The Elusive Hypercoagulable Patient Benjamin Brainard VMD, Dipl ACVAA, ACVECC Critical Care, University of Georgia, Athens, GA ([email protected]) Many diseases in veterinary medicine are complicated by the occurrence of micro or macro-thrombosis, which can magnify the morbidity and mortality of the condition. In diseases where hypercoagulability is suspected, anti-platelet or anticoagulant drugs are frequently recommended, without clear evidence of the effect on outcome or on the incidence of coagulopathy, consumptive or otherwise. Frequently, the choice for either anti-platelet or anti-coagulant drug is made due to convenience and owner compliance rather than an extensive knowledge of the origin of the hypercoagulability. The consequence of the presence of macrothrombi is significant disruption of blood flow to large areas of the body (e.g. lungs, hind limbs), while the upshot of microthrombi is more insidious organ dysfunction caused by thrombi lodged in small capillaries that gradually cause widespread organ hypoperfusion. Thrombotic disease has been reported in dogs suffering from immune-mediated hemolytic anemia (IMHA), sepsis, pancreatitis, heartworm disease, parvoviral enteritis, and neoplasia, among other diseases.1-3 In cats, pancreatitis, sepsis, and altered blood flow due to cardiomyopathy are associated with thrombus formation.4-6 Horses with colic, especially those with strangulating intestinal lesions or colitis, may have a presdisposition for venous thrombosis.7 Given the heterogeneity of diseases associated with thrombosis, the diagnosis of hypercoagulabity in a patient prior to the onset of coagulopathy is critical. Identification of the hypercoagulable animal will allow delivery of targeted therapy to the animals most at risk, but the best technique to achieve an antemortem, pre-thrombosis diagnosis of hypercoagulability is elusive, and may vary depending on thrombotic triggers in the different disease states. Hypercoagulability is generally suspected in animals with diseases that have a strong inflammatory component, because systemic inflammation can result in endothelial cell (EC) and monocyte activation and exposure of tissue factor (TF), shedding of TF-expressing microparticles from ECs and monocytes, increased circulating fibrinogen concentration, and generation of thrombin. Activation of platelets by inflammatory stimuli can also play a role, resulting in exposure of membrane P-selectin (CD62P) and phosphatidylserine (PS) on the platelet membrane that can interact with circulating mononuclear cells and coagulation factors, respectively. In addition to systemic inflammation, conditions that result in abnormal blood flow or blood stasis such as neoplasia, arterio-venous fistulae, or cardiac disease and arrhythmias (eg., atrial fibrillation8) may also presdispose to clot formation. The presence or absence of endogenous and exogenous substances (eg., prednisone therapy,9 low levels of antithrombin [AT]10) may drive animals towards a hypercoagulable state. The contributions of endothelial damage, blood stasis, and a hypercoagulable state towards thrombus formation was first described in the 1850’s by Rudolph Virchow, and are referred to today as Virchow’s triad. Identification of a hypercoagulable state: plasma testing Traditional coagulation tests, such as platelet count, activated partial thromboplastin time (aPTT), and prothrombin time (PT), are most useful for the demonstration of hypocoagulability; they do not reliably identify hypercoagulability. Prolongations of coagulation times in concert with a decreased platelet count may appear in patients with a consumptive coagulopathy. In clinical practice, a drop in circulating platelet count accompanied by a prolongation of at least 20% in baseline aPTT in an at-risk patient is generally the first indication of a consumptive coagulopathy.11 Another option for identification of a hypercoagulable state focuses on the measurement of specific procoagulant or anticoagulant molecules in circulation. These molecules can include fibrinogen concentration (when elevated contributing to a procoagulant state) or AT activity (decreased in hypercoagulable states). Other