scholarly journals Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1281-1285
Author(s):  
EA Thompson ◽  
MA Howard
Keyword(s):  
Von Willebrand Factor ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Polymorphonuclear Cells ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Von Willebrand ◽  
Willebrand Factor

In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio- CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.

scholarly journals Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1281-1285 ◽  
Author(s):  
EA Thompson ◽  
MA Howard
Keyword(s):  
Von Willebrand Factor ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Polymorphonuclear Cells ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio- CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.

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.

Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3919-3919
Author(s):  
Zhou Zhou ◽  
Han Hyojeong ◽  
Miguel A. Cruz ◽  
Jose A. Lopez ◽  
Jing-fei Dong ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Elevated Level ◽  
Dependent Manner ◽  
Cell Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

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.

N-Acetyl Cysteine Reduces von Willebrand Factor Multimer Size In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 420-420
Author(s):  
Junmei Chen ◽  
Francisca C. Gushiken ◽  
Leticia Nolasco ◽  
Joel F. Moake ◽  
Jose A. Lopez
Keyword(s):  
Von Willebrand Factor ◽  
Chronic Obstructive Lung Disease ◽  
Chronic Obstructive ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acetyl Cysteine ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) shares a similar domain structure with many polymeric mucins, including the presence of D domains and a C-terminal cysteine knot, which allows these molecules to form polymeric structures that can reach immense sizes. Precipitation of polymeric lung mucins complicates the clinical course of cystic fibrosis and chronic obstructive lung disease, and in both cases the viscosity of the inspissated mucus can be reduced by treatment with N-acetyl cysteine (NAC), which presumably reduces the size of the mucin polymers by reducing sensitive disulfide bonds. Because of the similarity of VWF and mucin multimers, we examined whether NAC could also reduce VWF multimer size both in vitro and in vivo. In vitro, we incubated NAC at different concentrations and for different times with ultra-large VWF multimers (ULVWF) isolated from endothelial cell supernatant and examined the effect on multimer size using agarose gel electrophoresis. NAC reduced ULVWF size in a time- and concentration-dependent manner, with the peak effect reached at 5 min and at concentration of 0.5 mM. We then examined the effect of NAC on ULVWF/platelet “strings” formed on the surface of histamine-activated endothelial cells by perfusing the strings with NAC solutions. At 1 mM, NAC eliminated almost all of the adherent strings within 5 minutes. We next examined the effect of NAC in vivo by following VWF multimer size with time in C57B/6 injected with NAC either intraperitoneally or intravenously. NAC, at a single dose of 500 mg/kg, induced a sustained reduction in VWF multimer size in the treated mice within 4 hours after injection. The effects lasted up to 8 hours. These results suggest that NAC may be a rapid, safe, and effective treatment for patients suspected of suffering from thrombotic thrombocytopenic purpura, a disorder characterized by a failure to process ULVWF.

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 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.

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.

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