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3 CE CREDITS CE Article 2 Hemostasis At a Glance ❯❯ Cody Alcott, DVM ❯❯ Charles Brockus, DVM, ❯❯ Brett Sponseller, DVM, Response to initial David M. Wong, DVM, PhD, DACVIM, DACVP PhD, DACVIM MS, DACVIM Charles River Labs Iowa State University vascular injury Sparks, Nevada Page 78 Iowa State University plug formation Abstract: Hemostasis, the cascade, and clot formation present a daunting list of and primary hemostasis Page 79 factors, pathways, and interactions to equine clinicians. A basic knowledge of hemostasis is necessary to evaluate various disease processes in horses. Initial injury to the vascular en- Endothelial cell reactions Page 80 dothelium results in local vasoconstriction and formation of a platelet plug. This initial response reduces loss from the damaged vessel but may be inadequate at maintaining hemostasis Secondary hemostasis alone. Therefore, the intrinsic, extrinsic, and common coagulation pathways interact with one Page 80 another to form and ultimately stabilize the platelet plug. Thrombin accomplishes this The coagulation cascade: by converting soluble to insoluble , resulting in clot stabilization. Alternatively, the extrinsic pathway promotes resolution of clot formation. Because of multiple positive and negative Page 80 feedback interactions as well as multiple circulating mediators and inhibitors, coagulation and The coagulation cascade: fibrinolysis are finely controlled systems in healthy patients. However, perturbations of the the intrinsic pathway coagulation cascade in disease states can result in severe complications or death. Page 81 Common pathway lterations in coagulation and Response to Initial Vascular Injury Page 81 fibrinolysis are relatively uncom- The main function of the coagulation sys- Clot formation Amon in horses, but when they tem is to control hemorrhage from the Page 82 occur, especially in critically ill patients, vascular endothelium when it has been T h r o m b i n general knowledge of the associated damaged by trauma or . Page 82 pathways is vital to equine clinicians to Conversely, and fibrinolytic Clot prevention properly diagnose, monitor, and treat mechanisms maintain patency and ade- Page 82 primary or secondary disease processes quate blood flow. The regulation of these involving these systems (Table 1 lists mechanisms is a tightly knit system of local Fibrinolysis coagulation factor synonyms). Clinicians and systemic feedback control pathways Page 83 must also be able to interpret and under- that maintains a delicate balance between Diagnostic testing stand the available diagnostic tests used pro- and processes. of coagulation to evaluate these systems. This article Hemostasis is defined as the arrest of Page 83 reviews coagulation, fibrinolysis, and bleeding by vasoconstriction and coagu- Adult versus neonatal diagnostic testing to evaluate these sys- lation and is an essential part of normal differences in diagnostic tems in horses. functioning of the cardiovascular system. tests Page 86 FIGURE 1

Primary and secondary hemostasis. (A) Initial vessel injury results in local vasocon- striction via reflex myogenic spasm and release of endothelin and thromboxane. (B) Formation of the platelet plug results from platelet adhesion, activation, recruitment, and aggregation. (vWF = von Willebrand’s factor)

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table 1 Coagulation Factor Synonyms and initiator of platelet adherence and forma- tion of the platelet plug, known as primary hemostasis. Likewise, damaged endothelium FACTOR SYNONYM activates the extrinsic and intrinsic coagula- I Fibrinogen tion cascades via and collagen fiber exposure. The direct result of II Prothrombin activating either or both coagulation cascades III Tissue factor, thromboplastin is conversion of prothrombin to thrombin; this subsequently results in the conversion of IV Calcium soluble fibrinogen to insoluble fibrin and sta- V Proaccelerin, labile factor bilization of the clot (secondary hemostasis). Platelet adherence and activation combined VI — with activation of the coagulation cascades result in a blood clot or formation to VII Proconvertin, stable factor stop hemorrhage. VIII Antihemophilic factor Platelet Plug Formation IX Christmas factor and Primary Hemostasis X Stuart-Prower factor Essential to progression of the clotting pro- cess is the development of a platelet plug via XI Plasma thromboplastin antecedent platelet adhesion, activation, recruitment, and aggregation (Figure 1). are circulating XII Hageman factor c y topla sm ic fr ag ment s of megak ar yoc y te pseu- XIII Fibrin-stabilizing factor, transglutaminase dopods. They contain actin–myosin molecules and thrombosthenin for platelet contraction, an endoplasmic reticulum and Golgi apparatus Hemostasis is usually initiated by vascular for enzyme production and calcium storage, trauma but can be induced by inflammation and a variety of enzymatic systems and secre- or sepsis.1 Smooth muscle contraction marks tory granules.2 Glycoproteins on the platelet the beginning of the hemostatic process surface and within platelet granules help medi- and begins instantaneously after endothe- ate adherence to the vascular endothelium and lial damage (Figure 1). Myogenic spasm of serve as a means of communication and acti- smooth muscle is followed by endothelial and vation among platelets. After vascular injury, platelet release of autocoid vasoconstrictive adherence is initiated largely by the binding substances—endothelin and thromboxane, of platelets to exposed subendothelial matrix respectively.2,3 Further vasoconstriction caused via von Willebrand’s factor (vWF).4 Fibrinogen by sensory nerve impulses results in a neuro- binding to platelet glycoprotein complexes genic contractile reflex of smooth muscle.2 enhances the activation of additional plate- Damaged endothelium serves as a mediator lets.5 Platelet aggregation ensues with further

(C) Secondary hemostasis involves activation of the intrinsic and extrinsic path- ways, culminating in the activation of prothrombin to thrombin via the common pathway of coagulation. (D) Clot stabilization occurs via fibrin fiber formation within the clot and subsequent clot stabilization by fibrin stabiliz- ing factor (XIII).

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platelet activation, forming the platelet plug. lial damage lead to exposure of subendothelial Prothrombin attaches to platelet surface recep- matrix, which is largely composed of collagen. tors as well, accumulating within the newly The combined endothelial expression of vWF formed platelet plug. Platelet activation results and tissue factor, in addition to subendothelial in platelet swelling and the development of collagen exposure, activates the coagulation membrane surface pseudopods containing cascade and supports platelet adhesion and alpha granules and dense bodies. Alpha gran- activation.5 vWF mediates the adhesion of plate- ules contain substances essential to hemostasis, lets to exposed collagen fibrils while stabilizing including adhesive for vWF, fibrinogen, factor VIII; the latter plays a role in secondary plasminogen, , and ; hemostasis. Tissue factor initiates secondary plasma proteins IgG and ; cellular mito- hemostasis via the extrinsic pathway. Collagen gens of platelet-derived growth factor; coagula- exposure simultaneously causes a change in tion factors V and VIII; and inhibitors configuration of the proteolytic enzyme factor 6 α2- and α2-antiplasmin. Dense XII, which signals the activation of the intrinsic bodies activate and recruit locally circulating pathway. In summary, the endothelium plays platelets via release of ADP, ATP, serotonin, a key role in initiating, amplifying, and modu- ionized calcium, histamine, epinephrine, and lating the coagulation process in response to pyrophosphate.7 Fibronectin and thrombo­ many conditions, including trauma, inflamma- spondin help reinforce and stabilize aggre- tion, sepsis, and endotoxemia.9,10 gated platelets. Platelet-derived growth factor increases smooth muscle growth while con- Secondary Hemostasis trolling tissue repair. These factors facilitate Secondary hemostasis begins with the activa- and regulate the continual adherence, activa- tion of the extrinsic and intrinsic pathways tion, and aggregation of platelets until a loose immediately after vessel damage. These path- but reversible plug forms. Further platelet plug ways converge to form prothrombin activator, stabilization is attained through the activation which catalyzes the formation of thrombin of coagulation pathways and the generation of from prothrombin. Thrombin converts soluble CriticalPo nt thrombin. fibrinogen to insoluble fibrin fibers that inter- The hemostatic pro- act with platelets, blood cells, and plasma for Endothelial Cell Reactions final formation of a stable clot. cess begins imme- Endothelial cells are responsible for initiating diately following coagulation in response to local vascular injury The Coagulation Cascade: vascular endothelial or systemic inflammation.8 Intact endothelium the Extrinsic Pathway damage. maintains normal function and limits activation of Activation of the extrinsic pathway occurs via coagulation by maintaining the smooth luminal expression of tissue factor, a cellular lipopro- surface of vessels. Endothelial production of tein, resulting from endothelial damage and the glycocalyx, a mucopolysaccharide, limits subsequent subendothelial matrix exposure platelet adherence to the endothelium and platelet (Figure 1). Activated factor VII (VIIa), alone or activation. Vessel trauma and resulting endothe- in combination with calcium and tissue fac- tor, forms a proteolytic complex composed of phospholipids and lipoproteins that enzymati- TO LEARN MORE cally activates .11 In addition to tissue factor, factor VII can be activated by factor IXa, Xa, XIIa, or XIIIa or thrombin and, when Therapeutics in Practice: Acute Blood activated, catalyzes the activation of additional 12 Loss (March 2008) fac tor X . The common pathway proceeds with Therapeutics in Practice: Treating the activation of factor X to produce thrombin Disseminated Intravascular Coagulation and, subsequently, fibrin. Thrombin’s activa- (July/August 2008) tion of factor VII is concentration dependent, with activation and inactivation occurring at Related content on lower and higher concentrations, respectively.13 CompendiumEquine.com Thrombin production is further regulated by the production of tissue factor pathway inhibi-

