Hematopoiesis

Hematopoiesis occurs through differentiation of stem cells that are formed early in embryonic life. Differentiation occurs in a series of steps in which burst-forming units (BFUs) and colony-forming units (CFUs) are formed for each of the major cell lines. These undifferentiated cells continue to proliferate and differentiate under the influence of a number of cellular and humoral factors that are produced by bone marrow and peripheral tissues.

Erythropoietin (EPO) is the most important regulator of the proliferation of committed erythroid cells (see the discussion of erythropoiesis-stimulating agents [ESAs]) (Figure 15-1).² The kidney is the major site of EPO production, where it is released in response to anemia or hypoxia. Among the more important regulators of the myeloid series are granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Several vitamins are needed for red and white blood cell formation (hematopoiesis and granulopoiesis, respectively).^2,3

Figure 15-1 Red blood cell formation is stimulated in the kidney. Erythropoietic factor release is stimulated by hypoxia. Erythropoietin stimulates erythroid precursors and the release of reticulocytes into circulation.

Vitamin B₁₂

Vitamin B₁₂ (cyanocobalamin), the “maturation factor,” is essential for DNA synthesis, and its deficiency inhibits nuclear maturation and division. Because cells that rapidly multiply are affected first, reduced RBC proliferation and a “maturation arrest” in the bone marrow are among the first indications of a vitamin B₁₂ deficiency. The erythroblast cannot continue to divide, becomes very large, and is referred to as a megaloblast (megaloblastic anemia). The mature erythrocyte is also large and is referred to as a macrocyte. Although its oxygen-carrying capacity is adequate, the enlarged cell is very fragile because of its large size, and it has a reduced life span.^2,3

Vitamin B₁₂ is a porphyrin-like compound with a ring containing a centrally located cobalt.² It can be acquired from both the diet and microbes in the gastrointestinal tract. Most microbial production is in the large intestine, however, where B₁₂ is not readily absorbed because it is dependent upon receptor mediated uptake. Dietary deficiency of B₁₂ is unlikely and usually results from poor absorption (including pancreatic enzyme deficiency) from the gastrointestinal tract. Absorption of B₁₂ is complicated (Figure 15-2) and depends on several factors.² Gastric acid and pancreatic enzymes are needed to release B₁₂ from dietary and salivary binding proteins.² To avoid being digested, B₁₂ is protected by binding to intrinsic factor (excreted by the exocrine pancreas in cats⁴) and R protein, which are secreted by the parietal cells. The bound B₁₂ complex is carried to the ileum, where B₁₂ is adsorbed to highly specific receptor sites on the brush border. Vitamin B₁₂ enters the cell by pinocytosis and then enters the blood, where it is bound with transcobalamin, the plasma carrier. Excessive vitamin B₁₂ is stored in large quantities in the liver and is slowly released as needed. Vitamin B₁₂ is excreted into the bile but undergoes enterohepatic cycling. Interference with absorption by the ileum will result in continuous depletion of B₁₂. Many months of defective vitamin B₁₂ absorption are necessary, however, before vitamin B₁₂ deficiency occurs. The anemia resulting from B₁₂ deficiency is also referred to as pernicious anemia. Exclusive uptake of cobalamine in the ileum as led to its detection in serum as a tool for diagnosing disease of the ileum.⁴ Deficiency has been described (in cats) by increased serum methylmalonic acid (MMA) coupled with serum cobalamine at or below 100 ng/mL (the lowest detectable concentration). However, the description of clinical and biochemical signs associated with deficiency focused on the gastrointestinal tract and did not address tests indicative of anemia.

Figure 15-2 The absorption of vitamin B₁₂ from the gastrointestinal tract is complicated and depends on the release of intrinsic factor and hydrochloric acid from the stomach. The drug complexes with a receptor. This complex is protected as it passes to the ileum, where it is absorbed by receptor-mediated pinocytosis.

Vitamin B₁₂ is available as a parenteral preparation in the pure form of cyanocobalamin or the more highly protein bound hydroxocobalamin. Hydroxocobalamin may provide more sustained effects than cyanocobalamin when given by injection.² Vitamin B₁₂ is also available for oral administration in the pure form or in combination with other vitamins and minerals. Methylcobalamin is another congener of vitamin B₁₂ and represents one of the two intracellular active forms of the vitamin (the other being deoxyadenosylcobalamin).² Foods high in vitamin B₁₂ include selected microbial sources and animal (meat) products. There are no significant toxicities associated with therapy. Indications for B₁₂ therapy are limited to situations of B₁₂ malabsorption such as ileectomy, gastrectomy, malabsorption syndromes, or chronic administration of cimetidine or other antisecretory drugs because an acid environment is necessary for release of B₁₂ from the diet and for intrinsic factor activity. In one study⁴ response to cobalamin treatment (250 μg subcutaneously once weekly in cats (n = 19) with gastrointestinal disease was prospectively studied, with a focus on indicators of gastrointestinal health. Indicators of cobalamin deficiency (serum MMA, cysteine) improved and were correlated with weight gain and folic acid use, however, improvements in indices of red blood cell health were not addressed.

Folic Acid

Folic acid (pteroylglutamic acid) is a cofactor needed for DNA synthesis because it promotes the formation of a nucleotide necessary for DNA formation. Folic acid is also necessary for RNA synthesis, and it serves as a methyl donor for the formation of vitamin B₁₂.² Folic acid is acquired from the diet, although it can also be formed by microbes. Dietary sources include yeast, liver, kidney, and green vegetables. Folic acid is also stored in the liver but not as avidly as is vitamin B₁₂. It undergoes enterohepatic circulation but is destroyed daily by catabolic processes. Daily requirements are high, and serum levels will fall rapidly several days after dietary deficiency. Gastrointestinal absorption of folic acid is not as complex as that of vitamin B₁₂, although it requires protein digestion and the presence of dihydrofolate reductase in the small intestine. Jejunal pathology can result in folate deficiency. The degree of folic acid binding to plasma proteins is not well understood.

Folic acid is available as both a parenteral and an oral (pure or combined product) form. The minimum daily requirement in humans is 50 μg/day, but this can increase to 100 to 200 or more in patients with high cell turnover rates (e.g., hemolytic anemia).² In humans the most popular form of folic acid supplementation is as part of a multivitamin preparation containing 400 to 500 μg of pteroylglutamic acid. In high disease states, 1 to 2 1-mg tablets are consumed. This dose may be the source of the recommended dose for cats and dogs. Compared with commercially available products, the dose recommended for small animals seems excessive, and it is not clear that such a high dose is necessary. There are, however, no apparent significant toxicities associated with therapy.

Indications for therapy are inadequate intake as a result of the administration of several drugs (methotrexate, potentiated sulfonamide antibiotics, some anticonvulsants such as phenytoin), liver diseases, malabsorption, or other chronic debilitating diseases. Folinic acid (leucovorin) is a formyl derivative of tetrahydrofolic acid and as such does not require the action of dihydrofolate reductase in order to act as a folate by contributing a carbon moiety. In humans, administration of folinic acid increases serum folate activity owing to 5-methyltetrahydrofolate. Folinic acid apparently serves as a substrate for inhibitors of dihydrofolate reductase such as methotrexate, or diaminopyrimidines such as trimethoprim or ormetoprim, and as such, can resolve some deficiencies associated with folic acid deficiency. However, it will not replace deficient folic acid or any of its derivatives prior to the tetrahydrofolate step in folic acid use.² As such, leucovorin can be used clinically to circumvent the actions of dihydrofolate reductase (e.g., methotrexate) and replace materials in the folic acid pathway beyond tetrahydrofolate, but can not prevent folic acid deficiency. Leucovoran also inhibits thymidylate synthtase and as such, facilitates anticancer efficacy of 5-fluorouracil.

