Abstract-On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin. T hrombin is a multifunctional serine protease generated at sites of vascular injury. Thrombin plays a key role in blood coagulation and thrombotic disorders. It acts as the central enzyme of the coagulation cascade by cleaving fibrinogen into fibrin and favoring its own production by activating several coagulation factors by limited proteolysis. Thrombin also regulates its own formation after binding to thrombomodulin (TM), an integral membrane-bound glycoprotein expressed on endothelial cells. TM acts as a cofactor of thrombin to activate protein C, a serine protease ensuring proteolytic inactivation of 2 coagulation factors, factor Va and factor VIIIa. Thrombin also interacts with a variety of cells mediating inflammatory and proliferative responses to vascular injury. 1 For all protein and cellular interactions, thrombin has a recognition site and a catalytic active site. By the former, called the anion-binding exosite (ABE1), thrombin binds to specific negatively charged sequences. By the catalytic site, thrombin exerts its proteolytic activity. 2 On vascular endothelial cells, thrombin ABE1 binds to TM. This binding occurs through the epithelial growth factor (EGF)-like domains 4 and 5 of TM. 3 Thrombin ABE1 also binds to the typical heptahelical thrombin receptor, the first member of pr...

Volume 39, September 1999 TRANSFUSION 951 BACKGROUND: The effect on platelets of two standard methods of platelet concentrate (PC) preparation was studied by flow cytometry. The findings were correlated with those obtained in an experimental in vitro perfusion model. STUDY DESIGN AND METHODS: PCs were prepared from whole blood by the platelet-rich plasma (PRP) or buffy coat (BC) method and placed on a flatbed platelet agitator at 22°C for up to 5 days. Platelet glycoproteins (GP)Ibα, GPIIb/IIIa, and GPIV, p-selectin and lysosomal integral membrane protein, and the binding of von Willebrand factor, fibrinogen, fibronectin, and coagulation factor Va were measured with the corresponding specific conjugated antibodies. Perfusions were carried out in an annular chamber with citrated blood depleted of platelets and white cells by filtration, to which samples from PCs were added. RESULTS: PRP-PC production provoked intense platelet activation. In contrast, in BC-derived PCs, platelet activation was milder, and only a significant increase in bound fibrinogen was seen. After 1 day of storage, differences between the methods that had been observed immediately after separation had almost disappeared. During the remaining storage period, increases in activation-dependent antigens and in procoagulant activity were measured. Of the studied platelet GPs, only GPIIb/ IIIa decreased by 25 percent in PRP-PCs. Differences in covered surface were not significant in perfusion studies performed on Day 0 and after 5 days of storage in PRP-PCs (26.8 ± 6.9 vs. 20.5 ± 5.8) or BC-PCs (23.8 ± 11 vs. 24.8 ± 10.2). CONCLUSION: Platelet activation occurred during the separation and storage of PCs prepared by both methods, and it was higher in PRP-PCs only in samples obtained immediately after preparation. Despite these changes, platelet adhesive and cohesive functions were similar in both types of PCs and remained basically unchanged after storage. ABBREVIATIONS: BC = buffy coat; FITC = fluorescein isothiocyanate; GP(s) = glycoprotein(s); LIMP = lysosomal integral membrane protein; MFI = mean fluorescence intensity; MoAb(s) = monoclonal antibody(ies); PC(s) = platelet concentrate(s); PE = phycoerythrin; PRP = platelet-rich plasma; vWF = von Willebrand factor.From the

These data indirectly suggest that the stress induced by the preparation method has an activating effect on platelet function that may imply a delayed platelet response to further stimuli. This effect may result in a deficient redistribution of signaling molecules within platelets.

Our present data support the concept that repeated platelet stress during hemodialysis has a deleterious effect on the organization of platelet cytoskeleton, which seems to impair the translocation of signal transduction proteins within platelets compromising the platelet function in uremia.