80 Compendium Equine: Continuing Education for Veterinarians® | March 2009 | CompendiumEquine.com FREE Hemostasis CE tor by the vascular endothelium when factor vitamin K–dependent epoxide reductase and Xa is formed via the extrinsic pathway.8 Tissue carboxylation of coagulation factors, thus limit- factor pathway inhibitor inhibits the extrinsic ing factor synthesis in the and activation at pathway through binding of the tissue factor/ the site of vascular injury.15 The combination of calcium/factors VIIa and Xa complex. The factor IXa, factor VIIIa, platelet phospholipid, small amount of thrombin that is produced tissue factor, and calcium ions completes the rapidly catalyzes its own formation via activa- intrinsic pathway process through activation of tion of and interacts with the intrinsic factor X. The complex of calcium and factors pathway by activating factor VIII. An inter- VIIIa, IXa, and X is found on the surface of connection between the extrinsic and intrin- platelets and is known as the tenase complex. sic pathways exists through the activation of Exposure of platelet phospholipids allows the factor IX of the intrinsic pathway by a tissue tenase complex to form. The entire complex factor–factor VIIa complex.14 Factor VII, which is then regulated by activation of factor VIII is produced by the liver, has the shortest half- by thrombin. Assembly of the tenase complex life of the coagulation factors and is, therefore, on a platelet phospholipid foundation in the significant during diagnostic evaluation of the presence of calcium allows clot formation to extrinsic pathway and of liver function.15 remain localized. Factor VIII is considered a in the The Coagulation Cascade: coagulation cascade and is activated by the pres- the Intrinsic Pathway ence of thrombin. Factor VIII works as a recep- Coagulation factors VIII, IX, XI, and XII are tor for factors IXa and X. As described above, essential for proper function of the intrinsic the conversion of factor VIII to VIIIa depends pathway (Figure 1). Calcium ions, platelet on the thrombin concentration, with a high con- phospholipids, and the proteins centration resulting in factor VIIIa cleavage and and high-molecular-weight (HMWK) inactivation. This inherent mechanism serves as are cofactors necessary for coagulation fac- a local means of coagulation regulation. tor activation and positive feedback. Contact between factor XII and collagen fibers exposed Common Pathway CriticalPo nt by endothelium damage is the primary initia- The intrinsic and extrinsic pathways converge Platelet adhesion tor of the intrinsic pathway. This stimulates a at the common pathway, resulting in prothrom- conformational change and activation of the bin activation, which occurs on platelet surfaces and aggregation proteolytic enzyme to factor XIIa. Conversion and requires formation of the prothrombinase at the site of initial of prekallikrein to by factor XIIa can complex. Components of the prothrombi- vascular injury also activate factor XII, and additional factor nase complex include phosphatidylinositol form the platelet XIIa results in further conversion of prekal- and phosphatidylserine (procoagulant platelet plug, which marks likrein to kallikrein. This positive feedback phospholipids), tissue phospholipids (from tis- the beginning of mechanism results in amplification of the sue factor), calcium ions, factors Va and Xa, primary hemostasis. 12 intrinsic pathway. In the presence of HMWK, and prothrombin. Within a few seconds, This is essential for factor XIIa converts factor XI; and this process prothrombinase catalyzes the conversion of is accelerated by prekallikrein. is prothrombin to thrombin in the presence of the coagulation pro- released from HMWK in this process, acting as calcium. Factor Va functions as a cofactor in cess to proceed. a potent vasodilator, and counterbalances other prothrombinase complex formation and is vasoconstrictive substances released with ves- bound to the surface of platelets. Factor X rep- sel damage. The enzymatic action of factor XIa resents the actual protease responsible for the activates factor IX. Factor IX contains vitamin conversion of prothrombin to thrombin, which K–dependent γ-carboxyglutamate (Gla) and is can be accelerated by factor Va and platelet calcium dependent (calcium binds and activates phospholipid. The proteolytic action of throm- Gla). Vitamin K is a cofactor in the carboxyla- bin to form factor Va catalyzes the formation tion of glutamate of coagulation proteins; it is of additional thrombin. Factor V is activated essential for the conformational change and cal- by a low thrombin concentration and is gradu- cium chelation that occur with factor activation, ally inactivated as the thrombin concentration specifically factors II (prothrombin), VII, IX, increases. This demonstrates a local feedback and X.16 Coumarin-type anticoagulants inhibit mechanism of coagulation.

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Clot Formation Thrombin The final phases of clot formation are char- Thrombin plays a pivotal role in all aspects acterized by the conversion of fibrinogen to of hemostasis. Thrombin has multiple “ave- fibrin by thrombin. Thrombin acts through nues” of positive feedback to accelerate fur- proteolytic cleavage of fibrinogen to a fibrin ther thrombin production. The catalysis of monomer. The polymerization of fibrin mono- factors V, VII, VIII, and XI drives the extrin- mers leads to fibrin fiber formation. Initially, sic and intrinsic coagulation pathways. Other weak fibrin bonds progress to form the long functions of thrombin include platelet activa- fibers of the clot reticulum. The conversion and tion, aggregation and secretion, and smooth activation of factor XIII (fibrin-stabilizing fac- muscle contraction.13,17 The ability of thrombin tor) by thrombin help develop covalent bonds to influence these reactions is concentration between fibrin fibers to further strengthen the dependent, as described above, and serves as clot (Figure 1). Fibrin-stabilizing factor is sup- a mechanism of local feedback to control clot plied by platelets and circulating plasma glob- formation. As the formation of thrombin accel- ulins in the clot reticulum. erates, so does the production of anticoagulant As the meshwork of fibrin fibers grows, cir- reactions necessary for clot dissolution. The culating red blood cells, platelets, and plasma binding of thrombin to thrombomodulin, tis- become trapped in damaged areas. This com- sue (TPA), and activated CriticalPo nt bination leads to the recruitment of platelets and C are examples of thrombin’s ability to Secondary hemo- coagulation factors necessary for clot expan- initiate clot breakdown.18 stasis further sta- sion and aggregation, eventually stopping ves- sel hemorrhage. Clot retraction begins within 20 Clot Prevention bilizes the platelet to 60 minutes after formation.2 Platelet release Endogenous anticoagulants maintain vessel plug via thrombin’s of procoagulants from alpha granules, as well patency by limiting intravascular coagula- conversion of fibrin- as factor XIII, further stabilizes the bonding of tion caused by daily trauma to the vascular ogen to insoluble fibrin fibers. Platelet contractile proteins throm- endothelium. Induction of the contact phase fibrin fibers. bosthenin, actin, and myosin are stimulated to and tissue factor pathway of the intrinsic and contract platelet spicules attached to adjacent extrinsic pathways, respectively, is limited by fibrin fibers. Calcium ions from platelet stores the endothelial production of the glycocalyx, and thrombin production facilitate further clot which lines the endothelium to produce a contraction. This process continues as the edges smooth surface. Tissue factor pathway inhibi- of the damaged vessel are pulled together in con- tor serves as an important endogenous mech- junction with smooth muscle contraction until anism of controlling hemostasis by directly hemorrhage ceases and hemostasis is achieved. inhibiting the formation of the factor Xa–factor

