Effective hemostasis depends on an adequate number of functional platelets, an adequate concentration and activity of plasma coagulation and fibrinolytic proteins, and a normally responsive blood vasculature. The diagnosis, treatment, and monitoring of hypo- and hypercoagulable animals is difficult with regard to both progression of disease and monitoring blood component and/or anticoagulation therapy. Citrated plasma samples are often used in veterinary medicine to determine fibrinogen concentration, activated partial thromboplastin time (APTT), prothrombin time (PT), and d-dimer or fibrinogen degradation product (FDP) concentration.
The introduction of the cell-based, tissue factor (TF)/Factor VII–dependent model of hemostasis has increased understanding of the complex biochemistry of physiologic hemostasis, leading to reevaluation of the traditional understanding of physiologic hemostasis divided into the intrinsic and extrinsic pathways of coagulation. Although citrated plasma contains many of the factors involved in coagulation, whole blood contains both the soluble factors and intravascular cells active in physiologic and pathologic hemostasis, incorporating TF and phospholipid-bearing cells, such as platelets and leukocytes.
A cell-based model of hemostasis has been introduced that explains physiologic hemostasis through a complex process in which the interaction of vascular tone, blood flow, endothelial cells, platelets, leukocytes, coagulation factors, and fibrinolytic factors and their cofactors and inhibitors result in balanced hemostasis and formation of a clot at the injured site. This dynamic model involves cellular regulation of coagulation in three phases: initiation, amplification, and propagation.
TF-bearing cells initiate hemostasis. TF is a transmembrane glycoprotein receptor found in extravascular tissues, including organ capsules and the adventitia of blood vessel walls. It is constitutively expressed on fibroblasts and, on cellular activation, on vascular smooth muscle cells, monocytes, and neutrophils. The TF-bearing cells and platelet surfaces act as the main cellular surfaces for assembly of the procoagulant complexes. Any vessel injury leads to TF exposure. Factor VII binding to TF results in activation to Factor VIIa. Factor VIIa bound to TF on the cell surface activates Factor IX to Factor IXa and Factor X to Factor Xa. Initially, the formed Factor Xa is limited to the TF-bearing cell, because Factor Xa that diffuses away from the cell is rapidly inhibited by TF pathway inhibitor (TFPI) or antithrombin.
Together with formed Factor Va, Factor Xa is assembled into the prothrombinase complex on the surface of the TF-bearing cell. An initial small amount of thrombin close to the cell independent of the presence of platelets is generated and is responsible for activation of platelets, release of Factor V from the platelets, activation of Factor V, activation of Factor VIII and release of Factor VIII from von Willebrand factor, and activation of Factor XI. Platelets also are activated by other mechanisms, including vessel wall collagen and von Willebrand factor, leading to adhesion and aggregation at the injured site.
As an essential part of the platelet activation process, the procoagulant phospholipid phosphatidylserine becomes available. The initially generated Factor IXa binds to the activated platelet surfaces, promoting formation of the “tenase” complex; this results in major Factor Xa formation and amplification of the coagulation process. The formed Factor IXa diffuses to the platelets, because it is not inhibited by TFPI and is inhibited slowly by antithrombin. The formed Factor Xa complexes with Factor Va on the activated platelet surface, forming the “prothrombinase” complex that leads to cleavage of prothrombin and to the major subsequent burst of thrombin responsible for cleaving fibrinogen and forming the hemostatic plug. Additional Factor IXa is supplied by Factor XIa on the platelet surface. Factor XIa activates the antifibrinolytic pathway.
The second wave of thrombin activates plasmin, thereby initiating fibrinolysis. This keeps the clot controlled at the site of injury. To control fibrinolysis, the antifibrinolytic pathway is activated by thrombin activation of thrombin-activatable fibrinolysis inhibitor (TAFI). TAFI slows the fibrinolytic process by inhibiting plasmin activity; this prevents premature clot lysis and allows clot propagation. The balance of fibrin formation and fibrinolysis regulates the size and quality of the fibrin plug and localizes it to the site of injury. The quality of the clot has a significant impact on how effectively it provides hemostasis.
Although the traditional division between primary and secondary hemostasis is not biologically accurate, it is still a useful diagnostic approach to hemostasis in animals with acquired or hereditary hemostatic disorders. Primary hemostasis is accomplished by interaction of platelets with exposed subendothelial surfaces. Simultaneously, plasma coagulation proteins are activated in a sequential cascade that depends on the phospholipid provided by the activated platelets and calcium ions from plasma to form a stable clot (secondary hemostasis). Circumstances that activate platelets and the coagulation proteins also activate plasma fibrinolytic proteins, which ensure localization of the clot and its timely dissolution.
