Department of Companion Animal Clinical Studies, Section Clinical Pathology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Pretoria, South Africa
Thrombosis is the partial or complete obstruction of a blood vessel by a thrombus (blood clot). Thromboembolism (TE) refers collectively to obstruction from local thrombus formation or translocation from a distant site (embolism). Virchow's triad describes three major factors promoting thromboembolism: endothelial injury, blood flow abnormalities, and hypercoagulability. Although a single factor may predominate, in most disease conditions combined abnormalities act synergistically to overcome haemostatic balance.
Our knowledge and understanding of the complex process of haemostasis and regulation have vastly improved over the past decade. The previous "cascade" model has largely been replaced by the "cell-based" model which has shed new light on haemostatic derangements. The cell-based model suggests that in vivo haemostasis occurs in distinct overlapping phases that require the participation of two different cell types: tissue factor (TF)-bearing cells and platelets. Several research reports have confirmed the key role of TF in the initiation of haemostasis. Tissue factor is a transmembrane glycoprotein receptor found in extravascular tissues including organ capsules and the adventitia of blood vessel walls (the extravascular location prevents initiation of haemostasis under normal flow circumstances with an intact endothelium). When injury occurs, the deeper layers of the vessel wall are disrupted and TF is exposed to flowing blood resulting in initiation of the haemostatic pathways. It is also expressed on fibroblasts and upon cell activation on vascular smooth muscle cells, monocytes, and neutrophils.
The cell-based model of haemostasis is a dynamic model that involves cellular regulation of haemostasis in three overlapping phases:
Initiation: Flowing blood is exposed to TF-bearing cells resulting in complex formation with preexisting traces of FVIIa (extrinsic pathway) and subsequent production of an initial small amount of thrombin through activation of FX (common pathway). This process is limited to the surface of the TF-bearing cell; any FXa that diffuses away from the cell is rapidly inhibited by tissue factor pathway inhibitor (TFPI) or antithrombin (AT).
Amplification: Thrombin binding to platelet surface receptors causes extreme changes in the surface of the platelet, resulting in shape change, shuffling of membrane phospholipids to create a procoagulant membrane surface, and release of granule contents. Thrombin-induced activation of FV and FVIII during this phase is also important to enhance coagulation.
Propagation: The release of platelet granule contents recruits additional platelets to the site of injury. The propagation phase occurs on the surface of these platelets. The intrinsic "tenase" complex (FIXa, FVIIIa) rapidly generates large amounts of FXa on the platelet surface. The generated FXa complexes with FVa forming the "prothrombinase" complex leading to cleavage of prothrombin and a subsequent burst of thrombin production responsible for cleaving fibrinogen and formation of the haemostatic plug.
Fibrinolysis is mediated by the proteolytic enzyme plasmin, converted from plasminogen. Tissue- type plasminogen activator [tPA] is the main plasminogen activator in the vasculature and is produced by the endothelial cells. Plasminogen activator inhibitor type 1 (PAI-1) is the major inhibitor of plasminogen activators. Many different cell types (megakaryocytes, endothelial cells, hepatocytes) synthesize PAI-1. α2-Antiplasmin is the primary inhibitor of plasmin and is produced by the hepatocytes. Insoluble crosslinked fibrin is a substrate for plasmin. D-dimers are the resultant degradation products of fibrin during fibrinolysis. Fibrin(ogen) degradation products (FDPs), on the other hand, are produced by the action of plasmin on fibrinogen or soluble fibrin and released during fibrinogenolysis, which may occur independent of thrombosis.
Endogenous anticoagulants limit activation of coagulation to the site of injury and thus prevent the development of a clot into a thrombus. Defective or deficient anticoagulants may result in pathologic thrombosis.
Tissue factor pathway inhibitor (TFPI) - Inhibitor of the formation of TF complexes and thus initiation of haemostasis. Tissue factor pathway inhibitor inhibits factors Xa, IXa, and the TF-VIIa complex. Tissue factor pathway inhibitor is predominantly produced by the endothelium.
Antithrombin (AT) - Synthesised by the hepatocytes. Comprises > 70% of the natural anticoagulant activity of blood.
Activated protein C (APC) pathway - Thrombin that escapes into the circulation from a site of injury must be controlled to prevent clot formation. It is either inhibited by AT in circulation or binds to thrombomodulin (TM), expressed on endothelial cells, which makes it more effective at activating protein C than stimulating the cleaving of fibrinogen or the activation of platelets. Activated protein C forms a complex with protein S (cofactor) that cleaves and inactivates any FVa and FVIIIa resulting in inhibition of the tenase and prothrombinase complexes, limiting further thrombin formation. Both protein C and S are produced by the hepatocytes and are vitamin K-dependent.
Haemostasis and Inflammation
It has become increasingly clear that inflammation and haemostasis are intricately linked and that both play roles in host defence. Cells and inflammatory mediators of the immune system are capable of triggering coagulation pathways, and coagulation proteases, on the other hand, have significant immunomodulatory effects. Inflammatory cytokines initiate coagulation events at sites of inflammation through various mechanisms that include expression of TF, altered thrombogenicity of endothelial surfaces, and activation of platelets. Tissue factor is the primary physiologic initiator of coagulation and endothelial cells, neutrophils, and monocytes express TF on their surfaces in response to a variety of stimuli including endotoxin, IL-6, tumour necrosis factor (TNF), IL-1, and immune complexes.
