deficiency of vitamin K-dependent factors. Which vitamin K-dependent coagulation factor has the shortest half-life? Which coagulation screening test becomes prolonged first with rodenticide toxicity? Compare the coumarin compounds to the indanedione compounds in terms of half-life, prognosis, and treatment. 9. Give an example of a congenital extrinsic platelet function defect; an intrinsic platelet function defect. 10. What is von Willebrand's Disease? How is the disease diagnosed? Is the bleeding pattern typical of a coagulation or a platelet disorder? Would you expect clot retraction to be normal in this disorder? 11. What is the mediator of physiologic fibrinolysis? What are three mechanisms for converting plasminogen to plasmin? Compare TPA, streptokinase, and urokinase in their origin and their mechanism of plasminogen activation. 12. Given the following coagulation test results in the face of a bleeding disorder, which diagnoses can be ruled out?

a. Hemophilia A

b. Hemophilia B

c. Von Willebrand's Disease

d. Canine thrombopathia

e. Thrombasthenic thrombopathia

f. Hageman's Disease

g. Warfarin toxicity

h. DIC

i. Chediak-Higashi Syndrome

6.3 NORMAL HEMOSTASIS 

Normal hemostasis is dependent upon the complex interaction of plasma coagulation and fibrinolytic proteins, platelets, and the blood vasculature (Figure VI.1). Following injury to the vessel wall vasoconstriction not only retards extravascular blood loss, but also slows local blood flow, enhancing the adherence of platelets to exposed subendothelial surfaces and the activation of the coagulation process. The adherence of platelets at sites of vascular injury is an important early event in hemostasis and is mediated by a plasma protein known as von Willebrand factor. Following adhesion additional platelets are recruited in response to components released from platelet granules including adenosine diphosphate (ADP) and serotonin. Platelets also synthesize thromboxane A2 (TXA2) and platelet activating factor which are potent platelet aggregating agonists and, in the case of TXA2, enhance vasoconstriction. Exposure of subendothelial surfaces and/or the generation of tissue factors also leads to the sequential activation of a series of coagulation proteins (Figure VI.2). These proteins, a series of enzymes and cofactors, ultimately convert prothrombin to thrombin. Thrombin not only cleaves fibrinogen into fibrin monomers but also activates factor XIII which serves to crosslink and stabilize the resulting fibrin network. The activity of many of the coagulation factors is not only dependent upon calcium but also the phospholipid provided by the surface of activated platelets. Binding of coagulation proteins to a common surface in close proximity enhances the efficiency of serial activation of the coagulation factors. Simultaneous to the activation of the coagulation cascade, plasminogen is converted to plasmin, the agent of fibrinolysis, not only by coagulation contact factors such as kallikrein, but also by tissue plasminogen activator. The latter activation requires fibrin thus limiting the production of plasmin to the site of thrombus formation. The localization of plasmin is important since the enzyme is fairly non-specific in its activity and will not only destroy fibrin but also fibrinogen, factor V and factor VIII.

In general, following vascular injury, platelets and the vasculature serve to temporarily stem blood flow while the coagulation proteins provide a more permanent repair. In turn, the fibrinolytic system aids in the containment of the thrombus locally and serves to slowly destroy the fibrin network as healing ensues. Qualitative or quantitative platelet defects are usually manifested by superficial ecchymotic or petechial bleeding especially from mucous membranes whereas coagulation protein abnormalities are characterized by delayed deep tissue hemorrhage and hematoma formation. 

6.4 DISORDERS OF HEMOSTASIS 

6.4.1 Coagulopathies

Disorders of hemostasis may be due to congenital or acquired defects in coagulation proteins, platelets, and/or the vasculature. Congenital coagulopathies are usually first recognized between birth and six months. Defects in coagulation at this time are exacerbated by a normally immature hemostatic mechanism (Buchanan, 1978) related to a young, developing liver, the site of synthesis of the majority of coagulation proteins. Newborn animals are likely to be deficient in vitamin K and oral intake of vitamin K is minimal in nursing animals. Animals with major coagulopathies due to deficiencies in factors vital for hemostasis are often stillborn or die of massive hemorrhage shortly after birth. Major coagulopathies include the existence of less than 1% of normal activity of factors VIII, IX, II and I. Animals with less severe coagulopathies (factor activities between 5 and 10% of normal) usually survive the neonatal period but encounter hemostatic difficulties between one and six months of age. Prior to six months kittens and puppies are subjected to a number of routine procedures including vaccination, deworming, tail docking and dew claw removal, ear cropping, and declawing. In addition, normal physiologic processes such as the shedding of deciduous teeth and the onset of estrus also occur, events not tolerated well by individuals with a compromised hemostatic response.

Acquired coagulopathies are more common than congenital coagulopathies and can occur at any age. However, because congenital coagulopathies are usually first apparent prior to six months, the origin of a bleeding diathesis at this young age may be difficult to discern at initial examination. Also, minor congenital hemostatic defects may not become apparent unless superimposed with an acquired coagulopathy. A thorough history and evaluation of coagulation screening tests and a platelet count will be helpful in determining the reason for a bleeding problem. 

6.4.2 COAGULATION SCREENING TESTS 

6.4.2.1 ACTIVATED COAGULATION TIME (ACT)

The ACT is a useful screen for evaluation of the intrinsic and common pathways. Whole blood (2 mls) is added to a tube containing diatomaceous earth as a contact activator and the tube gently agitated at specific intervals while timing until clot formation. A 37oC heating block is recommended for consistent results (Middleton and Watson, 1978). If a heating block is not available, a styrofoam cup and 37oC tap water is preferable to performing the assay at room temperature. Unlike the APTT, the ACT relies on platelet phospholipid to support the reaction, therefore, platelet counts of less that 10,000/ul may result in a prolonged ACT. Published reference ranges should not be used for this assay. Ideally a reference range should be established for your hospital based on times you obtain by repeatedly performing the test on animals you know to be normal (set up different ranges for each species and possibly separate ranges for animals under 2 months). 

6.4.2.2 ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

This assay is an indicator of the function of coagulation factors in the intrinsic and common pathways. A surface activator such as kaolin, phospholipid, and calcium are added to citrated plasma at 37oC and the time to clot formation is recorded. The test is a useful screen for the function of a number of clotting factors, however, specific factor activity must usually be less than 30% for a prolonged APTT to occur and the detection of a prolonged APTT is not always clinically significant. Deficiencies of factor XII and kallikrein will prolong the APTT without an associated hemorrhagic diathesis. A prolonged APTT in conjunction with overt bleeding without a history of rodenticide toxicity warrants the evaluation of specific coagulation factors. This requires the careful collection of citrated blood (1 part 3.8% trisodium citrate to 9 parts blood) and isolation and immediate freezing of plasma for factor analysis by a specialized hemostasis laboratory. Because young animals may normally have decreased factor activity compared to adults, age-matched controls are important not only for evaluating the APTT but also in evaluating specific factor activity (Dodds et al., 1975a). Young animals may normally have a slight prolongation in the APTT (1 to 3 seconds) compared to adults, however, they should not have markedly prolonged APTT's (> 5 seconds) or severe deficiencies in individual factors (less than 10% of normal activity). Identification of a carrier state by specific factor analysis may be difficult in young animals and require retesting at 6 to 12 months of age. 

6.4.2.3 PROTHROMBIN TIME (PT)

An indicator of the functional activity of factors in the extrinsic and common pathways, the prothrombin time assay requires citrated plasma, a source of tissue factor and calcium. The PT is a sensitive indicator of warfarin toxicity; since factor VII is the vitamin K-dependent factor with the shortest half-life the PT may be prolonged within 24 hours following warfarin ingestion, prior to a significant alteration in the APTT or the appearance of clinical signs. As with the APTT, age-matched control are important and specific factor activity must usually be less than 30% for a prolongation in the PT to occur. Because factor VII deficiency is usually not accompanied by hemorrhage, a bleeding diathesis of longer that 24 hours duration is not likely to be characterized solely by a prolonged PT. 