Hemoglobin Synthesis: Iron

Hemoglobin consists of a heme portion and a globin portion. The heme portion is formed from a pyrrole ring, four of which combine to form a protoporphyrin compound. This in turn combines with iron to form heme. Four hemes combine with the globulin globin to form hemoglobin. Several factors are necessary for hemoglobin formation.

Iron is a component of hemoglobin, myoglobin, and other substances, such as those found in cytochrome and electron transport systems.^2,3 Approximately 65% of total body iron is present in hemoglobin, 4% as myoglobin, and 1% in cytochromes and electron transport systems. The remaining 15% to 30% is stored as either ferritin, the soluble form of iron stores, or hemosiderin, the insoluble stores. Oral absorption of iron is slow, complicated, and not well understood (Figure 15-3). It is available in the diet in either a heme form, which makes up a small percentage of the total but readily absorbed form, or in a nonheme (ferric oxide) form. The nonheme form represents the largest dietary fraction, but its absorption is profoundly affected by dietary factors. Nonheme iron must be converted to the ferrous form for absorption to occur; conversion depends on an acidic environment. Absorption of iron is increased by hydrochloric acid and decreased in situations that decrease acid production in the stomach (e.g., chronic use of antisecretory drugs).

Figure 15-3 Absorption of usable iron from the gastrointestinal tract is maximized in an acidic environment. Iron combines with apoferritin in cells to form ferritin, the soluble form of iron. Iron is bound to transferrin in plasma. Saturation of apoferritin leads to saturation of transferrin. Excessive iron is stored as hemosiderin, a nonsoluble form of iron. In the presence of saturation, iron is eliminated in the gastrointestinal tract.

Iron is absorbed primarily from the proximal jejunum, where it immediately combines in the enterocyte with apoferritin to form ferritin. When transferred out of the enterocyte, ferritin dissociates, and iron combines with the globulin transferrin. Iron is transported in the plasma in this form, but the binding is loose so iron can be easily transferred to tissues. Iron is transferred to cells by way of specific receptors that interact with transferrin, especially in the liver. In cells, iron again combines with apoferritin to become ferritin. Small quantities are also stored as the very insoluble hemosiderin; the quantity of this storage form increases when the total quantity of iron in the body is much more than apoferritin can accommodate. When apoferritin is saturated, transferrin cannot release iron, and it thus becomes close to 100% bound (estimated iron stores).

There is no mechanism for the excretion of iron other than by the gastrointestinal tract. Gastrointestinal tract elimination occurs by (1) exfoliation of enterocytes containing iron, (2) biliary elimination, and (3) elimination in diet of iron not absorbed. Total body iron is regulated by altering the rate of absorption. If all the apoferritin in the enterocyte is combined with iron, the amount of iron in the enterocyte is high, and absorption from the diet is slowed. Absorption is faster if iron stores are depleted. This mechanism has been referred to as the mucosal block phenomenon.

Erythropoiesis-Stimulating Agents

EPO, an endogenous glycosylated protein hormone (MW 30,000 daltons), is the most important regulator of committed erythroid progenitor proliferation.² Extensive glycosylation is responsible for the activity of the molecule.⁶ The kidney (peritubular cell) is the primary source of endogenous EPO, although the liver produces a small amount in some species. Insufficient oxygen delivery to tissues is the primary stimulus for promotion of the transcription and thus production and secretion of EPO² (see Figure 15-1). Hypoxia or anemia will increase synthesis by a hundredfold or more. However, renal disease, damage to the bone marrow, or deficiencies of iron or other essential vitamins can impair synthesis. Inflammatory cytokines also impair secretion (as well as iron delivery). Prostaglandins appear to increase and nonsteroidal anti-inflammatory drugs appear to decrease EPO production.

Mechanism of Action

Once released from the renal cell, EPO travels to the bone marrow, where it binds to receptors on the surface of committed erythroid progenitors. The influence of EPO (including ESAs) on RBC production occurs at several steps. EPO binding initiates changes in intracellular phosphorylation.² Additionally, EPO stimulates proliferation and differentiation of erythroid precursors, including BFU–erythroid CFUs: erythroid, erythroblasts, and reticulocytes.² Recombinant human EPO also stimulates the release of reticulocytes from the bone marrow into the blood, where they subsequently mature.^7-11

Preparations

Human EPO has been isolated and cloned and can be synthesized in large quantities with recombinant techniques using a mammalian (hamster cell line). ESAs include two recombinant EPO (rEPO) products. Epoetin is a recombinant human product (rhEPO), available in two products that differ in their glycosylated carbohydrate patterns: alpha (Epogen, Eprex) and beta (Procrit). A second ESA is darbepoetin, a recombinant hyperglycosylated analog of the human protein, often referred to as a second-generation EPO. Hyperglycosylation increases stability. Darbepoetin is dosed in μg/kg, whereas epoetin is dosed as units/kg for epoetin: a dose of 6.25 to 12.5 μg/kg of darbepoeitin is equivalent in humans to a dose of 2500 to 5000 units/kg of rhEPO (0.0025 μg darbepoetin to 1 unit epoetin).¹² Albumin is included as a vehicle in both epoetin alpha and darbepoetin. However, because of the concern regarding potential transmission of disease carried by albumin, alternative vehicles such as polysorbate 80 have been used to formulate epoetin beta.¹³ Products are intended for intravenous or subcutaneous administration. Both canine (rcEPO)¹⁴ and feline (rfEPO)¹⁵ recombinant products have been developed but are not yet commercially available. Canine EPO is more closely homologous to feline EPO compared with rhEPO and will be preferred in cats should rcEPO approval precede the feline product.

Disposition

The disposition of ESAs has not been well characterized in either humans or animals. Glycosylation (addition of carbohydrates) prolongs elimination compared with the endogenous proteins. In humans the elimination half-life of rh-EPO is about 6 to 9 hours after intravenous administration, with a very small volume of distribution (0.055 L/kg). As with most endocrine products, plasma half-life may not necessarily reflect biological half-life.^9,11 Glycosylation of darbepoetin is greater than that of epoetin, leading to an accordingly longer (threefold) biological half-life in humans. Dosing subsequently can be reduced to every other day (3 times weekly).²

Adverse Effects

Indications and Clinical Use

The primary indication for ESAs in humans is anemia of chronic renal disease (CRD).^7,8,21,23,28 Most human patients receiving renal dialysis also receive ESAs. Indications in animals are likely to be similar to those in humans, particularly for renal disease, because clinical signs of weakness, somnolence, depression, and poor appetite associated with CRD in cats (and presumably dogs) is due to anemia. Decisions must be made regarding the product (epoetin, alpha or beta, versus darbepoetin), route (intravenous versus subcutaneous), and dosing regimen (dose and interval). Subcutaneous administration of epoetin—which can cause pain on administration—has been associated with better response in humans, although it appears to be more immunogenic. In contrast, darbepoetin appears to be equally efficacious regardless of the route.²⁹ Preference among the ESA products is not yet clear; evaluation of darbopoetin currently is ongoing.³⁰ Advantages of darbopoetin may include faster response time, particularly with front end loading, decreased dosing intervals, and decreased risk of immunogenicity. The average dose of darbepoetin used in human patients with CRD or cancer ranged from 3.5 to 5 μg/kg 1 to 3 times per week; preliminary studies indicate that administration as infrequent as once every 3 weeks may be sufficient.

