scholarly journals The Flow Dependent Adhesion of von Willebrand Factor (VWF)-A1 Functionalized Nanoparticles in an in Vitro Coronary Stenosis Model

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2679 ◽  
Author(s):  
Yathreb Asaad ◽  
Mark Epshtein ◽  
Andrew Yee ◽  
Netanel Korin
Keyword(s):  
Von Willebrand Factor ◽  
Coronary Stenosis ◽  
Nano Particles ◽  
Adhesive Properties ◽  
Polystyrene Nanoparticles ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

In arterial thrombosis, von Willebrand factor (VWF) bridges platelets to sites of vascular injury. The adhesive properties of VWF are controlled by its different domains, which may be engineered into ligands for targeting nanoparticles to vascular injuries. Here, we functionalized 200 nm polystyrene nanoparticles with the VWF-A1 domain and studied their spatial adhesion to collagen or collagen-VWF coated, real-sized coronary stenosis models under physiological flow. When VWF-A1 nano-particles (A1-NPs) were perfused through a 75% stenosis model coated with collagen-VWF, the particles preferentially adhered at the post stenotic region relative to the pre-stenosis region while much less adhesion was detected at the stenosis neck (~ 65-fold less). When infused through collagen-coated models or when the A1 coating density of nanoparticles was reduced by 100-fold, the enhanced adhesion at the post-stenotic site was abolished. In a 60% stenosis model, the adhesion of A1-NPs to collagen-VWF-coated models depended on the location examined within the stenosis. Altogether, our results indicate that VWF-A1 NPs exhibit a flow-structure dependent adhesion to VWF and illustrate the important role of studying cardiovascular nano-medicines in settings that closely model the size, geometry, and hemodynamics of pathological environments.

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

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4425-4437 ◽  
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.

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1535-1535
Author(s):  
Suzana M. Zorca ◽  
Emma C. Josefsson ◽  
Viktoria Rumjantseva ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Lectin Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation. The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

The A/T1381 polymorphism in the A1-domain of von Willebrand factor influences the affinity of von Willebrand factor for platelet glycoprotein Ibα

2007 ◽  
Vol 98 (07) ◽  
pp. 178-185 ◽  
Author(s):  
Tímea Szántó ◽  
Ágota Schlammadinger ◽  
Stephanie Staelens ◽  
Simon De Meyer ◽  
Kathleen Freson ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Association Studies ◽  
Platelet Glycoprotein ◽  
Platelet Receptor ◽  
Increased Risk ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMany polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphis mA/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbα. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbα under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbα than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbα, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.