FIGURE 2

Inhibition of occurs via multiple mechanisms. Thrombin can bind thrombomodulin, resulting in activation of and of factors Va and VIIIa. In addition, inhibits thrombin and factors XIIa, XIa, Xa, and IXa. Antithrombin also binds heparin-like molecules (i.e., heparin sulfate) that are expressed on endothelial surfaces, which subsequently inhibits thrombin and factors Xa and IXa.

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VIIa–tissue factor complex.14 The removal of min (Figure 3). Binding of TPA to fibrin is thrombin can also control coagulation. Fibrin inhibited by plasminogen–activator inhibitors fibers themselves incorporate large amounts of types 1 and 2. , which is produced thrombin within the clot reticulum.13 In addi- by epithelial cells lining excretory ducts, is tion, the binding of free thrombin by throm- also responsible for plasmin activation, to a bomodulin activates plasma protein C, which lesser degree, and assists in dissolution of inactivates factors Va and VIIIa via proteoly- fibrin clots that may form in these ducts.22 sis, representing another aspect of negative Plasmin causes proteolytic digestion of fibrin feedback control of coagulation and down- fibers, fibrinogen, prothrombin, and factors V, regulation of thrombin19 (Figure 2). Protein VIII, and XII. The digestion of fibrinogen and C has many anticoagulant, profibrinolytic, fibrin fibers results in the release of soluble and antiinflammatory actions and has been fibrin degradation products (FDPs). The diges- shown to decline during periods of sepsis.20 tion by plasmin of insoluble cross-linked fibrin Antithrombin inactivates the remaining throm- fibers during fibrinolysis causes the release of bin in the immediate area of clot formation. D-fragments or cross-link remnants, known as Antithrombin is responsible for inactivation of D-dimers. The action of plasmin is the final factors IXa, Xa, XIa, and XIIa through thrombin step in clot dissolution and can reopen pre- binding. Endogenous or exogenous heparin viously blocked vessels. Renal and hepatic binding to antithrombin results in a conforma- production of α2-antiplasmin inactivates free CriticalPo nt tional change of the and heightened plasmin.23 Endogenous antico- affinity for thrombin. Further thrombin activ- agulants antithrom- ity can also be limited by α2-macroglobulin, Diagnostic Testing of Coagulation 21 bin and protein C heparin cofactor II, and α1-antitrypsin. Cytology Examination of a peripheral blood smear or limit intravascular Fibrinolysis automated quantification of platelet numbers coagulation from Hemostasis is followed by the return to normal is one avenue of assessing primary hemostasis daily vascular vascular flow around, or adjacent to, the newly (table 2). Reported and established reference trauma. formed clot. Returning the damaged vessel to intervals for platelet concentration in an adult normal function requires the dissolution of the horse generally range from 100,000 to 300,000 clot by fibrinolysis. Circulating plasminogen platelets/µL. Six to 20 platelets per high-pow- (profibrinolysin) becomes ensnared within ered field (magnification: ×1000) is considered the newly formed clot, binding to fibrin and adequate when examining a peripheral blood fibrinogen. Trapped plasminogen is activated smear, if clumping of platelets is not observed. by endothelially synthesized TPA to form plas- Ten different fields must be examined and the

FIGURE 3

Fibrinolysis occurs through the conversion of plasminogen to plasmin by tissue plasminogen activator, which is released from damaged endothelial cells. This conversion results in proteolytic digestion of fibrin fibers within the clot and the production of fibrin degradation products. Circulating plasmin is neutralized byα 2-antiplasmin. (TPA = tissue plasminogen activator)

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average number of platelets per field deter- let function may not be normal, and platelet mined to adequately evaluate platelet concen- function testing is currently being evaluated in tration by this method. Platelet numbers must equine patients. be interpreted carefully in horses because of the effects of splenic sequestration and col- Template Bleeding Time lection methods. Splenic contraction can Template bleeding time evaluates endothelial mildly increase circulating platelets. Also, the interaction with circulating platelets. Template use of EDTA anticoagulant has been shown bleeding time is a cost-effective and easily to falsely lower the platelet concentration.24,25 performed evaluation of primary hemostasis, Therefore, the use of a sodium ion citrate blood whereas other functional tests are specific to tube (light blue–top tube) is recommended. secondary hemostasis. Hair is clipped over the Thrombocytopenia is classified as platelet skin distolateral to the accessory carpal bone, concentration below 100,000/µL, and clini- and a template bleeding device is placed and cal bleeding has been associated with plate- discharged. Filter paper is used to absorb let concentrations below 30,000/µL.24 Platelet blood that is discharged from the incision 1 characteristics, granularity, size, shape, and to 2 mm below the incision site. Timing starts clumping can also be evaluated on peripheral when the incision is made and ends when the blood smear examination. However, granules bleeding stops. The reported reference range in equine platelets are very faint compared for template bleeding time in healthy horses is with those of other species, making platelets 2 to 6 minutes.26 Thrombocytopenia and the more difficult to identify and granularity diffi- functional platelet disorders von Willebrand’s cult to assess in horses. In addition, even if the disease and Glanzmann’s thrombasthenia can platelet count is within normal limits, plate- be screened using this test.24,26

