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Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

Objective. To study the intracellular mechanism involved in the up-regulation of tissue factor (TF) on endothelial cells (ECs) by antiphospholipid antibodies (aPL), we examined the effects of aPL on the transcription, expression, and function of TF, the expression of interleukin-6 (IL-6) and IL-8, the induction of inducible nitric oxide synthase (iNOS), and the phosphorylation of p38 MAPK on human umbilical vein ECs (HUVECs).Methods. Cultured HUVECs were treated with IgG aPL (from patients with antiphospholipid syndrome [APS]) or with control IgG (from normal human serum). Phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS) were used as positive controls. TF expression was determined on the surface of HUVECs using an enzyme-linked immunosorbent assay (ELISA). TF activity was determined with the use of a chromogenic assay in cell lysates, and TF messenger RNA (mRNA) was determined by real-time quantitative polymerase chain reaction. Phosphorylation of p38 MAPK and induction of iNOS were determined by Western blotting, and levels of IL-6 and IL-8 were determined by ELISA.Results. PMA, LPS, and aPL significantly increased the expression of TF compared with controls. This up-regulation was significantly inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by MG132 (a specific inhibitor of NF-B). TF activity was significantly increased by treatment with IgG aPL and this effect was also inhibited by SB203580. Incubation of HUVECs with aPL increased TF mRNA 2-15-fold; these effects were abrogated by SB203580. IgG aPL induced significant phosphorylation of p38 MAPK and produced iNOS on HUVECs in a time-dependent manner. Treatment with IgG aPL also induced increased expression of IL-6 and IL-8 on HUVECs.Conclusion. Our data show that aPL induces significant increases in TF transcription, function, and expression, in IL-6 and IL-8 up-regulation, and in iNOS expression on HUVECs and that these processes involve phosphorylation of p38 MAPK and activation of NF-B.

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The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms ( The glycosaminoglycan (GAG)2 hyaluronic acid (HA) is a protein-free polymer of disaccharide units containing glucuronic acid and N-acetylglucosamine (1, 2). HA is involved in many physiological processes (3), such as wound healing, development, and metastasis of some cancers (4 -8). The typical molecular mass of the polysaccharide ranges from just a few thousand Da (tens of sugars) that are thought to be important in cellular signaling (6) to several million Da (tens of thousands of sugars). These larger forms of HA are present throughout the body and are particularly concentrated within the bursa of major joints, such as the knee, where they help to provide shock absorbance in cartilage or lubrication in synovial fluid (9, 10), and the eye, where HA maintains structural integrity of the vitreous humor (11). The adult human body contains ϳ15 g of HA, of which about 5 g are turned over daily (12). Partially degraded HA perfuses from extracellular matrices (ECMs) and enters the lymphatic and vascular circulation systems, where it is catabolized to shorter fragments. This active maintenance of HA turnover must be efficient in order to maintain homeostatic conditions for total body HA.All of the other GAGs, including the chondroitin sulfates (CSs), heparan sulfate (HS), and keratan sulfate, are linked to core proteins (as proteoglycans) that help to form ECMs, such as the basement membranes of tissues, or structural components of organs, such as the vitreous humor. There are over 30 known core proteins that are essential for a diverse array of functions, such as neural development, growth factor signaling, and pathogen recognition (13). These core proteins are found as prevalent components of tissue ECMs or as specialized components needed for the development of microenvironments that interface a specialized tissue cell type with the ECM. Both the proteoglycans and their attached GAG chains may combinatorially interact with ligands and contribute to modulation of the functional aspects of a particular microenvironment (e.g. CS interacting with apolipoprotein E for uptake of ␤-very low density lipoprotein in hippocampal neurons) (14). Although numerous studies have focused on how the inhibition of some CS proteoglycans enhances neural development, especially in injured spinal cord models, there is very little information on how CS and HS are catabolized. The current model is that extracellular chondroitinases, heparinases, and proteases initially break down these GAGs and proteoglycans, and their final digestion can then take place intracellularly at the local tissue * This research was supported by NIGMS, National Institutes of Health, Grant GM69961. The costs of publication of this article were defrayed in part by the payment of page charges. This article ...

SummaryProthrombotic properties of antiphospholipid (aPL) antibodies may be explained in part by their ability to enhance the activation of platelets pre-treated with low doses of ADP or thrombin. The antimalarial drug hydroxychloroquine (HQ) has been used successfully in prevention of postoperative thrombosis and in treatment of patients with SLE or APS. In one study, administration of HQ reversed the thrombogenic properties of aPL in mice. However, the mechanism of action of HQ in preventing thrombosis is not clearly understood. In order to explore this further, the effects of HQ on activation of platelets by aPL in the presence of a thrombin agonist was studied. The changes in the expression of GPIIb/IIIa (CD41a) and GPIIIa (CD61) on platelet membrane by flow cytometry were used as indicators of platelet activation. Citrated whole blood from a healthy donor was treated at room temperature with suboptimal doses of a thrombin agonist receptor peptide (TRAP) and affinity-purified aPL antibodies, in the presence and in the absence of hydroxychloroquine (1 mM). TRAP increased the expression of GPIIb/IIIa and GPIIIa on platelet surface. The treatment of the platelets with the six aPL antibodies in the presence of 12 nMol/ml TRAP further increased the expression of GPIIb/IIIa by 42.3 ± 12.3% and the expression of GPIIIa was further incremented by 46.8 ± 13.5%. The effects of aPL and TRAP on expression of platelet surface markers of activation was completely abrogated by HQ in a dose-dependent fashion and was effective at concentrations of HQ as low as 25 µg/ml (0.0125 mM). This suggests at least one possible mechanism by which HQ may prevent thrombosis. This may have important implications in treatment of thrombosis in APS patients.

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.