scholarly journals Binding of fibronectin to alpha-granule-deficient platelets.

1983 ◽  
Vol 97 (2) ◽  
pp. 571-573 ◽  
Author(s):  
M H Ginsberg ◽  
J D Wencel ◽  
J G White ◽  
E F Plow
Keyword(s):  
Surface Expression ◽  
Plasma Fibronectin ◽  
Platelet Factor ◽  
Fibronectin Receptor ◽  
Platelet Binding ◽  
Protein Functions ◽  
Fibronectin Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Molecular Nature

Most of the proposed functions for fibronectin involve its interaction with cells, yet the molecular nature of cellular fibronectin binding site(s) has remained obscure. Thrombin induces saturable platelet binding sites for plasma fibronectin and concurrently stimulates surface expression of a number of platelet alpha-granule constituents including thrombospondin and fibrin which are known to interact with fibronectin. To test the hypothesis that these (or other alpha-granule proteins) mediate plasma fibronectin binding, we used platelets of patients with the Gray Platelet Syndrome. These cells were deficient in thrombospondin, beta-thromboglobulin, platelet factor 4, fibronectin, and fibrinogen as measured in radioimmunoassay. They also had reduced von Willebrand factor content as judged by immunofluorescence. At plasma fibronectin inputs from 0.03 to 3 times the apparent kilodalton, these Gray platelets bound virtually identical quantities of fibronectin as normal cells. Thus, platelets containing 1,500 molecules of thrombospondin per platelet could bind more than 100,000 molecules of plasma fibronectin per cell following thrombin stimulation. These data preclude any simple model in which newly surface expressed thrombospondin (or other alpha-granule protein) functions as the major thrombin-stimulated plasma fibronectin receptor in this cell type.

scholarly journals Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2016-2021 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Von Willebrand Factor ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Platelet Surface ◽  
Granule Secretion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Shape ◽  
Stimulation Of

Abstract Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.

scholarly journals Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2736-2744 ◽  
Author(s):  
John H. Cleator ◽  
Wen Qin Zhu ◽  
Douglas E. Vaughan ◽  
Heidi E. Hamm
Keyword(s):  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Differential Regulation ◽  
Cell Surface Expression ◽  
Protease Activated Receptors ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links protease-activated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca2+ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Dose-response studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP–induced robust stimulation of intracellular Ca2+ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca2+-independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A led to a significant attenuation of PAR2-AP–mediated release of VWF. Confocal microscopy studies revealed that PAR2 and forskolin caused preferential release of a population of Weibel-Palade bodies (WPBs) consisting of only VWF. Thus, WPBs are pharmacologically and morphologically heterogeneous, and distinct granule populations are susceptible to differential regulation.

scholarly journals von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

Studies of α-granule proteins in cultured human megakaryocytes

2003 ◽  
Vol 90 (11) ◽  
pp. 844-852 ◽  
Author(s):  
Dragoslava Veljkovic ◽  
Elisabeth Cramer ◽  
Gulie Alimardani ◽  
Serge Fichelson ◽  
Jean-Marc Massé ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Protein Processing ◽  
Platelet Factor ◽  
Protein Storage ◽  
Granule Protein ◽  
Granule Proteins ◽  
Processing And Storage ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

Summaryα-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized α-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. α-Granule protein storage in vitrohas been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34+cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal α-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the α-granule proteins multimerin, von Willebrand factor, thrombospondin-1, β-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the α-granule protein in megakaryocyte cultures was contained within the cells (averages: 41 – 71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that α-granule proteins were processed to mature forms and were predominantly located in the α-granules of cultured megakaryocytes. Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying α-granule protein processing and storage.

scholarly journals Comparison of von Willebrand factor platelet‐binding activity assays: ELISA overreads type 2B with loss of HMW multimers

2020 ◽  
Vol 18 (10) ◽  
pp. 2513-2523
Author(s):  
Attila Szederjesi ◽  
Luciano Baronciani ◽  
Ulrich Budde ◽  
Giancarlo Castaman ◽  
Paola Colpani ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Activity ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3562-3573 ◽  
Author(s):  
AD Michelson ◽  
SE Benoit ◽  
MH Kroll ◽  
JM Li ◽  
MJ Rohrer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Surface Expression ◽  
Platelet Surface ◽  
Kinase C ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

scholarly journals Neutrophil cathepsin G modulates the platelet surface expression of the glycoprotein (GP) Ib-IX complex by proteolysis of the von Willebrand factor binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 158-168 ◽  
Author(s):  
CA LaRosa ◽  
MJ Rohrer ◽  
SE Benoit ◽  
MR Barnard ◽  
AD Michelson
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Cytochalasin B ◽  
Surface Expression ◽  
Cathepsin G ◽  
Prostaglandin I2 ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Content

Abstract The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb- IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin- induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed against the macroglycopeptide portion of GPIb alpha). In contrast to its proteolytic effect on GPIb alpha, the cathepsin G-induced decrease in platelet surface GPIX and the remnant of the GPIb-IX complex (defined by MoAbs FMC25 and AK1) was via a cytoskeletal-mediated redistribution, as determined by lack of change in the total platelet content of GPIX and the GPIb-IX complex; complete inhibition by cytochalasin B, prostaglandin I2, and prior fixation of platelets. Experiments with Serratia protease-treated and Bernard- Soulier platelets showed that neither platelet surface GPIb nor cathepsin G-induced proteolysis of GPIb were required for the cathepsin G-induced redistribution of the remnant of the GPIb-IX complex or the cathepsin G-induced increase in platelet surface P-selectin. In summary, neutrophil cathepsin G modulates the platelet surface expression of the GPIb-IX complex both by proteolysis of the vWF binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex. Prior studies show that, although thrombospondin 1, antiserine proteases, and plasma are all inhibitors of cathepsin G, the effects of cathepsin G on platelets, including an increase in surface GPIIb-IIIa, occur during close contact between neutrophils and platelets in a protective microenvironment (eg, thrombosis and local inflammation).(ABSTRACT TRUNCATED AT 400 WORDS).

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