scholarly journals Recurrent Arterial Thrombosis Linked to Autoimmune Antibodies Enhancing von Willebrand Factor Binding to Platelets and Inducing FcγRII Receptor-Mediated Platelet Activation

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2810-2817 ◽  
Author(s):  
Marc F. Hoylaerts ◽  
Chantal Thys ◽  
Jef Arnout ◽  
Jos Vermylen
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Low Dose ◽  
Antithyroid Drug ◽  
Fetal Loss ◽  
Thyroid Peroxidase ◽  
Spontaneous Aggregation ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

A patient with a history of recurrent late fetal loss associated with multiple placental infarcts and cerebrovascular ischemia at the age of 36, followed a year later by a myocardial infarction, was referred for further investigation. Coronary angiography was normal. Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies, and other thrombophilia parameters were negative, but there was moderate hyperthyroidism with positive thyroid peroxidase antibodies. Platelet numbers and von Willebrand factor (vWF) were normal. Her platelets showed spontaneous aggregation that disappeared with aspirin intake. However, aggregation still was induced by low levels of ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in patient platelet-rich plasma (PRP) was completely blocked by neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The monoclonal anti-FcγRII receptor antibody IV.3 inhibited partly, which suggests that PRP aggregation by low-dose ristocetin was elicited by vWF-immunoglobulin (Ig) complexes. Upon addition to washed human platelets, with vWF (10 μg/mL), purified patient Igs dose-dependently enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 μg/mL, an effect that disappeared again above 1 mg/mL. Aggregation was dependent on the vWF concentration and was blocked by IV.3 or neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous aggregation of normal platelets resuspended in patient plasma could be inhibited totally by IV.3 and partially by neutralizing anti-GPIb or anti-vWF antibodies. Perfusion with normal anticoagulated blood, enriched with 10% of control or patient plasma, over surfaces coated with vWF showed increased platelet adhesion and activation in the presence of patient antibodies. Treatment of the patient with the antithyroid drug thiamazol and temporary corticosteroids, aspirin, and ticlopidine did not correct the platelet hypersensitivity to ristocetin. These observations suggest that some autoantibodies to vWF may both enhance vWF binding to platelets and cause platelet activation through binding to the FcγRII receptor, and thereby may be responsible for a new form of antibody-mediated thrombosis.

Extracellular Hemoglobin Increases the Binding of the Active Form of Von Willebrand Factor to Glycoprotein Ibα and Platelet Activation Under Shear Stress: Implications for Sickle Cell Disease and Other Hemolytic Disorders

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2095-2095
Author(s):  
Miguel A. Cruz ◽  
Prasenjit Guchhait ◽  
Ryanne Ashley Brown
Keyword(s):  
Sickle Cell Disease ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Low Dose ◽  
Binding Capacity ◽  
Active Form ◽  
Cell Disease ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Abstract 2095 Sickle cell disease (SCD) is characterized by a hypercoagulable state that accelerates vaso-occlusive events in microcirculation, leading to acute and chronic organ damage. Studies have indicated that excessive release of hemoglobin from erythrocytes into plasma may contribute to platelet activation and thrombosis. This finding is likely due to the infusion of extracellular hemoglobin (ECHb), which causes platelet aggregation and adhesion on prothrombotic surfaces, and inhibits the metalloprotease ADAMTS13, the enzyme that reduces the size of the constitutively active ultra large (UL) von Willebrand factor (VWF) multimers into a smaller non-active form of VWF. In fact, we have described that the binding of ECHb to the A2 domain of VWF prevents the cleavage of VWF by ADAMTS13. This inhibition from ECHb may raise the levels of hyperreactive VWF multimers in blood, contributing to thrombosis. Interestingly, a recent study from another research group described a direct correlation between the rate of hemolysis and the levels of circulating hyperreactive VWF multimers in plasma from SCD patients. We further analyzed the functional consequences of ECHb binding to active VWF multimers and are now describing a novel role for ECHb in VWF-mediated thrombosis. With increasing concentrations of ECHb (0 – 600 μg/ml) mixed with a constant concentration of endothelial-derived ULVWF, we observed incremental increases in the binding capacity of ULVWF for platelet GPIbα. Maximal binding capacity was obtained at 200 μg/ml of ECHb. Identical results were obtained with plasma-derived VWF using a low dose of ristocetin to activate VWF. This finding is due to the preferential binding of ECHb to the active form of VWF. The anti-A2 domain monoclonal antibody VP-1 effectively inhibited the upregulated effect of ECHb on VWF-GPIbα binding by blocking the interaction between ECHb and the A2 domain in both ULVWF and plasma VWF. We then tested the effect of ECHb on VWF-mediated platelet activation/adhesion to a surface coated with fibrin(ogen) under high shear rates. This assay required the addition of a low dose of ristocetin (0.15 mg/ml) to whole blood prior to perfusion. The effect of ristocetin on platelet activation/adhesion was effectively blocked with either EDTA or antibodies against GPIbα and αIIbβ3. ECHb (200 μg/ml) or buffer was added to whole blood prior to perfusion over the fibrin(ogen)-coated surface. At a shear rate of 1500s−1, the amount of activated/adhered platelets observed with blood containing ECHb was significantly higher (300%) than that of blood containing buffer. Comparable results were obtained when ristocetin was substituted with a gain-of-function recombinant A1A2A3 mutant (R1450E, 100nM), which exposes the A2 domain and exhibits increased GPIbα-binding activity. The number of adherent platelets from blood containing only ECHb without ristocetin was <5% of that from blood containing only ristocetin. Previously, we have demonstrated that the dissociation of A1 and A2 domains in VWF increases the binding of A1 to GPIbα. Therefore, we speculate that the interaction of ECHb with the exposed A2 domain in active VWF influences the structure of the neighboring A1 domain, provoking A1 to adopt a conformation with a higher binding affinity for GPIbα. Furthermore, these results suggest an important role for the interaction between ECHb and hyperreactive VWF in SCD pathology, and most likely other conditions presenting with microangiopathic hemolytic anemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 1941: The Anti Von Willebrand Factor Aptamer ARC1779 Effectively Inhibits Platelet Activation and Adhesion

