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Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C–dependent and –independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-κB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.

Besides its well-recognized role in hemostasis and thrombosis, thromboxane A(2) synthase (TXAS) is proposed to be involved in thrombopoiesis and lymphocyte differentiation. To evaluate its various physiologic roles, we generated TXAS-deleted mice by gene targeting. TXAS(-/-) mice had normal bone marrow megakaryocytes, normal blood platelet counts, and normal CD4 and CD8 lymphocyte counts in thymus and spleen. Platelets from TXAS(-/-) mice failed to aggregate or generate thromboxane B(2) in response to arachidonic acid (AA) but produced increased prostaglandin-E(2) (PGE(2)), PGD(2), and PGF(2 alpha). AA infusion caused a progressive drop of mean arterial pressure (MAP), cardiac arrest, and death in wild-type (WT) mice but did not induce shock in TXAS(-/-) mice or in WT and TXAS(-/-) mice treated with antagonist to the thromboxane-prostanoid (TP) receptor. The TXAS(-/-) mice were able to maintain normal MAP upon AA insult when TP was present but were unable to do so when TP was blocked by an antagonist, suggesting a role of endoperoxide accumulation in influencing MAP. We conclude that TXAS is not essential for thrombopoiesis and lymphocyte differentiation. Its deficiency causes a mild hemostatic defect and protects mice against arachidonate-induced shock and death. The TXAS-deleted mice will be valuable for investigating the roles of arachidonate metabolic shunt in various pathophysiologic processes.

Hemophila B is an X-linked inherited disorder caused by the lack of a functional Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. However, the high cost associated with the protein concentrates poses a financial burden to protein replacement therapy. Other treatments, such as gene therapy and tissue implant techniques, are also under study but not yet developed for clinical use. An alternative approach is to develop a FIX molecule with higher specific activity that will reduce the amount of the clotting factor consumed in treatment. For this purpose, we used alanine-scanning mutagenesis and generated 7 alanine-replaced FIX variants that were expressed in HEK-293 cells and purified. These variants have either single (n=3), double (n=3) or triple (n=1) amino acid changes and each was characterized by ELISA and aPTT assay. We observed that the FIX triple mutation variant (R86A/E277A/R338A, FIX-Triple) exhibited 10-12 times higher clotting activity than wild-type FIX (FIX-WT). The affinity of IX-Triple to human Factor VIIIa increased 10-fold (Kd = 0.19 nM vs. 2.4 nM, FIX-Triple vs. FIX-WT) in a Factor Xa-generation assay, consistent with the fact that the amino acid substitutions were in two domains important for factor VIII interaction. Protein infusion into hemophilia B (HB) mice showed that recombinant FIX-Triple was also more effective than FIX-WT in shortening the clotting time in these HB mice. To further evaluate the medium and long-term efficacy of FIX-Triple expression, we used two methods of gene delivery in (HB) mice. For medium-term expression, we performed tail vein hydrodynamic plasmid injections of either FIX-WT or FIX-Triple, expressed from a liver-specific promoter. We assayed specific activity (clotting activity/antigen, U/mg) 24h post-delivery and found that FIX-Triple had a 3.5-fold higher specific activity than FIX-WT. For long-term expression, we performed tail vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple from a liver-specific promoter at vector doses of 4x1012 (high dose) and 8x1010 (low dose) vector genomes (v.g.)/kg. These mice were sacrificed 8 weeks post vector administration and blood samples were analyzed for FIX antigen (ELISA) and clotting activity (aPTT). Corroborating our previous observations, we found that the FIX-Triple variant had 7 times higher specific activity vs. FIX-WT, for either vector dose used. More importantly, mice that received AAV8-FIX-Triple exhibited activity of 23% of normal pooled human plasma (12-38%, n=6), in contrast to mice that received FIX-WT that only reached 4% (1-8%, n = 4), following 8x1010 v.g./kg vector administration. Activity and antigen level for both transgenes was dose-dependent. Lastly, we generated knock-in mice for FIX-WT and FIX-Triple (FIX-KI-WT and FIX-KI-Triple) by exchanging the mouse sequence for the human, keeping the endogenous promoter intact. Confirming our observations with hydrodynamic injection and AAV administration, mice with FIX-KI-Triple exhibited 7 fold higher specific activity than those with FIX-KI-WT (2040 ± 253 vs 279 ± 33 U/mg). Collectively, our results indicate that FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to Factor VIIIa, as demonstrated both in vitro and in vivo. Therefore, Factor IX-Triple is a good candidate for further evaluation in protein replacement therapy as well as other, gene-based therapeutic strategies.