procoagulant molecules, such as coagulation factor V or VIII activity, or levels of anticoagulant proteins C and S, may be used to discover the overall balance of hemostasis in an individual. While low AT levels may represent true pathology (eg., in patients with protein-losing nephropathy), they can also be indicative of utilization, in scenarios where large amounts of thrombin have been formed (eg., during growth of a thrombus) and it can sometimes be difficult to distinguish the chicken from the egg in terms of AT levels and hypercoagulability in patients with thrombi.12 A recent study of dogs with septic peritonitis described decreased AT and protein C activity in dogs who did not survive, although almost all of the dogs had AT lower than the reference interval.13 When AT combines with thrombin, a tertiary compound is created, the thrombin-antithrombin (TAT) complex. Elevated levels of circulating TAT complexes indicate that thrombin has been formed in the patient, and are a strong clue for an underlying hypercoagulable state. TAT is assayed using an ELISA (Dade/Siemens) that is relatively expensive and difficult to have available for use on individual clinical patients. TAT has been evaluated in dogs with hyperadrenocorticism,14 carcinoma,15 Babseiosis,16 and congestive heart failure17 and appears to be elevated in conditions associated with coagulation or inflammation. Other markers of ongoing thrombin generation include prothrombin activation fragments (F1+2), which are generated by the activity of factor Xa on factor II, and fibrinopeptides A and B. These have been rarely evaluated in dogs, and poor cross-reactivity to the reagents in the human-based assay has been noted.18 Whole blood viscoelastic testing: Coagulation testing that assesses more than one aspect of the coagulation cascade at the same time may be a useful adjunct in determining hypercoagulable states. Commonly available viscoelastic coagulation devices include thromboelastography (TEG) or rotational thromboelastometry (ROTEM), both of which can monitor coagulation kinetics in whole blood or plasma samples. Whole blood coagulation analysis has a theoretical benefit over plasma-based coagulation testing because the interaction of all cellular components of blood (minus the endothelium) contribute to the result. Viscoelastic testing evaluates the time to initial fibrin cross-linking, the rate of clot formation, and the strength and viscoelastic characteristics of the clot that is formed,19 Hypercoagulable samples start to clot more quickly, with a faster rate of clot formation, and greater clot strength. TEG has been used in an attempt to identify animals and people with hypercoagulability, with mixed results, partially due to the nature of the test. The TEG is sensitive to the RBC concentration in the sample; as hematocrit drops, the TEG tracing appears progressively hypercoagulable, even in normal animals.20,21 This complicates the TEG-based diagnosis of hypercoagulability in cases such as IMHA, where anemia is an important aspect of the disease itself. TEG has also been evaluated in veterinary medicine using a number of different protocols for rest time, rest temperatures, and activators. Most studies will use citrated blood that is rested for 30 minutes, either at room temperature or at 37° C (rest temperature does not significantly affect the results22). Some published studies have used TF as an activator (in at least 3 different concentrations) and others kaolin, while other studies have used no activator other than recalcification. As the blood sits during the rest period, the sample itself will become hypercoagulable through activation of factor XII and the contactactivation pathway; the use of a strong activator overcomes the slight changes that this may cause in the tracing. While activators (or not) affect the duration of time prior to initial fibrin formation (ie., the reaction, or R time), they do not substantially affect the maximum clot strength (“maximum amplitude”, MA), which is dependent primarily on the platelets and fibrinogen concentration in the sample. MA is sometimes transformed to the elastic shear modulus (“G”) which is calculated