The treatment of bleeding for haemophilic patients with inhibitors relies on the use of the bypassing agents, recombinant factor VIIa and factor eight inhibitor bypass activity (FEIBA). While both therapies are effective in the majority of bleeding episodes, there is a significant amount of interindividual variability when it comes to the response to therapy. As of yet, there is no reliable laboratory parameter that can predict the response to therapy in the same manner that factor VIII and factor IX levels predict response in non-inhibitor patients. Developing such a laboratory parameter is vital in order to maximize the clinical efficacy of these agents. Thromboelastography (TEG) is a device, which assesses clot formation over time in whole blood and has several characteristics which suggest it may be an effective way to monitor bypass agent therapy. We studied the ability of TEG to individualize the treatment regimens of three patients with high titre inhibitors assessing the response to recombinant factor VIIa, FEIBA, and when both were used sequentially. The TEG allowed for individualization of treatment for each of the three patients and resulted in more effective, convenient and less expensive treatment regimens. We thus believe that TEG is a promising device for monitoring of bypass agent therapy and should be studied further.

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Bleeding is the major adverse reaction to anticoagulants, leading to significant morbidity and even mortality. Protamine is a specific antidote for heparin yet is only partially effective for enoxaparin, and the activated factor X inhibitor fondaparinux and the direct thrombin inhibitors argatroban and bivalirudin lack specific antidotes. We evaluated the ability of recombinant activated factor VII (rFVIIa), a general hemostatic agent, to reverse the anticoagulant effects of heparin, enoxaparin, fondaparinux, argatroban, and bivalirudin, as measured by thromboelastography. Whole-blood samples containing each test anticoagulant, with or without rFVIIa 1.5-4.5 microg/ml, were prepared ex vivo (n >or= 48, each anticoagulant) and analyzed by thromboelastography. The thromboelastography parameters of clot initiation, propagation, rigidity and elasticity were compared for the ex-vivo samples for each anticoagulant. The reversal ability of rFVIIa was also assessed using the standard clinical assay used to monitor each anticoagulant. Thromboelastography was performed on blood from eight stably anticoagulated patients, with and without exogenous rFVIIa. For each anticoagulant, rFVIIa significantly improved and, in some cases, completely normalized all thromboelastography parameters (P < 0.001). rFVIIa significantly (P < 0.01) decreased the activated partial thromboplastin time for argatroban-containing, bivalirudin-containing, or heparin-containing blood yet did not affect the anti-activated factor X levels for enoxaparin-containing or fondaparinux-containing blood. By thromboelastography, rFVIIa exerted generally similar reversal effects on the anticoagulated patient samples as on the ex-vivo samples. In conclusion, rFVIIa effectively reverses the anticoagulant effects of heparin, enoxaparin, fondaparinux, argatroban, and bivalirudin, and should be considered for patients with excessive bleeding associated with these anticoagulants.

New anticoagulants, including the direct thrombin inhibitors (DTIs) and fondaparinux, are increasingly replacing unfractionated heparin and enoxaparin. We examined the effects of argatroban (n = 60), bivalirudin (n = 44), heparin (n = 14), enoxaparin (n = 22), and fondaparinux (n = 24) on clot formation utilizing thromboelastography. Blood samples containing anticoagulants at clinically relevant concentrations were prepared ex vivo and analyzed using kaolin or tissue factor activation. Thromboelastography parameters of clot initiation (R), clot propagation (K and angle), clot rigidity (maximum amplitude) and clot elasticity (G) were compared between anticoagulants. Thromboelastography was also performed on blood from eight patients receiving anticoagulants. Each anticoagulant exerted significant concentration-dependent effects on R, K and angle. Only heparin, enoxaparin, and fondaparinux significantly affected maximum amplitude and G. Significant differences existed for all parameters between heparin and each anticoagulant and between fondaparinux and each DTI (P < 0.001), and for angle, maximum amplitude, and G between enoxaparin and each DTI (P < 0.008). Thromboelastography responses in ex-vivo samples and patient samples were comparable. In conclusion, whereas argatroban, bivalirudin, heparin, enoxaparin and fondaparinux each delay clot formation, the DTIs do not alter clot rigidity or elasticity. The reduced bleeding reported with DTIs versus heparin may relate to the fact that clots form with normal rigidity and elasticity.