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Daniel Duerschmied ◽  
Patricia Wagner ◽  
Madaline Gilbert ◽  
James Gilbert ◽  
Denisa Wagner ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Carotid Arteries ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Occlusion Time ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Objectives Immediately after vessel wall damage, plasma von Willebrand factor (VWF) binds to exposed collagen and laminin, enabling the subsequent adhesion of platelets to immobilized VWF via glycoprotein (GP) Ibα. The GPIbα-VWF interaction is able to withstand high (arterial and intrastenotic) shear conditions, rendering this mechanism crucial for the initiation of hemostatic and thrombotic processes in the arterial circulation. In addition, released VWF promotes platelet activation. We have generated a nuclease-resistant anti-VWF A1 domain aptamer, ARC1779. Aptamers are nucleic acid molecules with high affinity and specificity due to the ability to fold into unique three dimensional structures. We examined the ability of ARC1779 to inhibit these platelet activation processes. Methods and Results Botrocetin-induced aggregation of platelet-rich plasma (IC90 ~100–300 nM) and shear force-induced platelet aggregation in whole blood (IC95 ~100 nM) were inhibited by ARC1779. Platelet adhesion was assessed under high shear conditions (1500/s) in a parallel plate flow chamber using calcein orange labeled platelets. ARC1779 at a concentration range of 7.5 nM − 375 nM produced a concentration dependent inhibition of platelet adhesion with an IC50 of 7.5 nM and IC90 of 75 nM. Almost complete abolition of platelet adhesion was achieved with 400 nM of the aptamer. Firm adhesion of platelets was assessed by monitoring time of adhesion of randomly selected adherent platelets treated with 200 nM of ARC1779. The time that ARC1779 treated platelets remained attached was significantly reduced compared to untreated platelets (p<0.001). The occlusion time after electrical injury of carotid arteries in cynomolgus macaques was assessed and found to be significantly prolonged during ARC1779 treatment. At a concentration of 700 nM, ARC1779 inhibited occlusion of the injured carotid arteries as effectively as the GPIIb/IIIa antagonist abciximab at 1300 nM. The bleeding time was significantly shorter with ARC1779 than with abciximab. Conclusions ARC1779 constitutes a promising novel anti-platelet agent for various thrombotic indications.

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.

Further Studies on Aggregation of Platelet-Type von Willebrand’s Disease Platelets by Human von Willebrand Factor

1986 ◽  
Vol 55 (03) ◽  
pp. 338-341 ◽  
Author(s):  
H Takahashi ◽  
W Tatewaki ◽  
M Hanano ◽  
R Nagayama ◽  
A Shibata
Keyword(s):  
Von Willebrand Factor ◽  
Ricinus Communis ◽  
Divalent Cations ◽  
Peanut Agglutinin ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Ricinus Communis Agglutinin ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryPlatelet-type von Willebrand’s disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF-induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation.

Interactions of Purified Rat Factor VIII/von Willebrand Factor with Rat and Human Platelets – Effect of Albumin and Ristocetin

1984 ◽  
Vol 52 (01) ◽  
pp. 057-059 ◽  
Author(s):  
E Dejana ◽  
M Furlan ◽  
B Barbieri ◽  
M B Donati ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rat Plasma ◽  
Human Platelets ◽  
High Concentrations ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Rat Platelets

SummaryRat platelets do not respond to ristocetin in their own plasma nor do they aggregate in the presence of bovine or porcine factor VIII von Willebrand factor (F VIII R:WF) or human F VIII R:WF in presence of ristocetin. However, rat plasma supports ristocetin induced aggregation of washed human platelets. In this study we report on purification of rat F VIII R:WF from cryoprecipitate. Similarly to porcine or bovine material, purified rat F VIII R:WF induced aggregation of human washed fixed platelets. This effect was enhanced by addition of ristocetin and was not modified by addition of albumin. Rat washed platelets were aggregated by ristocetin in the presence of rat or human F VIII R:WF provided that high concentrations of ristocetin are added in a system essentially free of extraneous proteins. Increasing concentrations of albumin dramatically reduced the ability of ristocetin to aggregate rat platelets while human platelet aggregation by human or rat F VIII R:WF was only moderately affected.These studies show that rat F VIII R:WF can interact with rat and human platelets. The lack of response of rat platelets to ristocetin in their own plasma is most likely due to a low sensitivity of rat platelets to this drug and to an inhibitory activity of plasma proteins on this reaction.

scholarly journals Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 827-831 ◽  
Author(s):  
EJ Harfenist ◽  
MA Packham ◽  
RL Kinlough-Rathbone ◽  
M Cattaneo ◽  
JF Mustard
Keyword(s):  
Von Willebrand Factor ◽  
Calcium Ion ◽  
Human Platelets ◽  
Ion Concentration ◽  
Albumin Solution ◽  
High Calcium ◽  
Calcium Ion Concentration ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Abstract To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.

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