6.4.2.4 THROMBIN TIME (TT)

This assay is a direct measurement of functional fibrinogen and entails the addition of thrombin to citrated plasma and the timing of clot formation. The rate of clot formation is directly proportional to the concentration of functional fibrinogen and is especially useful in the diagnosis and/or evaluation of individuals with disseminated intravascular coagulation (DIC). Although the test will also detect individuals with congenital hypo- or dysfibrinogenemia, these conditions are rare. Cats may normally have fibrinogen concentrations as low as 50 mg/dl (O'Rourke et al., 1982), therefore this assay may not be as useful in the cat for the evaluation of DIC. 

6.4.2.5 FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS (FDP's)

The assay detects the presence of elevated levels of circulating fibrinogen or fibrin degradation products by the use of specific antibodies coupled to latex beads. The sample requires serum taken from blood collected into a tube containing thrombin and a trypsin inhibitor (provided with the Thrombo-Wellcotest for FDP determination, Burroughs Wellcome). The test is useful when evaluating patients with known or suspected DIC. FDP's may also be elevated in patients with indwelling catheters, in severe warfarin toxicity, in horses with mesenteric artery thrombosis secondary to migration of Strongylus vulgaris larvae, and in horses with jugular vein thrombosis. 

6.4.2.6 ANTITHROMBIN III (ATIII)

Antithrombin III is a circulating protein inhibitor of several coagulation factors including XII, XI, X, IX, and thrombin (II). The activity of ATIII is greatly enhanced by heparin. Antithrombin III is an alpha2-globulin, synthesized by the liver, with a molecular weight of 62,000. Decreases in ATIII can occur secondary to hepatic failure, as a result of protein losing conditions, such as protein losing enteropathy or glomerulopathy, or secondary to consumption as a result of DIC. Individuals with ATIII concentrations of less than 70% are at risk for the development of thrombosis. Because ATIII is often depleted in DIC, the assay is useful in evaluating or diagnosing this syndrome. The test, however, was not useful in cats with DIC following experimental infection with feline infectious peritonitis virus (Boudreaux et al., 1988a; Boudreaux et al., 1988b). The test requires citrated plasma and age-matched controls are recommended. 

6.4.3 COAGULATION PROTEIN DISORDERS

Coagulation protein abnormalities may be either congenital or acquired. Acquired coagulopathies can be complicated by thrombocytopenia and/or vascular disorders and may progress if untreated to the development of DIC. Congenital coagulopathies (Table VI.3) are less common and if severe often require a specialized, isolated environment for successful management.

 6.4.4 CONGENITAL COAGULATION AND VASCULAR DISORDERS 

6.4.4.1 PREKALLIKREIN

Prekallikrein deficiency is a rare disorder in domestic animals and has only been described in a poodle (Chinn et al., 1986), a family of miniature horses (Turpentine et al., 1986) and a family of Belgian horses (Geor et al., 1990). The disorder is not usually associated with clinical bleeding and is often diagnosed fortuitously during the performance of routine coagulation screening tests. Affected animals have a prolonged APTT and ACT and a normal PT. The poodle with prekallikrein deficiency was 14 years old before the diagnosis was made and the miniature horses also did not have clinical bleeding. One of the Belgian horses did hemorrhage excessively after castration, however, this is the only report of a possible bleeding diathesis associated with prekallikrein deficiency. 

6.4.4.2 FACTOR XII

Factor XII deficiency or Hageman's disease is not associated with a bleeding diathesis (Bennett, 1984). Diagnosis of factor XII deficiency is often fortuitous during the conduction of routine screening procedures and is characterized by a prolonged APTT and ACT. The disorder has been described in cats (Kier et al., 1980) as well as in a German shorthaired pointer, a standard poodle (Dodds, 1984) and a family of miniature poodles (Randolph et al., 1986). The coexistence of factor XII deficiency with von Willebrand's disease (Randolph et al., 1986) or factor IX deficiency (Dillon and Boudreaux, 1987) did not seem to exacerbate bleeding. Although factor XII deficiency is not associated with apparent bleeding, affected individuals may be predisposed to infection and/or thrombosis. This may be related to the central role of factor XII in the activation of the complement cascade and the fibrinolytic pathway (Kane, 1984). Possible enhanced thrombosis and/or infection associated with factor XII deficiency has only been described in humans. This may be related to the relatively short life span of domestic animals compared to humans. Factor XII is normally lacking in the plasma of birds, marine mammals, and reptiles (Dodds, 1981). 

6.4.4.3 FACTOR XI

Individuals with severe factor XI deficiency characteristically have a minor bleeding diathesis which becomes major following trauma or surgery (Dodds and Kull, 1971). The disease is relatively rare and has only been described in the springer spaniel, Great Pyrenees, Weimaraner, and Kerry blue terrier (Dodds, 1984a). The occurrence of a circulating factor XI inhibitor, resulting in epistaxis, has been described in an adult cat (Feldman, et al., 1983).

The mode of inheritance is autosomal; it is not clear whether the gene is dominant or recessive. Affected individuals with factor XI levels less than 30 to 40% will usually have a prolonged APTT and ACT. Bleeding episodes can be curtailed by the intravenous infusion of fresh or fresh-frozen autologous plasma at 6 to 10 ml/kg of body weight. Factor XI deficiency has also been recognized in Holstein cattle (Kociba et al., 1969; Dodds, 1981).

6.4.4.4 FACTOR X

Factor X deficiency is a rare hemorrhagic disorder which to date has only been described in a family of American cocker spaniels (Dodds, 1973). The inheritance pattern is autosomal dominant with variable penetrance. Individuals homozygous for the gene are usually stillborn or die within the first weeks of life with massive pulmonary and/or abdominal hemorrhage. Heterozygotes have intermediate levels of factor X and have a mild to severe bleeding tendency. Individuals with factor X levels less than 30% usually have a prolonged PT, APTT and ACT. Treatment to control bleeding requires the intravenous infusion of fresh-frozen autologous plasma. 

6.4.4.5 FACTOR IX

Factor IX deficiency or hemophilia B is a sex-linked hemorrhagic disorder identical in presentation to factor VIII deficiency (hemophilia A). Although not as common as factor VIII deficiency the disorder has been described in one mixed breed dog (Littlewood et al., 1986) and nine purebred dogs (Sherding and DiBartola, 1980; Peterson and Dodds, 1979; Campbell et al., 1983; Verlander, 1984). In addition the defect has been recognized in one family of British shorthair cats (Dodds, 1981) and one family of Siamese-cross cats (Dillon and Boudreaux, 1987). In the latter group of cats, Danazol proved to be ineffective in raising factor IX activity (Boudreaux and Dillon, 1988). In the summer of 1989 the author acquired a factor IX deficient (3% factor IX activity) domestic medium haired cat donated from a small animal clinic in Columbus, Georgia. The veterinarian, a recent graduate from Auburn, had an unforgettable experience when she declawed and castrated the young, undiagnosed, hemophiliac. She was able to save the cat with repeated blood transfusions over a several week period. The cat is now an adult and in a household with 2 other cats. The only evidence of a hemostatic defect is the periodic occurrence of lameness associated with joint hemorrhage. These bleeds have been self-limiting, transfusions have not been required.