table 2 Diagnostic Testing of Hemostasis

PATIENT AGE

Diagnostic Test* < 24 Hours 4–7 Days 10–14 Days 25–30 Days Adult Factors Evaluated

PT (sec) 10.9 ± 0.6 9.6 ± 0.6 9.5 ± 0.4 9.4 ± 0.4 9.5 ± 0.3 Extrinsic and common39

aPTT (sec) 56.8 ± 6.3 39.8 ± 4.0 39.9 ± 4.8 40.8 ± 6.0 42.0 ± 8.9 Intrinsic and common39

Fibrinogen (mg/dL) 116.8 ± 39.1 196.8 ± 26.6 199.6 ± 50.0 221.1 ± 48.0 195 ± 54 Quantification of fibrinogen39

Platelet count (103/µL) 243 ± 170 181 ± 60 218 ± 57 245 ± 59 153 ± 49 Quantification of platelet numbers39

FDPs (µg/mL +1)1/2 8.2 ± 2.7 5.6 ± 3.4 4.5 ± 3.1 3.5 ± 2.6 1.8 ± 0.6 Presence of fibrin from action of plasmin on fibrin or fibrinogen39

Antithrombin activity (%) 107 ± 41 164 ± 35 170.9 ± 40.9 166.5 ± 40.6 202 ± 82 Evaluates antithrombin activity39

D-dimers (ng/mL) N/A N/A N/A N/A 677 ± 119 Specific evaluation for plasmin degradation of cross-linked fibrin1

Template bleeding time 4 ± 2 4 ± 2 4 ± 2 4 ± 2 4 ± 2 Hemostatic plug formation25,a (min)

Activated clotting time 5.8 ± 1.3 5.8 ± 1.3 5.8 ± 1.3 5.8 ± 1.3 2.6 ± 0.5 Secondary hemostasisa,26 (min)

Thrombin time (sec) N/A N/A N/A N/A 19.5 ± 5.5 Conversion time of fibrinogen to fibrinb

*Listed in order of most used to least used. aKnottenbelt D, Holdstock N, Madigan J. Equine Neonatology Medicine and Surgery. Philadelphia: Elsevier Science; 2004. bFeige K, Ehrat FB, Kastner SBR, et al. The effects of automated plasmapheresis on clinical, haematological, biochemical and coagulation variables in horses. Vet J 2004;169:102-107. aPTT = activated partial thromboplastin time; FDPs = fibrin degradation products;N/A = reference intervals for a horse of this age are not available; PT = prothrombin time.