An Essential Role of Factor VIII-Mediated Hemostasis in the Absence of von Willebrand Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3101-3101
Author(s):  
Yoshihiko Sakurai ◽  
Midori Shima ◽  
Shogo Kasuda ◽  
Shoko Omura ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Essential Role ◽  
Bolus Administration ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The replacement therapy with plasma-derived factor VIII (FVIII)/von Willebrand factor (VWF) concentrates is the first line treatment for the patients with type 3 von Willebrand disease (VWD). However, development of anti-VWF alloantibodies (inhibitor) is a major problem since the inhibitor neutralizes the VWF activity and may cause anaphylactic reactions. As an alternative treatment, the usage of FVIII concentrates has been reported but the mechanism of the hemostatic effects remains to be elucidated. Objectives: The purpose of this study is to address the role of FVIII in the hemostatic mechanism in the absence of VWF by in vitro and ex vivo analysis in the treatment for type 3 VWD with recombinant FVIII (rFVIII). Patient/Methods: The patient is a 55-year-old male with type 3 VWD. Blood samples were obtained before and 30 min after bolus administration. Rotating thromboelastometry (ROTEM) assay was performed to examine global interactions in hemostasis. To elucidate the effect on platelet activation, α-thrombin- and shear-induced platelet aggregation studies were performed. Further, α-thrombin-induced [Ca2+]i rise was assessed using fura2-AM loaded platelets. Results and Implications: The patient underwent two surgical procedures of multiple teeth extractions successfully with minimal bleeding by bolus administration of rFVIII (50 IU/kg) before procedure and followed by continuous infusion at rate of 10 IU/kg/h for 15 hours. FVIII:C was elevated from 1.0% to 20~30% 30 min after bolus infusion and maintained ~15% after 12 h-continuous infusion. ROTEM analysis showed that infusion of rFVIII shortened clotting time (preinfusion 2083.8±784.3 sec vs. post-infusion 1022.0±191.5 sec) and clot formation time (pre 1267.3±455.4 sec vs. post 705.8±261.8 sec) and increased α (pre 8.5±7.4 degree vs. post 23.5±4.4 degree). The α value and CFT indicate the rate of increase of elastic shear modulus. Addition of rFVIII to preinfusion blood in vitro corrected ROTEM parameters and thrombin-induced aggregation dose-dependently. Infusion of FVIII enhanced thrombin-induced platelet aggregation (% maximal aggregation: pre 26.3% vs. post 98.2%) as well as low shear-induced platelet aggregation (% maximal aggregation: pre 18% vs. post 52%). Furthermore, infusion of rFVIII meliorated thrombin-induced intracellular calcium flux of washed platelets (thrombin 10 nM, Ca flux: pre 414.0 nM vs. post 620.6 nM). Recently, the cell-based model of hemostasis provides a solid foundation for the relation between platelet and coagulation. Although coagulation initiation occurs normally via the extrinsic pathway, amplification mediated by the intrinsic pathway is seriously disturbed in type 3 VWD due to the marked decrease in FVIII. Therefore, correction of FVIII could result in the improvement of hemostasis. Our data demonstrated the effectiveness of FVIII in the surgical treatment for type 3 VWD and further suggested that FVIII molecules are incorporated into platelet phospholipids to facilitate platelet activation as well as act directly to intrinsic pathways to normalize coagulation. Conclusions: Our observations suggested that FVIII plays an essential role in hemostasis in the absence of VWF and provided the rationale for the usage of rFVIII in the hemostatic management of type 3 VWD.

