Abstract

ISTH07A1_P-S-083: Contact View [P-S-083] SUCCESSFUL PREGNANCY OUTCOME IN A WOMAN WITH HOMOZYGOUS ANTITHROMBIN DEFICIENCY (99 LEUPHE MUTATION) G. Alguel, K. Jochmans, R. Simanek, P. Quehenberger, M. Langer, I. Pabinger. Internal Medicine I, Medical University of Vienna, Vienna, Austria; Department of Hematology, Academic Hospital of the Free University of Brussels, Brussels, Belarus; Clinical Institute of Laboratory Medicine; Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria Introduction: Homozygous antithrombin (AT)-deficiency type II heparin-binding-site (HBS) increases the risk for venous thromboembolism (VTE) already in childhood and is associated with adverse pregnancy outcome. No cases of a successful pregnancy in a woman homozygous for AT deficiency had been described before. Methods: All exons and intron/exonjunctions were amplified by PCR. After a purification step, the PCR product was directly used for the sequence reaction using the Big Dye® Terminator Cycle Sequencing Ready Reaction kit v1.1 (Applied Biosystems). Results: AT-deficiency HBS with an AT plasma level of 25% was diagnosed after a deep vein thrombosis in a 23 year old Caucasian woman. Molecular analysis revealed homozygous G2759T mutation, which leads to a Leu to Phe change at amino acid position 99 conforming a type 2 HBS defect. The woman had two successful pregnancies without recurrent VTE, receiving therapeutic doses of low molecular weight heparin (LMWH) during her first and vitamin-K-antagonists during her second pregnancy. Both children were born by caesarean section at the 35th week, at first pregnancy due to fetal distress and at second due to amniorrhexis. AT replacement was given during the periand postpartal period. Intrauterine growth restriction (<10th percentile) was only observed during the first pregnancy. Conclusions: Women with homozygous AT-deficiency HBS defect are able to have a successful pregnancy even without AT replacement during pregnancy. Vitamin K antagonists seem to be superior compared to LMWH in these specific patients. Alguel G, Jochmans K, Simanek R, Quehenberger P, Langer M, Pabinger I. SUCCESSFUL PREGNANCY OUTCOME IN A WOMAN WITH HOMOZYGOUS ANTITHROMBIN DEFICIENCY (99 LEUPHE MUTATION). J Thromb Haemost 2007; 5 Supplement 2: P-S-083 Date: Sunday, July 8, 2007 Session Info: Poster: Protein C, Protein S, thrombomodulin Room: Exhibition Area file:///E|/working/jaymehra/Poster%20Protein%20C%20Protein%20S%20thrombomodulin/P-S-083.htm [1/16/2014 7:06:35 PM] Abstract ISTH07A1_P-S-084: Contact ViewISTH07A1_P-S-084: Contact View [P-S-084] ACTIVATED PROTEIN C HAS BOTH INTRACELLULAR AND EXTRACELLULAR EFFECTS ON BREAST CANCER AND ENDOTHELIAL CELL MIGRATION L.M. Beaulieu, M. Gramling, F.C. Church. Pathology and Laboratory Medicine, University of North Carolina Chapel Hill, Chapel Hill, NC, United States Introduction: Activated protein C (APC) is an anti-coagulant serine protease that regulates hemostasis. APC has other functions related to inflammation, proliferation, cell death, and migration. Methods: Using breast cancer and endothelial cells, we are investigating the role of APC in cell migration and invasion. For the breast cancer studies, we used the MDA-MB-231 cells in the transwell invasion, which was coated with Matrigel, and chemotaxis assay. Endothelial cells, specifically HUVEC, were used in the transwell invasion and chemotaxis assay and in the in vitro tube formation assay. Results: Treatment of cancer cells with APC increased migration in a concentration dependent manner. APC also increased endothelial cell migration and vessel formation but this effect occurred at lower concentrations. Only the active form of APC increased migration of endothelial and cancer cells. APC only affected migration when plated with the cells; thus APC is not a chemoattractant. APC-promoted cancer cell migration is attenuated in the presence of blocking antibodies to both endothelial protein C receptor (EPCR) and protease activated receptor-1 (PAR-1). Treatment with a proliferation inhibitor (Na butyrate) showed APC did not promote migration by increasing cell number. Cells treated with signal protein inhibitors showed APC increased migration through MAPK and PI3 kinase pathways, but not JNK pathway. Using blocking antibodies to uPA or PAI-1 did not alter the effects of APC to increase breast cancer cell invasion. However, using inhibitors to matrix metalloproteases (MMP)-2, -9, there was a loss of the effects on APC on invasion. Conclusions: Therefore, APC promotes cell migration by binding EPCR and PAR-1 and activating MAPK and PI3 kinase pathways intracellularly, while also interacting with MMP-2 and -9 extracellularly. Beaulieu LM, Gramling M, Church FC. ACTIVATED PROTEIN C HAS BOTH INTRACELLULAR AND EXTRACELLULAR EFFECTS ON BREAST CANCER AND ENDOTHELIAL CELL MIGRATION. J Thromb Haemost 2007; 5 Supplement 2: P-S-084 Date: Sunday, July 8, 2007 Session Info: Poster: Protein C, Protein S, thrombomodulin