Thrombin generation assay in a patient with acquired factor V inhibitor.

Abstract

Development of acquired inhibitor against factor V is a rare coagulation disorder that can either be asymptomatic or lead to a fatal hemorrhage. There is no standard treatment protocol. However, immunoadsorption and plasma exchange have proven to be effective. Spontaneous remission is also observed [1Favaloro E.J. Posen J. Ramakrishna R. Soltani S. McRae S. Just S. Aboud M. Low J. Gemmell R. Kershaw G. Coleman R. Dean M. Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation.Blood Coagul Fibrinolysis. 2004; 15: 637-47Crossref PubMed Scopus (0) Google Scholar]. As standard coagulation assays such as activated partial thromboplastin time (APTT), prothrombin time (PT) and plasma FV activity are of little help in predicting bleeding risk, there is still uncertainty as to when and to what extent hemostatic therapy should be initiated [2Streiff M.B. Ness P.M. Acquired FV inhibitors: a needless iatrogenic complication of bovine thrombin exposure.Transfusion. 2002; 42: 18-26Crossref PubMed Google Scholar]. An alternative diagnostic and monitoring approach may be the thrombin generation (TG) assay, which shows the amount and velocity of thrombin formation. This has been shown to adequately reflect hypercoagulable and hypocoagulable states [3Hemker H.C. Giesen P. Al Dieri R. Regnault V. De Smedt E. Wagenvoord R. Lecompte T. Béguin S. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability.Pathophysiol Haemost Thromb. 2002; 32: 249-53Crossref PubMed Scopus (554) Google Scholar]. In this case report, we present data obtained with TG over time in a patient with an acquired FV inhibitor treated with intravenous IgG (IVIG). A 56-year-old man with type 1 diabetes was admitted to our medical ward because of uncontrolled hyperglycemia. The diabetes was diagnosed at the age of 18 years, and the patient had already developed late complications, including diabetic angiopathy, nephropathy, neuropathy and retinopathy. Two months prior to the present admission, he had undergone suction drainage for a right-sided hemothorax following a fall due to a hypoglycemic episode. The patient was also known to have chronic anemia due to a β-thalassemia trait. On clinical examination, dullness to percussion and decreased breath sounds on the right lower hemithorax were found. Chest X-ray revealed signs of pneumonia and a pleural effusion. Routine laboratory workup on admission showed leukocytosis and elevated C-reactive protein. On the basis of the history, a diagnostic thoracenthesis was carried out, and revealed a hemorrhagic effusion. However, the results of microbiological investigations were negative. Because of the predominant pneumonia and the findings on basic coagulation assay of markedly prolonged PT (59.3 s) and APTT (112 s), surgical drainage was ruled out. The patient denied any history of bleeding diathesis or current anticoagulant treatment. There was also no sign of liver insufficiency. Further coagulation studies revealed markedly reduced FV activity (7.4%) as the cause of the prolonged PT and APTT. FV activity was measured with a one-stage clotting assay using FV-deficient plasma and Thromborel S on the Behring Coagulation System (all from Dade Behring, Marburg, Germany). The assay is sensitive enough to measure FV activity as low as 1%. Other coagulation factors that affect both global parameters were in the normal range. As both PT and APTT were normal during a previous hospital visit, acquired FV deficiency due to an inhibitor was suspected, and this was then confirmed with the Bethesda assay. The initial FV inhibitor titer was 2.2 BU mL−1. The patient did not show any clinical sign of ongoing bleeding during his hospital stay. However, because of the bloody pleural effusion and concern about further bleeding, a trial of IVIG therapy with a total dose of 1 g kg−1 body weight given over three consecutive days was started. To monitor the hemostatic changes during the course of the IVIG treatment, a TG assay using calibrated automated thrombography (CAT) was conducted. Blood was collected via antecubital venipuncture into tubes containing 0.129 m buffered trisodium citrate 3.8%. Platelet-poor plasma (PPP) was prepared by centrifugation at 2860 × g for 20 min and then stored at − 70 °C until analysis. TG was measured using the CAT PPP-Reagent LOW (Thrombinoscope BV; Maastricht, The