Because it has been available longer, more information is available regarding rhEPO use in both humans and animals. In humans with CRD, rhEPO normalizes the hematocrit, hemoglobin concentration, and RBCs after administration of 15 to 300 U/kg rhEPO 3 times weekly. Some human patients have required 500 U/kg. A stair-step approach has been used for nonresponding CRD in human patients in that the dosage continues to be increased until the desired response is achieved.⁸ When the desired response is achieved (a hematocrit of 30% to 40%), a smaller maintenance dose (25 to 100 U/kg three times a week) is given to maintain the hematocrit above 30%.⁸ Normalizing the PCV in patients with underlying renal disease may be associated with a greater risk of cardiovascular side effects.³¹ Because of convenience and cost, in humans the maintenance dose generally has been given subcutaneously, whereas the induction dose is often given intravenously. However, intravenous administration increasingly is being used for maintenance dosing to minimize the risk of immunogenicity (in human patients).¹⁸ The intravenous route in humans has the added advantage of convenience in those individuals with chronic access ports (i.e., human patients undergoing dialysis), a situation that is less likely in animals.

Erythropoetin therapy is indicated in dogs or cats with a PCV of 20% to 25% or less owing to renal disease (see Table 15-1). To date, of the commercially available products, reports (whether scientific or anecdotal) of use in animals are limited to alpha EPO. Recombinant human EPO has been used in small animals with chronic anemia.^21,22,23 When administered in uremic animals with CRD, the hematocrit of most patients normalizes within 3 to 4 weeks of therapy, and the clinical well-being of patients improves. Response to therapy is indicated by reticulocytosis, and an increase in hematocrit of 0.5% to 1% each day. Hypokalemia associated with uremia in cats should also resolve, in part because of improved appetite.²² White blood cells and platelets do not seem to be affected. Caution is indicated for patients that are hypertensive before rhEPO therapy. Informed consent should be obtained before use because of the high incidence of antibody formation. Initial treatment should begin at 100 U/kg (or, if a slower increase is acceptable, 50 to 75 U/kg) subcutaneously 3 times a week until the target hematocrit (originally 37% to 45% in dogs or 30% to 40% in cats; more recently 30% to 35% for dogs and 25% to 30% in cats) has been reached.²⁷ Generally, response requires approximately 4 weeks. The PCV should be measured twice weekly for the first several months of therapy. The dose is decreased to a maintenance dose of 50 to 100 U/kg and the frequency of administration to twice weekly once the target PCV has been achieved. The dose should be titrated to maintain the PCV in the mid-target range. If the PCV does not increase sufficiently, the dose of rhEPO can be increased in 25- to 50-U/kg increments. The maintenance dose will vary for each patient. A maximum dose has not been established in animals, although weekly doses of 300 to 1050 U/kg have been reported.²²

Among the more common causes of ESA therapeutic failure in humans is iron deficiency. Iron therapy generally is begun along with rhEPO. Iron therapy is not easily monitored. In humans serum ferritin and transferrin iron saturation are the most common tests used.³² An increasing percentage of hypochromic red blood cells and decreasing content of hemoglobin in reticulocytes may also reveal iron deficiency but will not reveal iron overload. Iron gluconate and iron sucrose are considered the safest of the intravenous iron medications.³² However, because intravenous iron dextran therapy is more expensive and is associated with anaphylactic therapy, oral iron therapy is preferred.^2,8 Further, the body can control iron content with oral but not intravenous administration. Differences in the gastrointestinal absorption of iron may alter response to rhEPO therapy. Addition of 200 mg of oral iron, however, appears to successfully maintain adequate iron stores in human patients with CRD receiving rhEPO. Oral iron supplementation in dogs is recommended at 100 to 300 mg of ferrous sulfate daily (20- to 60-mg elemental iron) and for cats 50 to 100 mg (10 to 20 elemental).²⁷ Iron dextran (10 to 20 mg/kg for dogs and 50 mg total dose for cats) is recommended by intramuscular administration (deep) for animals that cannot tolerate oral supplementation. Note that oral absorption of iron may not occur until serum ferritin is below 30 to 50 ng/mL (human patients). Erythropoiesis improves, however, even in human patients whose transferrin saturation is above that diagnostic of iron deficiency (16%).³³

Anemias other than that associated with CRD also may be associated with decreased levels of endogenous EPO in animals and thus might respond to supplementation. However, the potential application of these indications in veterinary medicine must be balanced by the lack of scientific support and the adversities that are more likely in animals compared with humans if rh-EPO is used (e.g., antibody production). These have been reviewed by Langston and coworkers²⁷ and others.^2,34 An enzyme-linked immunosorbent assay (ELISA) kit developed to detect canine EPO may be helpful in identifying the need for supplementation although access at the time of publication is not known.³⁵

The most common nonrenal use of ESAs in humans is anticipation of autologous blood transfusion; use of rhEPO increased the number of autologous units collected, and pretreatment may decrease the need for allogenic transfusion therapy. Although the need is less clear, a similar approach should be of benefit in animals; experimentally, pretreatment with iron and rhEPO in dogs resulted in higher hematocrit levels after transfusion than those in pretreatment with iron and saline.²⁷ Selected (nonmyeloid) cancer anemias associated with chemotherapy may respond to EPO supplementation, including multiple myeloma and myelodysplastic diseases (the latter defined on the basis of cytopenia, dysplastic hematopoietic precursor cells and less than 30% blasts in the bone marrow).^2,27,34 Anemia associated with cancer may reflect an inadequate EPO response; in humans the ratio of measured to predicted EPO (the latter based on population studies) of less than 0.9 is interpreted as an indication of potential response. Response may be related to dose: One retrospective study of multiple myeloma in humans reported a higher overall survival in patients who received a high dose (greater than 60,000 U/week) rather than a lower routine dose.³⁶ The use of ESAs in human cancer patients has been reviewed,³⁸ and guidelines for such use have been published.^38,39

A combination of rhEPO and immunosuppressive therapy apparently was not successful in cats with pure red cell aplasia (not associated with previous rhEPO therapy) but may have prolonged survival rates of dogs with myelofibrosis.²⁷ An interesting and potentially relevant application of ESA is for patients in the intensive care unit. In human patients in the intensive care unit, blood loss, including that associated with repetitive phlebotomy for diagnostic testing, can be significant. The veterinary patient—particularly the smaller one—may be similarly predisposed. Relative EPO deficiencies in these patients further predispose to anemias, as is indicated by transferrin saturations of less than 20% in the majority of patients. The use of rhEPO decreases the number of transfusions needed in these patients. The use of exogenous EPO in viral immunosuppressive diseases is tempting but controversial and might best be based on demonstration of endogenous EPO deficiency. Whereas endogenous EPO appears to be low and thus exogenous therapy is of benefit in human patients with HIV-associated anemia, EPO concentrations are increased in feline patients with feline leukemia virus.²⁷ However, the PCV increased after 2 weeks of rhEPO in nonanemic cats positive for feline immunodeficiency virus; antibodies did not appear with this short-course therapy. Other uses of rhEPO in dogs have been characterized by variable success. The use of rHEPO in racing animals is likely to be associated with the production of antibodies and therefore is discouraged not only for ethical and legal reasons but also for health reasons. Human growth factors (granulopoietin and EPO) were associated with bone marrow recovery in a dog with ehrlichiosis.⁴⁰ However, therapy took several months before response in peripheral counts were realized. The use of EPO in patients with pancytopenia associated with parvovirus is controversial, particularly in light of one case of fulminant infection in a human patient with immunodeficiency who was subsequently treated with rhEPO.⁴¹ Evaluating response of drug-induced bone marrow depression to rHEPO is complicated by the impact of discontinuing the inciting drug.