In cases of severe factor IX deficiency (<1%) puppies or kittens may die at or immediately following birth. Excessive bleeding from the umbilical cord or tails and feet at the time of tail docking and dew claw removal are common signs. Hemarthrosis, gingival bleeding during tooth eruption, and spontaneous hematoma formation are other typical manifestations. Individuals with factor IX levels between 5 and 10% may not be recognized as being hemophiliacs unless challenged with trauma or surgery. Small dogs and cats also often go unrecognized as having a bleeding disorder despite the presence of very low levels of factor IX, presumably due to their light weight and usually protected environments. Affected individuals will have a prolonged ACT and APTT. Carriers of hemophilia B usually have factor IX levels between 40 and 60% and cannot be detected on routine coagulation screening tests. Identification of a carrier state in animals less than 6 months of age by factor activity evaluation should be done with caution since young animals may normally have lower activity than adults. Age-matched controls are important but may not eliminate the necessity of reevaluating the individual at 8 to 12 months of age. As with factor VIII deficiency the disorder primarily affects males except in closely inbred families where females may be affected as well. Transfusion of fresh or fresh-frozen homologous plasma at 6 to 10 ml/kg every 12 hours is the recommended treatment for acute bleeding episodes. Although external hemorrhage is fairly easy to discern, affected individuals often bleed internally either into the thorax or abdomen, between fascial planes separating muscle groups or into the brain. These bleeding episodes are often not recognized until a crisis exists. Unchecked hemorrhage can result in muscle necrosis, paralysis, seizures and/or hypovolemic shock. 

6.4.4.6 FACTOR VIII

Factor VIII deficiency or hemophilia A is one of the most common inherited hemostatic defects in dogs and cats (Dodds, 1984a). The defect has also been recognized in Arabians, standardbreds, Quarter horses and thoroughbreds (Dodds, 1975c; Henninger, 1988). Due to the X-linked nature of the disorder males are usually affected while females are asymptomatic carriers. The birth of affected female progeny requires the mating of an affected male to a carrier female, a possibility in closely inbred families. The other possibility is the spontaneous development of hemophilia A as a result of a gene mutation. This may have been the cause of hemophilia in an adult female Cocker spaniel-poodle cross examined at Tufts University (Murtaugh and Dodds, 1988).

Puppies with factor VIII deficiency usually experience prolonged bleeding from the umbilicus at birth, the gingiva during tooth eruption, and following routine surgical procedures such as tail docking and ear cropping. Spontaneous hematoma formation, hemarthrosis and hemorrhagic body cavity effusions are also common manifestations. Individuals having less than 5% factor VIII activity usually experience the most severe bleeding diathesis. Factor VIII levels between 5 and 10% may not be associated with spontaneous hemorrhage. The hemostatic defect in these puppies is often not recognized prior to a traumatic or surgical event. A recurrent shifting leg lameness may be the only manifestation observed (Johnstone and Norris, 1984). Factor VIII deficiency in cats can be associated with spontaneous hematoma formation, but, presumably due to their light weight and agility, affected cats often do not experience prolonged hemorrhage except following trauma or surgery (Cotter et al., 1978). A similar presentation may also be observed in small breed dogs. Factor VIII deficiency is characterized by a prolonged APTT and ACT. Factor VIII activity is often less than 10% while factor VIII-related antigen (von Willebrand factor) levels are normal or greater than normal (Dodds, 1984a). Carriers of hemophilia A have intermediate levels of factor VIII (40 to 60%) but identification of carriers should be done with caution in animals less than 6 months of age. Carriers usually have a normal APTT and ACT. Treatment for hemophilia A requires repeated transfusions of fresh whole blood, plasma, or frozen plasma concentrates at 6 to 10 ml/kg of body weight 2 to 3 times a day until bleeding is under control. Plasma transfusions are preferred due to the possible sensitization of the animal to red blood cell antigens. 

6.4.4.7 FACTOR VII

Factor VII deficiency has been recognized in beagles, miniature snauzers, Alaskan malamutes, boxers, and bulldogs (Dodds, 1984a). The disorder is usually not accompanied by detectable bleeding although affected individuals may experience bruising or prolonged bleeding following surgery. Postpartum hemorrhage has also been described as a potential complication (Spurling et al., 1974). Factor VII deficiency is usually discovered fortuitously during screening tests for blood clotting ability. The disorder is characterized by a prolonged PT. The inheritance pattern is autosomal with incomplete dominance. 

6.4.4.8 FACTOR II

Disorders of prothrombin or factor II are extremely rare and have only been described in the English cocker spaniel (Hill et al., 1982) and boxer breeds (Dodds, 1979). In humans and in the boxer the defect has been characterized as having an autosomal recessive inheritance pattern. The disorder in the boxer was one of dysprothrombinemia, immunologic levels of the prothrombin molecule were normal. The existence of an abnormal prothrombin or an actual prothrombin deficiency was not determined in the cocker spaniel. Signs in affected puppies included epistaxis and gingival bleeding. In adults bleeding episodes became milder and individuals experienced easy bruising and/or dermatitis. Coagulation studies in untreated puppies were characterized by a prolonged ACT, APTT and PT. The TT was normal. Transfusion of fresh whole blood successfully arrested bleeding episodes for up to a period of three days. Fresh or fresh-frozen plasma transfusions, however, are preferred if the animal does not require red blood cells as well. 

6.4.4.9 FACTOR I (FIBRINOGEN)

Congenital afibrinogenemia has not been described in dogs or cats but dysfibrinogenemia has been recognized in one inbred family of Russian wolfhounds (Dodds, 1984a). Coagulation screening test results included a prolonged ACT, APTT, PT and TT. Fibrinogen could be detected by quantitative but not by qualitative methods. Affected animals experienced mild bleeding manifested by lameness and epistaxis; challenge with surgery or trauma resulted in life threatening bleeding. Hypofibrinogenemia has been reported in the St. Bernard and Vizsla. Bleeding was severe and coagulation screening tests were similar to dysfibrinogenemia, however, a quantitative reduction in fibrinogen was determined (Dodds, 1978). Congenital afibrinogenemia has been reported in one family of Saanen dairy goats (Dodds, 1984). Treatment to arrest protracted bleeding includes the intravenous infusion of fresh or fresh-frozen plasma or plasma cryoprecipitate. 

6.4.5 VITAMIN K-DEPENDENT MULTIFACTOR COAGULOPATHY

A congenital coagulopathy has been described in Devon Rex cats (Maddison et al., 1990) that is characterized by hematoma formation and hemorrhage into the conjunctiva, joints, and thoracic and abdominal cavities. Affected cats have prolonged PT and APTT tests and diminished levels of factors II, VII, IX, and X. Hemorrhage can be controlled with vitamin K1 treatment. The pathogenesis of the factor deficiencies is uncertain but includes a selective malabsorption of vitamin K that is overcome by large doses of vitamin K1 or an abnormality in vitamin K metabolism that impedes the gamma-carboxylation of vitamin K-dependent factors. The inheritance pattern appears to be autosomal. 

6.4.6 CUTANEOUS ASTHENIA (EHLERS-DANLOS SYNDROME)

Cutaneous asthenia, also known as rubber puppy disease, is an uncommon, congenital, inherited connective tissue disorder characterized by loose, hyperextensible, fragile skin (Muller et al., 1983). The defect has been recognized in both dogs and cats as well as in humans, mink, cows, sheep and horses. The underlying defect is in the synthesis and/or maturation of type I collagen and, as a result of lack of vascular support, affected animals often experience subcutaneous hematomas (Poulsen et al., 1985). A platelet function defect may also be present in this disease (George et al., 1984). There is no treatment. 

