Robert D. Rosenberg

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.

Activation of Coagulation after Administration of Tumor Necrosis Factor to Normal Subjects

Tumor necrosis factor has been implicated in the activation of blood coagulation in septicemia, a condition commonly associated with intravascular coagulation and disturbances of hemostasis. To evaluate the early dynamics and the route of the in vivo coagulative response to tumor necrosis factor, we performed a controlled study in six healthy men, monitoring the activation of the common and intrinsic pathways of coagulation with highly sensitive and specific radioimmunoassays. Recombinant human tumor necrosis factor, administered as an intravenous bolus injection (50 micrograms per square meter of body-surface area), induced an early and short-lived rise in circulating levels of the activation peptide of factor X, reaching maximal values after 30 to 45 minutes (mean +/- SEM increase after 45 minutes, 34.2 +/- 18.2 percent; tumor necrosis factor vs. saline, P = 0.015). This was followed by a gradual and prolonged increase in the plasma concentration of the prothrombin fragment F1+2, peaking after four to five hours (mean increase after five hours, 348.0 +/- 144.8 percent; tumor necrosis factor vs. saline, P less than 0.0001). These findings signify the formation of factor Xa (activated factor X) and the activation of prothrombin. Activation of the intrinsic pathway could not be detected by a series of measurements of the plasma levels of factor XII, prekallikrein, factor XIIa-C1 inhibitor complexes, kallikrein-C1 inhibitor complexes, and the activation peptide of factor IX. The delay between the maximal activation of factor X and that of prothrombin amounted to several hours, indicating that neutralization of factor Xa activity was slow. We conclude that a single injection of tumor necrosis factor elicits a rapid and sustained activation of the common pathway of coagulation, probably induced through the extrinsic route. Our results suggest that tumor necrosis factor could play an important part in the early activation of the hemostatic mechanism in septicemia.

TARGETED DISRUPTION OF CD39/ATP DIPHOSPHOHYDROLASE RESULTS IN DISORDERED HEMOSTASIS AND THROMBOREGULATION

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

The separation of active and inactive forms of heparin

Abstract Heparin has been fractionated into two distinct forms. The isolation of these species was accomplished by sucrose density gradient centrifugation of heparin mixed with antithrombin-heparin cofactor. Approximately 1 3 of this mucopolysaccharide was bound to antithrombin-heparin cofactor and had potent anticoagulant activity. This component was clearly separated from the remaining 2 3 of the heparin which could not form a stable complex with antithrombin-heparin cofactor and had minimal anticoagulant activity.

PR39, a peptide regulator of angiogenesis

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin–proteasome-dependent degradation of hypoxia-inducible factor-1α protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1α degradation may underlie the mechanism of inflammation-induced angiogenesis.

Actions and interactions of antithrombin and heparin.

THE coagulation of blood occurs via two pathways in which activation of specific plasma proteins initiates a cascade of reactions that ultimately lead to a fibrin clot. In the first pathway, injury...

Management of heparin therapy: Controlled prospective trial.

Among 100 consecutive patients receiving heparin in therapeutic dosage, major bleeding occurred in 21, and minor bleeding in 16. Two patients died from bleeding, and two had recurrent pulmonary embolism. Major bleeding occurred in 21% when therapy was regulated with whole-blood clotting time and in 20% when heparin was given without clotting tests. In a subsequent prospective trial patients received heparin by intermittent intravenous injection with or without laboratory control according to the partial thromboplastin time or continuously by intravenous infusion. Recurrent thromboembolism occurred once in each group. Major bleeding was seven times more frequent with intermittent injection than with continuous infusion. Control with the partial thromboplastin time did not prevent major bleeding in patients receiving intermittent injections. With continuous infusion, one-fourth less heparin was required than with intermittent injections. Administration of heparin by continuous infusion appears safer than intermittent injection with or without laboratory control and is no less effective for prevention of thromboembolism.

Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells.

Tumor necrosis factor (TNF) dramatically alters the levels of various surface components of the blood vessel wall, such as blood coagulation enzyme receptors, leukocyte-adhesive receptors, and class 1 major histocompatibility complex antigens, which may have relevance to its effects in septic shock, angiogenesis, and tumor growth. However, the precise mechanism by which the cytokine is able to accomplish this remodeling of the endothelial cell surface has not been defined. We have demonstrated that exposure of bovine and human endothelial cells to TNF leads to suppression of the functional cell surface thrombin receptor, thrombomodulin (TM), and TM mRNA of virtually identical magnitude. The cytokine has no significant effect on the stability of TM mRNA or endothelial receptor turnover. Nuclear run-on studies reveal that the treatment of endothelial cells with TNF for short periods reduces TM gene transcription to as little as 3% of control values and that this inhibition does not require new protein synthesis.

Systematic Review and Meta-Analysis of the Role of Defunctioning Stoma in Low Rectal Cancer Surgery

Summary Background Data:The role of a defunctioning stoma in patients undergoing low anterior resection for rectal cancer is still the subject of controversy. Recent studies suggest reduced morbidity after low anterior rectal resection with a defunctioning stoma. Methods:Retrospective and prospective studies published between 1966 and 2007 were systematically reviewed. Randomized controlled trials (RCTs) comparing anterior resections with or without defunctioning stoma were included in a meta-analysis. The pooled estimates of clinically relevant anastomotic leakages and of reoperations were analyzed using a random effects model (odds ratio and 95% confidence interval, CI). Results:Relevant retrospective single (n = 18) and multicenter (n = 9) studies were identified and included in the systematic review. Analysis of incoherent data of the leakage rates in these nonrandomized studies demonstrated that a defunctioning stoma did not influence the occurrence of anastomotic failure but seemed to ameliorate the consequences of the leak. Four RCTs were included in the meta-analysis. The odds ratio for clinically relevant anastomotic leakage was 0.32 (95% CI 0.17–0.59), revealing a statistically significant benefit conferred through a defunctioning stoma (Z = 3.65, P = 0.0003). The odds ratio for reoperation because of leakage-caused complications was 0.27 (95% CI 0.14–0.51), with significantly fewer reoperations in patients with a defunctioning stoma (Z = 3.95, P < 0.0001). Overall mortality rates were comparable regardless of the presence of a defunctioning stoma. Conclusion:A defunctioning stoma reduces the rate of clinically relevant anastomotic leakages and is thus recommended in surgery for low rectal cancers.

Heparan sulfate proteoglycans of the cardiovascular system. Specific structures emerge but how is synthesis regulated

The cell surfaces and surrounding extracellular matrix of the cardiovascular system possess large quantities of heparan sulfate proteoglycans (HSPGs) 1 (1). These highly charged macromolecules consist of different core proteins with covalently linked heparan sulfate chains (HS) of varying monosaccharide sequence which serve as critical mediators of biologic processes (2, 3). For example, these components are involved in regulating mesodermal cell fate, positioning of the heart, vasculogenesis and angiogenesis after ischemic injury, interactions of cells with adhesive proteins and blood vessels, proliferation of smooth muscle cells during atherogenesis, metabolism of lipoproteins, nonthrombogenic characteristics of endothelial cells, etc. (4–9). Detailed investigations over the past decade have defined the structures of HSPGs, uncovered the molecular mechanisms by which these components carry out their diverse functions, and revealed that specific monosaccharide sequences of HS are required for interaction with biologic targets. In this review, we outline the current state of our knowledge about the structure and the biosynthesis of HSPGs as well as describe interactions of these components with growth factors, enzymes, and protease inhibitors. These observations provide a conceptual framework for elucidating the roles of HSPGs in other biologic systems. Structure and biosynthesis of HSPGs