Hemostatic capabilities are traditionally assessed by tests of primary hemostasis (platelet count and buccal mucosal bleeding time) and secondary hemostasis through plasma-based assays designed to further localize defects, such as the APTT for intrinsic (XII, XI, IX, VIII) and common (X, V, II, fibrinogen) pathways, and PT for extrinsic (VII) and common pathways). The fibrinolytic system is evaluated with measurements of degradation products such as FDP and d-dimer or by thromboelastography, and endogenous anticoagulant ability has been evaluated through antithrombin, protein C, and protein S. Additional specialized individual coagulation factor tests can further localize congenital defects.
Thus, plasma-based coagulation screening tests can help identify the defective or deficient coagulation protein. Although this traditional approach makes it possible to effectively and systematically localize the cause of bleeding, it may be difficult from a clinical perspective to evaluate general hemostatic capability and to predict or monitor the effect of anticoagulant or procoagulant treatment. This may partially be because plasma-based tests of the secondary and fibrinolytic systems target specific elements of the hemostatic system, thus potentially ignoring other factors that may contribute significantly to overall hemostatic capability in acquired disorders. Another plausible reason is the low sensitivity of APTT and PT; usually, activity of a coagulation protein must be <30% and sometimes <10% of normal before an abnormality is detected.
Tests for increased risk or tendency toward thrombosis are generally available only through some academic and research laboratories. Measurement of antithrombin activity is being offered by increasing numbers of laboratories. Tests for measurement of activities of plasminogen, protein C, α2-antiplasmin, tissue plasminogen activator, and plasminogen activator inhibitor have been established in some domestic animals.
Because whole blood contains all the intravascular factors and cells involved in physiologic and pathologic hemostasis, incorporating TF and phospholipid-bearing cells, whole blood assays may provide a more accurate reflection of in vivo hemostasis than traditional plasma-based hemostasis assays. However, few whole blood assays to assess both primary and secondary hemostasis have been used in veterinary studies to date.
The platelet function analyzer, PFA-100, is a method for quantitative, simple, and rapid in vitro assessment of primary platelet-related hemostasis at high shear stress. The test requires a small volume (0.8 mL) of citrated whole blood, which is drawn under vacuum through a 200-μm diameter stainless steel capillary and a 150-μm diameter aperture in a nitrocellulose membrane coated with collagen and epinephrine (CEPI) or collagen and ADP (CADP). A platelet aggregate forms that blocks blood flow through the aperture; the time taken to occlude the aperture is reported as the closure time. Prolonged closure times are seen with inherited platelet function disorders (eg, storage pool disorders), with aspirin ingestion, and with von Willebrand disease. The PFA-100 also has the ability to monitor response to treatment with both desmopressin acetate and glycoprotein IIb/IIIa antagonists.
Thromboelastography (TEG) allows for rapid assessment of hemostatic function in whole blood. It evaluates all of the steps in hemostasis, including initiation, amplification, and propagation, as well as fibrinolysis, including the interaction of platelets and leukocytes with the proteins of the coagulation cascade. Thus, TEG combines evaluation of the traditional plasma components of coagulation with the cellular components. TEG is performed on unstabilized fresh whole blood within 4–6 minutes of taking the blood sample. This is generally not practical in a routine clinical setting, and using citrated stabilized whole blood (with recalcification immediately before analysis) has been proposed to increase the time span from sampling to analysis. A TF-activated TEG assay on citrated whole blood has been validated in dogs and shown to have a low analytical variation and good correlation to clinical signs of bleeding compared with many traditional plasma-based coagulation assays.
Thromboelastography is the first modality available to clinicians that can evaluate hypercoagulability. It is especially useful in dogs with DIC, neoplasia, sepsis, and parvoviral infection, and to evaluate platelet dysfunction in dogs with hypothermia. TEG analysis is a valuable aid in the diagnostic evaluation of animals with abnormal hemostasis and a supplement to traditional coagulation tests such as PT, APTT, d-dimer, and fibrinogen assays. The use of TEG, especially in patients with suspected hypercoagulability, has become more widespread in hospitals with critical care specialists.
The primary stage of normal coagulation requires normal numbers and function of platelets to form a plug.
A fibrin clot then forms (secondary stage) utilizing the platelet plug and normal clotting factors. The extent of the clot is controlled by fibrinolysis.
Evaluation of coagulation requires a combination of tests of primary and secondary stages. Thromboelastography is the gold standard for evaluation of both primary and secondary coagulation.