Glycosaminoglycan backbone is not required for the modulation of hemostasis: effect of different heparin derivatives and non-glycosaminoglycan analogs.

Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.

Presence of asymmetric dimethylarginine gradients across high-grade lesions in patients with coronary artery disease.

BACKGROUND: Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is a systemic marker of endothelial dysfunction. Although experimental evidence indicates that asymmetric dimethylarginine may play an important role in atherogenesis, local asymmetric dimethylarginine levels have not been measured in vivo. OBJECTIVES: We sought to determine whether: (i) asymmetric dimethylarginine is elevated locally at sites of coronary lesions, (ii) systemic asymmetric dimethylarginine concentrations correlate with local levels, and (iii) percutaneous coronary intervention produces immediate local asymmetric dimethylarginine elevation. METHODS: In patients undergoing percutaneous coronary intervention (n=15), blood samples were obtained from a peripheral venous site, the coronary ostium proximal to the lesion and the coronary vessel distal to the lesion, before percutaneous coronary intervention. Samples were also obtained distal to the coronary lesion immediately after percutaneous coronary intervention and from the peripheral venous line 24 h after percutaneous coronary intervention. RESULTS: Asymmetric dimethylarginine gradients were present across the coronary bed: local asymmetric dimethylarginine (micromol/l) was significantly higher distal to coronary lesions compared with proximally (2.39+/-1.27 vs. 1.52+/-0.68, P=0.005), and to systemic venous levels (2.39+/-1.27 vs. 1.17+/-0.72, P=0.001). Local asymmetric dimethylarginine did not increase immediately after percutaneous coronary intervention (1.88+/-0.89 vs. 2.39+/-1.27, P=0.11). Peripheral venous percutaneous coronary intervention levels 24 h after percutaneous coronary intervention were similar to baseline values (1.17+/-1.2 vs. 1.17+/-0.72, P=0.98). CONCLUSION: Asymmetric dimethylarginine gradients exist across coronary lesions, suggesting asymmetric dimethylarginine release at the plaque site. Local asymmetric dimethylarginine accumulation may contribute to the endothelial dysfunction associated with high-grade coronary lesions. Peripheral asymmetric dimethylarginine is a marker of generalized endothelial dysfunction, but our findings highlight its limitation in detecting focal injury.

Randomized trial of different regimens of heparins and in vivo thrombin generation in acute deep vein thrombosis.

Low-molecular-weight and unfractionated heparins are frequently used to treat venous thromboembolism, but it is not known whether they are equally effective in inhibiting in vivo generation of thrombin. In this multicenter trial, 1048 patients were randomized to intravenous unfractionated heparin (group A), twice daily low-molecular-weight heparin (reviparin) for 1 week (group B), or once daily reviparin for 4 weeks (group C). All patients received vitamin K antagonists. Blood samples withdrawn at the baseline and at weeks 1 and 3 were analyzed using markers of in vivo thrombin generation and other coagulation parameters. During the first 3 weeks symptomatic recurrent deep vein thrombosis-pulmonary embolism (DVT/PE) occurred in 17 (4.5%) of 375 patients in group A compared with 4 (1.0%) of 388 patients in group B, and 9 (2.4%) of 374 patients in group C. Forty percent of patients in group A, 53.4% in group B, and 53.5% in group C showed 30% or greater reduction in thrombus size assessed by venography. Patients in group B had significantly greater reduction in D-dimer, prothrombin fragments 1 and 2 (F1 + 2), endogenous thrombin potential (ETP), and thrombin-antithrombin (TAT) complexes compared to groups A and C. Greater release of tissue factor pathway inhibitor (TFPI) and reduction in levels of thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were significantly more pronounced in group C patients. Reviparin administered twice daily plus vitamin K antagonist is more effective in inhibiting in vivo thrombin generation compared to intravenous unfractionated heparin plus vitamin K antagonist, and reviparin once daily produced significantly higher TFPI release and greater reduction in TAFI and fibrinogen levels.

Antithrombotic Agents in the Management of Sepsis.

Sepsis, a systemic inflammatory syndrome, is a response to infection and when associated with multiple organ dysfunction is termed, severe sepsis. It remains a leading cause of mortality in the critically ill. The response to the invading bacteria may be considered as a balance between proinflammatory and antiinflammatory reaction. While an inadequate proinflammatory reaction and a strong antiinflammatory response could lead to overwhelming infection and death of the patient, a strong and uncontrolled proinflammatory response, manifested by the release of proinflammatory mediators may lead to microvascular thrombosis and multiple organ failure. Endotoxin triggers sepsis by releasing various mediators including tumor necrosis factor-alpha and interleukin-1(IL-1). These cytokines activate the complement and coagulation systems, release adhesion molecules, prostaglandins, leukotrienes, reactive oxygen species and nitric oxide (NO). Other mediators involved in the sepsis syndrome include IL-1, IL-6 and IL-8; arachidonic acid metabolites; platelet activating factor (PAF); histamine; bradykinin; angiotensin; complement components and vasoactive intestinal peptide. These proinflammatory responses are counteracted by IL-10. Most of the trials targeting the different mediators of proinflammatory response have failed due a lack of correct definition of sepsis. Understanding the exact pathophysiology of the disease will enable better treatment options. Targeting the coagulation system with various anticoagulant agents including antithrombin, activated protein C (APC), tissue factor pathway inhibitor (TFPI) is a rational approach. Many clinical trials have been conducted to evaluate these agents in severe sepsis. While trials on antithrombin and TFPI were not so successful, the double-blind, placebo-controlled, phase III trial of recombinant human activated protein C worldwide evaluation in severe sepsis (PROWESS) was successful, significantly decreasing mortality when compared to the placebo group. Better understanding of the pathophysiologic mechanism of severe sepsis will provide better treatment options. Combination antithrombotic therapy may provide a multipronged approach for the treatment of severe sepsis.