6.5 ACQUIRED COAGULATION AND VASCULAR DISORDERS 

6.5.1 COUMARIN/INDANEDIONE TOXICITY

Rodenticide induced coagulopathies are fairly common in the dog and cat. Clinical signs can vary and may include lethargy, respiratory distress, lameness, petechial and ecchymotic hemorrhages, epistaxis and hemoptysis (Schulman, 1986). Occasionally sudden death occurs without prior signs of illness. Anticoagulant rodenticides interfere with the vitamin K-dependent carboxylation of factors II, VII, IX and X. The epoxide-reductase enzyme necessary for the recycling of vitamin K is inhibited resulting in depletion of body stores of vitamin K. As a result only nonfunctional precursors of the vitamin K-dependent factors are synthesized (Davis, 1982). Coumarin compounds including warfarin, coumafuryl, brodifacoum, and bromadiolone have a half-life of up to 55 hours. In contrast, indanedione compounds (pindone, valone, diphacinone and chlorophacinone) have a half-life as long as 15 to 20 days (Mount and Feldman, 1983). In addition the indanedione compounds may interfere with exocrine pancreatic function resulting in reduced intestinal absorption of vitamin K (Mount and Feldman, 1983). Coagulation screening tests in uncomplicated rodenticide toxicity are usually characterized by a prolonged ACT, APTT, and PT with a normal TT. Since factor VII has the shortest half-life of the vitamin K-dependent factors it is conceivable that with early detection only the PT will be prolonged. However, bleeding will likely not be present at this time and there will be a history of rodenticide ingestion within the past 24 hours. Untreated or high dose rodenticide toxicity can also be associated with thrombocytopenia and/or DIC. Due to the marked difference in the duration of action of coumarin versus indanedione compounds it is important to determine the source of toxicity prior to instituting therapy. The recommended treatment for coumarin toxicity is 0.25 mg to 2.5 mg of vitamin K1/kg for four to six days. In contrast, treatment for indanedione toxicity may require doses of vitamin K1 as high as 5 mg/kg for 3 to 6 weeks. High-dose vitamin K1 therapy, however, should be administered with caution since vitamin K1 was reported to induce Heinz body anemia in a dog when administered at 4 mg/kg for 5 days (Fernandez et al., 1984). In life threatening situations, initiation of therapy by subcutaneous or intramuscular injection followed by oral dosing may be required. Intravenous injection is not recommended nor is therapy with vitamin K3.

6.5.2 ROCKY MOUNTAIN SPOTTED FEVER (RMSF)

Rickettsia rickettsii, the etiologic agent of RMSF, is an obligate intracellular parasite transmitted principally by the vector ticks Dermacentor variabilis and Dermacentor andersoni. The organism invades vascular endothelial cells leading to cell necrosis, increased vascular permeability, and perivascular hemorrhage and edema (Greene and Phillip, 1984). The developing vasculitis is accompanied by thrombocytopenia and variable activation of the coagulation mechanism. Early signs may include petechial and ecchymotic hemorrhages of the skin and mucous membranes, retinal hemorrhage, epistaxis, melena, and hematuria. Severely affected individuals may develop DIC. The most useful test for diagnosing RMSF is the microimmunofluorescence (micro IF) test. Although cross reactions do develop between other rickettsial organisms the titer is usually highest for the specific rickettsia causing the infection. Paired acute and convalescent serum samples (10 to 14 days apart) are recommended for testing. Tetracycline or chloramphenicol is the recommended therapy. Chloramphenicol should be administered with caution to young dogs and cats, however, to avoid toxicity related to reduced drug metabolism by an immature liver. Intravenous fluid therapy should also be used with caution due to the presence of enhanced vascular permeability. Although RMSF has been recognized extensively in the dog its significance in the cat is unknown.

6.5.3 HERPESVIRUS INFECTION

Although the incidence is sporadic, herpesvirus infection can result in the rapid death of puppies usually between ages of 7 and 21 days. The disease is characterized by multiple hemorrhages throughout numerous tissues including the liver, kidney, brain, gastrointestinal tract and lung as a consequence of a viral-induced necrotizing vasculitis (Carmichael, 1977). Puppies usually die within 24 hours; treatment is often unsuccessful.

6.5.4 LIVER DISEASE

The liver is the major site of synthesis for most of the coagulation proteins. Because coagulation factors have a relatively short half-life (4 hours to 2 days) hepatic disease may result in a fairly rapid change in factor activity. Hepatic degeneration, inflammation, cirrhosis or neoplasia may result in decreases in factor activity, particularly factors XI, IX, X and VIII. Decreases in factor activity may or may not be sufficient to cause increases in coagulation screening tests since factor activity generally must be less than 30% to affect these tests. However, in two studies performed in dogs, the PT and/or APTT were abnormal in 50 to 66% of dogs with liver disease (Badylak et al., 1983; Badylak and Van Fleet, 1981). Although screening tests such as the PT and APTT may be fairly sensitive in detecting coagulation factor abnormalities associated with liver disease, they are not specific and cannot be used to predict the type of liver pathology present. Fibrinogen and vWF factor activity may actually be increased with liver disease. This may be related to the nature of fibrinogen (acute phase reactant) and the extrahepatic synthesis of vWF.

6.5.5 DISSEMINATED INTRAVASCULAR COAGULATION (DIC)

Disseminated intravascular coagulation is a pathologic process involving overwhelming activation and consumption of coagulation proteins and platelets often accompanied by enhanced fibrinolysis (Fruchtman and Aledort, 1986; Kane, 1984). The syndrome may occur secondarily to a variety of events including viral, bacterial, protozoal or rickettsial infections, parasite migration, heat stroke, burns, shock or trauma. In newborn humans the most common triggering events for the induction of DIC are hypothermia, hypoxia, shock and sepsis. Important contributing factors include the immaturity of the newborn's clotting mechanism and reduced or inadequate protective functions such as reduced ATIII activity and impaired immune-phagocytic clearance mechanisms (Buchanan, 1978). Gastrointestinal disease often results in hemostatic abnormalities in horses (Johnstone and Crane, 1986; Morris et al., 1988). This includes disease secondary to colitis, torsion/obstruction, impaction, or Ehrlichia risticii (Potomac horse fever; equine ehrlichial colitis). Laboratory abnormalities usually include increased fibrinogen concentrations and a prolonged APTT. FDP concentrations may or may not be increased. Recently, protein C and plasminogen were found to decrease in horses with intestinal ischemia and endotoxemia (Welles et al., 1991). In the future, protein C and plasminogen may be useful predictors for the outcome of equine colic. The occurrence of laminitis in horses with gastrointestinal disease may be secondary to activation of hemostasis resulting in microvascular thrombosis and digital ischemia.

Although DIC is not a primary event, if left unchecked it will often result in the death of the animal due to thrombosis and/or hemorrhage within vital organs. A key to the control of DIC is identification and elimination of the underlying cause. Acute DIC is characterized by prolongation of the ACT, APTT, PT, and TT, elevated FDP's (>40 ug/ml), a reduction in ATIII and thrombocytopenia. Chronic DIC is often more difficult to recognize; compensation by the liver and bone marrow in coagulation factor and platelet production, respectively, may result in normalization or even shortening of many of the coagulation screening tests. Horses commonly present with a chronic DIC type of pattern. Horses with DIC are rarely hypofibrinogenemic and are almost always hyperfibrinogenemic. This is probably related to the ability of the equine liver to rapidly synthesize this acute phase reactant protein in response to inflammation.

Treatment for DIC should be centered on the identification and treatment of the underlying cause. Supportive therapy including intravenous fluids to maintain fluid volume and organ perfusion are vital. Heparin therapy is controversial and often must be accompanied by plasma transfusions to obtain effective ATIII activity.

6.5.6 PLATELET DISORDERS

Platelet disorders can be generally divided into four categories (Table VI.2), congenital and acquired thrombocytopenias, and congenital and acquired functional platelet disorders. Congenital abnormalities in platelet function are further categorized as defects in adhesion, aggregation and/or secretion. These defects may be either intrinsic or extrinsic to the platelet. The prevalence of intrinsic congenital platelet function disorders in animals is not known mainly due to the scarcity of specialized laboratories equipped to evaluate platelet function in non- human species. The common practice by professional breeders of mating closely related individuals to maintain selected desirable traits also increases the risk of producing congenital defects. Intrinsic platelet function defects may be difficult to recognize but should be suspected in young nonmedicated animals experiencing mucosal or superficial bleeding in the presence of normal coagulation screening tests, normal levels of von Willebrand factor, and a normal platelet count. Although documentation is scarce, newborn puppies are believed to have platelet numbers comparable to adults (Earl et al., 1973). Platelet function is newborn dogs and cats is not known but is reported to be normal in human infants (Blifeld et al., 1986. Acquired platelet function disorders are also often characterized by thrombocytopenia.