Therapeutic Failure

The most likely reason for a patient not to respond to rhEPO is inappropriate therapy. If the anemia is not associated with CRD, rhEPO therapy may be ineffective if endogenous rhEPO concentrations are maximally increased. Failure in a patient with low endogenous rhEPO may occur for several reasons. The dosage may be insufficient. Failure of an anemic animal to respond to rhEPO may indicate the development of antibodies directed toward rhEPO, as previously discussed.²² Animals that develop antibodies should recover, with antibodies potentially resolving within 3 to 4 weeks in dogs. Recovery may depend in part on the severity of renal disease, with recovery less likely in those animals with severe renal disease. In humans pure red cell aplasia as a result of antibody formation may be treated with a low dose (3.3 mg/kg) of cyclosporine.^6,42 Differences in vehicles, stabilizers, and handling were identified as possible contributors to autoantibodies in human patients with renal disease who were receiving recombinant EPO; changes in response to these problems has dramatically reduced the incidence of antibody formation.¹⁶ Response to ESAs is likely to require iron supplementation.²² Chronic inflammatory disease decreases response to rhEPO, as do myelophthisic diseases that replace bone marrow with fibrous tissue. Alternatively, anemia may persist because patient nutrition is not sufficient to support increased EPO.

Chemistry

Anabolic steroids are synthetic compounds structurally related to testosterone. They have protein-anabolic activity similar to that of testosterone but ideally minimal androgenic effects (i.e., minimal masculinization) (Table 15-2).^51,52 Testosterone is the sole endogenous androgen in most mammals. Its androgenic effects and rapid elimination have led to the manufacture of synthetic compounds. Chemical manipulations that have produced clinically useful anabolic steroids include (Figure 15-4) (1) alkylation (addition of a methyl [CH₃] group) at the 17β position (e.g., methyl-testosterone, stanozolol), which impairs hepatic metabolism, thus prolonging elimination half-life; (2) esterification of the 17β hydroxyl group (e.g., Deca-Durabolin) such that absorption from parenteral sites is prolonged; and (3) modification of the steroidal ring structure. The sequelae of changes in the steroidal structure vary with the modification and include prolonged absorption or elimination.

Table 15-2 Anabolic and Androgenic Activity of Selected Steroids

Figure 15-4 Chemical structures of several anabolic steroids. Rings and their positions are indicated for testosterone. Esters on R-groups also alter absorption characteristics. Methylation (methyltestosterone, stanozolol) increases anabolic activity as well as toxicity. Nandrolone is a nonmethylated anabolic steroid. Mibolerone has little anabolic activity, whereas the double bond at C1 and C2 contributes to marked activity with boldenone. Nonmethylated products tend to be less efficacious but less hepatotoxic.

Mechanism of Action

The mechanism of hematinic action of anabolic steroids on the cellular level is typical of the steroidal compounds.^52-55 Anabolic steroids enter the RBCs, where they enhance glycolysis and the formation of steroidal 17-keto metabolites. These metabolites are delivered to tissues, including the bone marrow, where they interact with a cytosolic receptor in the appropriate cell and are transferred to the cell nucleus. In the nucleus they induce the formation of RNA and synthesis of an effector protein that brings about the pharmacologic effect.

The sequelae of steroid-induced nuclear transcription are manifold.^54,55 The proposed sequelae on RBC formation include (1) stimulation of EPO production by way of EPO-stimulating factor, (2) differentiation of stem cells into EPO-stimulating factor–sensitive cells (e.g., hemocytoblasts), and (3) direct stimulation of erythroid-progenitor cells. Anabolic steroids also increase intracellular concentrations of 2,3-bisphosphoglycerate in erythrocytes; oxygen release into tissues is subsequently increased.

Efficacy of the anabolic steroids on the RBC mass depends on the presence of supportive materials.^54,55 This includes adequate concentrations of androgen dehydrogenase in the RBC, adequate EPO concentrations, and sufficient bone marrow cellularity. Thus the effectiveness of anabolic steroids in treating anemia may be limited depending on the cause (i.e., renal disease accompanied by low EPO levels). Administration of high doses of these steroids may cause a negative feedback inhibition.

Pharmacologic Effects

The difference in the pharmacologic effects of these drugs among the various tissues and, specifically, whether an androgenic versus an anabolic effect will predominate cannot be attributed to differences in target tissue receptor structure because there appears to be only one androgen receptor type. Differences in response may be concentration dependent (e.g., reproductive tissues requiring higher concentrations than nonreproductive tissues), with drugs differing in their binding to the androgen receptor. However, mibolerone has little anabolic effect despite tight receptor binding and methylation; in contrast, boldenone, which is not methylated, has marked anabolic activity, attributed to a double bond at positions 1 and 2 (see Figure 15-4).⁵⁶

The effect of several anabolic steroids in renal EPO was studied in the perfused canine kidney exposed to 4 hours of hypoxia.⁵⁷ Activities important to secretion of EPO included a double bond at position 4 and 5 and the absence of a methyl group at position 10 (see Figure 15-4). Alternatively, differences in response may reflect conversion of the drug by target tissues to an active metabolite that subsequently causes the pharmacologic effect.^54,55 In the presence of continued administration, anabolic steroids initiate and maintain a positive nitrogen balance, although the response is short-lived in intact males.^52,54,55 The anabolic effects of stanozolol have been studied in dogs. Based on urine excretion of urea, retention of intravenously administered radiolabeled amino acid was increased in sled dogs (n = 10) receiving stanozolol either 2 mg/dog orally twice daily (increased from a baseline of 30% to posttreatment retention of 50%), or 25 mg weekly for 4 weeks (increased from 27% to 67%). The authors⁵⁸ concluded that such an effect might be beneficial with acute or chronic conditions characterized by protein loss. Anabolic steroids antagonize glucocorticoid-induced protein catabolism by competitively inhibiting glucocorticoid binding to glucocorticoid receptors. Although anabolic steroids appear to vary in their ability to antagonize the effects of glucocorticoids, protein anabolism may be induced in patients experiencing glucocorticoid-induced protein catabolism. Anabolic steroids also reduce urinary excretion of nitrogen, sodium, potassium, chloride, and calcium.

