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ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

ABSTRACT: Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is not dependent on ADP. Human fibrinogen inhibited in a competitive manner the ADP-induced binding of 125I-labeled vWF (9 micrograms/ml) with an IC50 of 25 micrograms/ml. Conversely, unlabeled vWF inhibited ADP-induced binding of 125I-labeled fibrinogen (60 micrograms/ml) with an IC50 of 16 micrograms/ml. A synthetic dodecapeptide (Mr, 1188), analogous with the specific platelet receptor recognition site of human fibrinogen gamma chain (gamma 400-411), inhibited binding of both 125I-labeled vWF and 125I-labeled fibrinogen to ADP-treated platelets, whereas it was without effect on binding of 125I-labeled vWF to ristocetin-treated platelets. These data indicate that vWF and fibrinogen have a common receptor mechanism for their interaction with human platelets that is dependent on ADP occupancy of its binding sites and is recognized by the sequence of 12 amino acid residues at the carboxyl terminus of the human fibrinogen gamma chain.

SUBMITTER: Timmons S

PROVIDER: S-EPMC391607 | biostudies-literature | 1984 Aug

REPOSITORIES: biostudies-literature

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ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

Timmons S S Kloczewiak M M Hawiger J J

Proceedings of the National Academy of Sciences of the United States of America 19840801 15

Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by pro ...[more]

PMID: 6087354

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Project description:Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.

Project description:Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress. SUMMARY:Objective When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by VWF, secreted vWbp and bacterial ClfA anchors S. aureus to vascular endothelium under shear stress.

Dataset Information

ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

Publications

ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

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