6.6 PLATELET SCREENING TESTS

6.6.1 PLATELET COUNT

Acquired thrombocytopenia is the most common cause of platelet-related bleeding disorders. Congenital thrombocytopenia is a rare disorder in animals, one example is the thrombocytopenia which accompanies the cyclic hematopoiesis of gray collies. There are many causes of acquired thrombocytopenia including vaccines, rickettsial agents, viruses, and drugs. Accurate platelet counts are important in the monitoring of these patients to determine if treatment has been successful. Platelet counts are usually performed in practice with the use of the Unopet system, a hemocytometer, and a microscope. In some practices and in most clinical pathology laboratories, platelet counts are performed with automated instruments. Many of these instruments will also determine the mean platelet volume (MPV). The MPV is an indication of the size of the circulating platelet population. A high MPV in the face of thrombocytopenia generally means that megakaryocytes are attempting to respond to the low platelet number by releasing larger platelets into the circulation, this is a favorable sign. A low MPV in the face of thrombocytopenia may indicate that there are insufficient numbers of megakaryocytes present or that they are failing to respond. The normal canine range for MPV (AU Clinical Pathology laboratory) is 6.2 - 10.6 fl for EDTA samples.

The sample of choice for the determination of platelet number is a blood sample collected into EDTA. Platelet counts can be performed on citrated whole blood prior to the centrifugation of the sample to remove plasma for coagulation testing. (You cannot remix blood that has been sedimented because the platelets in such a sample will be clumped and an accurate count cannot be performed.) Samples collected for platelet counts cannot be sent through the mail; platelet counts should be performed on samples within 2 to 4 hours of collection. Normal platelet counts are usually greater than 150,000/ul although there are species differences. In some horses and ponies platelet counts may consistently be as low as 90,000/ul, however these are unusual cases. Prediction of whether an animal will bleed excessively cannot be made based on circulating platelet number. Some individuals with platelet numbers as low as 10,000/ul will not spontaneously bleed while others with platelet numbers as high as 100,000/ul will have hemorrhagic tendencies. Variables such as platelet size and function, and vascular integrity, may all contribute to the variability observed clinically. Platelet number can be estimated by evaluating a blood smear, however, this technique is not recommended if the patient is suspected of having thrombocytopenia. Smear evaluation is too crude an estimate of platelet number for critical assessment of response to treatment.

6.6.2 BLEEDING TIME

The bleeding time is generally considered to be an indicator of primary hemostasis or platelet status. This procedure has much more applicability to humans but many techniques have been attempted and described in animals. The existence of haired, thick skin and relative lack of a consistent surface to perform bleeding times on are but two of the obstacles veterinarians face. The cuticle bleeding time, a procedure where the toenail is cut and the time it takes for bleeding to stop is measured, is not recommended. A wide variation in times can be obtained depending on how much of the toenail is severed not to mention the pain that can be inflicted with such a technique. The buccal mucosa bleeding time (Jergens et al., 1987; Parker et al., 1988) has been described in dogs and cats and appears to be a fairly reliable indicator of primary hemostasis. In this technique the bleeding time is performed on the oral mucosa using a spring-loaded cassette which delivers a precise depth and length of cut. The technique avoids the hair and thick skin normally encountered when attempting to perform a bleeding time. The technique does, however, usually required that the animal be anesthetized. Use of the bleeding time to evaluate primary hemostasis generally should be reserved for those patients in which platelet number is known to be adequate but platelet function is questioned. The technique would be especially useful in Doberman Pinschers just prior to ear cropping.

6.6.3 CLOT RETRACTION

Clot retraction is a test of platelet function than can be performed in a clinical setting. (The technique is described in a separate section.) As with the bleeding time, this technique should be reserved for animals known to have normal platelet numbers but have questionable platelet function. The clot retraction test should also not be performed in animals known to be on medication that will inhibit platelet function. The clot retraction test is a test of platelet function which relies on the normal interaction between thrombin, platelet receptors, and fibrinogen. Because it is a fairly specific test of platelet function, not all animals with a platelet function defect will have an abnormal clot retraction test. Basset hounds with canine thrombopathia have platelets which will interact with thrombin and express fibrinogen receptors, therefore the clot retraction test in these patients is normal. Otterhounds with thrombasthenic thrombopathia have platelets which either lack or have reduced amounts of the receptor needed for normal interaction with fibrinogen. These patients have abnormal clot retraction. Patients with von Willebrand's disease have normal clot retraction because their in vivo adhesion defect is extrinsic to the platelet; platelets of these individuals respond normally to thrombin.

6.6.4 PLATELET AGGREGATION

Platelet evaluation using a platelet aggregometer is presently the definitive means for evaluating platelet function. The technique is highly specialized and not readily available. Platelet function testing requires the collection of 10 to 20 mls of citrated, whole blood and the isolation of platelet rich plasma. Because testing must be completed within 4 hours of collection, the animal being tested must be on or near the premises, samples cannot be sent through the mail. Although human laboratories are more likely to be equipped for the performance of platelet function testing, use of one of these laboratories is not recommended unless the lab is well-versed in the isolation and testing of platelets in the particular species of interest. Techniques routinely used for human samples cannot be applied to veterinary samples and there is also much variation within the veterinary species. Platelet function testing, including aggregation and release, are available at AU for the horse, dog, cat, cow, sheep and goat.

6.7 CONGENITAL INTRINSIC PLATELET FUNCTION DISORDERS

6.7.1 CHEDIAK-HIGASHI SYNDROME (CHS)

Chediak-Higashi syndrome is an autosomal recessive genetic disorder characterized by abnormal leukocyte, melanocyte and platelet granulation (Meyers et al., 1982). Platelets of affected individuals lack discernible dense granules and are deficient or reduced in storage pools of adenine nucleotides, serotonin, and divalent cations. Studies of platelet ultrastructure indicate that CHS platelets do not form tight aggregates in response to ADP in vitro. The disease has been identified in a line of Persian cats; all of the affected animals exhibited a "blue smoke" hair color and pale irises with the development of bilateral nuclear cataracts in several individuals (Prieur et al., 1979; Kramer et al., 1977; Collier et al., 1979). Affected cats experienced prolonged bleeding at incision sites and the development of hematomas following venipuncture. Chediak- Higashi syndrome has also been diagnosed in mink, cattle, and mice.

6.7.2 CANINE THROMBOPATHIA

Canine thrombopathia is an hereditary, intrinsic platelet disorder which has only been described in basset hounds (Johnstone and Lotz, 1979). Affected individuals exhibit signs typical of quantitative or qualitative platelet defects, ie., epistaxis, gingival bleeding, and petechiation. The mode of inheritance for the defect is unknown but evaluation of affected families suggests an autosomal dominant inheritance with variable penetrance (Dodds and Catalfamo, 1986; Catalfamo et al., 1986). The platelet defect is characterized by abnormal fibrinogen receptor exposure and impaired dense granule release. The underlying dysfunction is related to defective stimulus-response-coupled platelet activation. Thrombopathic platelets have been shown to have elevated basal levels of cyclic AMP (Boudreaux et al., 1985). The presence of impaired phosphodiesterase activity in intact cell studies but normal phosphodiesterase activity in disrupted cell preparations implies the defect is one of regulatory control (Boudreaux et al., 1986a; Boudreaux et al., 1986b). Signs referable to platelet dysfunction in non-thrombocytopenic basset hounds with normal levels of von Willebrand factor is suggestive of canine thrombopathia. Unfortunately, reliable diagnosis of the disease requires that the affected individual be presented at specialized facilities equipped for the evaluation of platelet function; the clot retraction test in this disorder is normal. At present, a screening program is being developed to facilitate diagnosis of platelet function defects in the field (Catalfamo, 1986). This program has greatly enhanced the identification of thrombopathic basset hounds.

