Deborah A. Lewis

Whole blood gene expression analyses in patients with single versus recurrent venous thromboembolism

INTRODUCTION Venous thromboembolism may recur in up to 30% of patients with a spontaneous venous thromboembolism after a standard course of anticoagulation. Identification of patients at risk for recurrent venous thromboembolism would facilitate decisions concerning the duration of anticoagulant therapy. OBJECTIVES In this exploratory study, we investigated whether whole blood gene expression data could distinguish subjects with single venous thromboembolism from subjects with recurrent venous thromboembolism. METHODS 40 adults with venous thromboembolism (23 with single event and 17 with recurrent events) on warfarin were recruited. Individuals with antiphospholipid syndrome or cancer were excluded. Plasma and serum samples were collected for biomarker testing, and PAXgene tubes were used to collect whole blood RNA samples. RESULTS D-dimer levels were significantly higher in patients with recurrent venous thromboembolism, but P-selectin and thrombin-antithrombin complex levels were similar in the two groups. Comparison of gene expression data from the two groups provided us with a 50 gene probe model that distinguished these two groups with good receiver operating curve characteristics (AUC 0.75). This model includes genes involved in mRNA splicing and platelet aggregation. Pathway analysis between subjects with single and recurrent venous thromboembolism revealed that the Akt pathway was up-regulated in the recurrent venous thromboembolism group compared to the single venous thromboembolism group. CONCLUSIONS In this exploratory study, gene expression profiles of whole blood appear to be a useful strategy to distinguish subjects with single venous thromboembolism from those with recurrent venous thromboembolism. Prospective studies with additional patients are needed to validate these results.

Binding of factor VIII inhibitors to discrete regions of the factor VIII C2 domain disrupt phospholipid binding.

We characterized seven factor VIII inhibitors with epitopes in the C2 domain of factor VIII using a series of factor V C2 domain chimeras that substituted exon-sized fragments of the C2 domain of factor VIII for the corresponding regions of factor V. All inhibited co-factor activity of factor VIII and six inhibited binding of factor VIII to phosphatidylserine. Inhibitors Hz, JN and GK32 bound epitopes within amino acids S2173–K2281; inhibitors GK24 and TO bound epitopes within amino acids V2223–Y2332; and inhibitors UNC11 and UNC12 bound epitopes throughout the C2 domain (amino acids S2173–Y2332). Inhibitors Hz, JN and UNC12 inhibited the co-factor activity of chimera 5A, which substituted amino acids S2173–Q2222 of factor VIII for the corresponding region of factor V, in a prothrombinase assay. This inhibition could be partially reversed by pre-incubation of chimera 5A with phospholipid vesicles, suggesting that these antibodies interfered with phospholipid binding. Inhibitors UNC11 and UNC12, on the other hand, did not inhibit the binding of chimera 1A to phosphatidylserine, suggesting that binding to the segment spanning amino acids V2282–Y2332 does not necessarily block phospholipid binding. These results agree with the model of the phospholipid-binding site determined by crystal structure of the C2 domain of factor VIII.

The reactivity of paired plasma and serum samples are comparable in the anticardiolipin and anti‐β2‐glycoprotein‐1 ELISAs

inhibit VWF secretion at a distal step in exocytosis, common to [Ca]iand cAMP-raising agents, and are compatible with the notion that exogenous NO can inhibit granule-membrane fusion (although additional signaling mechanisms such as [Ca]i could be involved). However, endogenous NO is most unlikely to provide short-term autocrine inhibition of VWF secretion by this mechanism. NO may also inhibit endothelial VWF secretion in vivo via a cGMP/cGK-dependent pathway. The relative role of the two proposed NO-dependent inhibitory pathways cannot be delineated in a single cell system. Thus, these results also remind us of the limitations of cultured endothelial cells for the study of the physiological regulation of endothelial secretion.

Gene-expression patterns predict phenotypes of immune-mediated thrombosis (Blood (2006) 107, 4, (1391-1396))

The authors retract the 15 February 2006 paper cited above because they have been unable to reproduce the results that were performed independently by the first author, Anil Potti, regarding validation of predictive models for thrombotic phenotypes. It has also been recognized that multiple samples appear to be duplicated in the training and validation datasets pertaining to the analysis presented in Figure 2 of the paper, which was also performed by Anil Potti. Since these results are fundamental to the conclusions of the paper, the authors formally retract the paper. The authors deeply regret the impact of this action on the work of other investigators and apologize to the readers, reviewers, and editors of Blood.

Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII.

Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of beta-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V forma phospholipid binding site, but residues in the A3 and C1 domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvs WR1 and WR16 (epitope : E2181-M2199) with half saturation values of 23 and 47 muM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factor VIII to C2-specific scFvs WR13 and EL14 (epitope : K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factor VIII to the A3-C1-specific scFvs KM36 (epitope : Q1778-D1840) and KM38 (epitope : S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 microM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.