α2β1 integrin, GPVI receptor, and common FcRγ chain on mouse platelets mediate distinct responses to collagen in models of thrombosis.

Objective: Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ-/-) or the complex was depleted.

Proteolytic activation transforms heparin cofactor II into a host defense molecule.

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense.

The collection of NFATc1-dependent transcripts in the osteoclast includes numerous genes non-essential to physiologic bone resorption.

Osteoclasts are specialized secretory cells of the myeloid lineage important for normal skeletal homeostasis as well as pathologic conditions of bone including osteoporosis, inflammatory arthritis and cancer metastasis. Differentiation of these multinucleated giant cells from precursors is controlled by the cytokine RANKL, which through its receptor RANK initiates a signaling cascade culminating in the activation of transcriptional regulators which induce the expression of the bone degradation machinery. The transcription factor nuclear factor of activated T-cells c1 (NFATc1) is the master regulator of this process and in its absence osteoclast differentiation is aborted both in vitro and in vivo.

Thrombomodulin is required for the antithrombotic activity of thrombin mutant W215A/E217A in a mouse model of arterial thrombosis.

Introduction: The thrombin mutant W215A/E217A (WE thrombin) has greatly reduced procoagulant activity, but it activates protein C in the presence of thrombomodulin and inhibits binding of platelet glycoprotein Ib to von Willebrand factor and collagen under flow conditions. Both thrombomodulin-dependent protein C activation and inhibition of platelet adhesion could contribute to the antithrombotic activity of WE thrombin.

Materials And Methods: To assess the role of thrombomodulin, we administered WE thrombin to thrombomodulin-deficient (TM(Pro/Pro)) mice and measured the time to occlusive thrombus formation in the carotid artery after photochemical injury of the endothelium.

Inhibition of antithrombin by Plasmodium falciparum histidine-rich protein II.

Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner.

Dermatan sulfate and bone marrow mononuclear cells used as a new therapeutic strategy after arterial injury in mice.

Background Aims: Previously, we have demonstrated that administration of dermatan sulfate (DS) suppresses neointima formation in the mouse carotid artery by activating heparin co-factor II. A similar suppressive effect was observed by increasing the number of progenitor cells in circulation. In this study, we investigated the combination of DS and bone marrow mononuclear cells (MNC), which includes potential endothelial progenitors, in neointima formation after arterial injury.

Vascular dermatan sulfate and heparin cofactor II.

Heparin cofactor II (HCII) is a plasma protease inhibitor of the serpin family that inactivates thrombin by forming a covalent 1:1 complex. The rate of complex formation increases more than 1000-fold in the presence of dermatan sulfate (DS). Endothelial injury allows circulating HCII to enter the vessel wall, where it binds to DS and presumably becomes activated.

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.

Irreversible inactivation of alpha-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared.

A self-inactivating gamma-retroviral vector reduces manifestations of mucopolysaccharidosis I in mice.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in alpha-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a gamma-retroviral vector (gamma-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) gamma-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective.

Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II.

Heparin cofactor II (HCII)-deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect.

Mice lacking the extracellular matrix protein MAGP1 display delayed thrombotic occlusion following vessel injury.

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time.

Accelerated atherogenesis and neointima formation in heparin cofactor II deficient mice.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation.

Heparin cofactor II modulates the response to vascular injury.

Heparin cofactor II (HCII) has several biochemical properties that distinguish it from other serpins: (1) it specifically inhibits thrombin; (2) the mechanism of inhibition involves binding of an acidic domain in HCII to thrombin exosite I; and (3) the rate of inhibition increases dramatically in the presence of dermatan sulfate molecules having specific structures. Human studies suggest that high plasma HCII levels are protective against in-stent restenosis and atherosclerosis. Studies with HCII knockout mice directly support the hypothesis that HCII interacts with dermatan sulfate in the arterial wall after endothelial injury and thereby exerts an antithrombotic effect.

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate.

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS.

Placental dermatan sulfate: isolation, anticoagulant activity, and association with heparin cofactor II.

Pregnancy is associated with hemostatic challenges that may lead to thrombosis. Heparin cofactor II (HCII) is a glycosaminoglycan-dependent thrombin inhibitor present in both maternal and fetal plasma. HCII activity increases during pregnancy, and HCII levels are significantly decreased in women with severe pre-eclampsia.

Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants.

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets.

Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity.

A linear sulfated fucan with a regular repeating sequence of [3)-alpha-L-Fucp-(2SO4)-(1-->3)-alpha-L-Fucp-(4SO4)-(1-->3)-alpha-L-Fucp-(2,4SO4)-(1-->3)-alpha-L-Fucp-(2SO4)-(1-->]n is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid.

Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice.

The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo.

The alpha 2 beta 1 integrin serves as a receptor for collagens, laminin, and several other nonmatrix ligands. Many studies have suggested that the alpha 2 beta 1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury and that the interactions of the platelet alpha 2 beta 1 integrin with subendothelial collagen after vascular injury are required for proper hemostasis. We have used the alpha 2 beta 1 integrin-deficient mouse to evaluate the contributions of the alpha 2 beta 1 integrin in 2 in vivo models of thrombosis.

Heparin cofactor II deficiency.

Objectives: To review of the state of the art relating to congenital heparin cofactor II deficiency as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of laboratory assays for assessing this thrombotic risk in individual patients.

Data Sources: Review of the medical literature, primarily from the last 10 years.

Data Extraction And Synthesis: After an initial assessment of the literature, including review of clinical study design and laboratory methods, a draft manuscript was prepared and circulated to participants in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia.

Heparin cofactor II inhibits arterial thrombosis after endothelial injury.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate, heparan sulfate, or heparin. HCII has been proposed to regulate coagulation or to participate in processes such as inflammation, atherosclerosis, and wound repair. To investigate the physiologic function of HCII, about 2 kb of the mouse HCII gene, encoding the N-terminal half of the protein, was deleted by homologous recombination in embryonic stem cells.

Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II.

The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin(54-65) ([5F]Hir(54-65)(SO(3)(-))) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir(54-65)(SO(3)(-)) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation.