The vasoactive compound bradykinin (BK) is liberated by proteolytic cleavage from high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Expression of kininogens on cell surface receptors may affect the delivery of BK at sites of inflammation. Therefore, we investigated whether BK itself alters the expression of binding sites for its parent molecules, HK and LK, on the surface of cultured human umbilical vein endothelial cells (HUVEC). 125I-LK and 125I-HK each bind to a single class of sites on HUVEC in reactions that are saturable, reversible, and zinc-dependent (Bmax = 9.7 +/- 0.2 x 10(5) sites/cell; kd = 43.3 +/- 8 nmol/L; n = 5 and Bmax = 10.3 +/- 0.4 x 10(5) sites/cell; kd = 40.3 +/- 0.9 nmol/L; n = 3 for LK and HK, respectively). HK and LK compete for the same binding site (Ki = 19.4 +/- 5 nmol/L HK v 125I-LK; Ki = 24.5 +/- 4 nmol/L LK v 125I-HK, n = 3). Moreover, 50-fold molar excess light chain of HK inhibits 125I-LK binding 51% and 50-fold molar excess LK and the heavy chain of HK inhibit 125I-light chain of HK binding 92% and 76%, respectively. Preincubation of HUVEC with BK produces a transient, concentration- dependent increase in the binding of HK and LK, reaching a maximum 3 to 4 hours after addition of BK (46% increase over control for HK; 57% increase over control for LK; P < .005 for each ligand). Des-Arg9- bradykinin, a B1 receptor agonist, increases kininogen binding to the same extent as BK; the upregulation of kininogen binding sites by BK is partially blocked by a B1 but not by a B2 receptor antagonist. The protein kinase C inhibitors (PKC), sphingosine and H7, completely block the induction of HK receptors by BK. Phorbol 12-myristate 13-acetate (PMA), which also activates PKC, stimulates the binding of HK and the purified light chain of HK to HUVEC as well. However, unlike HK and its light chain, binding of LK and the heavy chain of HK are increased by PMA only in the presence of added calcium ion. These studies show that BK upregulates a common binding site for HK, LK, and each chain of HK on HUVEC. Induction of kininogen receptors on endothelial cells by BK may modulate the generation of this vasoactive compound at sites of vascular injury.

mbH (Stuttgart) 74 (6) 1604-12 (1995) thereafter. They suggested that a condition of rebound hypercoagula bility could occur in these patients, especially one week after interrup tion when prothrombin complex factors are already back to normal values but factor VIII remains high. The recorded changes in factor VIII:C levels after anticoagulation withdrawal are not easy to explain. One possible, though as yet un proven, explanation of this decreasing trend during the first weeks may be a transient consumption/degradation of factor VIII:C. Such a hypo thesis would seem to be corroborated by the presence of an inverse correlation between factor VIII:C levels and increased F l+ 2 values. We may surmise that, when anticoagulants are withdrawn, a complete prothrombin factor recovery (occurring in less than 7 days) associated with high factor VIII levels may trigger activation of blood coagulation, thus producing increased hypercoagulability marker levels and consumption/ degradation of factor VIII. Such a condition of hyper coagulability is usually transient and self-limiting (1). We believe that the time period for (and perhaps mode of) OAT withdrawal deserves further laboratory and clinical investigation with a view to better understanding clotting balance changes and, if possible, identifying early those patients at particularly high risk of thrombosis recurrence.

The processes of implantation and placentation are both dependent on the invasion and remodeling of the uterine endometrium and vasculature by trophoblasts. Because the secretion and autocrine binding of urokinase (uPA) appears to be a common mechanism used by cells to facilitate plasmin-dependent tissue invasion, we measured the production of uPA and expression of uPA receptors by trophoblasts. Prourokinase bound specifically, reversibly, and with high affinity to cultured trophoblasts, via the uPA epidermal growth factor-like domain. Trophoblasts derived from two first-trimester placentae bound more prourokinase than cells isolated from term placentae. Furthermore, in vitro differentiation of cultured cytotrophoblasts into syncytiotrophoblasts was associated with diminished expression of urokinase receptors and a parallel decrease in the cellular content of uPA receptor mRNA. Trophoblasts also secreted prourokinase and plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). Although prourokinase was secreted in amounts sufficient to endogenously saturate trophoblast uPA receptors, trophoblasts secreted greater amounts of PAI-1 and PAI-2 than uPA, and no net plasminogen activator activity was detected in trophoblast conditioned medium. In contrast, plasminogen added directly to cultured trophoblasts was readily converted to plasmin. Although the invasion and remodeling of uterine tissues by trophoblasts is a complex process dependent on several proteases of varying specificity, our findings suggest that the expression and modulation of urokinase receptors on the trophoblast cell surface may play an important role in this process.

Despite intensive effort, no effective pharmacological inhibitors of the Ras oncoprotein have been reached the clinic given the impression that Ras proteins are undruggable. However, progresses have been made in several directions to manipulate Ras.In this manuscript, we describe a novel and promising approach for specifically targeting the Ras/Raf interaction. We have identified the amino acid sequence involved in the binding of Ras to Raf. The amino acids of the binding site were coupled to an optimized penetrating peptide in order to generate a chimeric peptide, Mut3DPT-Ras, able to penetrate the cells and target Ras/Raf interaction. We demonstrate that this protease-resistant peptide has an in vitro apoptotic effect on several cancer cell lines and on primary cells isolated from chronic lymphocytic leukemia (CLL) as well as an antitumoral effect on chronic lymphocytic leukemialike and lymphoma xenograft model. The new generated peptide allows the modulation of the Ras/Raf interaction and might have a potential as a new therapeutic approach for cancer treatment.

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