The Ca 2؉ -sensitive 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A 2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA 2 , thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA 2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA 2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA 2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA 2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA 2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA 2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA 2 is not required for its receptor-mediated activation.On activation of platelets with physiological agonists such as thrombin, significant amounts of arachidonic acid are rapidly liberated for transformation to thromboxane A 2 via the cyclooxygenase-thromboxane synthase pathway. There is substantial evidence to indicate that this efficient receptor-mediated mobilization of arachidonic acid is mediated by a phospholipase A 2 pathway (1, 2) and that the involved phospholipase A 2 is the Ca 2ϩ -sensitive cytosolic phospholipase A 2 (cPLA 2 ) 1 (3, 4).Many studies with different cellular systems, including platelets, have documented that phosphorylation of cPLA 2 by receptor-mediated events accompanies the stimulated release of arachidonic acid from cellular phospholipids (5). Lin et al. (6) established that this phosphorylation is "activating" (i.e. it increases the catalytic activity of cPLA 2 severalfold) and occurs at Ser 505 residing within a MAP kinase consensus sequence (Pro-Leu-Ser 505 -Pro). In fact, cPLA 2 was phosphorylated and activated by the MAP kinase ERK2 in vitro and in vivo (i.e. in cultured cells overexpressing cPLA 2 and ERK2; Ref. 6), and accordingly, the ERKs were taken to be responsible for the proline-directed phosphorylation of cPLA 2 observed in various cellular systems. Surprisingly, we have noted that phosphorylation of cPLA 2 occurred in the absence of ERK activation in human platelets stimulated with the thrombin receptor agonist peptide SFLLRN (7). Furthermore, under conditions in which ER...

Major coagulation abnormalities were found after successful resuscitation of cardiac arrest. These abnormalities are consistent with secondary down-regulation of the thrombomodulin-endothelial protein C receptor pathway.

SummaryThe HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min−1 and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-012-9912-9) contains supplementary material, which is available to authorized users.

IntroductionPROWESS (Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis) was a phase III, randomized, double blind, placebo controlled, multicenter trial conducted in patients with severe sepsis from 164 medical centers. Here we report data collected at study entry for 1690 patients and over the following 7 days for the 840 patients who received placebo (in addition to usual standard of care).MethodsNineteen biomarkers of coagulation activation, anticoagulation, fibrinolysis, endothelial injury, and inflammation were analyzed to determine the relationships between baseline values and their change over time, with 28-day survival, and type of infecting causative micro-organism.ResultsLevels of 13 of the 19 biomarkers at baseline correlated with Acute Physiology and Chronic Health Evaluation II scores, and nearly all patients exhibited coagulopathy, endothelial injury, and inflammation at baseline. At study entry, elevated D-dimer, thrombin–antithrombin complexes, IL-6, and prolonged prothrombin time were present in 99.7%, 95.5%, 98.5%, and 93.4% of patients, respectively. Markers of endothelial injury (soluble thrombomodulin) and deficient protein C, protein S, and antithrombin were apparent in 72%, 87.6%, 77.8%, and 81.7%, respectively. Impaired fibrinolysis (elevated plasminogen activator inhibitor-1) was observed in 44% of patients. During the first 7 days, increased prothrombin time (which is readily measurable in most clinical settings) was highly evident among patients who were not alive at 28 days.ConclusionAbnormalities in biomarkers of inflammation and coagulation were related to disease severity and mortality outcome in patients with severe sepsis. Coagulopathy and inflammation were universal host responses to infection in patients with severe sepsis, which were similar across causative micro-organism groups.

Summary Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-017-0446-z) contains supplementary material, which is available to authorized users.