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 the progression of disease and the monitoring of blood component and/or anticoagulant 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 enhanced understanding of the complex biochemistry of physiological hemostasis, leading to reevaluation of the traditional cascade model of physiological hemostasis, which divides coagulation into intrinsic and extrinsic pathways.
Although citrated plasma contains many of the factors involved in coagulation, whole blood contains not only these soluble factors but also the cellular components (eg, TF and phospholipid) that participate in normal and pathological hemostasis.
Physiological Understanding of Hemostasis in Animals
The traditional model of hemostasis, known as the cascade or waterfall model, divides coagulation proteins into an extrinsic and an intrinsic arm or pathway, both of which lead to a final, common pathway ending in formation of fibrin. This model offers a simplified understanding of how factors activate one another; however, it might not accurately reflect complex and overlapping interactions of procoagulant elements, especially the importance of cell surfaces.
Pearls & Pitfalls
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The cell-based model of hemostasis has been introduced to explain hemostasis through the interaction of vascular tone, blood flow, endothelial cells, platelets, and leukocytes, as well as coagulation and fibrinolytic factors, cofactors, and inhibitors (1). These interactions, under normal circumstances, result in balanced hemostasis, ensuring that there is formation of a clot at an injured site, inhibition of clotting when not needed, and fibrinolysis of clots when appropriate.
This dynamic model involves cellular regulation of coagulation in three phases: initiation, amplification, and propagation.
Initiation: TF-bearing cells initiate hemostasis. TF (factor III) 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 upon cellular activation it is also expressed on vascular smooth muscle cells, monocytes, and neutrophils. The TF-bearing cells and platelet surfaces act as the main surfaces for assembly of procoagulant complexes.
Any vessel injury leads to TF exposure. Factor VII binds to TF, resulting in activation to factor VIIa. Factor VIIa that is bound to TF on the cell surface, the "extrinsic tenase complex," 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.
Amplification: Factors Va and Xa assemble into the "prothrombinase complex" on the surface of the TF-bearing cell. A small, priming amount of factor IIa (thrombin) is initially generated close to the cell. This small amount of thrombin is not enough to lead to clot formation; however, it amplifies clot-forming elements by activating platelets to release factor V, causing von Willebrand factor to release factor VIII, and activating factors V, VIII, and XI.
Platelets are also activated by other mechanisms, including exposure of vessel wall collagen and von Willebrand factor, leading to platelet adhesion and aggregation at the injured site. Activated platelets aggregate to form the initial platelet plug that precedes the fibrin clot.
Propagation: As an essential part of platelet activation, the procoagulant phospholipid phosphatidylserine becomes available on the exterior surface of platelets. In the propagation phase of the cell-based model, the binding of factor IXa to activated platelets promotes formation of the "intrinsic tenase complex" of factor IXa, factor VIIIa, phosphatidylserine, and calcium.
Factor IXa activated during the initiation phase by the interaction of factor VIIa and TF-bearing cells is able to diffuse to the surface of activated platelets because it is not inhibited by TFPI and is only slowly inhibited by antithrombin.
Factor IX can also be activated on the surface of platelets by factor XIa. Formation of the intrinsic tenase complex on the surface of activated platelets results in abundant factor Xa formation. As in the amplification phase, factor Xa complexes with factor Va, forming the "prothrombinase complex" on the surface of activated platelets and leading to the cleavage of factor II (prothrombin) and a major subsequent burst of factor IIa (thrombin). Thrombin cleaves factor I (fibrinogen) to factor Ia (fibrin), forming a fibrin clot.
The thrombin burst in the propagation phase activates plasmin, thereby initiating fibrinolysis, which 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, thereby preventing premature clot lysis and enabling clot propagation.
The balance of fibrin formation and fibrinolysis regulates the size and quality of the fibrin clot and localizes it to the injury site. The quality of the clot has a marked impact on how effectively it provides hemostasis.
Clinical Approach to Assessing Hemostasis in Animals
Although the traditional division between primary, secondary, and tertiary (fibrinolysis) hemostasis is not biologically accurate, it is still a useful approach to assessing hemostasis in animals with acquired or hereditary hemostatic disorders.
Primary hemostasis is accomplished by interaction of platelets with exposed subendothelial surfaces and results in a platelet plug. Simultaneously, plasma coagulation proteins are activated in a sequential cascade that depends on the phospholipid provided by the activated platelets and on calcium ions from plasma to form a stable fibrin 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 (tertiary hemostasis).
Tests of Primary Hemostasis
Tests of primary hemostasis include in-clinic tests such as platelet count and buccal mucosal bleeding time (BMBT), as well as tests offered by reference laboratories, such as platelet closure time and von Willebrand factor (vWF) measurement.
Platelet counts can be estimated from peripheral blood smears. Platelets can be activated upon blood collection, leading to clumping and underestimated machine platelet counts, which emphasizes the importance of manual blood smear analysis. Apparent thrombocytopenia caused by in vitro platelet agglutination should not cause spontaneous bleeding.
Platelet counts can be lower than the reference range in clinically normal Greyhounds and other sight hound breeds. EDTA-induced platelet clumping occurs occasionally in dogs and horses (pseudothrombocytopenia), and cat platelets activate and clump readily during blood collection.