As part of their anabolic activity, these compounds increase the circulating RBC mass (and possibly granulocytic mass). Red blood cell indices, hemoglobin, and hematocrit increase in various types of anemia. White blood cell mass may increase in cases of pancytopenia, although white blood cell response takes longer than the RBC response. Response by thrombocytes is slower and less predictable.^52,54,55

Pharmacokinetics

The absorption and disposition of anabolic steroids depend on the type of preparation, the presence of specific receptors, and the species to which it is administered.^52,54,55 Most anabolic steroids depend on hepatic metabolism for elimination, with those metabolites that are anaologs of the parent compound accompanied by metabolic activity.⁵⁶ The risk of abuse in athletes (including the racing animal) has led to metabolic profiling of some steroids. Among the steroids, Beagle hepatic microsomal metabolism of steroids yielded hydroxylated and oxidized metabolites testosterone, methyltestosterone, mibolerone, and boldenone, with testosterone a metabolite of boldenone and androstenedione a major metabolite of testosterone (see Figure 15-4).

Preparations

Anabolic steroids have been divided into two categories depending on the presence or absence of an alkyl (CH₃) group at the 17-carbon position, although the relevance of this division is probably less important than the impact of the individual drugs (see Figure 15-4).^54,55 Oral and parenteral preparations are available, with the alkylated products being better absorbed orally. Oil-based parenteral preparations are intended for slow release. Examples of alkylated anabolic steroids include methyltestosterone, (oral), oxymetholone (oral), stanozolol (oral and parenteral), methandrostenolone, ethylestrenol, and norethandrolone (see Table 15-1). Examples of nonalkylated anabolic steroids include testosterone cypionate or enanthate, oxymetholone, and nandrolone in its decanoate form (parenteral).

Toxicity Versus Efficacy

A review of risks in human athletes includes cardiovascular adversities, increased risk of cancer, behavioral disorders, and increased risk of tendon damage.⁵⁹ Hepatotoxicity is the most common serious adverse effect associated with androgenic or anabolic steroids.^54,55 Toxicity ranges from mild increases in clinical laboratory tests to hepatocellular carcinoma (in humans). Drugs alkylated at the 17-carbon position are more likely to cause hepatotoxicity (see Figure 15-4). Although the mechanism is not known, the drugs or their metabolites may be carcinogenic or may increase the metabolism of other drugs to carcinogenic or hepatotoxic compounds. Cholestatic liver damage occurs early and can be significant but is apparently reversible if the drug is discontinued before irreversible hepatic lesions develop. It is not clear if the hepatotoxic effects that occur in humans are likely in dogs or cats.

Masculinization is a major undesirable (or desirable) side effect of anabolic steroids in humans.^52,54,55,59 Virilization can occur but is seldom objectionable in dogs or cats. In dogs and cats, anabolic steroids increase libido in males, interfere with the female reproductive cycle, and cause masculinization of fetuses if administered during pregnancy. Other undesirable consequences of anabolic steroid therapy in dogs include hyperplasia of the perineal glands and stimulation of androgen-dependent tumors, such as anal carcinomas and prostatic carcinomas. In humans edema resulting from water retention occurs.^52,54,55

Clinical Indications

The use of anabolic steroids for treatment of renal disease and catabolic effects associated with cachexia are addressed in their respective chapters. Human recombinant EPO has largely replaced anabolic steroids in the treatment of chronic anemias, particularly those associated with renal disease. Anabolic steroids are, however, indicated for treatment of chronic, nonregenerative anemias that fail to respond to EPO; for animals that react adversely to EPO; or for hematopoietic diseases that are not typically associated with decreased concentrations of EPO.^54,55 Of human patients with aplastic anemias who have failed conventional therapy of anemia, 50% or more have responded to anabolic steroids.^54,55 Anabolic steroids have been used in conjunction with anticancer chemotherapeutic drugs to protect bone marrow production of RBCs, thus allowing longer and perhaps more toxic chemotherapy.^52,54,55 Anabolic steroids presumably help decrease the catabolic effects of cancer. Treatment several weeks before cancer chemotherapy may enhance response to anabolic steroids. Use of anabolic steroids for cancer has not been established in dogs and cats.

Hematopoietic response to anabolic therapy is variable, and the time to clinical improvement is long, frequently 3 or more months. Cellularity of the bone marrow appears to determine rate of response to anabolic steroids. Cessation of therapy may result in recurrence of the underlying diseases. Among the anabolic steroids, nandrolone decanoate has the greatest hematopoietic effect.^54,55 Leukocyte and thrombocyte counts may also increase after therapy with anabolic steroids, although these effects are slower in onset and less likely, particularly for thrombocytes. Danazol is the anabolic steroid of choice in the treatment of thrombocytopenia, especially that which is immune mediated. Beneficial effects probably include impaired clearing of IgG-labeled platelets and decreased antibody formation. Response to therapy may take several months, and relapse may occur when danazol therapy is discontinued. Anabolic steroids and, in particular, danazol, have been used with some benefit for patients with hemolytic anemia. Benefits include not only expansion of the RBC mass but also impaired complement activation and binding to RBCs. Human patients suffering from anemia caused by renal disease respond to continuous administration of anabolic steroids. RBC indices improve, as well as appetite, muscle mass, and strength.

In general, the following approaches should be used with anabolic steroids in dogs and cats (see Table 15-1). State restrictions regarding the use of these drugs should be observed. Nandrolone decanoate is the drug of choice except in cases of thrombocytopenia, for which danazol is the drug of choice. Several months of therapy may be necessary before a clinical response is observed. The dose should be decreased once the maximum therapeutic effect occurs. Relapse of disease may occur once the drug is discontinued, and hepatotoxicity is a potential contraindication for use of these drugs.

A final consideration regarding anabolic steroids is use in racing dogs. In addition to potential performance-enhancing abuse, testosterone, methyltestosterone, and mibolerone are used to prevent estrus.⁵⁶ Detection of urinary metabolites in racing animals is an ongoing field of study.⁵⁶

Topical

Lyophilized concentrates of one or more clotting factors are available as topical preparations. These preparations usually provide an artificial factor or structural matrix that facilitates clotting (Figure 15-6). An intact hemostatic mechanism is necessary for their efficacy. These are absorbable products and are indicated for capillary oozing from small, superficial vessels. Examples of concentrated factors include thromboplastin (prothrombin is converted to thrombin, used locally in surgery), thrombin (converts fibrinogen to fibrin; available as powder, solution, or sponge), and fibrinogen. Examples of artificial matrices include absorbable gelatin sponge and oxidized cellulose (treated surgical gauze that promotes clotting) (see Figure 15-6).

Figure 15-6 Examples of coagulants (A).Oxidized cellulose (Surgicel shown here) provides a matrix for clotting (B). Gelfoam also provides an artificial structural matrix that initiates the clotting process (C). Products that contain silver nitrate (shown here), tannic acid, or salicylic acid precipitate proteins and cause coagulation by destroying tissue. Their use is limited to small, topical lesions.

Astringents act locally by precipitating proteins. These agents do not penetrate tissues and thus are restricted to surface cells. They can be damaging to surrounding tissues. Examples include ferric sulfate, silver nitrate (e.g., sticks; see Figure 15-6), and combinations that include tannic acid (e.g., STA).

Epinephrine and norepinephrine are hemostatic drugs only by virtue of their vasoconstrictive effects. They may be included in topical medications or injectable local anesthetics to decrease blood flow to the tissues.

Systemic

Fresh blood or blood components are hemostatic drugs only in states of (coagulation) factor deficiency. Examples include plasma (contains electrolytes, albumin, and some coagulation factors), fresh frozen plasma (plasma in which factors V and VII are stable), cryoprecipitate, and platelet-rich plasma.

