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.

scholarly journals Adhesive properties of the carbohydrate-modified von Willebrand factor (CHO-vWF)

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 947-952 ◽  
Author(s):  
AB Federici ◽  
C De Romeuf ◽  
PG De Groot ◽  
B Samor ◽  
R Lombardi ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Significant Loss ◽  
Total Carbohydrate ◽  
Adhesive Properties ◽  
Normal Platelet ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this cooperative study, we explored the role of the carbohydrate moiety (CHO) of von Willebrand factor (vWF) in supporting platelet adhesion. Because of previous discrepant results, all purification steps and CHO modifications by various enzymes were critically evaluated. Under our conditions, CHO-modified vWF preparations contained less than 5% of the initial sialic acid ([Neu]-ase-vWF) and less than 45% ([Neu-Gal]-ase-vWF) or 21% ([Neu-Gal-eF]-ase-vWF) of the D-galactose. These preparations usually showed increased electrophoretic mobility but no significant loss of high-mol-wt multimers when proteolysis had been prevented. Some degree of proteolysis was noted in some carbohydrate-modified vWFs, but the degree of degradation observed did not correlate with the removal of D- galactose. Platelet adhesion to various matrices increased after removal of the terminal sialic acid ([Neu]-ase-vWF) and approximately 45% of the D-galactose ([Neu-Gal]-ase-vWF), but returned to normal values when greater than 70% of the total carbohydrate had been removed by endoglycosidase F [Neu-Gal-ef]-ase-vWF). These changes in reactivity were also reflected in the spontaneous aggregation in normal platelet- rich plasma (PRP) after CHO removal.

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 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.

Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

1981 ◽  
Author(s):  
F Fauvel ◽  
Y J Legrand ◽  
N Gutman ◽  
J P Muh ◽  
G Tobelem ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Human Artery ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
Aggregation Of Platelets

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.

scholarly journals Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 625-628 ◽  
Author(s):  
DM Peterson ◽  
NA Stathopoulos ◽  
TD Giorgio ◽  
JD Hellums ◽  
JL Moake
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Shear Stresses ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.

scholarly journals Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 625-628 ◽  
Author(s):  
DM Peterson ◽  
NA Stathopoulos ◽  
TD Giorgio ◽  
JD Hellums ◽  
JL Moake
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Shear Stresses ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Abstract Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.

ASIALO VON WILLEBRAND FACTOR ENHANCES PLATELET ADHESION TO VASCULAR SUBENDOTHELIUM

1987 ◽  
Author(s):  
A Ordinas ◽  
E Bastida ◽  
M Garrido ◽  
J Monteagudo ◽  
L de Marco ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Albumin ◽  
Flow Conditions ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Native Von Willebrand factor (NvWF) binds to platelets activated by thrombin, ADP or ristocetin, and also supports the adhesion of platelets to subendothelium at high shear rates. In contrast, asialo von Willebrand factor (AvWF) induces platelet aggregation in absence of platelet activators. We investigated the role of AvWF in supporting the adhesion of platelets to rabbit vessel subendothelium under flow conditions at a shear rate of 2000 sec-1 for 5 min using the Baumgartner perfusion system. We also studied the effects of blockage of platelet GPIb or GPIIb/IIIa on platelet adhesion using monoclonal antibodies (Mabs),and we measured the rate of binding of 111I-labeled NvWF and AvWF to subendothelium. Perfusates consisted of washed platelts and red cells resuspended in a 4% human albumin solution to which increasing concentrations of NvWF or AvWF had been added. Platelets interacting with the perfused vessels were evaluated morphometrically using a computerized system. At a concentration of 1.2 /ig/ml the percentage of total coverage surface was 21.3 ± 4.8% and 40.0±14.6%, for NvWF and AvWF, respectively (p<0.01). Addition of either Mab against GPIb (LJlbl) or against GPIIb/IIIa (CP8) to the perfusates, reduced platelet deposition (p <0.01). The rates of binding of 111I-labeled NvWF and AvWF to perfused vessel subendothelium were similar (0.83±0.1μg and 0.95±0.1 μg ,respectively).Our results indicate that AvWF enhances the interaction of washed platelets with the vessel subendothelium under flow conditions. Furthermore, they suggest that this effect is related to the interaction of AvWF with platelets and not to an increased affinity of AvWF for subendothelium.

INCREASED ADHESIVE PROPERTIES OF THE CARBOHYDRATE-MODIFIED VON WILLEBRAND FACTOR (CHO-VWF): CRITICAL ROLE OF THE MULTIMERIC STRUCTURE AND CORRELATION WITH PLATELET AGGREGATION

1987 ◽  
Author(s):  
A B Federici ◽  
C De Romeuf ◽  
P G De Groot ◽  
P M Mannucci ◽  
B Samor ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Critical Role ◽  
Freezing And Thawing ◽  
Adhesive Properties ◽  
Spontaneous Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

We have reexplored the role of the carbohydrate moiety (CHO) on the von Willebrand Factor (vWF) structure and function by critically evaluating its different purification steps and modifications in CHO content by specific enzymes. Structural and functional assays have been evaluated separately in each laboratory (Milano and Lille) and jointly in Utrecht during several organized experiments. Under our conditions, the CHOVWFs obtained were characterized by less than 5% of sialic acid "(Neu)asevWF" and about 45% of D-Galactose "(Neu-Gal)ase-vWF" remaining, by increased electrophoretic mobility without any significant losses of the high molecular weight multimers and by their capacity to induce spontaneous aggregation in normal platelet rich plasma (PRP). Platelet adhesion to these different CHO-vWFs was tested in the flat chamber devised by Sakariassen in the presence of different subendothelial matrices and data expressed as the percentage of the surface covered by platelets. The blood reconstituted with different plasma samples showed the following percentual values of surface coverage (mean ± SD):- Normal plasma = 15 ± 3.8- Severe vWd plasma = 4 ± 1.9- SvWd pl + Native vWF = 14 ± 2.8- SvWd pl + (Neu) ase-vWF = 23 ± 3.5- SvWd pl + (Neu-Gal) ase-vWF = 19 ± 2.9This significantly increased adhesion to the subendothelium of the CHO-vWFs corresponded to the spontaneous aggregation present in normal PRP but it disappeared when the multimeric structure was damaged by in vitro proteolysis and/or by storage conditions (changes in temperature and freezing and thawing). From these results we may conclude that removal of terminal sugars enhances not only platelet-vWF interactions, but also platelet adhesion to the subendothelium.

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

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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