Room: Exhibition Area file:///E|/working/jaymehra/Poster%20Protein%20C%20Protein%20S%20thrombomodulin/P-S-084.htm [1/16/2014 7:06:35 PM] Abstract ISTH07A1_P-S-085: Contact ViewISTH07A1_P-S-085: Contact View [P-S-085] EVIDENCE OF PROTEIN C (PC) GLA DOMAIN AMINO ACIDS CRUCIAL FOR INTERACTION WITH PROTEIN S (PS) F. Saller, S. Gandrille, B. Villoutreix, T. Kaabache, M. Aiach, J. Emmerich, D. Borgel. Inserm U765, Université Paris 5, paris, France Introduction: We have previously shown that the PS Gla domain was involved in interaction with activated protein C (APC). In the present study, we used computational methods to delineate a face (R3) of solvent-exposed residues (N33, D35 and L38) in the PC Gla domain that is potentially involved in interaction with PS. Methods: Three chimeras between PC and prothrombin (FII) were stably expressed in HEK293 cells. In GlaFII-PC, the PC Gla domain was substituted by that of FII. In a second chimera, R3FIIPC, the three R3 residues of the PC Gla domain were replaced by the corresponding residues of FII. In R3PC-GlaFII-PC, the PC R3 residues were finally reintroduced into the Gla domain of the GlaFIIPC chimera. All chimeras were tested for their binding to phospholipids and their PS-dependent anticoagulant activity using an APTT-based assay. Results: GlaFII-PC, R3PC-GlaFII-PC and R3FII-PC bound to phospholipids with comparable affinities (Kd app=53, 18, and 26 nM, respectively) when compared to wild-type PC (Kd app=57nM). All activated chimeras expressed, at least, the same anticoagulant activity as wild-type APC. The ability of PS to exert its cofactor effect to the activated chimeras was then evaluated by measuring a ratio between the clotting time with and without PS (ratio=2.96 for wild-type APC). As previously reported, the anticoagulant activity of GlaFII-APC was completely independent of PS (ratio=1.03). Similarly, the anticoagulant activity of R3FII-GlaPC-APC is insensitive to PS (ratio=1.13). Interestingly, the anticoagulant activity of R3PC-GlaFII-APC significantly recovered (more than 65%) dependence on PS (ratio=2.04). Conclusions: Our results provide a direct and strong evidence for an implication of PC Gla residues N33, D35 and L38 in interaction with PS. These residues are part of a PC region that has been proposed to be involved in interaction with PS in a previous study. Saller F, Gandrille S, Villoutreix B, Kaabache T, Aiach M, Emmerich J, Borgel D. EVIDENCE OF PROTEIN C (PC) GLA DOMAIN AMINO ACIDS CRUCIAL FOR INTERACTION WITH PROTEIN S (PS). J Thromb Haemost 2007; 5 Supplement 2: P-S-085 Date: Sunday, July 8, 2007 Session Info: Poster: Protein C, Protein S, thrombomodulin Room: Exhibition Area file:///E|/working/jaymehra/Poster%20Protein%20C%20Protein%20S%20thrombomodulin/P-S-085.htm [1/16/2014 7:06:36 PM] Abstract ISTH07A1_P-S-086: Contact ViewISTH07A1_P-S-086: Contact View [P-S-086] SELECTIVE ENHANCEMENT OF PROTEIN S REAGENT SENSITIVITY BY BOVINE GAMMA GLOBULIN Z. Cao, R. Bottenus, O. Safa, J. Vasquez, M. Triscott. Research and Development, Instrumentation Laboratory, Orangeburg, NY, United States Introduction: Bovine serum albumin (BSA) and bovine gamma globulin (BGG) are two commonly used carrier proteins in reagent manufacturing. New findings indicate that these proteins not only stabilize the products, but may also play important roles in the product activity. We investigated the role BGG plays in the enhancement of protein S reagent sensitivity. Methods: Synthetic phospholipids (sPL) and recombinant tissue factor (rTF) were used to formulate protein S (ProS) reagent in the presence or absence of BGG with cations added at different stages of formulation. Two sPL blends were prepared, a 20%Phosphatidyl Serine (PS) blend, and a 34%PS blend. These were used to formulate a protein S reagent, similar to the one used for the HemosIL ProS assay. Testing was performed using the reagents with or without activated protein C (APC) on the ACL Futura. Phospholipid vesicle sizes were compared using a Submicron Particle Sizer, model NICOMP 380. Results: We found that BGG does not influence the sensitivity of ProS reagent when the 20%PS lipid blend was used in reagent formulation. However, BGG selectively enhances protein S sensitivity when the 34%PS was used. The effect of BGG on the sensitivity of protein S assay can be regulated by switching the order of cation addition when the 34%PS blend liposomes are used for reagent formulation. When sPL and rTF were blended into buffer containing BGG and cations, no enhancement from BGG was observed; however, if sPL and rTF were blended into BGG-containing buffer and cations were added afterwards, the sensitivity of ProS reagent was dramatically enhanced. Our studies suggest that such enhancement in protein S sensitivity might be correlated with liposomal structures and vesicle sizes. Conclusions: BGG enhances protein S reagent sensitivity, but such augmentation depends on the content of sPL and the formula