Vitamin K

Vitamin K is a hemostatic drug only in instances of vitamin K deficiency. It is necessary for hepatic synthesis of coagulation factors II, VII, IX, and X (Figure 15-7). Several forms of vitamin K are available for therapeutic use. Vitamin K₁ (phytonadione), the plant form, can be given parenterally and orally. It is more effective and more rapid in onset compared with other vitamin K analogs. However, anaphylactic reactions have been reported with intravenous administration, particularly with those preparations containing polysorbate 80, a known releaser of histamine in dogs. The drug can also be given intramuscularly. The effects of vitamin K₁ occur within 1 hour of administration,^3,70 although several hours are needed to resolve bleeding tendencies as coagulation proteins are replaced.

Figure 15-7 Vitamin K catalyzes the carboxylation of factors II, VII, IX, and X. As part of the reaction, the oxidized vitamin must be reduced to continue activation of coagulation factors. Coumarin derivatives block the reduction of vitamin K, rendering it incapable of activating coagulation factors. The arrow on warfarin identifies the chiral carbon that leads to S and R enantiomers. Both isomers are highly (>95%) bound to plasma proteins. Enterohepatic circulation was evident in cats 4 hours after administration.

Vitamin K₂ (menaquinone) is the natural animal and microbial form of vitamin K. Vitamin K₃ (menadione) is one of several synthetic forms. It must be metabolized to the active state. Vitamin K₃ usually is absorbed too slowly to be used effectively in acute conditions, but it can be used for chronic therapy once the acute crisis has been resolved. In states of hypocoagulation, vitamin K₁ is the preferred form, administered either subcutaneously or orally (3 mg/kg subcutaneously in multiple sites followed by 1 mg/kg every 24 hours parenterally or orally).⁷¹ Fat will enhance the oral absorption of vitamin K.

The most common use of vitamin K is treatment of rodenticide toxicity (see later discussion of anticoagulants). Duration of treatment for rodenticide poisoning will vary according to the toxicant. Toxicities from rodenticides containing coumarin or warfarin should be treated for 4 to 6 days; intoxication with diphacinone or brodifacoum requires treatment for at least 14 days.⁷¹ For toxicity with longer-acting rodenticides (i.e., indandiones), the dose of vitamin K is often reduced for subsequent weeks:⁷¹ 0.5 mg/kg, week 2; 0.25 mg/kg, weeks 3 and 4. Prothrombin time should be monitored for 2 days after vitamin K is discontinued to detect residual rodenticide toxicity. Screening tests should remain normal for 3 to 4 days after therapy has been discontinued.

Heparins

Heparin is a heterogeneous mixture of sulfated (anionic) polysulfated glycosaminoglycans (PSGAG) (Figure 15-8). Glycosaminoglycans are a family of structurally diverse and distinct polyanionic complex carbohydrates. Included in this group are heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratin sulfate, and hyaluronic acid. Each of the glycosaminoglycans is composed of repeating polysaccharides consisting of an amino sugar (e.g., glucosamine or galactosamine) and uronic (e.g., glucuronic or iduronic) acid or a hexose (galactose; keratin sulfate only). Each is ubiquitous in location. Among them, heparan sulfate proteoglycans are diverse in location with those that are endothelial derived serving as the primary endogenous anticoagulant. They are an integral part of stromal matrices, basement membranes, and almost all cell surfaces where they provide cohesion between vessels and vascular stroma.⁴³ Endogenous synthesis results in multiple compounds that vary in the lengths of carbohydrate chains Heparin and heparan sulfate are the most complex members of this class of compounds and are characterized by the greatest biologic diversity.⁴³ Proteoglycans are formed when proteins, which serve as the core for the PGAG, are posttranslationally (in the Golgi apparatus) modified with the addition of glycosaminoglycan chains. Each is sulfated to variable degrees, depending on the repeating subunits of which the disaccharide is composed.

Figure 15-8 Heparin is a heterogeneous mixture of sulfated, anionic, and polysulfated glycosaminoglycans. The subunits include uronic acid, sulfonaminoglycosamine, N-acetylglucosamine, and a pentasaccharide sequence. Heparin is one of many polysulfated glycosaminoglycans (PGAGs) found throughout the body. Other structurally similar PGAGS include hyaluronate, heparin sulfates, chondroitin sulfates, keratan sulfates, and dermatan sulfates.

Heparin was so named because of its initial discovery in high concentrations in the liver. It is often located attached to serine residues of core proteins (e.g., coagulation factors). Commercially available heparin is prepared from porcine intestinal mucosa and bovine lung. It is stored in mast cells, along with other PGAGs, including chondroitin and dermatan sulfate. Heparin also is stored in the body in basophils and, to a lesser extent, in vascular endothelium. Tissues with high concentrations of mast cells serve as a source of heparin. Heparin is a highly sulfated (generally 2 to 3 per subunit) glycosaminoglycan composed of alternating sequences of sulfoaminoglycosamine and uronic acid units and smaller amounts of N-acetylglucosamine (see Figure 15-8). Its structure also includes a unique and specific sulfated glucosamine pentasaccharide sequence that binds to antithrombin (AT) III and thus serves as the active site of the molecule. The number of active sites in a molecule of heparin is variable, but it is present in only about 30% of the molecules. In its native state, the molecular weight is quite variable, ranging from 3 to 50 kDa. The molecular weight of commercial unfractionated heparin (UFH) generally varies from 1800 to 30,000 daltons (mean 15,000, representing about 45 monosaccharide chains).⁸⁰ However, heparin also has been prepared through filtration methods as fractionated or low-molecular-weight (LMW) heparins, which normally make up less than 5% of endogenous heparin. The LMW heparins generally are only 1000 to 10,000 daltons in size and are specifically defined as an average weight of 8000 daltons or less, with at least 60% of the product being less than 8000 daltons in size (see Figure 15-8). A variety of LMW heparins have been made, with each varying by the method of fractionation or depolymerization. The LMW heparins have much higher affinity for AT (by way of Xa) compared with UFH and thus are more potent, providing 70 units/mg of antifactor Xa and a ratio of antifactor Xa to AT activity of 1.5 or more.⁸⁰ Doses of fractionated LMW preparations are thus lower than unfractionated products (Table 15-3). Preparations of UFH also contain contaminants such as dermatan sulfate; these contaminants may be present in LMW heparin preparations but in diluted form. Intentional contamination of heparins with oversulfated chondroitin sulfates is addressed with adverse reactions. Some of these compounds also have anticoagulant activity, albeit less than heparin.⁸¹

Table 15-3 Doses of Unfractionated Heparin and Low Molecular Weight Heparin

Mechanism of action

Heparin interacts with many proteins, probably by electrostatic forces between the polyionic groups of the glycosaminoglycan and the cationic groups of proteins. Example proteins include proinflammatory chemokines, growth factor, extracellular matrix proteins, and leukocyte proteins. Intracellular effects reflect interaction of heparin or heparan sulfates with cell surface-binding proteins (which are not necessarily true receptors) followed by endocytotic internalization.