as: (5000*MA)/(100-MA) and reported in dynes/sec. The coagulation index (CI) is a unitless number calculated by an algorithm that integrates the main TEG parameters to arrive at a single number (0.1227R + 0.0092K + 0.1655MA – 0.0241(angle) – 5.0220, derived for dogs using citrated whole blood),19 and those patients with a CI higher than the normal range are hypercoagulable, and those below the range are hypocoagulable. In general, the reference interval for CI is -3 – 3. In terms of actual prediction of thrombotic tendencies, very little outcomerelated data exists in the human or veterinary TEG literature. Some studies have related the TEG variable MA to the occurrence of myocardial infaction (MI) in humans undergoing general surgery and following trauma,23,24 and others have related this value to the likelihood of coronary stent thrombosis following PCI. 25 Other studies in various populations of people have not found this correlation.26 An early study of 9 dogs with parvoviral enteritis described hypercoagulability in all dogs (based on increased MA), and 4 of these dogs developed jugular venous thrombi.27 In dogs and humans with trauma, the presence of hypocoagulability on the TEG is correlated with morbidity and mortality, but hypercoagulability on the TEG was not associated with risk for venous thromboembolism. In dogs with septic peritonitis, the preoperative MA was hypercoagulable and higher in those dogs who survived hospitalization, which may also represent a more compensated coagulation state (vs. those who were relatively hypocoagulable due to consumption). A recent necropsy-based study failed to find a association between the presence of hypercoagulability on the TEG and thrombosis in 26 dogs, but the criteria had the TEG performed within 7 days of necropsy, so may not have reflected the underlying state at the time of thrombus formation.28 With the relatively low incidence of definitively-diagnosed thrombotic events in veterinary medicine, a standardized multi-center study may be necessary to determine whether TEG, alone or in concert with other testing, can identify those animals at high risk for thromboembolic disease, and to see beyond the inherent heterogeneity of the underlying diseases. Calibrated automated thrombin generation (CAT) has been less studied in dogs, but shows promise as a test that can demonstrate the kinetics of thrombin formation in a sample of citrated plasma or PRP. Through the use of a fluorogenic substrate, thrombin production is tracked and graphed as a thrombin generation curve. The area under the curve indicates the total capacity for thrombin generation in the sample and is called the endogenous thrombin potential (ETP). The slope of the curve gives the rate of thrombin generation and it is possible to calculate the total amount of thrombin formed as well. CAT has been investigated in veterinary medicine in the context of therapeutic monitoring for anticoagulant treatment,29,30 but has been used in human medicine to identify hypercoagulability in certain patient populations. Flow cytometric determination of hypercoagulability Assays for markers of platelet activation such as P-selectin expression or platelet-neutrophil aggregates may indicate a propensity towards platelet activation in specific patients.31 Flow cytometric techniques also can also be used to document the presence of procoagulant MPs and PS externalization on platelet membranes. Standardization of techniques is necessary to assay for MPs because of their small size (less than 1.5 microns). Increased expression of platelet P-selectin has been reported in patients with IMHA,32 and has been studied as a marker of therapeutic effect in patients given platelet inhibiting drugs.33 In humans and pigs, high levels of soluble P-selectin have been associated with inflammation and venous thrombosis. The Advia 120 hemostasis analyzer (Bayer Healthcare, Shawnee Mission, KS) reports a parameter called mean platelet component (MPC), which is related to the granularity of the circulating platelets. After activation, the granularity of platelets decreases, and thus a decreased MPC may represent activated platelets, although a recent study