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Activated Clotting Time tor (III); coagulation factors V, VII, and X; Secondary hemostasis can be functionally prothrombin; and fibrinogen result in prolon- assessed using the activated clotting time gation of PT. The test is conducted by mixing (ACT). A deficiency in factors V, VIII, IX, X, XI, citrated plasma with tissue factor and calcium. and XII; prothrombin (factor II); and fibrino- The time until fibrin fiber or clot formation is gen prolongs the ACT. The ACT is not affected calculated automatically. Citrated whole blood by the blood concentration of factor VII or the or plasma can be used up to 3 days after collec- blood platelet concentration and thus evalu- tion with the concurrent submission of a con- ates the intrinsic and common pathways.27 trol sample from a nor mal horse. 29 Prolongation However, severe thrombocytopenia can result of PT occurs when the fibrinogen concentra- in a prolonged ACT because of decreased tion decreases below 100 mg/dL, the coagu- availability of phospholipid. The test is con- lation factors listed above are reduced more ducted by mixing whole blood directly with than 50%,30 prothrombin decreases 30%,30 or diatomaceous earth, incubating it at 98.6°F vitamin K deficiency is present. Clinically rel- (37°C), tilting the sample back and forth, and evant prolongation has been described as a recording the time to clot formation. The refer- 20% increase in clotting time compared with ence time for clot formation has been reported that of healthy horses.30 as 2 minutes and 38 seconds (± 29 seconds).27 Variations in the testing protocol, including Thrombin Time syringe versus vacuum collection, incubation The thrombin time test is an assessment of the period and temperature, and frequency of terminal common pathway of secondary hemo­ monitoring for clot formation, account for the stasis and is a direct assessment of fibrinogen general low sensitivity of this test. quality and quantity. FDPs interfere with fibrin CriticalPo nt p ol y m e r i z at io n a nd c a n pr olo ng t h r o m bi n t i m e. Partial thrombo- Partial Thromboplastin Time Hypofibrinogenemia also prolongs thrombin plastin time is used Evaluation of the intrinsic and common path- time. The test is conducted by the addition of to evaluate the ways via the partial thromboplastin time thrombin to citrated plasma and is interpreted intrinsic and com- (PTT) is similar to evaluation by the ACT, but by the time interval for clot formation. mon pathways. the PTT has a higher sensitivity. Deficiency of factors V, VIII, IX, X, XI, and XII; prothrom- Proteins Induced by Vitamin K bin; and fibrinogen causes prolongation of the Absence or Antagonism PTT. The test is conducted by adding citrated The proteins induced by vitamin K absence or plasma to a glass tube with a phospholipid antagonism (PIVKA) test is an evaluation of the and calcium chloride substrate. The time until proteins awaiting conversion to coagulation clot formation is then determined. factors as well as proteins that inhibit coag- ulation. Factors II, VII, IX, and X and the Activated Partial Thromboplastin Time anticoagulant proteins C and S depend on The activated partial thromboplastin time (aPTT) vitamin K carboxylation of glutamate from test is similar to that for the PTT, except that a their protein precursor for normal hemostatic contact activator (e.g., kaolin, silicates, ellagic function. In the absence of vitamin K, con- acid) is added to the specimen when deter- tinual synthesis of protein precursors causes mining the aPTT. The aPTT is most commonly a buildup within hepatocytes and a decline used to evaluate the intrinsic and common of functional coagulation factor production. pathways. Prolongation of the PTT and aPTT The leakage of PIVKA into the peripheral in horses has been associated with hemophilia circulation is quantified through the PIVKA A (factor VIII deficiency) and plasma prekal- test, which determines the clotting time with likrein deficiency.28 the addition of thromboplastin to the sam- ple. Deficiencies in prothrombin and factors Prothrombin Time VII, IX, and X as well as anticoagulant poi- The prothrombin time (PT) or one-stage PT test soning can cause prolongation of the PIVKA is an analysis of the extrinsic and common test. The PIVKA test, in conjunction with the pathways, specifically their ability to convert PT, has been widely used in diagnosing and fibrinogen to fibrin. Deficiencies of tissue fac- confirming anticoagulant poisoning in dogs

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when results exceed 150 seconds.31 The use of Antithrombin the PIVKA test in horses has been limited to The hepatocyte-produced α-globulin antithrom- suspected cases of anticoagulant toxicity but bin is considered one of the most important could also be indicated in cases of feed toxic- endogenous inhibitors of coagulation. The ity (moldy sweet clover).32 function of antithrombin is the neutralization of thrombin; activated factors IX, X, XI, and XII; Fibrinogen kallikrein; and plasmin. A functional assay of Evaluation of the fibrinolytic system through antithrombin (chromogenic assay) is preferred the fibrinogen concentration is less sensitive to simply detecting its presence (immunoassay) in horses than in other species.30 A decreased in plasma.24 Chromogenic assays determine fibrinogen concentration can indicate dis- the amount of functional antithrombin present seminated intravascular coagulation (DIC), but by adding the test plasma to a reagent contain- given the capacity of the equine liver to pro- ing heparin and excess thrombin or factor Xa, duce large amounts of acute-phase proteins which also contains the chromogen-labeled (fibrinogen) in response to inflammation, the substrate for thrombin or factor Xa.36 The more fibrinogen level may be increased or remain antithrombin present in the test specimen, the unaltered despite profound inflammation.26,33 less activity of thrombin or factor Xa and thus The fibrinogen concentration can be deter- less color, which is measured spectrophoto- mined by a heat precipitation test that com- metrically. A decreased plasma concentration pares the plasma protein concentration before of antithrombin can be caused by widespread and after heating. A more sensitive mechanical coagulation or DIC,1 decreased hepatic func- CriticalPo nt method can be conducted by many coagula- tion,37 or protein-losing enteropathies and tion analyzers. nephropathies.33 Increased antithrombin pro- Prothrombin time is duction has been implicated with acute-phase used to evaluate the Fibrin Degradation Products protein production in cases of acute inflamma- extrinsic and com- FDPs are the result of the consumption of tion24 and has been suggested to be one of the mon pathways. fibrino­gen or cross-linked fibrin fibers by plas- most accurate prognostic indicators in horses min. Fibrin fragments are detected by mixing with colic.30 Antithrombin I complexes in a 1:1 a blood sample with thrombin and a protease ratio with thrombin, and thrombin–antithrom- inhibitor. An increased FDP concentration sug- bin complex has been used as an indirect gests increased activities of fibrinolysis or fibrin- means to evaluate thrombin production/acti- ogenolysis. Increased serum FDPs can occur vation during hypercoagulable states.38 when fibrin deposition exceeds normal clear- ance mechanisms, and they have been linked Adult Versus Neonatal Differences to DIC, severe inflammatory processes, hemor- in Diagnostic Tests rhagic disorders, and postoperative states.1 Clear differences in the reference intervals between adult horses and neonatal foals are D-dimers present as demonstrated in Table 2. The most D-dimer production is the end result of fibrin- striking differences are noted when comparing olysis caused by plasmin activity. Specifically, coagulation times of foals younger than 24 hours it is the breaking of thrombin-induced fibrin of age with those of adults. Differences include fiber bonds with the release of D-fragments higher platelet concentrations, increased FDPs, (dimers). D-dimer identification differs from prolonged PT and aPTT, and lower fibrinogen testing for FDPs in that it is specific for dis- concentrations. With the exception of the plate- solution, by plasmin, of fibrin generated from let concentration, hemostatic parameters in foals active thrombin production, whereas FDPs are indistinguishable from healthy adult horse indicate general fibrinolysis and fibrinogenoly- values by 1 month of age.39 sis. D-dimer testing in conjunction with FDP analysis has been used in assessing DIC in Conclusion horses,34 but it is important to note that the Clot formation and dissolution is a dynamic D-dimer concentration alone is not specific for balance of procoagulant and anticoagulant the process or diagnosis of DIC35 and, there- processes necessary in establishing hemosta- fore, should not be overinterpreted. sis while preserving normal tissue perfusion.