The most recognized sequelae of protein–heparin interaction are the anticoagulant actions of heparin.⁴³ However, this effect is indirect, reflecting facilitation of the actions of endogenous anticoagulant proteins. The most notable is AT, one of several endogenous serine proteinase inhibitors (serpins) whose thrombin inhibition occurs only in the presence of heparin. Long chains of heparin bind to specific lysine residues on AT. At low heparin concentrations (0.1 to 1 U/mL), AT rapidly inhibits the activity of the clotting factors IIa (thrombin), Xa, and IXa, resulting in prolongation of activated partial thromboplastin time (APTT) and thrombin time (TT); prothrombin time (PT) is only mildly affected. At high heparin concentrations (>5 U/mL), heparin interacts with heparin cofactor II (HCII), which binds only to thrombin, causing further inhibition. Binding of heparin to AT or HCII results in a stable complex (Figure 15-9). Binding induces conformational change in the anticoagulant complex such that the active sites of the inhibitor is exposed, facilitating binding to the clotting factors. As such, heparin increases the velocity, but not magnitude, of interaction between the endogenous anticoagulants and clotting factors. The rate of interaction can increase 10,000-fold in the presence of heparin. The anticoagulant and clotting factor complex will remain intact after activity is inhibited; as such AT is a “suicide” substrate.⁵⁴ In contrast, heparin will be released to interact with other molecules. Other inhibitors, whose concentrations are less than one hundredth of that of AT are also inhibited at high concentrations; these include plasminogen activator inhibitor, protein C inhibitor, and tissue factor pathway factor inhibition of Xa. Heparin also inhibits factors IXa and to some degree Xia; the kallikrein–kinin system and, through binding to complement C1, the complement system are inhibited. Factor VIIa is minimally inhibited.

Figure 15-9 The pentasaccharide sequence of heparin interacts with the amino residues of antithrombin III, changing the conformation of antithrombin II and its ability to interact with factors IXa, XIa, XIIa, and, in particular, II and Xa. Heparin may directly interfere with factor X.

Because the specific AT-binding site of heparin is generally absent in other glycosaminglycans (limited in heparin sulfates and absent in chondroitin and dermatan sulfates), these latter compounds are characterized by minimal anticoagulant activity.

Simultaneous inhibition of both AT and thrombin requires molecules greater than 5400 MW. LMW heparins generally act through AT inhibition of Xa rather than IIa. Thus LMW heparin fractions are more potent anticoagulants (a lower concentration inhibits Xa) compared with high-molecular-weight (HMW) fractions,⁸¹ but HMW heparins may be more effective anticoagulants because their large structure facilitates simultaneous binding of both AT and thrombin simultaneously.⁷² Whereas the ratio of antiXa:IIa activity for UFH is generally 1:1, the same ratios range from 2 to 4:1 up to 9:1 for LMW heparins in humans.⁸² Lower ratios might be expected for dogs because LMW heparin appears to have less effect on AT.

KEY POINT 15-5

Although fractionated low-molecular-weight heparins are more potent inhibitors of Xa, products that contain high-molecular-weight fractions may actually be more effective anticoagulants because their large structure facilitates simultaneous binding of both antithrombin and thrombin.

The effect of heparin on thrombin and Xa occurs only when the factors are not bound to platelets or fibrin; platetet factor IV blocks the interaction between heparin and AT. However, heparins have an antiplatelet effect because of a high affinity for platelet factor IV. Most heparin preparations also inhibit platelet aggregation, negating effects of platelet-derived growth factor on vascular smooth muscle and leading to their descriptor as platelet-active anticoagulants. vWF (platelet factor VIII) is among the factors bound and inhibited by heparin.⁸⁰ Heparins also bind to vascular endothelium, causing release of two other endogenous PGAGs and altering vascular endothelial permeability.⁸¹

Heparin has other effects not related to its anticoagulant activity. In human patients heparin may prove useful for prevention of atherosclerosis and for accelerated formation of collateral circulation in the presence of thrombosis (angiogenesis).^81,83 Heparin can inhibit or modulate selected targets of an allergic inflammatory response, including neutrophils and T cells. Heparins appear to decrease leukocyte adherence to vascular endothelium and facilitate leukocyte migration along a chemoattractant gradient to sites of inflammation. Derivatives of low anticoagulant activity block superoxide anion generation, probably by interacting with superoxide dismutase. Heparin appears to impair mast cell degranulation by altering intracellular calcium release through blockade of inositol 1,4,5-triphosphate receptors.⁴³ Heparin also appears able to influence tumor cell metastasis. Tumor cell invasion from the vasculature probably involves host degradative enzymes, including heparinases, which target both heparin and heparan sulfate. Exogenous heparin inhibits the hydrolysis of heparan sulfate. Heparins with low anticoagulant activity have reduced the metastasis of several tumor types in various experimental models.⁴³ Smooth muscle cell proliferation such as that which accompanies atherosclerosis and bronchial asthma also is influenced by heparin and heparan sulfate.⁸⁴ Heparin inhibits capillary endothelial growth but binds with high affinity to fibroblast growth factors and potentiates their growth-promoting effects.⁵⁴ As such, heparin facilities growth of smooth muscle of the vasculature (including pulmonary), airways, intestine, and contractile cells such as fibroblasts and renal glomerular mesangial cells. Effects can be correlated with molecular size and sulfation of heparin; heparin and other heparans may serve as reservoir for growth factors. Endogenous heparin has been implicated in diseases characterized by smooth muscle proliferation; manipulation of selected members of the heparins may ultimately prove to be a therapeutic alternative to treatment.⁴³ Finally, heparin liberates lipoprotein lipase and can decrease serum triglyceride concentrations, thus “clearing” lipemia.^72,54

Drug disposition

The complex chemistry of heparin and its binding to proteins and cells leads to complex disposition. Absorption and distribution of heparin are limited by the large size and polarity. Studies characterizing heparin elimination are generally based on response to therapy (i.e., changes in the APTT). However, more recent studies in animals have detected the drug chemical in blood.⁸⁵Absorption of heparin after oral administration or by aerosolization is negligible; as such, heparin is a parenteral anticoagulant. Subcutaneous absorption is rapid; intramuscular administration is generally contraindicated because of the risk of hemorrhage. Heparin is metabolized by heparinase in the liver and by reticuloendothelial cells. Metabolites of heparinase activity are excreted in the urine. The anticoagulant activity of the metabolites apparently has not been addressed. The elimination half-life of heparin appears to depend on molecular weight. The LMW fractions are cleared less rapidly compared with larger molecules, leading to a half-life two to three times longer than that of endogenous heparin. As such, molecules with a higher affinity for AT activity will be cleared more rapidly than those responsible for anti-Xa activity.⁸⁶ It is perhaps for this reason that UFH remains the preferred heparin in situations in which a rapid resolution of response upon discontinuation of therapy is desired (e.g., surgical patients or other patients at risk for bleeding).

The pharmacodynamics and elimination half-time of response (based on anti Xa activity) of dalteparin (Fragmin; anti-Xa activity of 2500 U/mL) have been reported in dogs (Table 15-4). Targeted therapeutic ranges based on anti-Xa activity approximates 0.3 U/mL for UFH and 0.5 U/mL for LMW heparin.⁸⁷ Dose response indicates increasing effects with increasing dose, with response more rapid in onset and of greater magnitude with intravenous compared with subcutaneous administration. Response occurs in 2 minutes with intravenous administration, compared with 2 hours with subcutaneous administration. Peak APTT and TT at 2 hours after subcutaneous administration of 200 IU/kg were similar to that measured at 1 minute after 25 IU administered intravenously. Peak effects after intravenous administration approximated 1.75 times baseline for APTT and up to 2.5 times baseline (TT); return to baseline occurred at approximately 1 hour. The impact of subcutaneous administration on APTT and TT was muted: peak effect was no more than 1.25 times baseline for either APTT or TT at all doses. Return to baseline appeared to be dose dependent for both routes but generally was faster (1 to 4 hours, but up to 8 hours) after intravenous compared with SC administration (6 to 10 hours). The authors noted that significant anticoagulant activity lasted up to 3 hours, but even the highest subcutaneous doses failed to affect either APTT or TT to a clinically significant degree. The authors also concluded that monitoring LMW heparin administration in dogs would best be accomplished by measuring anti-FXa activity with chromogenic substrates.⁸⁸ The influence of LMW heparin on APTT and TT in dogs appears to be less than that in humans, suggesting species differences in coagulation response. However, this may reflect a shorter APTT in normal dogs compared with normal humans.

