ADP-induced inhibition of von Willebrand factor-mediated platelet agglutination

1976 ◽  
Vol 230 (5) ◽  
pp. 1406-1410 ◽  
Author(s):  
RA Grant ◽  
MB Zucker ◽  
J McPherson
Keyword(s):  
Human Plasma ◽  
Von Willebrand Factor ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Normal Human ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed ◽  
Platelet Shape

Human plasma von Willebrand factor (vWF) plus the antibiotic ristocetin, or bovine or porcine vWF alone, agglutinates platelets in either normal human ethylenediaminetetraacetate (EDTA)-treated citrated platelet-rich plasma (PRP) or citrated PRP from patients with the congenital platelet defect thrombasthenia. The prior addition of 1-10 muM ADP, which causes platelet shape change but not aggregation under these conditions, inhibited vWF-mediated agglutination. Inhibition was prevented by 200 muM ATP. Addition of ADP caused prompt reversal of established vWF-mediated agglutination, which resumed when the ADP was enzymatically removed. EDTA-treated, Formalin-fixed, washed normal platelets also underwent vWF-mediated agglutination. ADP was inhibitory only when added before fixation. Epinephrine (40 muM), prostaglandin E1 (7 muM), or serotonin (2 muM) added before fixation caused slight to moderate inhibition but always less than ADP. Platelets from blood chilled before fixation were fully active. Platelets fixed in freshly prepared PRP did not agglutinate as well as those fixed after incubation of PRP, probably because centrifugation exposes the platelets to ADP. It concluded that ADP causes a reversible decrease in the accessibility of the membrane receptor to vWF.

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 Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1804-1809 ◽  
Author(s):  
JL Miller ◽  
ZM Ruggeri ◽  
VA Lyle
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Normal Platelet ◽  
High Affinity Binding Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed

Abstract The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

Some Immunofluorescent Observations on Factor VIII/von Willebrand Factor in Dogs

1980 ◽  
Vol 44 (02) ◽  
pp. 056-061 ◽  
Author(s):  
Elizabeth V Potter ◽  
Martha A Shaughnessy ◽  
David Green
Keyword(s):  
Factor Viii ◽  
Blood Vessels ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Human Blood ◽  
Normal Human ◽  
Canine Plasma ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Normal Human Blood

SummaryFactor VIII/von Willebrand factor (vWF) was sought by immunofluorescence in or on canine platelets and blood vessels. None was found on normal canine platelets and little was present in normal canine arteries, veins and capillaries compared with normal human blood vessels. However, free granules of vWF were scattered in platelet-rich canine plasma and occasional granules appeared on small clumps of platelets when ristocetin or collagen was added to the plasma. When the same platelets were suspended in human plasma and ristocetin or collagen was added, more clumps were formed and more vWF (human) was associated with these clumps. When thrombin was added to canine platelets in either canine or human serum, more solid, small clumps of platelets were formed and stained with the anti-vWF sera. When thrombin was added to canine platelets in either canine or human plasma, a single large clot was formed which stained brightly for vWF.

Partial amino acid sequence of purified von Willebrand factor–cleaving protease

Blood ◽  
2001 ◽  
Vol 98 (6) ◽  
pp. 1654-1661 ◽  
Author(s):  
Helena E. Gerritsen ◽  
Rodolfo Robles ◽  
Bernhard Lämmle ◽  
Miha Furlan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Normal Human Plasma ◽  
High Molecular Weight Complex ◽  
Normal Human ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor–cleaving protease (vWF-cp) is responsible for the continuous degradation of plasma vWF multimers released from endothelial cells. It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in plasma. Purified vWF-cp may be useful for replacement in these patients, who are now treated by plasma therapy. In this study, vWF-cp was purified from normal human plasma by affinity chromatography on the IgG fraction from a patient with autoantibodies to vWF-cp and by a series of further chromatographic procedures, including affinity chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. Four single-chain protein bands, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, showed Mr of 150, 140, 130, and 110 kd and were found to share the same N-terminal amino acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence (Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel filtration as a high-molecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by the use of concentrated chaotropic salts. vWF-cp in normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37°C; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was even a temporary increase in protease activity during the first 3 days of incubation.

scholarly journals Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1804-1809
Author(s):  
JL Miller ◽  
ZM Ruggeri ◽  
VA Lyle
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Normal Platelet ◽  
High Affinity Binding Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

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

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.

Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF

1999 ◽  
Vol 82 (11) ◽  
pp. 1386-1389 ◽  
Author(s):  
Helena Gerritsen ◽  
Peter Turecek ◽  
Hans Schwarz ◽  
Bernhard Lämmle ◽  
Miha Furlan
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Protease Activity ◽  
Plasma Sample ◽  
Functional Assay ◽  
Normal Human Plasma ◽  
Normal Human ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPatients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with non-familial TTP.

Lack of Stability of Aggregates after Thrombin-Induced Reaggregation of Thrombin-Degranulated Platelets

1992 ◽  
Vol 67 (04) ◽  
pp. 453-457 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Dennis W Perry ◽  
J Fraser Mustard ◽  
Marco Cattaneo
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Aggregate Stability ◽  
Relative Importance ◽  
Platelet Aggregates ◽  
The Stability ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryThe stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2C1 was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 μM) and chymotrypsin(10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymo-trypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.Electron microscope gold immunocytochemistry using antihuman fibrinogen IgG, anti-von Willebrand factor and anti-fibronectin showed a) that fibrinogen in the vacuoles of degranulated platelets was visible at focal points of platelet contact in the aggregates, but that large areas of platelet contact had no fibrinogen detectable between them; and b) in comparison to fibrinogen, little fibronectin or von Willebrand factor (vWf) was detectable in the platelets.Since the linkages between thrombin-degranulated platelets reaggregated by thrombin can be disrupted either by raising cAMP (thus making glycoprotein IIb/IIIa unavailable) or by proteolysis, these linkages are less stable than those formed between normal platelets. It might therefore be expected that platelets that take part in thrombus formation and then recirculate are likely to form less stable thrombi than platelets that have not released their granule contents.

scholarly journals Humanized GPIbα–von Willebrand factor interaction in the mouse

2018 ◽  
Vol 2 (19) ◽  
pp. 2522-2532 ◽  
Author(s):  
Sachiko Kanaji ◽  
Jennifer N. Orje ◽  
Taisuke Kanaji ◽  
Yuichi Kamikubo ◽  
Yosuke Morodomi ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Von Willebrand Factor ◽  
Mouse Strain ◽  
Platelet Rich Plasma ◽  
Platelet Glycoprotein ◽  
Transgenic Mouse Strain ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Knockin Mouse

Abstract The interaction of platelet glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIbα binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with Vwf exon 28–encoding domains A1 and A2 replaced by the human homolog (VWFh28). VWFh28 mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIbα on platelets (mGPIbαnull;hGPIbαTg; H1MA) to generate a new strain (H1HA) with humanized GPIbα-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIbα-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIbα-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIbα-VWFA1 binding, which also inhibited FeCl3-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIbα-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.

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