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Calcium, an essential ion for hemostasis, is extension of vessel clotting. Normal function used in all reactions of clotting factors, except of this dynamic process leads to maintenance the first two steps of the intrinsic pathway. of vessel patency while simultaneously con- The separation of the intrinsic and extrinsic trolling excessive blood loss. Knowledge of coagulation pathways has greatly facilitated these basic mechanisms of clot formation and the understanding of secondary hemostasis. the diagnostic means to evaluate coagulation However, it is apparent that these two systems should aid equine clinicians. function simultaneously to produce thrombin. Tissue factor stimulates the induction of the extrinsic pathway, while factor XII interacts TO LEARN MORE with exposed collagen fibers to initiate the intrinsic pathway. The extrinsic pathway is very rapid, while the intrinsic pathway proceeds at Watch for a related article titled “Modification a slightly slower rate. Along with clot forma- of the Coagulation Cascade: Available tion, activation of fibrinolysis occurs, serving Medications” in an upcoming issue. as a “check and balance” to limit detrimental

References 1. Monreal L, Angles A, Espada Y, et al. Hypercoagulation and 4. Nakamura F, Pudas R, Heikkinen O, et al. The structure of the hypofibrinolysis in horses with colic and DIC. Equine Vet J Suppl GPIb-filamin A complex.Blood 2006;107(5):1925-1932. 2000;32:19-25. 5. Nakamura T, Kambayashi J, Okuma M, et al. Activation of the 2. Guyton AHJ. Textbook of Medical Physiology. 11th ed. Philadel- GP IIb-IIIa complex induced by platelet adhesion to collagen is phia: Elsevier; 2006. mediated by both alpha2beta1 integrin and GP VI. J Biol Chem 3. Turk JR. Physiologic and pathophysiologic effects of endothelin: im- 1999;274(17):11897-11903. plications in cardiopulmonary disease. JAVMA 1998;212(2):265-270. 6. Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev

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1993;7(1):52-62. 24. Reed SMW, Bayly M, Sellon DC. Equine Internal Medicine. 2nd 7. McNicol A, Israels SJ. Platelet dense granules: structure, function ed. Philadelphia: Elsevier; 2004. and implications for haemostasis. Thromb Res 1999;95(1):1-18. 25. Hinchcliff KW, Kociba GJ, Mitten LA. Diagnosis of EDTA-depen- 8. Levi M, ten Cate H, van der Poll T. Endothelium: interface be- dent pseudothrombocytopenia in a horse. JAVMA 1993;203(12): tween coagulation and inflammation. Crit Care Med 2002;30(5 1715-1716. suppl):S220-S224. 26. Lassen ED, Swardson CJ. Hematology and hemostasis in the 9. Aird WC. Vascular bed-specific hemostasis: role of endothelium horse: normal functions and common abnormalities. Vet Clin North in sepsis pathogenesis. Crit Care Med 2001;29(7 suppl):S28-S34. Am Equine Pract 1995;11(3):351-389. 10. Marshall JC. Inflammation, coagulopathy, and the pathogenesis 27. Rawlings CA, Byars TD, Van Noy MK, et al. Activated coagula- of multiple organ dysfunction syndrome. Crit Care Med 2001;29(7 tion test in normal and heparinized ponies and horses. Am J Vet suppl):S99-S106. Res 1975;36(5):711-713. 11. Bom VJ, Bertina RM. The contributions of Ca2+, phospholipids 28. Zimmel DN. Current Therapy in Equine Medicine. 5th ed. St. and tissue-factor apoprotein to the activation of human blood-coag- Louis: Elsevier Science; 2003. ulation factor X by activated factor VII. Biochem J 1990;265(2):327- 29. Wagner AE. Transport of plasma for prothrombin time 336. testing in monitoring warfarin therapy in the horse. JAVMA 12. Roncales FJ. Schalm’s Veterinary Hematology. 5th ed. Philadel- 1981;178(3):306. phia: Lippincott Williams & Wilkins; 2000. 30. Dallap BL. Coagulopathy in the equine critical care patient. Vet 13. Fenton 2nd JW, Ofosu FA, Brezniak DV, et al. Understand- Clin North Am Equine Pract 2004;20(1):231-251. ing thrombin and hemostasis. Hematol Oncol Clin North Am 31. Mount ME, Kim BU, Kass PH. Use of a test for proteins in- 1993;7(6):1107-1119. duced by vitamin K absence or antagonism in diagnosis of anti- 14. Hack CE. Tissue factor pathway of coagulation in sepsis. Crit coagulant poisoning in dogs: 325 cases (1987-1997). JAVMA Care Med 2000;28(9 suppl):S25-S30. 2003;222(2):194-198. 15. Sheafor SE, Couto CG. Anticoagulant rodenticide toxicity in 21 32. Beasley V. Toxicants that interfere with the function of vitamin dogs. JAAHA 1999;35(1):38-46. K. Veterinary Toxicology. Ithaca, NY: International Veterinary Infor- 16. Vermeer C. Gamma-carboxyglutamate-containing proteins and mation Service (www.ivis.org); 1999. the vitamin K–dependent carboxylase. Biochem J 1990;266(3):625- 33. Dolente BA, Wilkins PA, Boston RC. Clinicopathologic evidence 636. of disseminated intravascular coagulation in horses with acute coli- 17. Walz DA, Anderson GF, Ciaglowski RE, et al. Thrombin-elicited tis. JAVMA 2002;220(7):1034-1038. contractile responses of aortic smooth muscle. Proc Soc Exp Biol 34. Stokol T, Erb HN, De Wilde L, et al. Evaluation of latex aggluti- Med 1985;180(3):518-526. nation kits for detection of fibrin(ogen) degradation products and 18. Mann KG. Thrombin formation. Chest 2003;124(3 suppl):4S- D-dimer in healthy horses and horses with severe colic. Vet Clin 10S. Pathol 2005;34(4):375-382. 19. Good LI. Thromboembolic disease: physiology of hemostasis 35. Stokol T, Brooks MB, Erb HN, et al. D-dimer concentrations in and pathophysiology of thrombosis. Compend Contin Educ Vet healthy dogs and dogs with disseminated intravascular coagula- 2003;25(9):650-659. tion. Am J Vet Res 2000;61(4):393-398. 20. Esmon CT. The protein C pathway. Chest 2003;124(3 suppl): 36. Stockham SL, Scott MA. Fundamentals of Veterinary Clinical 26S-32S. Pathology. Ames: Iowa State University Press; 2002. 21. Lau A, Berry LR, Mitchell LG, et al. Effect of substrate and fibrin 37. Johnstone IB, Petersen D, Crane S. Antithrombin III (ATIII) activ- polymerization inhibitor on determination of plasma thrombin gen- ity in plasmas from normal and diseased horses, and in normal ca- eration in vitro. Thromb Res 2007;119(6):667-677. nine, bovine and human plasmas. Vet Clin Pathol 1987;16(1):14-18. 22. Sappino AP, Huarte J, Vassalli JD, et al. Sites of synthesis of 38. Topper MJ, Prasse KW, Morris MJ, et al. Enzyme-linked immu- urokinase and tissue-type plasminogen activators in the murine nosorbent assay for thrombin-antithrombin III complexes in hors- kidney. J Clin Invest 1991;87(3):962-970. es. Am J Vet Res 1996;57(4):427-431. 23. Menoud PA, Sappino N, Boudal-Khoshbeen M, et al. The kid- 39. Barton MH, Morris DD, Crowe N, et al. Hemostatic indices in ney is a major site of alpha(2)-antiplasmin production. J Clin Invest healthy foals from birth to one month of age. J Vet Diagn Invest 1996;97(11):2478-2484. 1995;7(3):380-385.

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1. Platelet adherence to the vascular c. production of tissue factor a. V, X, IX, and XII. endothelium during primary d. antithrombin release b. II, VII, IX, and X. hemostasis is initiated via c. VII, VIII, IX, and XI. a. platelet glycoprotein. 3. Tissue factor combines with . II, VIII, X, and XII. b. IgG. ______of the extrinsic pathway c. vWF. to activate factor X, thereby initiating 5. The ability of antithrombin to bind d. thrombosthenin. the common pathway. thrombin, thus controlling the rate a. IXa c. XII of clot formation, can be greatly 2. How does the vascular endothelium b. II d. VIIa enhanced by prevent platelet adherence in healthy a. endogenous or exogenous heparin. vessels? 4. Vitamin K is an essential cofactor in the b. TPA. a. exposure of subendothelial collagen carboxylation of glutamate during the c. plasmin. b. production of the glycocalyx activation of factors d. thrombomodulin.

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6. During fibrinolysis, plasmin is best determined by responsible for the proteolytic a. PT and ACT. digestion of b. ACT and PTT. a. factors IX, X, XI, and XII and c. thrombin time and PT. thrombin. d. PTT and aPTT. b. factors V, VIII, and XII; fibrin; and prothrombin. 9. During clot dissolution, which c. TPA and factors V, VII, IX, of the following tests best and X. quantifies the activity of d. plasma proteins C and S, TPA, plasmin? and factors II, VIII, and XII. a. D-dimer b. FDP 7. What are the two most c. PTT important endogenous d. antithrombin anticoagulants? a. protein C and antithrombin 10. A deficiency of vWF, resulting b. fibrinogen and plasmin in coagulopathy with a normal c. thrombin and Christmas factor platelet concentration, PT, and d. factor VII and vWF aPTT, can be attributed to a. factor VIII deficiency. 8. Functional assessment of the b. prekallikrein deficiency. intrinsic and common pathways c. platelet adhesion. of secondary hemostasis is d. vitamin K deficiency.

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