ABSTRACT
It has been shown that ß(2) -glycoprotein I (ß(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that ß(2) GPI might play a role in TTP. We found that ß(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that ß(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of ß(2) GPI to VWF by surface plasmon resonance, and determined that domain I of ß(2) GPI is the binding site of VWF A1 domain. Adhesion of ß(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that ß(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that ß(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.
Subject(s)
Purpura, Thrombotic Thrombocytopenic/blood , beta 2-Glycoprotein I/blood , Antibodies, Monoclonal, Murine-Derived/immunology , Blood Platelets/drug effects , Blood Platelets/metabolism , Blood Platelets/pathology , Case-Control Studies , Cells, Cultured , Erythrocytes/metabolism , Erythrocytes/pathology , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Platelet Aggregation , Ristocetin/pharmacology , beta 2-Glycoprotein I/antagonists & inhibitors , beta 2-Glycoprotein I/immunology , von Willebrand Factor/metabolismABSTRACT
Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis.
Subject(s)
Endothelial Cells/chemistry , Insulin-Like Growth Factor Binding Proteins/chemistry , Proteomics/methods , Weibel-Palade Bodies/chemistry , Endothelial Cells/physiology , Exocytosis , Genetic Vectors , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Immunohistochemistry , Mass Spectrometry , Neovascularization, Physiologic , P-Selectin/chemistry , Protein Structure, Tertiary , Protein Transport , Transfection , Weibel-Palade Bodies/physiology , von Willebrand Factor/chemistryABSTRACT
The natural anticoagulant protein S contains a so-called thrombin- sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro . The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.