did not find an association between MPC and SIRS criteria or general disease type (inflammatory vs. non-inflammatory).34 Fibrinolysis Another indirect measure of hypercoagulability is fibrinolysis; decreased fibrinolysis can contribute to the persistence of thrombi, and the morbidity may be indistinguishable from patients with hypercoagulability. Hypofibrinolysis is also common in patients with systemic inflammation, who demonstrate increased levels of α2-antiplasmin, plasminogen activator inhibitor-1, and thrombinactivatable fibrinolysis inhibitor (TAFI). In a necropsy study of horses with severe gastrointestinal disease, persistent fibrin deposition was described in multiple organs.35 The evaluation of fibrin(ogen) degradation products (FDP) and ddimers (produced by breakdown of cross-linked fibrin) do not reliably support or undermine the presence of a thrombus, at least in the context of pulmonary thromboembolic disease (PTE),36 although other studies of patients with presumed hypercoagulable conditions (e.g. septic peritonitis) have demonstrated persistent elevations in d-dimers, although these levels were not predictive of survival.37 TEG can also be used to assess fibrinolysis in dogs, but requires added tPA to force the fibrinolysis to occur within a reasonable time. This technique has been used to assess the presence of fibrinolysis in dogs that have an acute coagulopathy secondary to hemoperitoneum.38 There are many tools and assays that con contribute to a global assessment of the coagulation status of a patient. Used in together, and used for serial assessments, these parameters, in addition to a good physical examination, may give a leg up on the ante-thrombosis diagnosis of hypercoagulability. As with any disease, the clinician must weigh the pros and cons of anticoagulation, with a view towards the expected duration and resolution of the illness. If a rapid resolution is not in sight, thromboprophylaxis may be indicated. References: 1. Sinnott VB, Otto CM: Use of thromboelastography in dogs with immunemediated hemolytic anemia: 39 cases (2000-2008), J Vet Emerg Crit Care 19(5):484-488, 2009. 2. Boswood A, Lamb CR, White RN: Aortic and iliac thrombosis in six dogs, J Small Anim Pract 41(3):109-114, 2000. 3. LaRue MJ, Murtaugh RJ: Pulmonary thromboembolism in dogs: 47 cases (1986-1987), J Am Vet Med Assoc 197(10):1368-1372, 1990. 4. Schoeman JP: Feline distal aortic thromboembolism: a review of 44 cases (1990-1998), J Feline Med Surg 1(4):221-231, 1999. 5.Stokol T, Brooks M, Rush JE, et al: Hypercoagulability in cats with cardiomyopathy, J Vet Intern Med 22(3):546-552, 2008. 6. Norris CR, Griffey SM, Samii VF: Pulmonary thromboembolism in cats: 29 cases (1987-1997), J Am Vet Med Assoc 215(11):1650-1654, 1999. 7. Epstein KL, Brainard BM, Giguere S, Vrono Z, Moore JN. Serial viscoelastic and traditional coagulation testing in horses with gastrointestinal disease. J Vet Emerg Crit Care. 2013;23(5):504-516. 8. Usechak PJ, Bright JM, Day TK: Thrombotic complications associated with atrial fibrillation in three dogs, J Vet Cardiol 14(3):453-458, 2012. 9. Rose LJ, Dunn ME, Allegret V, et al: Effects of prednisone administration on coagulation variables in healthy Beagle dogs, Vet Clin Pathol 40(4):426-434, 2011. 10. Lennon EM, Hanel RM, Walker JM, et al. Hypercoagulability in dogs with protein-losing nephropathy as assessed by thromboelastography. J Vet Intern Med. 2013;27(3):462-8. 11. Ralph AG, Brainard BM. Update on disseminated intravascular coagulation: when to consider it, when to expect it, when to treat it. Top Companion Anim Med. 2012;27(2):65-72. 12. de Laforcade AM, Rozanski EA, Freeman LM, et al: Serial evaluation of protein C and antithrombin in dogs with sepsis, J Vet Intern Med 22(1):26-30, 2008. 13. Bentley AM, Mayhew PD, Culp WT, Otto CM .Alterations in the hemostatic profiles of dogs with naturally occurring septic peritonitis. J Vet Emerg Crit Care. 2013;23(1):14-22. 14. Klose TC, Creevy KE, Brainard BM. Evaluation of coagulation status in dogs with naturally occurring canine hyperadrenocorticism. J Vet Emerg Crit Care. 2011;21(6):625-32. 