Buccal mucosal bleeding time is a point-of-care screening test commonly used to assess primary hemostasis in dogs. The lip is inverted, and a spring-loaded, automated lancet device produces a standardized incision on the mucosal side. Blood adjacent to the wound is gently blotted with absorbing paper every 5 seconds until bleeding stops. Canine BMBT is reported as normal (< 4 minutes) or abnormal. BMBT can also be measured in cats; however, the test is uncommonly used clinically, it usually requires sedation, and the technique and interpretation are not well standardized or documented (2).
Platelet closure time, measured by a platelet function analyzer, provides a quantitative, simple, and rapid in vitro assessment of primary platelet-related hemostasis at high shear stress. The test requires a small volume of citrated whole blood, which is drawn under vacuum through a 200-mcm-diameter stainless steel capillary and a 150-mcm-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 platelet closure times occur with inherited and acquired platelet function disorders, including von Willebrand disease. Platelet function analyzers also can be used to monitor response to treatment with both desmopressin acetate and glycoprotein IIb/IIIa antagonists.
Assays of canine vWF are typically required for diagnosis of von Willebrand disease and include measurement of vWF antigen concentration and collagen-binding assays for measurement of vWF activity.
Tests of Secondary Hemostasis
Tests of secondary hemostasis include plasma-based assays measuring activated clotting time (ACT), activated partial thromboplastin time (aPTT), and prothrombin time (PT).
aPTT assesses factors of the intrinsic (XII, XI, IX, VIII) and common (X, V, II, I) pathways.
PT assesses factors of the extrinsic (VII) and common pathways.
Measurement of the activity of individual coagulation factors can identify additional specific factor deficits, helping to localize and define the problem in animals suspected of having hemostatic disease.
Tests of Tertiary Hemostasis
The fibrinolytic system (tertiary hemostasis) is traditionally evaluated with measurements of clot breakdown products, such as FDPs and D dimers.
Endogenous anticoagulant ability has been evaluated via the measurement of antithrombin, protein C, and protein S activity.
Viscoelastic Testing
Plasma-based coagulation screening tests can help identify defective or deficient coagulation proteins. Although this traditional approach makes it possible to effectively and systematically localize causes of bleeding, it can be difficult from a clinical perspective to globally evaluate clotting disturbances that affect multiple stages of hemostasis, identify hypercoagulable states, and monitor the effect of anticoagulant or procoagulant treatment.
One reason for this difficulty might be that plasma-based tests of the secondary and tertiary systems target specific elements of the hemostatic system, thus potentially ignoring other factors that could substantially contribute 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.
Viscoelastic testing, such as thromboelastography or thromboelastometry, enables rapid assessment of hemostatic function in whole blood. Because whole blood contains the soluble factors and cellular components involved in hemostasis, whole blood assays can provide a more accurate reflection of in vivo hemostasis than can traditional plasma-based hemostasis assays. These modalities evaluate all the steps of hemostasis, including initiation, amplification, and propagation, as well as fibrinolysis, which includes the interaction of platelets and leukocytes with the proteins of the coagulation cascade.
Viscoelastic testing modalities evaluate both the traditional plasma components of coagulation and the cellular components. Viscoelastic testing is performed on unstabilized fresh whole blood within minutes of taking a blood sample, and coagulation pathways are assessed after chemical activators have been added to the sample. Viscoelastic testing modalities analyze the dynamics of clot formation time, clot strength, and clot breakdown.
Viscoelastic testing is the first modality available to clinicians that can evaluate hypercoagulability. It 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.
Point-of-care viscoelastic testing machines are becoming more widely available, and abundant emerging research seeks to translate observations from various viscoelastic testing modalities into clinically meaningful information for a variety of hyper- and hypocoagulable disorders in a variety of species.
Key Points
The primary stage of hemostasis requires normal number and function of platelets and von Willebrand factor to form a plug.
In secondary hemostasis, a fibrin clot forms via TF-bearing cells, activated platelet surfaces, and clotting factors. The extent of the clot is controlled by fibrinolysis (tertiary hemostasis).
Traditional evaluation of coagulation could involve a combination of tests of primary, secondary, and tertiary stages. Viscoelastic testing enables a more global assessment of interacting hemostatic pathways.
For More Information
eClinPath.com (Cornell University): Hemostasis
Veterinary Hematology, Clinical Chemistry, and Cytology. 3rd ed. Wiley-Blackwell; 2022:201-219.
Also see pet owner content regarding bleeding disorders of dogs, cats, and horses.
References
Hoffman M. A cell-based model of coagulation and the role of factor VIIa. Blood Rev. 2003;17(suppl 2):S1-S5. doi:10.1016/s0268-960x(03)90000-2
Alatzas DG, Mylonakis ME, Kazakos GM, Kostoulas P, Kritsepi-Konstantinou M, Polizopoulou ZS. Reference values and repeatability of buccal mucosal bleeding time in healthy sedated cats. J Feline Med Surg. 2014;16(2):144-148. doi:10.1177/1098612X13502973