BACKGROUND: The novel anticoagulants fondaparinux (Fond), argatroban (Arg), and bivalirudin (Biv) are being used increasingly for a variety of indications, even replacing heparin and warfarin in certain settings. While heparin and warfarin have antidotes (protamine and vitamin K, respectively), the newer agents lack known antidotes. Recombinant factor VIIa (rFVIIa) has reversed the effects of some novel anticoagulants in in vitro and animal studies. We evaluated the ability of rFVIIa to reverse the anticoagulant effects of Fond, Arg, Biv, unfractionated heparin (Hep), and enoxaparin (Enox) in whole blood using thromboelastography (TEG). METHODS: TEG was performed using dilute tissue factor as an activator on native whole blood from healthy adults within 4 minutes of atraumatic venipuncture and the following parameters measured: time to clot initiation (R, in mins), rate of clot propagation (K, in mins and angle in degrees), clot rigidity (MA, in mm), and clot strength (G, in dynes/cm2). For each experiment the blood was split 4 ways before analysis and the following was added: nothing (baseline), anticoagulant, anticoagulant plus rFVIIa, and anticoagulant plus rFVIIa placebo (control). Multiple experiments using 9 volunteers were done for each anticoagulant at a therapeutic concentration and rFVIIa at a final concentration of 1.5, 3, 4.5, and 9 mcg/mL (1.5–3 mcg/mL is a therapeutic concentration in hemophilia). Protamine reversal served as a positive control for heparin. RESULTS: Each anticoagulant delayed clot initiation and propagation. The direct thrombin inhibitors (Arg, Biv) exerted lesser effects than the antithrombin-dependent agents (Hep, Enox, Fond) on clot rigidity and strength. rFVIIa at each test concentration reversed the anticoagulant effect of each agent as measured by TEG (placebo had no effect). A dose response was noted for the 9 mcg/mL rFVIIa concentration but not for the 3 lower concentrations. We thus grouped the results for the 3 lower concentrations. The data for the 9 mcg/mL rFVIIa concentration (not shown) was in line with the grouped data yet with values closer to baseline. The reversal effect was statistically significant for each parameter with the antithrombin-dependent agents and for clot initiation and propagation for the direct thrombin inhibitors. See tables for details. Protamine successfully reversed heparin’s effect (data not shown). CONCLUSIONS: rFVIIa successfully reversed the anticoagulant effects in whole blood of therapeutic concentrations of Arg, Biv, and Fond, which lack known antidotes, Enox, for which protamine reversal is only partially effective, and heparin. These findings support rFVIIa as a potential non-specific antidote for newer anticoagulants. TEG parameters: Antithrombin-dependent agents with and without addition of rFVIIa Baseline Hep Hep+rFVIIa Enox Enox+rFVIIa Fond Fond+rFVIIa Mean values; changes in parameters between anticoagulant and anticoagulant + rFVIIa are all statistically significant (p<0.05). R 8.6 23 14 20.5 7.5 29.6 9.2 K 3.1 11.3 4.9 9.7 3 17 4.8 Angle 50.9 18.8 38 20.7 43.6 12.5 41.6 MA 60.5 44.3 54.6 39 54.2 31.1 58.3 G 7.3 4 6.1 3.3 6 2.6 7.1 TEG parameters: Direct thrombin inhibitors with and without addition of rFVIIa Baseline Arg Arg+rFVIIa Biv Biv+rFVIIa Mean values; changes in parameters R, K and angle between anticoagulant and anticoagulant + rFVIIa are statistically significant (p<0.05). R 8.6 14.8 10.8 26.2 21.2 K 3.1 4.6 3 7.4 4 Angle 50.9 40.9 53.2 31.5 43.1 MA 60.5 57.1 59.6 49 53.4 G 7.3 6.7 7.4 5.3 5.6