Adenovirus Induced Thrombocytopenia: The Role of P-Selectin and von Willebrand Factor in Virus- Mediated Platelet Clearance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 222-222 ◽  
Author(s):  
Maha Othman ◽  
Andrea Labelle ◽  
Ian Mazzetti ◽  
David Lillicrap
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Critical Role ◽  
Flow Cytometric Analysis ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Acute thrombocytopenia has been consistently reported following IV administration of adenoviral vectors (Ad) but the mechanism responsible for this phenomenon has not been elucidated. Thrombocytopenia appears 24 hours after IV administration of Ad and is vector dose dependent. In this study, we have assessed the potential roles of the adhesive proteins P-selectin and von Willebrand Factor (VWF) on the aggregation and clearance of platelets following virus administration. We have addressed the question of whether the thrombocytopenia is due to a direct effect of the virus on platelets or an indirect effect related to interaction of platelets with other proteins or cells modified by the virus. We assessed platelet count in a group of Balb/c and C57Bl/6 mice over 1 week period following Ad administration and performed a detailed examination of the events within the first 24 h after Ad injection, the period that precedes the appearance of thrombocytopenia. We examined the effect of Ad on expression of the platelet activation marker P-selectin and the formation of platelet leukocyte aggregates (PLA) by means of flowcytometry after incubation of adenovirus with mouse platelets in vitro, and following Ad administration in vivo. To assess the role of VWF in Ad-induced thrombocytopenia we measured plasma VWF levels one hour after injection of Ad. Further investigations involved comparison of platelet counts, platelet activation, and the formation of PLA in a group of VWF KO mice. All studies have been performed with a replication deficient E1/E3-deleted Ad 1x 1011 viral particles/mouse. Our in vitro studies have shown that Ad directly activates mouse platelets as shown by increased expression of P-selectin. The average index of platelet activation for platelets stimulated by Ad was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. Our in vivo studies have confirmed the development of significant thrombocytopenia in both Balb/c as well as C57Bl/6 WT mice (n=8, p=0.00001, n= 6, p=0.002) 24 hours following Ad administration. Significant P-selectin expression was documented in both strains (n=4,p=0.0003; n=3, p=0.0008 respectively) as well as significant PLA one hour following Ad (n=4, p=0.01; n=3, p=0.007). The VWF KO mice showed non-significant thrombocytopenia (n= 6, p=0.063) at 24 hours following Ad, significant P-selectin expression (n=3, p=0.0003), but no significant PLA formation at one hour (n=3 p=0.12) relative to pre-injection levels. Plasma VWF levels were significantly elevated in both Balb/c and C57Bl/6 WT mice one hour following administration of the virus (n= 3, p=0.02; n= 3, p= 0.001). The average plasma VWF levels were 48.1 U/mL at 1h compared to 5.7 U/mL pre injection in Balb /c mice and 85.9 U/mL compared to 6.1 U/mL in C57Bl/6 mice. These studies have shown that Ad can act as an inducer of mouse platelet activation and as a promoter for platelet-leukocyte association both in vitro and in vivo. We have demonstrated a role for Ad in stimulating VWF release from the endothelium, and have shown that VWF has a critical role in platelet activation and clearance following Ad administration. We conclude that P-selectin and VWF proteins are directly involved in interactions between endothelial cells, platelets and leukocytes, a complex interaction that can explain at least in part the mechanisms underlying Ad-mediated thrombocytopenia.

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

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4425-4437
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

Platelet 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.

scholarly journals Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism.

1989 ◽  
Vol 109 (1) ◽  
pp. 367-375 ◽  
Author(s):  
E Dejana ◽  
M G Lampugnani ◽  
M Giorgi ◽  
M Gaboli ◽  
A B Federici ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Direct Role ◽  
Platelet Receptor ◽  
Adhesion Plaques ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Receptor Antibodies

Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF. Using antibodies to the beta chains of fibronectin (beta 1) and vitronectin (beta 3) receptors it was found that ECs adherent to vWF show clustering of both receptors. The beta 1 receptor antibodies are arranged along stress fibers at sites of extracellular matrix contact while the beta 3 receptor antibodies were sharply confined at adhesion plaques. ECs release and organize endogenous fibronectin early during adhesion to vWF. Upon blocking protein synthesis and secretion, ECs can equally adhere and spread on vWF but, while the beta 3 receptors are regularly organized, the beta 1 receptors remain diffuse. This suggests that the organization of the beta 1 receptors depend on the release of fibronectin and/or other matrix proteins operated by the same cell. Antibodies to the beta 3 receptors fully block EC adhesion to vWF and detach ECs seeded on this substratum. In contrast, antibodies to the beta 1 receptors are poorly active. Overall these results fit with an accessory role of beta 1 receptors and indicate a leading role for the beta 3 receptors in EC interaction with vWF. To identify the EC binding domain on vWF we used monoclonal antibodies produced against a peptide representing the residues Glu1737-Ser1750 of the mature vWF and thought to be important in mediating its binding to the platelet receptor glycoprotein IIb-IIIa. We found that the antibody that recognizes the residues 1,744-1,746, containing the Arg-Gly-Asp sequence, completely inhibit EC adhesion to vWF whereas a second antibody recognizing the adjacent residues 1,740-1,742 (Arg-Gly-Asp-free) is inactive. Both antibodies do not interfere with EC adhesion to vitronectin. This defines the molecular domain on vWF that is specifically recognized by ECs and reaffirms the direct role of the Arg-Gly-Asp sequence as the integrin receptor recognition site also in the vWF molecule.

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