Down-regulation and redistribution of GPV/GPVf2
, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human 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.

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8504585 Method of inhibiting binding of von Willebrand factor to human platelets and inducing interaction of platelet with vessel walls

Biotechnology Advances ◽

10.1016/0734-9750(86)90585-9 ◽

1986 ◽

Vol 4 (2) ◽

pp. 348-349

Keyword(s):

Von Willebrand Factor ◽

Human Platelets ◽

Vessel Walls ◽

Von Willebrand ◽

Willebrand Factor

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The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

10.1055/s-0039-1687415 ◽

1979 ◽

Author(s):

H.R. Gralnick ◽

D.K. Morisato

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Binding Sites ◽

Human Platelets ◽

Galactose Oxidase ◽

Scatchard Analysis ◽

Human Fibrinogen ◽

Number Of Binding Sites ◽

Von Willebrand ◽

Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

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

Blood ◽

10.1182/blood.v70.3.827.827 ◽

1987 ◽

Vol 70 (3) ◽

pp. 827-831 ◽

Cited By ~ 12

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

10.1055/s-0038-1652578 ◽

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.

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The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

10.1055/s-0038-1652985 ◽

1981 ◽

Author(s):

P Silber ◽

T H Finlay

Keyword(s):

Von Willebrand Factor ◽

Binding Sites ◽

Binding Constant ◽

Specific Binding ◽

Human Platelets ◽

Scatchard Analysis ◽

Binding Data ◽

Number Of Binding Sites ◽

Von Willebrand ◽

Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

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Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor

Blood ◽

10.1182/blood.v90.11.4425 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4425-4437 ◽

Cited By ~ 60

Author(s):

Jan Schulte am Esch ◽

Miguel A. Cruz ◽

Jonathan B. Siegel ◽

Josef Anrather ◽

Simon C. Robson

Keyword(s):

Von Willebrand Factor ◽

Conformational Changes ◽

Human Platelets ◽

Pathological Process ◽

Xenograft Rejection ◽

Modifying Factors ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

AbstractPlatelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

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Asialo-von Willebrand factor inhibits platelet adherence to human arterial subendothelium: discrepancy between ristocetin cofactor activity and primary hemostatic function

Blood ◽

10.1182/blood.v70.4.1084.1084 ◽

1987 ◽

Vol 70 (4) ◽

pp. 1084-1089 ◽

Cited By ~ 6

Author(s):

JB Lawrence ◽

HR Gralnick

Keyword(s):

Von Willebrand Factor ◽

Whole Blood ◽

Specific Activity ◽

Human Platelets ◽

Primary Hemostasis ◽

Platelet Adherence ◽

Wall Shear ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor

Abstract Platelet adherence at high wall shear rates requires plasma von Willebrand factor (vWF). Clinically, the ristocetin cofactor (RCof) activity is the only widely available assay for vWF function. When purified vWF is treated with neuraminidase to yield asialo-vWF (AS- vWF), its RCof activity is increased by 20% to 40%. AS-vWF binds to normal human platelets independently of ristocetin and induces platelet aggregation in the presence of fibrinogen. To determine whether AS-vWF also shows an enhanced capacity to support platelet adherence to subendothelium, we used the Baumgartner technique. Intact vWF, AS-vWF, or AS-vWF treated with beta-galactosidase (asialo, agalacto-vWF; AS,AG- vWF) was added to normal citrated whole blood before perfusion over human umbilical artery segments (wall shear rate, 2,600 sec-1). Four micrograms per milliliter AS-vWF caused a 69% reduction in total platelet adherence compared with citrated whole blood (P less than .001), and 4 micrograms/mL AS,AG-vWF led to a 48% reduction (P less than .005). With 4 micrograms/mL intact vWF, the platelet adherence values were not significantly different from the controls. No significant differences in subendothelial platelet thrombi or postperfusion platelet counts were evident among any of the groups. In reconstituted afibrinogenemic perfusates, 4 micrograms/mL AS-vWF caused a 42% reduction in platelet adherence (P less than .05). Thus, AS-vWF is a potent inhibitor of platelet adherence, despite its enhanced RCof specific activity. Abnormalities in vWF carbohydrate may play a role in impaired primary hemostasis in some patients with von Willebrand's disease.

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Absence of tubular structures and immunolabeling for von Willebrand factor in the platelet alpha-granules from porcine von Willebrand disease [published erratum appears in Blood 1987 Feb;69(2):707]

Blood ◽

10.1182/blood.v68.3.774.774 ◽

1986 ◽

Vol 68 (3) ◽

pp. 774-778 ◽

Cited By ~ 19

Author(s):

EM Cramer ◽

JP Caen ◽

L Drouet ◽

J Breton-Gorius

Keyword(s):

Von Willebrand Factor ◽

Human Platelets ◽

Von Willebrand Disease ◽

Tubular Structures ◽

Electron Microscopic ◽

Dense Part ◽

The Matrix ◽

Cytoplasmic Microtubules ◽

Von Willebrand ◽

Willebrand Factor

Abstract The electron microscopic localization of von Willebrand factor (vWF) was studied in platelets from normal and von Willebrand disease (vWD) pigs. In normal pig platelets, immunolabeling for vWF was far more intense and extensive than in human platelets and was either localized at one pole of the alpha-granule or all along its periphery or long axis. As in human platelets, this immunolabeling coincided with the presence of tubules about 200 nm in diameter. These structures were more numerous than in human platelets, with up to 30 tubules per alpha- granule. They were easily identified either in transverse sections, usually grouped in a less electron-dense part of the matrix at one pole of the alpha-granule, or in longitudinal sections parallel to the long axis of the elongated granules, or coiled around the alpha-granule core. They closely resemble those structures found in Weibel-Palade bodies. In platelets from pigs with severe vWD, these structures were absent, as was the immunolabeling for vWF; however, cytoplasmic microtubules were normally present in these platelets. Thus, the granule-associated tubules can be distinguished from the microtubules, which are larger in diameter (250 nm), are present in both normal and vWD platelets, and do not stain for vWF. These results strongly suggest that the tubular structures present in the alpha-granules of normal porcine platelets correspond to the vWF molecule itself.

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Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽

10.1182/blood.v99.12.4486 ◽

2002 ◽

Vol 99 (12) ◽

pp. 4486-4493 ◽

Cited By ~ 109

Author(s):

Gregor Theilmeier ◽

Carine Michiels ◽

Erik Spaepen ◽

Ingrid Vreys ◽

Désiré Collen ◽

...

Keyword(s):

Von Willebrand Factor ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Perfusion Studies ◽

Von Willebrand ◽

Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

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