Herbert C. Whinna

Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross‐sectional pharmacodynamic study based on peak and trough plasma levels

Knowledge of anticoagulation status during dabigatran therapy may be desirable in certain clinical situations.

Coagulation activation and inflammation in sickle cell disease-associated pulmonary hypertension.

Patients with sickle cell disease-associated pulmonary hypertension have increased endothelial dysfunction, coagulation activation and inflammation compared with patients without pulmonary hypertension. Endothelial dysfunction and coagulation activation appear to be the result of chronic hemolysis. See related perspective on page 1. Background Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD. Design and Methods This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD. Results Patients with SCD (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+2, D-dimer) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SCD patients with PHT (n=26) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation. Conclusions SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.

The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis

The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the protease-factor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4- to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2- to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl(3)-induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P </= .003), and more than 45 minutes. These data support the role of the factor IXa heparin-binding exosite as a critical regulator of coagulation and novel antithrombotic target.

Role of Thrombin Anion-binding Exosite-I in the Formation of Thrombin-Serpin Complexes

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (k on) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77 a Q, K81Q, K109Q, K110Q, and K149 e Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereask on values were decreased 2–3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal k on values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77 a Q, where k on values were decreased 2–4-fold, compared with rIIa. For HCII/heparin, k onvalues for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149 e Q) were 2–3-fold lower than rIIa. Larger decreases in k on values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149 e Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had k onvalues reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3–7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the “hirudin-like” domain of HCII with exosite-I of thrombin.

Interference of Monoclonal Antibody Therapies with Serum Protein Electrophoresis Tests

To the Editor: During routine serum protein electrophoresis (SPE), we recently detected an apparent IgG heavy-chain immunoglobulin in a 51-year-old woman with a 5-year history of IgDκ multiple myeloma. Other relevant laboratory values at the time included the following: serum creatinine, 159 μmol/L (reference interval, 62–97 μmol/L); IgG, 2.55 g/L (reference interval, 6.00–17.00 g/L); IgM, <0.25 g/L (reference interval, 0.35–2.90 g/L); and IgA, 0.40 g/L (reference interval, 0.40–4.00 g/L). The rarity of an IgG heavy-chain gammopathy in combination with an IgDκ and a low quantitative IgG value raised suspicion of an interference. Consultation with the treating physician revealed that the patient was enrolled in a phase II clinical trial of dexamethasone and siltuximab for the treatment of refractory myeloma. Siltuximab (Centocor), a high-affinity antibody to human interleukin-6, is currently being used in clinical trials for the treatment of a number of malignancies, including multiple myeloma. Siltuximab is a chimeric immunoglobulin comprising the variable antigen-binding region of a mouse antibody and the constant region of human IgG1κ (1). It binds to human interleukin-6, a survival factor for myeloma cells (2). We obtained siltuximab from the manufacturer …

Interlaboratory agreement in the monitoring of unfractionated heparin using the anti-factor Xa-correlated activated partial thromboplastin time

Summary.  Background: In an effort to improve interlaboratory agreement in the monitoring of unfractionated heparin (UFH), the College of American Pathologists (CAP) recommends that the therapeutic range of the activated partial thromboplastin time (APTT) be defined in each laboratory through correlation with a direct measure of heparin activity such as the factor Xa inhibition assay. Whether and to what extent this approach enhances the interlaboratory agreement of UFH monitoring has not been reported. Objectives: We conducted a cross‐validation study among four CAP‐accredited coagulation laboratories to compare the interlaboratory agreement of the anti‐FXa‐correlated APTT with that of the traditional 1.5–2.5 times the midpoint of normal (1.5–2.5:control) method for defining the therapeutic APTT range. Patients and methods: APTT and FXa inhibition assays were performed in each laboratory on plasma samples from 44 inpatients receiving UFH. Results: Using the anti‐FXa‐correlation method, there was agreement among all four laboratories as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in seven (16%) patient samples. In contrast, consensus was achieved in 23 (52%) samples when the 1.5–2.5:control method was employed. Conclusions: The anti‐FXa‐correlation method does not appear to enhance interlaboratory agreement in UFH monitoring as compared with the traditional 1.5–2.5:control method. Adoption of the anti‐FXa‐correlation method produces considerable disparity in UFH dosing decisions among different centers, although the clinical impact of this disparity is not known.

Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia

Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins

We studied the RNA aptamer Toggle‐25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3‐fold by Toggle‐25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle‐25, but it was only reduced 3‐fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin‐binding site of thrombin, anion‐binding exosite‐2 (exosite‐2). We localized the Toggle‐25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite‐2. We conclude that a RNA aptamer to thrombin exosite‐2 might provide an effective clinical reagent to control heparins anticoagulant action.

Towards a standardization of the murine ferric chloride‐induced carotid arterial thrombosis model

A. P . OWE N S I I I , * 1 Y . LU ,* 1 H . C . WHINNA, § C . G ACHET ,– W. P . FA Y** and N . MACKMAN* *Division of Hematology/Oncology, Department of Medicine, UNC McAllister Heart Institute, §Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; –UMR S949 INSERM-Université de Strasbourg, Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg Cedex, France; **Department of Internal Medicine and Medical Pharmacology and Physiology, and Dalton Cardiovascular Research Center, School of Medicine, University of Missouri, Columbia, MO, USA

Overview of murine thrombosis models

There are a myriad of options on where and how to perform thrombosis studies in mice. Models have been developed for systemic thrombosis, larger and smaller vessels of both the arterial and venous systems as well as several different microvascular beds. However, there are important differences between the models and investigators need to be careful and thoughtful when they choose which model to use.