In large studies of patients with cardiac disease, it was observed that up to 9% are ASA resistant (ASAR) and 23% are ASA semi responders using the platelet function analyzer (PFA-100), which measures whole blood platelet adhesion and aggregation under high shear. The prevalence of ASAR is of great clinical importance. With an estimated 20 million patients in the USA taking ASA for prevention of atherosclerotic events, even a 10% incidence equates to more than two million patients receiving inadequate anti-platelet therapy. Given the widespread use of ASA, more reliable and rapid methods to measure aspirin sensitivity are needed. Importantly, future large scale studies to determine the effect of monitoring ASA sensitivity to optimize therapy are compromised due to current lack of uniformity in assessing ASAR from center to center. The classical method of platelet aggregometry is labor-intensive and not readily adaptable to the clinical setting. Moreover, platelet aggregometry measures platelet responses under low shear, which does not simulate the high shear conditions expected to be involved in arterial thrombosis. In contrast, the PFA-100 does measure thrombus formation under high shear. The new TEG platelet coagulation assay uses whole blood, includes high shear, and is a point of care method. We compared these three techniques to assess aspirin effects on platelet function and clot formation: PFA-100 with collagen-epinephrine cartridges, TEG platelet coagulation assay, and standard optical aggregation in platelet rich plasma using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation. Twelve normal individuals previously defined as ASA sensitive or ASAR, using the PFA-100, were studied at baseline and following three days of oral aspirin at 81mg/day. We found that all four patients determined to be ASAR by PFA-100 were found to be sensitive in TEG and aggregometer assays when using AA as the agonist. Furthermore, some participants showed a gain of platelet function following ASA when studied on the TEG and aggregometer using ADP as the agonist. Currently, it is unclear which response to ASA treatment is most important to predict cardiovascular complications in normal individuals or patients placed on aspirin. Gum et al, showed that increased urinary secrection of thromboxane metabolites, suggesting ASA insensitivity, is associated with a higher incidence of cardiovascular events. Unfortunately, this test may simply indicate poor patient compliance rather than true ASAR, so a clear demonstration of platelet sensitivity will also be necessary. Our data confirms the need for a collaborative trial comparing different platelet assays to assess true ASA sensitivity together with concurrent measurements of urinary thromboxane metabolite levels. Knowing which assay best links aspirin sensitivity to disease outcome will allow physicians to better manage normal individuals and patients at risk for significant cardiovascular disease.

INTRODUCTION: Large prospective studies must be performed to measure the true effect of clopidogrel (Plavix) or aspirin (ASA) in prevention of vascular disease. An easy and accurate point of service instrument will facilitate these studies and confirm effectiveness and compliance with medications. With such a variety of equipment available for analysis of platelet dysfunction, it is important to understand the sensitivity and efficacy of each assay when evaluating patients. We have found significant differences in our comparative studies of the PFA-100, optical aggregation, and Thrombelastograph (TEG) platelet coagulation in normal individuals using Plavix or ASA. METHODS: We have compared these three techniques to evaluate their effectiveness in assessing ASA and Plavix inhibition of platelet function. Eighteen normal individuals, who had been examined previously for ASA effect on these instruments, were studied at baseline and after three days of Plavix 75mg daily. All samples were drawn and run simultaneously. The TEG platelet coagulation assay used whole blood in a sodium heparin tube instead of a 3.2 % citrate tube which was used for aggregation and the PFA-100. The PFA-100 whole blood assays included both collagen-epinephrine (EPI) and collagen-adenosine diphosphate (ADP) cartridges. The TEG platelet coagulation assays were run with arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. The optical aggregation used platelet rich plasma with AA and ADP agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation in measurement of Plavix effect. Optical aggregation and the TEG platelet assay did show the effects of Plavix, with the aggregometer being more sensitive than the TEG platelet assay although much more time consuming. In additional studies, the Plavix inhibition became more striking in the TEG by extending treatment to 6 days, suggesting that future studies should be done after a week (rather than 3 days) to see full effect. In contrast, Plavix effect was absent on the PFA-100. All three assays did show the effects of ASA in previously identified aspirin- responsive individuals. Ideally, one instrument could be used to assess response to both agents. However, the sensitivity of the instrument and the pharmacology of these and future medications must play an important role in the selection of which assay will be most informative for these large population studies in the prevention of vascular disease. Figure Figure