Expression and translation of the COX-1b gene in human cells--no evidence of generation of COX-1b protein.

Cyclooxygenase 1b (COX-1b) is a splice variant of COX-1, containing a retained intron 1 within the signal peptide sequence. COX-1b mRNA is found in many species, but the existence of a functionally active protein, which is possibly related to different species-dependent lengths of intron 1, is controversially discussed. The human intron 1 comprises 94 bp, and the resulting frameshift at the intron 1-exon 2 junction creates a premature stop codon.

Complete downmodulation of P-selectin glycoprotein ligand in monocytes undergoing apoptosis.

Objective: Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)-mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells.

Methods And Results: PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding.

Membrane environment rather than tissue factor expression determines thrombin formation triggered by monocytic cells undergoing apoptosis.

Monocyte apoptosis is an important determinant of atherothrombosis. Two major mechanisms for apoptosis-associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up-regulation of tissue factor (TF). However, the relative importance of these mechanisms is unclear.

Alternative splicing of platelet cyclooxygenase-2 mRNA in patients after coronary artery bypass grafting.

Recently, we cloned from platelet mRNA a novel cyclooxygenase (COX)-2 splice variant, designated COX-2a, which is characterized by a partial deletion of exon 5. Preliminary studies of mRNA distribution of COX-2 isoforms in platelets from coronary artery bypass grafting (CABG) patients showed a variable increase in COX-2a mRNA expression after cardiac surgery. Thus, we assessed whether this variant may play a functional role in these patients.

Cholesterol enhances thrombin-induced release of fibroblast growth factor-2 in human vascular smooth muscle cells.

Objective: The mitogenic response to the G protein-coupled receptor agonist thrombin in human vascular smooth muscle cells (SMCs) depends on release of fibroblast growth factor-2 (FGF-2). Yet, intracellular mechanisms triggering FGF-2 release are unknown. The present study investigates possible effects of cholesterol enrichment and depletion, which have been shown to influence FGF-2-dependent signaling and SMC mitogenesis, on thrombin-induced FGF-2 release.

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant.

It has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells.

Human cyclooxygenase-1b is not the elusive target of acetaminophen.

A cyclooxygenase-1 splice variant (cyclooxygenase-1b), cloned from canine brain, was proposed to be an acetaminophen-sensitive enzyme. Unlike in canines, the retention of intron 1 in the human sequence results in a frame shift and predicts a truncation of the protein. We have sought to answer the question whether human cyclooxygenase-1b, if expressed, is a target of acetaminophen.

Rapid release of active tissue factor from human arterial smooth muscle cells under flow conditions.

Circulating tissue factor (TF) is an important determinant of coronary thrombosis. Among other cell types, such as monocytes, vascular smooth muscle cells (SMCs) are capable of releasing TF. When studied under static conditions, SMCs do release TF, but this process is slow and, thus, cannot explain the elevated levels of circulating TF, as observed in patients with acute coronary syndromes.

Regulation of thrombomodulin expression in human vascular smooth muscle cells by COX-2-derived prostaglandins.

There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI2) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI2. Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost.

Thermodynamics of apocalmodulin and nitric oxide synthase II peptide interaction.

The Ca2+-free form of calmodulin (CaM), apocalmodulin (ApoCaM), regulates a variety of target proteins including nitric oxide synthase II (NOS-II). The CaM-binding site of NOS-II can bind ApoCaM with high affinity. Substitution of hydrophobic amino acids by charged amino acids at crucial positions 3, 9 and 13 within the CaM-binding motif did not abolish the ApoCaM interaction that occurred with significant affinity, though the affinity of the interaction was decreased remarkably.

Cyclooxygenase COX-2a, a novel COX-2 mRNA variant, in platelets from patients after coronary artery bypass grafting.

There are two principal cyclooxygenase isoforms referred to as COX-1 and COX-2. Recently, COX-3 has been identified. We have demonstrated the expression of COX-2 in platelets from patients after coronary artery bypass grafting (CABG).

Reaction rate constants of superoxide scavenging by plant antioxidants.

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce.

Target recognition of apocalmodulin by nitric oxide synthase I peptides.

An increasing number of proteins are found that are regulated by the Ca(2+)-free state of calmodulin, apocalmodulin. Many of these targets harbor a so-called IQ motif within their primary sequence, but several target proteins of apocalmodulin lack this motif. We investigated whether the Ca(2+)-dependent calmodulin-binding site of nitric oxide synthase I could be transformed into a target site of apocalmodulin.