6.7.3 THROMBASTHENIC THROMBOPATHIA

Thrombasthenic thrombopathia is an autosomal, inherited, intrinsic platelet disorder of otterhounds (Raymond and Dodds, 1979). The defect is distinguished by the presence of bizarre, giant platelets (30-80%) resembling those described in the Bernard-Soulier syndrome as well as by reductions in the membrane glycoproteins II and III similar to platelets of individuals with Glanzmann's thrombasthenia. Platelets of affected individuals fail to support normal clot retraction, have reduced retention on glass bead surfaces, and fail to aggregate normally in response to ADP, collagen or thrombin. The existence of an abnormal clot retraction test in this disorder greatly facilitated its identification in the field. Affected otterhounds have prolonged bleeding times and readily form hematomas at sites of injury or venipuncture.

6.7.4 SPITZ THROMBOPATHIA

An intrinsic platelet disorder has been described in 2 Spitz dogs that presented at the Auburn University Small Animal Clinic (Boudreaux et al., 1992). Both dogs were female and presented at separate times (approximately 18 months apart) with histories of chronic epistaxis and gingival bleeding. One of the dogs had a shifting leg lameness. both dogs were anemic from chronic blood loss at the time of presentation. The platelets of the dogs did not aggregate in response to ADP, collagen, or PAF. The platelets did aggregate in response to thrombin but there was a lag phase. So far the platelet disorder is very similar to the one described in basset hounds (Canine Thrombopathia).

6.7.5 BOVINE THROMBOPATHIA

Bovine thrombopathia is an autosomally inherited function defect of Simmenthal cattle. Clinical bleeding varies from mild to severe and is exacerbated by trauma or surgery.

6.8 CONGENITAL EXTRINSIC PLATELET FUNCTION DISORDERS

VON WILLEBRAND'S DISEASE (VWD)

Von Willebrand's disease due to defective or deficient factor VIII-von Willebrand factor (FVIII-vWF) is the most common inherited bleeding disorder of dogs (Dodds, 1984b). Two inheritance patterns exist, one is autosomal recessive while the other is autosomal with an incompletely dominant expression (Dodds, 1975b). The latter form is more common and has been recognized in nearly all dog breeds. Signs include mucosal bleeding primarily manifested by gingival bleeding, epistaxis, and hematuria. Stillbirths, neonatal deaths, prolonged bleeding at tail docking, ear cropping or dew claw removal are other common manifestations. Factor VIII-vWF circulates as a complex with factor VIII-coagulant, the protein deficient in hemophilia A. Factor VIII-coagulant activity may also be reduced in severe forms of VWD resulting in prolongation of the APTT and ACT. However, in most cases of VWD all coagulation screening tests are normal. Because factor VIII-vWF is important in mediating the adhesion of platelets to subendothelial surfaces the presentation mimics an intrinsic platelet function defect or thrombocytopenia. However, because platelets are normal in this syndrome, vWD is referred to as an extrinsic platelet function disorder. Puppies experiencing a bleeding diathesis in the absence of abnormal coagulation screening tests or thrombocytopenia should be tested for VWD. At present most assays are quantitative and involve electroimmunodiffusion techniques (Benson et al., 1983), however, a qualitative assay has also been developed (Johnson et al., 1985) and an ELISA technique is being developed to replace the electroimmunodiffusion technique. Citrated plasma samples to be analyzed for factor VIII-vWF should be frozen immediately and sent frozen in plastic tubes to a veterinary diagnostic laboratory within two weeks of collection. Puppies should not have been vaccinated or received medication within 2 weeks of sampling. Affected puppies may only experience bleeding problems following vaccination or surgical procedures. The administration of drugs known or suspected to alter platelet function should be avoided. Hemorrhagic crises can be arrested by the transfusion of autologous fresh whole blood or plasma at 6 to 10 ml/kg. Plasma is preferred especially if cross-matching cannot be performed since these dogs may require repeated transfusions. Desmopressin acetate (DDAVP), a synthetic analogue of vasopressin, has been administered intravenously in humans to raise the concentration of circulating vWF (Warrier and Lusher, 1983). A maximal response (2-fold or greater rise in vWF) is usually reached within 1 to 2 hours after a dosage of 0.3 ug/kg body weight. Unfortunately equivalent responses have not been seen in normal healthy dogs or in Doberman Pinschers with vWD even at dosages as high as 3 ug/kg body weight (Giger and Dodds, 1989). In spite of the lack of observable rise in vWF activity, the bleeding times of dogs with vWD did shorten to the normal range 2 hours after administration of DDAVP. Possibly DDAVP may be useful in some dogs as a transient relief to a bleeding episode. DDAVP has also been administered to canine plasma donors within 2 hours of the collection of blood to enhance the efficacy of the plasma transfusion. Von Willebrand's disease is rare in the cat and only one case has been described (French et al., 1987).

6.9 ACQUIRED PLATELET FUNCTION DISORDERS

6.9.1 FELINE LEUKEMIA VIRUS INFECTION (FeLV)

Viral replication and accumulation within the cytoplasm of megakaryocytes results in the infection of circulating platelets (Beck et al., 1986). Feline leukemia virus associated thrombocytopenia may be due to aplasia or atrophy of bone marrow stem cells, immune-mediated clearance of infected cells or extravascular sequestration within lymphoid tissues. Thrombocytopenia, thrombocytosis and/or impairment of platelet function may accompany FeLV induced myeloproliferative disease (Boyce et al., 1986). Hemorrhage occurring in the face of a normal or elevated platelet count and a normal coagulation profile is an indication of impaired platelet function. Abnormal platelet function has been demonstrated in numerous cases of myeloproliferative disease in humans (Schafer, 1984) and in a dog with radiation-induced megakaryoblastic leukemia (Cain et al., 1986).

6.9.2 DRUG-INDUCED

Drugs can impair platelet function by inhibiting receptor binding of agonists, by inhibiting transduction of messages received at the platelet surface, or by inhibiting the execution of the platelet response (Table VI.1) including aggregation, secretion, or the generation of TXA2 (Cowan, 1982). Drug-induced impairment of platelet function may not be clinically significant unless coupled with another underlying platelet function defect such as von Willebrand's disease.

6.10 CONGENITAL THROMBOCYTOPENIA

CYCLIC HEMATOPOIESIS

Cyclic hematopoiesis is an autosomal recessive disorder described in gray collies characterized by cyclic fluctuations in the number of circulating neutrophils, reticulocytes and platelets (Cheville, 1975; DiGiacomo et al., 1983). The basis for the disease is a bone marrow stem cell defect resulting in neutropenic and thrombocytopenic episodes occurring approximately every twelve days. Mortality is high, most puppies die prior to six months of age due to fulminating infection. Since thrombocytopenia occurs concurrently with neutropenia, excessive bleeding is a potential complication.

6.11 ACQUIRED THROMBOCYTOPENIA

6.11.1 EHRLICHIA PLATYS INFECTION

The rickettsial organism Ehrlichia platys is the causative agent of canine infectious cyclic thrombocytopenia (Harvey et al., 1978). The disease is characterized by thrombocytopenic episodes occurring at one to two week intervals following cycles of enhanced parasitemia in acutely affected individuals. Single to multiple round or oval basophilic inclusions may be observed in infected platelets immediately prior to periods of thrombocytopenia, however the percentage of infected platelets declines with each succeeding wave of parasitemia. Chronic infections may not be cyclic and may present as slowly resolving thrombocytopenias. Ehrlichia platys infection alone has not been reported to be associated with overt bleeding, however, concomitant infection with other disorders may exacerbate an already impaired hemostatic mechanism. Diagnosis of Ehrlichia platys, as with Ehrlichia canis, is most reliably made by an indirect fluorescent antibody test (French and Harvey, 1983). Paired serum samples ten days to two weeks apart are ideal. The antibody test for Ehrlichia platys does not cross react with that for Ehrlichia canis, however, there is a high incidence of concomitant infection. Although the mode of transmission has not been determined, the tick is a likely vector. Tetracycline is the recommended therapy.