15. Vilar Saavedra P, Lara García A, Zaldívar López S, et al. Hemostatic abnormalities in dogs with carcinoma: a thromboelastographic characterization of hypercoagulability. Vet J. 2011;190(2):e78-83. 16. Rafaj RB, Matijatko V, Kis I, et al. Alterations in some blood coagulation parameters in naturally occurring cases of canine babesiosis. Acta Vet Hung. 2009;57(2):295-304. 17. Tarnow I, Falk T, Tidholm A, et al. Hemostatic biomarkers in dogs with chronic congestive heart failure. J Vet Intern Med. 2007;21(3):451-7. 18. Ravanat C, Freund M, Dol F, et al: Cross-reactivity of human molecular markers for detection of prothrombotic states in various animal species, Blood Coagul Fibrinolysis 6(5):446-455, 1995. 19. Donahue SM, Otto CM: Thromboelastography: a tool for measuring hypercoagulability, hypocoagulability, and fibrinolysis, J Vet Emerg Crit Care 15(1):9-16, 2005 20. Epstein KL, Brainard BM, Lopes MA, Barton MH, Moore JN. Thrombelastography in 26 healthy horses with and without activation by human recombinant tissue factor. J Vet Emerg Crit Care, 2009;19(1):96-101. 21. Brooks AC, Guillaumin J, Cooper ES, Couto CG. Effects of hematocrit and red blood cell-independent viscosity on canine thromboelastographic tracings. Transfusion. 2014;54(3):727-34. 22. Ralph AG, Brainard BM, Pittman JR, et al. Effects of rest temperature, contact activation, and sample collection technique on canine thrombelastography. J Vet Emerg Crit Care, 2012; 22(3):320-6 23. McCrath DJ, Cerboni E, Frumento RJ, et al. Thromboelastography maximum amplitude predicts postoperative thrombotic complications including myocardial infarction. Anesth Analg. 2005;100(6):1576-83. 24. Kashuk JL, Moore EE, Sabel A, et al. Rapid thrombelastography (r-TEG) identifies hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146(4):764-72. 25. Gurbel PA, Bliden KP, Kreutz RP, et al. The link between heightened thrombogenicity and inflammation: pre-procedure characterization of the patient at high risk for recurrent events after stenting. Platelets. 2009 Mar;20(2):97-104. 26. Paarup Dridi N, Lønborg JT, Radu MD, et al. Hypercoagulation Assessed by Thromboelastography is Neither Related to Infarct Size nor to Clinical Outcome After Primary Percutaneous Coronary Intervention. Clin Appl Thromb Hemost. 2013 May 1. PMID: 23613040 27. Otto CM, Rieser TM, Brooks MB, Russell MW. Evidence of hypercoagulability in dogs with parvoviral enteritis.J Am Vet Med Assoc. 2000;217(10):1500-4. 28. Vince Thawley, personal communication. 29. Conversy B, Blais MC, Dunn M, et al.Rivaroxaban demonstrates in vitro anticoagulant effects in canine plasma. Vet J. 2013 Nov;198(2):437-43. 30. Allegret V, Dunn M, Bédard C. Monitoring unfractionated heparin therapy in dogs by measuring thrombin generation. Vet Clin Pathol. 2011;40(1):24-31. 31. Wills TB, Wardrop KJ, Meyers KM. Detection of activated platelets in canine blood by use of flow cytometry. Am J Vet Res. 2006;67(1):56-63. 32. Weiss DJ, Brazzell JL. Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia. J Vet Intern Med. 2006;20(3):682-6. 33. Brainard BM, Epstein KL, Lobato DN, et al. Treatment with aspirin or clopidogrel does not affect equine platelet expression of P selectin or plateletneutrophil aggregates. Vet Immunol Immunopathol. 2012;149(1-2):119-25. 34. Smith JR, Smith KF, Brainard BM. Platelet parameters from an automated hematology analyzer in dogs with inflammatory clinical diseases. Vet J 2014;in press. doi: 10.1016/j.tvjl.2014.07.009. [Epub ahead of print] 35. Cotovio M, Monreal L, Navarro M, et al. Detection of fibrin deposits in tissues from horses with severe gastrointestinal disorders. J Vet Intern Med. 2007;21(2):308-13. 36. Epstein SE, Hopper K, Mellema MS, Johnson LR. Diagnostic utility of Ddimer concentrations in dogs with pulmonary embolism. J Vet Intern Med. 2013;27(6):1646-9. 37. Bentley AM, Mayhew PD, Culp WT, Otto CM. Alterations in the hemostatic profiles of dogs with naturally occurring septic peritonitis. J Vet Emerg Crit Care. 2013;23(1):14-22. 38.Fletcher DJ, Rozanski E, deLaforcade A, Brainard BM Coagulopathy and hyperfibrinolysis in dogs with spontaneous hemoperitoneum is associated with lactate concentrations and depletion of protein C. J Vet Emerg Crit Care. 2014; in press.