ALBERT

Description: Introduction Hepatic veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) are important and serious thrombotic complications after hematopoietic stem cell transplantation (HSCT), but the early diagnosis remains difficult since their clinical manifestations are similar to those of other transplantation-related complications. Our aim in this study is to illustrate the early alteration of hemostatic parameters in recipients of hematopoietic stem cell transplantation and then determine its value in transplantation-related thrombotic complications and other post-HSCT clinical settings, such as acute graft-versus-host disease (aGVHD) and infection. Methods Plasma from 95 patients undergoing HSCT was collected prior to conditioning therapy and then weekly until five weeks after HSCT. Hemostatic parameters were evaluated prospectively in our institution. 1. Plasminogen activator inhibitor-1(PAI-1), tissue-plasminogen activator(t-PA), protein C(PC), von Willebrand factor(vWF)and thrombomodulin(TM)were investigated by enzymimmunoassay. Other hemostatic parameters such as activated partial thromboplastin time(APTT), prothrombin time(PT), fibrinogen (Fg), antithrombin III(AT III) and D-dimer(D-Di) were measured with hemagglutinin equipment in the same time. 2. According to the different settings after transplantation, three groups of transplant associated complications were classified as thrombus group (VOD n=5, TMA n=1), aGVHD group (n=29) and infection group (n=19). Systemic analyses were carried out for the hemostatic parameters and transplantation-related thrombotic complications or other clinical settings. Results Significant increase was observed in the levels of fibrinogen, t-PA and PAI-1 after transplant, while Protein C and ATIII decreased significantly(P0.05). All the patients with three different complications presented with significantly increased PAI-1 and lower level of Protein C compared with those who had no complication (P0.05). However, 6 patients with thrombotic complications (VOD5, TMA1) extremely showed elevated PAI-1 levels after the clinical onset of thrombotic complications by comparison with highest post-HSCT values in the aGVHD patients or infection patients (P

Description: Patients with myeloproliferative disorders (MPD) are at a high risk of developing thrombotic events. We hypothesize that one of the contributory factors to this thrombotic tendency is the involvement of vascular endothelial cells (EC) by the malignant process. In vitro and in vivo assays were used to determine the involvement of EC in patients with MPD. Endothelial progenitor cells (EPC) were assayed from the peripheral blood (PB) mononuclear cells (MNCs) of 3 normal controls (NC) and 16 patients with MPD (12 polycythemia vera (PV), 4 primary myelofibrosis (PMF). MNC were cultured for 2 days in EC growth media on fibronectin(FN)-coated plates. The non-adherent cells were then harvested and transferred to a secondary FN-coated plate for additional 5–14 days. EC colonies were identified by their morphological appearance. The colonies were plucked and analyzed for PECAM-1(CD31), VE-Cadherin(CD144), VEGFR-2, vWF, Endoglin(CD105), ULEX-1, CD45, CD14 by flow cytometry and acetylated LDL(Ac-ADL) uptake. EC colonies were CD31+CD144+VEGFR2+ULEX-1+vWF+CD105+CD45+CD14+ and capable of taking up Ac-LDL and when exposed to TNF-α and IL-1β, expressing ICAM(CD54) and E-selectin(CD62e). MPD MNC formed fewer numbers of EC colonies than normal MNC (31.1±34.2 vs 78.8±28.9; p

Description: Object In order to compare the functions of protein kinase C (PKC) and calcium (Ca2+) in platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of Gq signal transmission pathway in the course of thrombin receptors activation. Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet at different time point (0, 1, 2, 5, 10, 30min), then the alterations of platelet aggregation and GPIb were analyzed in the involvement of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator). Results Either PAR1 or PAR4 peptide can induce absolute platelet aggregation, together with a reversible internalization of GPIb. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA/AM while the shape change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreases GPIb centralisation upon PAR1 stimulation (P

Description: Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.