Netherlands), which contains 1 pm tissue factor (TF) and 4 μm phospholipid in the final concentration. The assay was carried out in duplicate using a Fluoroscan Ascent (Thermo Labsystems, Helsinki, Finland) with an excitation filter at 390 nm and an emission filter at 460 nm. A dedicated software program (Thrombinoscope BV software package 3.0.0.29) was used for data analysis. Measurements were carried out on day 1 (before IVIG infusion) and on days 3, 6 and 13 after initiation of IVIG therapy. Prior to the IVIG treatment (day 1), virtually no thrombin generation could be detected (Fig. 1A). The assay was repeated with a final TF concentration of 5 pm. However, the higher TF concentration did not have any influence on this finding (data not shown). Three days after initiation of IVIG therapy, there was only a slight increase in FV activity to 8.6% and a decrease in PT to 31.3 s, but a steep increase in TG was observed. On follow-up, further reductions in lag time (from 25.4 min on day 3 to 8.8 min on day 13) and time to thrombin peak (from 57 to 11.9 min) were observed. However, there was no more obvious increase in the endogenous thrombin potential (ETP) beyond day 3. On day 13, plasma FV activity rose to 41.3%, and the FV inhibitor was no longer detectable. As a comparison, Fig. 1B shows a TG curve from healthy volunteers collected under the same assay conditions. The patient was then discharged in a stable condition. Because he moved to another city a few weeks after hospital discharge, a repeat TG assay was not possible. A telephone interview conducted 6 months later revealed that he did not experience any clinically obvious clotting abnormality again. In our patient, treatment with IVIG led to a normalization of hemostasis as confirmed by the TG assay, the disappearance of FV inhibitor, and the rise in FV activity. IVIG was given on the basis of its reported efficacy in patients with acquired hemophilia A. However, a spontaneous recovery cannot be ruled out with certainty. The amount of thrombin generated, the lag phase and the time to thrombin peak are influenced by FV, as FV is part of every step in the process of TG [4Hoffman M. Monroe 3rd, D.M. A cell-based model of hemostasis.Thromb Haemost. 2001; 85: 958-65Crossref PubMed Scopus (1088) Google Scholar]. Platelet FV rather than plasma FV plays a significant role in the amplification phase of coagulation. FV inhibitor leads to an impaired initiation phase, and this in turn results in impaired platelet activation. On the other hand, FV on the surface of activated platelets becomes neutraliszed by FV inhibitor, which leads to a reduction in prothrombinase activity and thrombin burst. The amount of FV needed to produce an adequate thrombin burst is still unclear. In our patient, minimal changes in FV activity from 7.4% to 8.6% led to a considerable increase in thrombin formation. Higher concentrations of FV did not result in any further increase in ETP, but did result in further reductions in the lag phase and the time to peak. Al Dieri et al. [5Al Dieri R. Peyvandi F. Santagostino E. Giansily M. Mannucci P.M. Schved J.F. Béguin S. Hemker H.C. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding.Thromb Haemost. 2002; 88: 576-82Crossref PubMed Google Scholar] observed no severe impairment in ETP at FV levels of 2% in patients with congenital FV deficiency. However, they used much higher TF concentrations (15 pm) in their assay. Therefore, their data are hardly comparable to ours. Furthermore, a measurable amount of the coagulation factor in the presence of an acquired autologous inhibitor may not necessarily correlate with its hemostatic function. Studies in patients with acquired autoantibody hemophilia A have shown that the inactivation has a type II pattern, with some residual FVIII activity being identifiable even after incubation at high concentrations of antibody. The presence of the inhibitor rather than the measurable coagulation factor activity is thus important in assessing the hemostatic state. In conclusion, the TG assay could be useful for monitoring hemostatic changes in patients with FV deficiency due to an inhibitor. However, there are issues of standardization to be dealt with. Clinical correlation with TG cut-off values is also lacking. These issues have to be addressed before the assay can be used in clinical routine practice. The authors state that they have no conflict of interest.