Activation of coagulation in inflammation ultimately leads to the production of thrombin (FIIa) which has both procoagulant and anticoagulant properties and can stimulate inflammation and cellular proliferation. It is also the most potent activator of platelets. At the site of injury, platelets exert their effect through a wide array of interactions both in response to external signals from sentinel cells, such as platelet activating factor from macrophages, but also through release of mediators such as chemokines stored in the α granules. Hyperfibrinogenemia is a common finding in inflammation and is consistent with an acute phase response. Platelet surfaces are densely populated with receptors, of which the glycoprotein IIb/IIIa complex (GPIIb/IIIa) is the most prominent. Studies have shown that even with severe thrombocytopenia, thrombin-activated platelets bind to large amounts of fibrinogen via GPIIb/IIIa receptors, resulting in increased clot strength. Endothelial surfaces are normally antithrombotic, mainly because of expression of TM, but at sites of inflammation the injury to the endothelium will expose the blood to TF. Circulatory inflammatory mediators, such as TNF, have the effect of reducing endothelial expression of TM and receptors for protein C.
Antithrombin (AT) has significant anti-inflammatory properties by modulating the interaction between endothelial cells and leukocytes, reducing leukocyte activation and adhesion. Antithrombin-stimulated prostacyclin release from endothelial cells reduces platelet aggregation and decreases proinflammatory cytokine production. Antithrombin also directly inhibits IL-6 release and TF expression of endothelial cells. The APC pathway has significant anti-inflammatory actions, directly through inhibition of cytokine production in leukocytes, and indirectly by the inactivation of thrombin and the inhibition of further thrombin generation. Treatment with APC in human sepsis patients has a reported 20% reduction in relative risk of mortality and these patients had significantly lower IL-6 activity.
Laboratory Assays of Hypercoagulability
The complex pathogenesis of hypercoagulability and its associated disease states complicate laboratory diagnosis of TE.
Although variable shortening or prolongation of PT and aPTT are often found in patients with TE, these changes are nonspecific and rarely aid in the diagnosis or management of TE.
Hyperfibrinogenemia has been reported to be an independent risk factor for TE in people. Hyperfibrinogenemia is a common finding in canine IMHA, feline cardiomyopathy, and sepsis. The predictive value of fibrinogen for animal TE has yet to be determined.
Antithrombin activity relates to TE risk in conditions such as PLN, where AT deficiency plays a major role in the pathogenesis of hypercoagulability. While risk of TE has been attributed to AT values below 60%, there are few empiric data in animals at this time to define a specific AT threshold.
D-dimer is a distinct fibrin degradation product formed by plasmin proteolysis of crosslinked fibrin. Quantitative D-dimer assays are reported to be proven diagnostic assays for ruling out PTE in human thrombotic syndromes.
Fibrin(ogen) Degradation Products (FDPs)
FDP measurement is primarily used to detect increased fibrinolysis associated with excessive coagulation. It may also be used to detect increased fibrinogenolysis.
Thrombin-Antithrombin complex (TAT)
Free thrombin in plasma is neutralised by irreversible complex formation with AT. Plasma concentration of TAT is an indirect measure of in vivo thrombin generation. High TAT is evidence of an active hypercoagulable state.
A measure of the viscoelastic properties of a fibrin clot in whole blood, generating qualitative tracings and numeric values depicting clot strength and stability, and the dynamics of clot formation and breakdown. Thromboelastography can be used to test for hypo- or hypercoagulability, monitor anticoagulant effects, guide transfusion therapy, and predict postoperative TE. TEG features of hypercoagulability have been described in various inflammatory and infectious conditions in dogs.
Platelet Function Analysis
Platelet aggregometry measures platelet aggregation in response to agonists (e.g., ADP, collagen); however, it is generally not useful for clinical characterisation of TE. Flow cytometry enables the detection of expression of activation markers and the presence of bound fibrinogen. Automated haematology analysers measure several variables of platelet function. The mean platelet component concentration (MPC) is a measure of platelet granularity and if decreased may be due to platelet degranulation, indicating activation. Mean platelet volume (MPV) is a measure of platelet size. Large platelets are known to have a greater potential for aggregation and secretion, and therefore suggests an increased risk of thrombosis.
Canine thrombotic syndrome typically causes arterial and venous TE, with multiorgan involvement. Although the frequency of TE is less common in cats, aortic thromboembolism (ATE) secondary to cardiomyopathy is by far the most common feline thrombotic syndrome.
Table. Specific disorders with increased risk of thrombosis or thromboembolism
Hypercortisolism (hyperadrenocorticism & iatrogenic corticosteroid therapy)
Immune-mediated haemolytic anaemia (IMHA)
Lymphocytic enteritis (IBD)
PLN (Nephrotic syndrome)
1. Hoffman M, Monroe DM. A cell-based model of hemostasis. Thromb Hemost. 2001;85:958–965.
2. Smith SA. Overview of hemostasis. In: Weiss DJ, Wardrop KJ, eds. Schalm's Veterinary Hematology. 6th ed. Ames, IA: Wiley-Blackwell; 2010.
3. Hackner SG, Schaer BD. Thrombotic Disorders. In: Weiss DJ, Wardrop KJ, eds. Schalm's Veterinary Hematology. 6th ed. Ames, IA: Wiley-Blackwell; 2010.
4. Mischke R. Overview of haemostasis. In: Day MJ, Kohn B, editors. BSAVA Manual of Canine and Feline Haematology and Transfusion Medicine. 2nd ed. Gloucester, UK: British Small Animal Veterinary Association; 2012.
5. Smith SA. Thrombosis. In: Day MJ, Kohn B, eds. BSAVA Manual of Canine and Feline Haematology and Transfusion Medicine. 2nd ed. Gloucester, UK: British Small Animal Veterinary Association; 2012.
6. Weiss DJ, Rashid J. The sepsis-coagulant axis: a review. J Vet Intern Med. 1998;12:317–324.
7. Licari LG, Kovacic JP. Thrombin physiology and pathophysiology. J Vet Emerg Crit Care. 2009;19(1):11–22.