Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a serious condition associated with sepsis. Clinical management of DIC is hampered by lack of clear diagnostic criteria. The International Society on Thrombosis and Haemostasis (ISTH) has proposed a diagnostic scoring algorithm for overt DIC based on routine laboratory tests. The objective was to assess a modified version of the ISTH scoring system and determine the effect of drotrecogin alfa (activated) (DrotAA, recombinant human activated protein C) on patients with DIC. The large database from the PROWESS clinical trial in severe sepsis was retrospectively used to assess a modified ISTH scoring system. Baseline characteristics and treatment effects of DrotAA were evaluated. At baseline, 29% (454/1568) of patients had overt DIC. Overt DIC was a strong predictor of mortality, independent of APACHE II score and age. Placebo-treated patients with overt DIC had higher mortality than patients without (43 vs. 27%). DrotAA-treated patients with overt DIC had a trend towards greater relative risk reduction in mortality than patients without (29 vs. 18%, P = 0.261) but both groups had greater relative risk reduction than placebo-treated patients. Serious bleeding rates during DrotAA infusion in patients with and without overt DIC were slightly increased (P = 0.498), compared with placebo, while clinically overt thrombotic events during the 28-day period were slightly reduced (P = 0.144). Modified ISTH overt DIC scoring may be useful as an independent assessment for identifying severe sepsis patients at high risk of death with a favorable risk/benefit profile for DrotAA treatment. Patients without overt DIC also received significant treatment benefit.

Gene expression profile of antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis.

Human protein C is a natural anticoagulant factor, and a recombinant activated form of the molecule (rhAPC) is completing clinical evaluation for treatment of severe sepsis. Because of the pathophysiologic role of endothelial dysfunction in severe inflammatory disease and sepsis, we explored the possibility that rhAPC might directly modulate endothelial function, independent of its anticoagulant activity. Using broad transcriptional profiling, we show that rhAPC directly modulates patterns of endothelial cell gene expression clustering into anti-inflammatory and cell survival pathways. rhAPC directly suppressed expression of p50 and p52 NFkappaB subunits, resulting in a functional decrease in NFkappaB binding at target sites. Further, rhAPC blocked expression of downstream NFkappaB regulated genes following tumor necrosis factor alpha induction, including dose-dependent suppression of cell adhesion expression and functional binding of intracellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. Further, rhAPC modulated several genes in the endothelial apoptosis pathway, including the Bcl-2 homologue protein and inhibitor of apoptosis protein. These pathway changes resulted in the ability of rhAPC to inhibit the induction of apoptosis by the potent inducer, staurosporine. This new mechanistic understanding of endothelial regulation and the modulation of tumor necrosis factor-induced endothelial dysfunction creates a novel link between coagulation, inflammation, and cell death and provides insight into the molecular basis for the efficacy of APC in systemic inflammation and sepsis.

17beta-estradiol, but not raloxifene, decreases thrombomodulin in the antithrombotic protein C pathway.

Raloxifene is a nonsteroidal selective estrogen receptor modulator (SERM) that mimics the effects of estrogen on some plasma lipids and may have direct effects on the vascular wall. The objective of this study was to determine the effects of 17beta-estradiol, raloxifene, and LY139,478 (a related benzothiophene SERM) on the anticoagulant protein C pathway. In human vascular endothelial cells activated with interleukin-1 (IL-1), we demonstrated decreased thrombomodulin-dependent protein C activation. 17beta-estradiol reduced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells by decreasing thrombomodulin expression. In contrast, raloxifene and LY139,478 enhanced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells through upregulation of thrombomodulin. Regulation of the protein C pathway via thrombomodulin on vascular endothelium may be a novel mechanism by which SERMs could potentially confer cardioprotective effects and reduce the thrombotic risk associated with HRT in compromised patients.

Functional interactions between the thrombin receptor and the T-cell antigen receptor in human T-cell lines.

The proteolytically activated thrombin receptor (TR) is expressed by T lymphocytes, which suggests that thrombin may modulate T-cell activation at sites of hemostatic stress. We examined the relationship between TR function and T-cell activation in the Jurkat human T-cell line and in T-cell lines with defined defects in T-cell antigen receptor (TCR) function. Stimulation with thrombin or the synthetic TR peptide SFLLRN produced intracellular Ca2+ transients in Jurkat cells. As the concentration of TR agonist was increased, peak Ca2+ mobilization increased, but influx of extracellular Ca2+ decreased. TR signaling was enhanced in a TCR-negative Jurkat line and in T-cell lines deficient in the tyrosine kinase lck or the tyrosine phosphatase CD45, both of which are essential for normal TCR function. TCR cross-linking with anti-CD3 IgM desensitized TR signaling in Jurkat cells, but not in CD45-deficient cells. A proteinase-activated receptor (PAR-2)-specific agonist peptide, SLIGKV, produced small Ca2+ transients in both MEG-01 human megakaryocytic cells and Jurkat cells, but was less potent than the TR-specific agonist TFRIFD in both cell types. Like TR signaling, PAR-2 signaling was enhanced in TCR-negative or lck-deficient Jurkat clones. These findings provide evidence for functional cross-talk between proteolytically activated receptors and the TCR.