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Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently avail-able antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents., Introduction Protein disulfide isomerase (PDI) is the prototypical member of an extended family of oxidoreductases, best known as endoplasmic reticulum-resident enzymes. These enzymes catalyze posttranslational disulfide bond formation and exchange [...]

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex., Introduction Hypercholesterolemia describes the presence of increased lipids in blood that is caused either by a predisposed genetic condition, such as a mutation of the LDL receptor (LDLR), or by [...]

The recent advances in knowledge and information about thrombus formation are discussed. Some of the new hypothesis and treatment methodologies for the prevention of thrombosis are also highlighted.

Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates thrombus formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet thrombus formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein--coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4-/- mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet thrombus formation., Introduction Evidence is mounting that important steps in the process of thrombus formation may be regulated by the oxidation states of labile disulfide bonds in critical hemostatic proteins (1). The [...]

Bile salt-dependent lipase (BSDL) is an enzyme involved in the duodenal hydrolysis and absorption of cholesteryl esters. Although some BSDL is transported to blood, the role of circulating BSDL is unknown. Here, we demonstrate that BSDL is stored in platelets and released upon platelet activation. Because BSDL contains a region that is structurally homologous to the V3 loop of HIV-1, which binds to CXC chemokine receptor 4 (CXCR4), we hypothesized that BSDL might bind to CXCR4 present on platelets. In human platelets in vitro, both BSDL and a peptide corresponding to its V3-like loop induced calcium mobilization and enhanced thrombin-mediated platelet aggregation, spreading, and activated [[alpha].sub.IIb][[beta].sub.3] levels. These effects were abolished by CXCR4 inhibition. BSDL also increased the production of prostacyclin by human endothelial cells. In a mouse thrombosis model, BSDL accumulated at sites of vessel wall injury. When CXCR4 was antagonized, the accumulation of BSDL was inhibited and thrombus size was reduced. In BSDL-/- mice, calcium mobilization in platelets and thrombus formation were attenuated and tail bleeding times were increased in comparison with those of wild-type mice. We conclude that BSDL plays a role in optimal platelet activation and thrombus formation by interacting with CXCR4 on platelets., Introduction Pancreatic cholesterol esterase or bile salt-dependent lipase (BSDL; E.C.3.1.1.13) is an enzyme involved in the duodenal hydrolysis and absorption of cholesteryl esters (1), (2). BSDL is synthesized in the [...]

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and [...]

Research has been conducted on the phospholipid membranes, an important factor in blood coagulation protein activation. The role of the phosphatidylethanolamine in factor X activation by the enzymatic complex composed of serine protease, tenase complex, has been investigated.

An amphopathic peptide from the C2 domain of blood coagulation factor VIII can activate factor IXa in the absence of a phospholipid membrane. This domain forms a helix, and a minimum length of helix is required to activate factor IXa.

Hydrophobic amino acids are involved at the site of gamma-carboxylation in the propeptide form of a conotoxin from the marine cone snail Conus. This snail is the only invertebrate that synthesizes gamma-carboxyglutamic acid.

Tandem mass spectrometry (MS/MS) of 28 residue peptides harboring gamma-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the gamma-CO(sub 2) moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/MS using electron capture dissociation (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO(sub 2), allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO(sub 3) loss. ECD thus is a unique complement to conventional methods for MS/MS, causing less undesirable loss of side-chain functionalities as well as more desirable backbone cleavages.

An examination of the role of free cysteine residues in vitamin K-dependent carboxylase catalysis has been conducted using recombinant vitamin K-dependent carboxylase that has been purified without propeptide and glutamic acid-containing substrate using a FLAG epitope tag. The results confirmed the previous observation that the binding of a substrate containing glutamic acid is responsible for vitamin K epoxidation and that the binding to a carboxylatable substrate makes the enzyme catalytically competent.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a [Gamma]-carboxyglutamic acid-containing peptide, conotoxin [Epsilon]-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic [Ca.sup.2+] channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of [Ca.sup.2+] in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two [Gamma]-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.

A grasp of the molecular kinetic parameters for adhesion molecule interactions is necessary to gauging the relative contribution of various receptor pairs to the rolling of leukocytes in the vasculature. A study was conducted to investigate the affinity of soluble P-selectin for the soluble P-selectin glycoprotein ligand-1 in a simple, chemically defined system where two components could be observed in the solution phase.

The function of gamma-carboxyglutamic acid (Gla) in the calcium-induced structural transition of conantokin G was examined. Conantokin G is a Gla-containing conotoxin isolated from the venom of the marine cone snail conus geographus. The three-dimensional structure of the conantokin G/Ca2+ complex was determined and compared to the high-resolution structure of conantokin G in the absence of metal ions. Findings showed that upon binding of Ca2+ to Gla, conantokin G undergoes a conformation transition from a distorted curvilinear 3(sub 10) helix to a linear alpha-helix.