Search

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.

Calyculin A (CLA) and okadaic acid (OA), specific and potent inhibitors of protein phosphatase 1/2A, inhibit platelet aggregation. However, their inhibitory mechanisms remain unknown. We investigated the effects of CLA on the exposure of fibrinogen receptor in thrombin-stimulated platelets, using flow cytometry with a monoclonal antibody against the fibrinogen receptor of activated glycoprotein (Gp)IIb/IIIa complex (PAC-1). CLA inhibited the exposure of fibrinogen receptor in a dose related manner when added either before or 3 min after thrombin stimulation. In contrast, CLA had no significant effect when the expression of GpIIb/IIIa complex was examined in resting platelets, using a monoclonal antibody recognizing non-activated GpIIb/IIIa complex (NNKY1-32). These results suggest that protein phosphatase 1/2A may be directly involved in the exposure of platelet fibrinogen receptor.

Abstract: Urokinase-type plasminogen activator (u-PA), which cleaves plasminogen to yield plasmin, is a serine protease of fibrinolysis and is presumed to play a key role in extracellular proteolysis and facilitate the migration of cancer cells. This study was conducted prospectively to evaluate the prognostic significance of u-PA antigen level in breast cancer tissues. u-PA concentrations in the cytosol of 226 breast cancer tissues were determined prospectively by enzyme-linked immunosorbent assay using cytosol fractions prepared for steroid hormone assay. The median follow-up period of the patients was 60 months. Various prognostic factors were evaluated by univariate analysis or multivariate analysis using the Cox proportional-hazards method. Patients with primary breast cancer containing high levels of u-PA had a significantly shorter disease-free survival than patients with low levels of u-PA antigens. In multivariate analysis, a high level of u-PA was an independent risk factor for disease-free survival, being independent of age, axillary node status, and estrogen receptor status. Among the major prognostic factors, a high u-PA antigen level, lymph node involvement, and a positive estrogen receptor status were the most important for predicting relapse-free survival (P=0.044, P<0.0001, P=0.0039). This first prospective study confirmed the prognostic significance of the u-PA antigen level in association with other major prognostic factors. The results of our present study suggest that u-PA in breast cancer tissue might be involved in breast cancer invasion and metastasis.

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.