6.11.2 EHRLICHIA CANIS INFECTION

Hemorrhage manifested by hematoma formation or prolonged bleeding from venipuncture or surgical incision sites, epistaxis, gingival bleeding, retinal hemorrhages, melena, petechiae or ecchymoses is common in canine ehrlichiosis (Greene and Harvey, 1984). Affected individuals may or may not present with thrombocytopenia. Abnormal platelet function and vasculitis have been proposed as the cause of bleeding in individuals with normal platelet counts since in most cases the PT and APTT are normal. As a consequence the severity of bleeding does not always correspond to circulating platelet number. Thrombocytopenia is associated with decreased platelet survival; enhanced platelet consumption being mediated by immune, inflammatory or coagulatory mechanisms (Smith et al., 1975; Pierce et al., 1977; Lovering et al., 1980). Impaired platelet function may be due to an acquired platelet membrane defect associated with marked elevations in serum globulin concentrations as seen in some cases of multiple myeloma (Kuehn and Gaunt, 1985).

6.11.3 BOVINE VIRUS DIARRHEA

Thrombocytopenia was seen in 15 of 146 cases of clinically acute bovine viral diarrhea virus (BVDV) infection in adult cattle (Rebhun et al., 1989) and in 12 of 18 veal calves inoculated with BVDV (Corapi et al., 1990). Platelet counts ranged from 2,000 to 33,000/ul (thrombocytopenia occurred with 3 to 11 days in experimentally inoculated calves). Bloody diarrhea, petechial and ecchymotic hemorrhage, epistaxis, and abnormal bleeding from injection sites were described in affected animals. Although BVDV was detected on the surface of platelets, surface-bound immunoglobulin was not found suggesting that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela of BVDV infection.

6.11.4 DRUG-INDUCED

Drugs can induce thrombocytopenia in humans and animals by enhancing peripheral platelet destruction or by suppressing marrow production. Increased platelet consumption can be related to a direct toxic effect of the drug or to an immune-mediated destruction. Drug-induced immune thrombocytopenia may occur due to binding of the drug to the platelet surface with subsequent antibody production to the drug-platelet complex or as a result of nonspecific absorption of immune complexes on the platelet membrane. Many drugs are suspected or have been demonstrated to induce thrombocytopenia (Table VI.1). Drug-induced immune thrombocytopenia is often idiosyncratic making prediction of a reaction to a drug impossible. Removal of the drug usually results in a rapid return to normal platelet numbers. Rarely drugs can induce an auto-immune phenomenon and thrombocytopenia persists even following discontinuation of the drug. Drug-induced marrow suppression usually occurs at the stem cell level resulting in a pancytopenia and hypocellular bone marrow.

6.11.5 VACCINE-INDUCED

Modified-live adenovirus and paramyxovirus vaccines have been reported to induce thrombocytopenia in some individuals (Jones, 1984). The mechanism is unknown but is likely an immune-mediated event either associated with antibody production against viral antigens adhered to the platelet membrane or with the non-specific binding of antibody-viral complexes to the surface of platelets. The phenomenon occurs within 3 to 10 days following a challenge administration of vaccine and is therefore most likely to occur in young animals receiving routine booster injections. Vaccine-induced thrombocytopenias are usually transitory and may not be recognized unless superimposed on another platelet or coagulation defect. Routine surgical procedures such as tail docking and ear croppinping should be avoided for two weeks following a repeat vaccination.

6.11.6 IDIOPATHIC THROMBOCYTOPENIC PURPURA

Idiopathic thrombocytopenic purpura (ITP) is a thrombocytopenic condition that is probably immune-mediated. ITP has been described in horses and dogs (Byars and Green, 1982; Larson VL et al., 1983) and is characterized by blood platelet concentrations of less than 100,000/ul in the absence of other hematologic abnormalities. Megakaryocytes may be increased or absent in these patients depending on the target of the immune response. Treatment for ITP in humans has included corticosteroids, danazol, splenectomy, and ascorbate (Brox et al., 1988). Danazol was an effective treatment for cortivcosteroid-resistent immune-mediated thrombocytopenia in a dog (Blood et al., 1989).

6.12 TABLE VI.1

DRUGS MEDIATING QUALITATIVE AND QUANTITATIVE PLATELET DISORDERS

---

I. Impairment of Platelet Function

• A. Receptor Blockade or Alteration in Membrane Charge or Permeability.
  • 1. Furosemide
  • 2. Phentolamine
  • 3. Chlorpromazine
  • 4. Lidocaine
  • 5. Penicillin, Carbenicillin

B. Impairment of Signal Transduction

• 1. Papaverine
• 2. Dipyridamole
• 3. Caffeine
• 4. Theophylline

C. Impairment of Execution

• 1. Aspirin
• 2. Phenylbutazone
• 3. Indomethacin
• 4. Pentobarbital
• 5. Sulfinpyrazone
• 6. Ticlopidine
• 7. Acetaminophen

II.Thrombocytopenia

A. Marrow Suppression

• 1. Chloramphenicol
• 2. Phenylbutazone
• 3. Diphenylhydantoin
• 4. Sulfonamides
• 5. Estrogen

B. Platelet Consumption or Destruction

• 1. Ristocetin
• 2. Acetaminophen
• 3. Aspirin
• 4. Diphenylhydantoin
• 5. Levamisole
• 6. Methicillin, Penicillin
• 7. Sulfisoxazole

6.13 TABLE VI.2

QUALITATIVE AND QUANTITATIVE PLATELET DISORDERS

I. Functional Disorders of Platelets

A. Congenital (Intrinsic)

• 1.Chediak-Higashi Syndrome (cats, cattle, mink, mice)
• 2.Canine Thrombopathia (Basset hounds)
• 3.Thrombasthenic Thrombopathia (Otterhounds)
• 4.Bovine Thrombopathia (Simmenthal)
• 5.Cutaneous Asthenia? (Many dog breeds, cats)
• 6.Spitz Thrombopathia
• 7.Cyclic Hematopoiesis (Grey collies)

B. Congenital (Extrinsic)

• 1.von Willebrand's Disease

C. Acquired

• 1.Feline Leukemia Virus
• 2.Ehrlichia platys?
• 3.Ehrlichia canis?
• 4.Drug-induced

II. Thrombocytopenia

A. Congenital

• 1.Cyclic Hematopoiesis

B.Acquired

• 1.Ehrlichia platys
• 2.Ehrlichia canis
• 3.Feline Leukemia Virus
• 4.Drug-induced
• 5.Vaccine-induced
• 6.Rocky Mountain Spotted Fever
• 7.Herpesvirus
• 8.Bovine Viral Diarrhea Virus
• 9.Disseminated Intravascular Coagulation

6.14 TABLE VI.3

CONGENITAL COAGULATION PROTEIN DISORDERS

6.15 Figure VI.1

Normal hemostasis is dependent upon the complex interaction of plasma coagulation proteins, platelets and the blood vasculature.

6.16 Figure VI.2

A series of enzymes and cofactors ultimately convert prothrombin to thrombin. Thrombin cleaves fibrinogen into fibrin and activates factor XIII resulting in the formation of a stable fibrin network.

6.17 Figure VI.3

The factor VIII complex is composed of factor VIII-coagulant and von Willebrand factor. The two proteins are distinct and are under separate genetic control. Factor VIII-coagulant is approximately 1.0% of the mass of the total complex.

