diamide and Thrombosis

CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca2+-dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG-GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca2+ mobilisation and protein kinase C activation. PAO also prevented the Ca2+ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein system, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca2+-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.

Topics: Arsenicals; Blood Platelets; Diamide; Dimercaprol; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Molecular Targeted Therapy; Oxidation-Reduction; Platelet Aggregation; Protein Binding; rap1 GTP-Binding Proteins; Thrombin; Thrombosis; Toluene

Intravascular thrombus formed under low shear conditions consists of red cells enmeshed within a fibrin network. Since red cells reduce the permeability of fibrin network by surface drag and by volume occupancy the significance of red cell aggregability and deformability in network permeability needs examination. In this study networks were developed by the addition of thrombin to washed red cells suspended in platelet free plasma. The effects of the polymers polyvinylpyrrolidone (PVP) and poloxamer 188 on network permeability were compared to gauge the influence of red cell aggregation. Both polymers increase network permeability by an action on fibrin polymerisation but PVP alone enhances red cell aggregation. PVP was found to increase network permeability significantly both by increasing the permeability of the fibrin component of the network and by increasing red cell aggregation and thus reducing red cell surface drag. In separate experiments red cells were pre-treated with heat, glutaraldehyde, or diamide to reduce cell deformability. Decreased cell deformability caused significant reductions in network permeability. This was ascribed to the reduced aggregability of hardened red cells. Red cell aggregation during coagulation enhances molecular transport through modifying the network. This may have implications for the penetration of fibrinolytic agents.

Topics: Blood Coagulation; Blood Viscosity; Diamide; Erythrocyte Aggregation; Erythrocyte Deformability; Fibrin; Glutaral; Hot Temperature; Humans; Poloxalene; Povidone; Thrombosis