scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366 ◽  
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3322-3328 ◽  
Author(s):  
Patrick André ◽  
Cécile V. Denis ◽  
Jerry Ware ◽  
Simin Saffaripour ◽  
Richard O. Hynes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Glycoprotein ◽  
Ionophore A23187 ◽  
Stop And Go ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Low Shear

Abstract With the use of intravital microscopy, a new type of platelet–endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds−1) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm2·s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded withA23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in β3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet–endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibα (GPIbα) because the adhesion of murine platelets expressing exclusively the human GPIbα could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbα. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbα. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.

scholarly journals Multimeric composition of endothelial cell-derived von Willebrand factor

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2074-2076 ◽  
Author(s):  
HM Tsai ◽  
RL Nagel ◽  
VB Hatcher ◽  
II Sussman
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Culture Media ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Buffer System ◽  
Ionophore A23187 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3322-3328 ◽  
Author(s):  
Patrick André ◽  
Cécile V. Denis ◽  
Jerry Ware ◽  
Simin Saffaripour ◽  
Richard O. Hynes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Glycoprotein ◽  
Ionophore A23187 ◽  
Stop And Go ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Low Shear

With the use of intravital microscopy, a new type of platelet–endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds−1) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm2·s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded withA23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in β3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet–endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibα (GPIbα) because the adhesion of murine platelets expressing exclusively the human GPIbα could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbα. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbα. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.

scholarly journals Relationship between human development and disappearance of unusually large von Willebrand factor multimers from plasma

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1851-1858 ◽  
Author(s):  
JA Katz ◽  
JL Moake ◽  
PD McPherson ◽  
MJ Weinstein ◽  
KJ Moise ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Gestational Age ◽  
Calcium Ionophore ◽  
Sampling Period ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Control Mechanisms ◽  
Older Children ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF. There was no significant relationship between either gestational age at time of delivery or birth weight and likelihood of finding ULvWF multimers in cord PPP samples. No maternal PPP sample contained ULvWF multimers. Serial heelstick samples from 16 preterm and term neonates were analyzed for 8 weeks. ULvWF multimers disappeared from the PPP of ten of the neonates during this time. The PPP of four neonates had vWF patterns similar to those in normal adult PPP throughout the sampling period. The ULvWF multimeric forms of fetal and neonatal PPP samples were similar to those constitutively released from endothelial cells. They were not as slowly migrating in a very porous 0.5% agarose gel system as the ULvWF multimers released from Weibel-Palade bodies in response to the calcium ionophore A23187. A vWF protomer was present in 97% of fetal samples, 83% of cord blood specimens, and 11% of neonatal heelstick samples, but was not found in any maternal sample. These results indicate that control mechanisms operative in older children and adults to prevent circulation of ULvWF multimers and vWF protomeric forms are normally acquired late in uterine life or during the neonatal period. ULvWF multimers, which are normal components of fetal, most cord, and some neonatal plasma samples, may contribute to in utero and postnatal hemostasis.

scholarly journals Multimeric composition of endothelial cell-derived von Willebrand factor

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2074-2076 ◽  
Author(s):  
HM Tsai ◽  
RL Nagel ◽  
VB Hatcher ◽  
II Sussman
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Culture Media ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Buffer System ◽  
Ionophore A23187 ◽  
Von Willebrand ◽  
Willebrand Factor

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

scholarly journals Relationship between human development and disappearance of unusually large von Willebrand factor multimers from plasma

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1851-1858
Author(s):  
JA Katz ◽  
JL Moake ◽  
PD McPherson ◽  
MJ Weinstein ◽  
KJ Moise ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Gestational Age ◽  
Calcium Ionophore ◽  
Sampling Period ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Control Mechanisms ◽  
Older Children ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF. There was no significant relationship between either gestational age at time of delivery or birth weight and likelihood of finding ULvWF multimers in cord PPP samples. No maternal PPP sample contained ULvWF multimers. Serial heelstick samples from 16 preterm and term neonates were analyzed for 8 weeks. ULvWF multimers disappeared from the PPP of ten of the neonates during this time. The PPP of four neonates had vWF patterns similar to those in normal adult PPP throughout the sampling period. The ULvWF multimeric forms of fetal and neonatal PPP samples were similar to those constitutively released from endothelial cells. They were not as slowly migrating in a very porous 0.5% agarose gel system as the ULvWF multimers released from Weibel-Palade bodies in response to the calcium ionophore A23187. A vWF protomer was present in 97% of fetal samples, 83% of cord blood specimens, and 11% of neonatal heelstick samples, but was not found in any maternal sample. These results indicate that control mechanisms operative in older children and adults to prevent circulation of ULvWF multimers and vWF protomeric forms are normally acquired late in uterine life or during the neonatal period. ULvWF multimers, which are normal components of fetal, most cord, and some neonatal plasma samples, may contribute to in utero and postnatal hemostasis.

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.

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