Novel soluble Flt-1 isoforms in plasma and cultured placental explants from normotensive pregnant and preeclamptic women.

Pregnant women who develop preeclampsia exhibit higher circulating levels of the soluble VEGF receptor-1 (sFlt-1). Recent findings suggest that soluble Flt-1 may contribute to the pathogenesis of preeclampsia by binding and neutralizing vascular endothelial growth factors (VEGF) and placental growth factor (PlGF). Existing literature identifies sFlt-1 as a 100 kDa glycoprotein, a product of an mRNA splice variant. We hypothesized that sFlt-1 expression may be more complex with multiple variants of sFlt-1 as well as multiple sources during normal pregnancy and preeclampsia. Using a combination of affinity purification of sFlt-1 by heparin-agarose and epitope specific antibodies, we performed Western blot analysis with epitope specific antibodies for sFlt-1. Plasma of preeclamptic women exhibits significantly higher amounts of a novel 145 kDa variant of sFlt-1, along with the 100 kDa isoform. We identified sFlt-1 variants in the conditioned medium from placental explant cultures that are hypoxia responsive with varying sizes, including 185, 145,100 and 60 kDa forms, as well as antigenicity. The 145 kDa was similar in antigenicity to the 100 kDa found in plasma whereas the 185 and 60 kDa sFlt-1 demonstrated different epitopes. Deglycosylation studies also confirm that there are multiple sFlt-1 polypeptides. Co-immunoprecipitation with VEGF suggests that these different sFlt isoforms can bind VEGF and therefore, may be of functional importance. Finally, comparison of sFlt-1 in the conditioned medium obtained from cultured cytotrophoblasts, peripheral blood mononuclear cells (PBMCs) and human uterine microvascular cells (HUtMVECs) exhibit mainly the100 kDa sFlt-1. Collectively these data suggest the presence of multiple isoforms of sFlt-1 in the circulation of women with preeclampsia as well as in uncomplicated pregnancies and the possibility of multiple sources. Placental hypoxia may contribute to sFlt-1 over expression but other regulatory mechanisms cannot be ruled out.

Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis.

Glycoprotein (GP) IIb/IIIa is pivotal in homotypic platelet aggregation and may also be involved in the heterotypic adhesion of leukocytes and tumor cells to platelets. This study was primarily undertaken to compare the antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist, XV454, to that of abciximab in 2 flow models of platelet thrombus formation: (1) direct shear-induced platelet aggregation imposed by a cone-and-plate rheometer and (2) platelet adhesion onto von Willebrand factor (vWF)/collagen I followed by aggregation in a perfusion system. XV454 inhibited platelet aggregation in a concentration-dependent manner in both experimental models. Maximal inhibition of aggregation was achieved by XV454 at approximately 70% receptor occupancy, which is lower than the >/=85% previously reported for abciximab. At similar levels of receptor blockade (approximately 45%), XV454 appeared to be relatively more effective than abciximab in suppressing platelet aggregation. Neither XV454 nor abciximab inhibited platelet adhesion to collagen. Pretreatment of surface-adherent platelets with either XV454 or abciximab inhibited the attachment of monocytic THP-1 cells under flow. In contrast, the rapidly reversible GPIIb/IIIa inhibitor orbofiban failed to suppress these heterotypic interactions. These findings demonstrate that XV454 is a potent GPIIb/IIIa antagonist with a long receptor-bound lifetime like abciximab and may be beneficial for the treatment/prevention of thrombotic complications.