6.19 Factors Involved in Contact Activation

6.19.1 Factor XII

Factor XII is a single chain beta-globulin with a molecular weight of 80,000 to 90,000. Factor XII can be activated by contact with a negatively charged surface (autoactivation; solid phase activation) or by protease induced enzymatic cleavage (enzymatic activation; fluid phase activation). The physiologic solid phase activator is subendothelial vascular basement membrane. Physiologic enzymatic activators include plasmin and kallikrein. The rate of factor XII cleavage and activation by kallikrein is much faster than the autoactivation pathway. Once activated, factor XII can activate factor XI and prekallikrein.

6.19.2 Kallikrein

Plasma kallikrein circulates in blood as a precursor, prekallikrein. Seventy-five percent of prekallikrein is bound to high molecular weight kininogen (HMWK). Activated factor XII can convert prekallikrein to kallikrein which in turn reciprocally activates factor XII.

6.19.3 Factor XI

Factor XI circulates in plasma as a bimolecular complex with HMWK. Factor XI is activated by activated factor XII. Kallikrein does not directly activate factor XI.

6.19.4 High Molecular Weight Kininogen

High molecular weight kininogen (HMWK) is cleaved by kallikrein, releasing bradykinin, and producing a disulfide-linked molecule containing a heavy and light chain. The light chain binds to surfaces and directs prekallikrein binding to surfaces such that it orders the cleavage of prekallikrein and exposure of the active site for coagulation of kallikrein. A similar function for the light chain of HMWK has been shown for factor XI. Activated factor XII cleaves HMWK in a manner similar to kallikrein.

6.19.5 Vitamin K-Dependent Factors

The vitamin K-dependent factors include II, VII, IX, and X, and proteins C and S. These factors are similar in that they are all synthesized within the liver via a vitamin K-independent mechanism to an inactive precursor form. Conversion of these inactive precursors to functional proteins is vitamin K-dependent and occurs during a post-translational modification. Vitamin K serves as an essential cofactor for the enzyme that carboxylates protein-bound glutamic acid residues on the precursor proteins to gamma-carboxyglutamic acid. Upon acquisition of these gamma-carboxyglutamic acid residues, the vitamin K-dependent factors achieve an overall negative charge which allows them to interact with calcium, an event vital to their normal function. During the carboxylationstep, vitamin K is converted to an inactive epoxide and must be recycled via a series of enzymes back to the active quinone form.

 

 

6.19.6 Causes of Deficiencies of Vitamin K-dependent Factors

• 1.Dietary inadequacy - of significance in newborns or individuals without normal gut microflora.
• 2.Biliary obstruction - vitamin K is fat soluble.
• 3.Malabsorption - secondary to neoplasia, severe parasitemia, ulcerative colitis, sprue, lymphocytic-plasmacytic enteritis, etc.
• 4.Liver disease - with severe parenchymal damage the liver will be unable to use vitamin K in synthesizing vitamin K-dependent factors (vitamin K treatment will not be useful in this instance). However, many patients with liver disease respond favorably to vitamin K treatment.
• 5.Drugs
  • a.coumarin anticoagulants - vitamin K antagonism.
  • b.antibiotics - destruction of vitamin K synthesizing microflora in the gut.
• 6.Toxins
  • a.warfarin, indanedione - vitamin K antagonism.
  • b.plants - a fungal metabolite, dicumarol, is produced from substrates in spoiled sweet clover hay. Vitamin K antagonism results.

  6.19.6.1 Protein C

  Protein C is a vitamin K-dependent protein with a molecular weight of 62,000. Protein C is activated by thrombin via thrombomodulin on the surface of endothelial cells. (Thrombin forms a 1:1 complex with thrombomodulin, and the complex, in the presence of calcium, activates protein C.) Thrombin bound to thrombomodulin can still be neutralized by antithrombin III at a rate equivalent to free enzyme. However, bound thrombin has a greatly diminished ability to clot fibrinogen, activate factor V, or trigger platelet activation. Therefore thrombomodulin acts as an anticoagulant by:

  • 1) Stimulating thrombin-dependent protein C activation;
  • 2) Directly inhibiting thrombin's procoagulant activities.

  Activated protein C is a potent inhibitor of factors V & Va, and VIII & VIIIa. Protein C can prevent assembly of the prothrombinase complex thereby suppressing thrombin production. Protein C also inhibits factor Xa production by inhibiting factor VIII. Thrombomodulin-mediated protein C activation and thrombin clearance occurs primarily in the microcirculation. The rate of inactivation of activated factors V and VIII (Va and VIIIa) by protein C is 30 times faster than inactivation of V and VIII in their unactivated forms. The rate of inactivation is greatly enhanced by phospholipid and calcium (a property of all vitamin K-dependent proteins). Protein C also enhances fibrinolysis by enhancing tissue type plasminogen activator release from vascular endothelial cells.

  Protein C has a physiologic circulating inhibitor. Combined factor V & VIII deficiency, reported rarely in humans, may be, in some instances, due to deficiency of this inhibitor.

  Patients with decreased protein C (<50%) are at risk of thrombosis. Patients with DIC develop a decrease in protein C activity and a decrease in protein C inhibitor. This often is not accompanied by a decrease in protein C antigen until later (if the DIC continues and is severe). This is thought to be due to the measurement of inactive protein C either bound to its inhibitor or some other complex.

  Patients with homozygous protein C deficiency develop fatal neonatal purpura fulminans (fulminant thrombosis).

  6.19.6.2 Protein S

  Protein S is a vitamin K-dependent protein but it is not the zymogen of a serine protease. Protein S greatly accelerates the inactivation of factor Va by protein C, presumably by increasing the affinity of protein C for phospholipids. Protein S deficiency predisposes individuals to venous thromboembolism.

  Sixty percent of protein S is bound to C4b-binding protein and 40% is free. Free protein S is the cofactor for protein C. The concentration of free protein S is determined by subtracting the concentration of protein S bound to C4b-binding protein from the total protein S concentration.

6.19.7 The Coagulation Pathways - Summary

The activation of the contact system is believed to be initiated by the binding of plasma factor XII to a negatively charged surface, where autoactivation of factor XII (XIIa) occurs, converting it to an active serine protease. The binding of factor XIIa on the surface is associated with the activation and adsorption of HMWK. Since the majority of plasma prekallikrein and factor XI exists in bimolecular complexes with HMWK, activation of HMWK to allow surface binding will bring prekallikrein and factor XI to the surface. On the surface factor XIIa can cleave prekallikrein to kallikrein and factor XI to XIa. Kallikrein can initiate reciprocal activation, generating additional factor XIIa from factor XII. Factor XIa converts factor IX to factor IXa in the presence of calcium. Factor IXa, in concert with factor VIII, phospholipid, and calcium (Factor X Activation Complex) converts factor X to Xa. Factor Xa, together with factor V, phospholipid and calcium (Prothrombinase Complex) converts factor II (prothrombin) to factor IIa (thrombin). Thrombin in turn cleaves fibrinogen into fibrin monomers which are cross-linked and stabilized by factor XIIIa. Factor XIII is activated by thrombin in the presence of calcium.

The extrinxic system is much shorter than the intrinsic system and involves the exposure of blood to tissue factor (factor III), a lipoprotein which in addition to calcium and factor VII, converts factor X to factor Xa. Factor X marks the initiation of the common pathway.

 

-	Factor X Activation Complex	Prothrombinase Complex
Enzyme	IXa	Xa
Ion cofactor	calcium	calcium
Surface	phospholipid	phospholipid
Protein	-	-
cofactor	VIII	V
Substrate	X	II

• 1.Substrates (X and II) in both reactions are vitamin K-dependent (contain gamma-carboxyglutamic acid residues responsible for binding, via calcium, to negatively charged phospholipid surfaces).
• 2.Enzymes (