scholarly journals Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions

2020 ◽  
Vol 4 (13) ◽  
pp. 2953-2961 ◽  
Author(s):  
Magdolna Nagy ◽  
Gina Perrella ◽  
Amanda Dalby ◽  
M. Francisca Becerra ◽  
Lourdes Garcia Quintanilla ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Transmembrane Domain ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Glycoprotein Vi ◽  
Heterozygous Variant ◽  
Platelet Aggregates ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are ∼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

2014 ◽  
Vol 112 (12) ◽  
pp. 1159-1166 ◽  
Author(s):  
Viviana Daidone ◽  
Giovanni Barbon ◽  
Elena Pontara ◽  
Grazia Cattini ◽  
Lisa Gallinaro ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

ROLE OF RELEASED ADP IN THE STABILIZATION OF PLATELET AGGREGATES: STUDIES IN PATIENTS WITH DEFICIENCY IN AMINE STORAGE GRANULES CONTENTS

1987 ◽  
Author(s):  
M Cattaneo ◽  
M T Canciani ◽  
J F Mustard
Keyword(s):  
Von Willebrand Factor ◽  
Human Platelets ◽  
Small Extent ◽  
Amine Storage ◽  
Storage Granules ◽  
Platelet Aggregates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Amine Storage Granules

Human platelets aggregated by thrombin (T) under conditions in which the release reaction (RR) occurs to only a small extent can be deaggregated by agents that dissociate 125I-fibrinogen bound to platelets. In contrast, when platelets undergo the RR, they cannot be reading deaggregated even though combinations of inhibitors cause 125I-fibrinogen to dissociate. Therefore, material released from platelet granules seems to stabilize aggregates. T-induced aggregates of washed platelets deficient in fibrinogen or von Willebrand factor cannot be deaggregated readily by deaggregating agents, implying that released fibrinogen or von Willebrand factor do not have a major role in stabilizing aggregates. To examine the role of platelet (δ-granule contents in stabilizing platelet aggregates, aggregation and deaggregation were studied with platelets from patients with (δ- Storage Pool Deficiency (δ- SPD). Platelet aggregation and the release ofβ-TG in response to T (1 U/ml) were similar for platelets from patients and controls. Platelets from patients (but not from controls) could be deaggregated by PGE1 (10 uM) plus chymotrypsin (10 U/ml), with hirudin (5 U/ml) added to block further effects of T. Addition of ADP (20 uM) to the (5-SPD platelets 5 sec after T abolished the ability of this combination of inhibitors to deaggregate the platelets. The addition of serotonin (2 uM) 5 sec after T did not prevent inhibitors from deaggregating δ-SPD platelets. When apyrase was added to normal platelets immediately before they were aggregated by T, the combination of inhibitors readily deaggregated the platelets. Therefore, released ADP may stabilize platelet aggregates through a mechanism that could be independent of released fibrinogen and von Willebrand factor.

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 The role of von Willebrand factor in breast cancer metastasis

2021 ◽  
Vol 14 (4) ◽  
pp. 101033
Author(s):  
Chia Yin Goh ◽  
Sean Patmore ◽  
Albert Smolenski ◽  
Jane Howard ◽  
Shane Evans ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Von Willebrand Factor ◽  
Cancer Metastasis ◽  
Breast Cancer Metastasis ◽  
Von Willebrand ◽  
Willebrand Factor

Role of Platelet Membrane Glycoproteins and Von Willebrand Factor in Adhesion of Platelets to Subendothelium and Collagen

1987 ◽  
Vol 516 (1 Blood in Cont) ◽  
pp. 52-65 ◽  
Author(s):  
KJELL S. SAKARIASSEN ◽  
EDITH FRESSINAUD ◽  
JEAN-PIERRE GIRMA ◽  
DOMINIQUE MEYER ◽  
HANS R. BAUMGARTNER
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

scholarly journals Partial characterization of a binding site for von Willebrand factor on glycocalicin

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 19-26 ◽  
Author(s):  
AD Michelson ◽  
J Loscalzo ◽  
B Melnick ◽  
BS Coller ◽  
RI Handin
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Binding Activity ◽  
Proteolytic Fragment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N- acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta- galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.

scholarly journals Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1214-1217
Author(s):  
E Fressinaud ◽  
D Baruch ◽  
C Rothschild ◽  
HR Baumgartner ◽  
D Meyer
Keyword(s):  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

scholarly journals Regulation of plasma von Willebrand factor

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 96 ◽  
Author(s):  
Karl C Desch
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Vascular Wall ◽  
Healthy Human ◽  
Venous Thromboembolic ◽  
Artery Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a central role in the initiation of blood coagulation. Through interactions between its specific functional domains, the vascular wall, coagulation factor VIII, and platelet receptors, VWF maintains hemostasis by binding to platelets and delivering factor VIII to the sites of vascular injury. In the healthy human population, plasma VWF levels vary widely. The important role of VWF is illustrated by individuals at the extremes of the normal distribution of plasma VWF concentrations where individuals with low VWF levels are more likely to present with mucocutaneous bleeding. Conversely, people with high VWF levels are at higher risk for venous thromboembolic disease, stroke, and coronary artery disease. This report will summarize recent advances in our understanding of environmental influences and the genetic control of VWF plasma variation in healthy and symptomatic populations and will also highlight the unanswered questions that are currently driving this field of study.

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