Table 15-4 Pharmacodynamic Response to Plasma Anti-factorXa (Dalteparin) in Healthy Dogs^88,89

The pharmacodynamics (based on anti-Xa activity) of 5 days’ treatment of dalteparin (100 IU/kg subcutaneously twice daily) or enoxaparin (1 mg/kg subcutaneously twice daily) was compared with that of UFH (250 IU/kg subcutaneously every 6 hours) in cats (n = 5) using a randomized (for the first treatment) crossover design with a 14-day washout between treatments. Peak activity for all three products occurred at approximately 2 to 4 hours, with the maximum effect of each product in anti-Xa (U/mL) approximating 0.6 (UFH), 0.45 (enoxaparin) and 0.3 (dalteparin). Concentrations reached the human therapeutic range of 0.5 to 1 U/mL anti-Xa activity in only one cat receiving dalteparin. For each drug mean activity was not detectable before the next dose (trough). These data led the investigators to conclude that higher doses than those studied are indicated,⁸⁷ Vargo and coworkers drew similar conclusions in their study of rhe effect of dalteparin (100 IU/kg SC, b.i.d, for 13 days) in healthy cats (n = 8) (based on anti-Xa activity). Activity was measurable in only 4 cats; activity present by 4 hr after a single dose (the first sampling time) and returned to baseline by 6 hr; in these cats. Prothrombin time and APTT were not significantly altered by dalteparine administration; although sample size was small, clinical values were essentially unchanged in the cats.^88a The half-life of heparin is prolonged in renal or liver failure.⁸¹ Bioavailability (subcutaneously) of the LMW heparins appears to be similar in humans, approximating 80% to 90%,⁸² and approximates 100% in dogs.⁸⁹

Disseminated Intravascular Coagulaopathy

Heparin is used with blood and plasma for the treatment of DIC. The use of heparin for treatment of DIC remains controversial.⁷¹ Its efficacy for DIC depends on adequate concentrations of AT (≥40% of normal). Replacement therapy with either whole fresh blood or fresh or fresh frozen plasma is indicated if AT levels are not normal or for actively bleeding patients. A loading dose is generally followed by a maintenance dose; the loading dose can be preincubated with blood or plasma in order to maximize effects on AT Maintenance dosing should be based on changes in APTT rather than on a fixed dose. This is particularly true in patients suffering from DIC because the synthesis of cofactors varies among patients with DIC. Although normal dogs respond rapidly to heparin therapy, identification of changes in APTT in DIC patients may be difficult. Neutralization of heparin with polybrene (hexadimethrine bromide)⁷¹ can be used to distinguish changes in the APTT due to heparin from those due to DIC once a baseline APTT has been established. The recommended dose of heparin is markedly variable, ranging from a low of 75 to 100 U/kg administered subcutaneously (a dose unlikely to change APTT but likely to be sufficient to target AT) to a high of 750 to 1000 U/kg (severe disease with organ damage caused by microthrombosis) every 8 hours. An intravenous bolus of 5000 U or 80 U/kg followed by constant-rate infusion of 5 to 18 U/kg/hr also has been recommended. For a hypercoagulable state, a subcutaneous dose of 200 to 300 U/kg every 6 hours is recommended. The APTT should be monitored every 6 hours. In general, if higher doses are used, the APTT should be prolonged by 1.5-fold to twofold. Once a therapeutic response is established, the APTT and platelet count should be monitored daily. Heparin should not be abruptly discontinued in patients with DIC because of the risk of rebound hypercoagulability associated with AT deficiency. Nonanticoagulant uses of heparin are increasing in human medicine. Anecdotal reports in humans indicate that heparin can be beneficial for the treatment of asthma and allergic inflammation.

DIC has been reviewed in cats,¹⁰⁹ including its treatment. Of the 46 cats studied retrospectively, 43 died or were euthanized, precluding conclusions regarding the most effective therapies.

Vitamin K Antagonists (Oral Anticoagulants)

The oral anticoagulants differ primarily in their duration and magnitude of effect. Studies of their importance in veterinary medicine have focused primarily on their toxic rather than their therapeutic indications,¹¹ although these drugs are being used increasingly to treat thromboembolic diseases.

Streptokinase and Streptodornase

Streptokinase and streptodornase are synthesized by streptococcal organisms. Urokinase is prepared from cultures of human renal cells. Streptokinase, streptodornase, and urokinase are used in the treatment of wounds that do not respond to antibacterial therapy, burns, ulcers, chronic eczemas, ear hematomas, otitis externa, osteomyelitis, chronic sinusitis, or other chronic lesions. They are available as powders for local or systemic administration. Streptokinase may be useful for the treatment of feline thromboembolic disease. In an experimental model, streptokinase reduced mean thrombus weight after administration of a loading dose (90,000 IU/cat) and a constant maintenance infusion of 45,000 IU (studied for only 3 hours). Clinical use of streptokinase has not been reported.⁷⁰

Tissue Plasminogen Activator

Tissue-type plasminogen activator (tPA) activates plasminogen bound to fibrin. Its use should be limited to cases of thrombosis (rather than prevention). Some studies have indicated a potential use for tPA for dissolution of thrombi in cats, but side effects should be minimized before general use of the drug can be accepted. Although 50% of cats with spontaneous aortic thrombosis treated with tPA (0.25 to 1 mg/kg per hour for a total dose of 1 to 10 mg/kg) had resolution of clinical signs, 70% of the cats treated died from reperfusion injury, heart failure, or other side effects.⁷⁰

Clinical Use

Cost and lack of refined doses and selectivity have limited the use of thrombolytic drugs for treatment of (pulmonary) thromboembolism in dogs and cats.

Homocysteine

The recognition of arterial damage in two infants with inborn errors of metabolism resulting in an increase of homocysteine led to the investigation of the possible role of homocysteine in thromboembolic disease.¹²⁷ However, homocysteine accumulation generally is accompanied by an increase of its precursor, S-adenosylhomocysteine (SAH). This demethylated product of numerous S-adenosylmethionine (SAM)-dependent transmethylation reactions may cause a potent feedback inhibition, resulting in hypomethylation of reactions critical to thrombogenic control. In homocystinuria, lowered plasma homocysteine is accompanied by lowered SAH concentrations and restored transmethylation reactions. It is not clear if increased homocysteine (or SAH) is a cause or effect (and thus a marker) of thromboembolic disorders. Because the kidneys are responsible for clearing up to 70% of homocysteine, concentrations increase in concert with creatinine in patients with renal disease, reaching increases of threefold to fivefold above baseline. Other factors that increase homocysteine include decreased folate and vitamin B concentrations and cardiovascular disease itself.