Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow

2008 ◽  
Vol 99 (06) ◽  
pp. 1068-1078 ◽  
Author(s):  
Marjolijn van Lier ◽  
Sandra Verhoef ◽  
Sandra Cauwenberghs ◽  
Johan Heemskerk ◽  
Jan-Willem Akkerman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Calcium Signalling ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Signalling Molecules ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySeveral studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca2+ signalling and secretion during the interaction of platelets with VWF and collagen.VWF/ ristocetin-induced platelet aggregation was delayed after treatment with methyl β-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca2+ response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca2+ responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A2 and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca2+ signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Maximum fluid concentrations of materials released from platelets at a surface

1983 ◽  
Vol 244 (1) ◽  
pp. H109-H114 ◽  
Author(s):  
G. A. Adams ◽  
I. A. Feuerstein
Keyword(s):  
Platelet Aggregation ◽  
Empirical Data ◽  
Maximum Amount ◽  
Inhibit Platelet Aggregation ◽  
Fluid Concentration ◽  
Diffusion And Convection ◽  
Platelet Accumulation ◽  
Von Willebrand ◽  
Willebrand Factor

We examine the estimation of local concentrations of materials that are released from the dense and alpha-granules of platelets during accumulation of platelets upon collagen-coated glass. Platelet/red blood cell suspensions were perfused through a 1.3-mm-ID tube. Empirical data were used in a calculation procedure, based on diffusion and convection, designed to yield an upper bound on the interfacial fluid concentration (IFC) for each substance considered. The necessary empirical data are the rate of platelet accumulation and the maximum amount of material in the platelet capable of secretion. It was found that the IFC is dependent on the shear rate at the surface (G) and is proportional to G0.27. This means that an eightfold increase in flow rate would increase the IFCs approximately twofold. Serotonin, pyrophosphate, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate (ATP) were found not to be present in sufficient quantities to produce IFCs that could influence platelet aggregation if used alone at the IFC. A second set of materials, fibrinogen, fibronectin von Willebrand factor, and calcium, had IFCs less than their concentrations normally found in plasma. A third category, containing adenosine 5'-diphosphate (ADP) alone, had an IFC close to those known to affect platelet aggregation. The role of metabolites of arachidonic acid, which may promote or inhibit platelet aggregation, awaits further description.

IMPORTANCE OF LARGE VON WILLEBRAND FACTOR (vWF) MULTIMERS IN vWF INTERACTION WITH PLATELET GLYCOPROTEIN IIb/IIIa

1987 ◽  
Author(s):  
M Yamamoto ◽  
Y Ando ◽  
K Watanabe ◽  
H Iri ◽  
Y Araki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Glycoprotein ◽  
Induce Platelet Aggregation ◽  
Maximum Extent ◽  
Physiological Relevance ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

Recently it has been reported that, in addition to binding to glycoprotein (GP) lb, vWF also interacts with GPIIb/IIIa, although the physiological relevance of this interaction is not completeley clear. In this paper, we have investigated the role of different size of vWF multimers in vWF-mediated platelet aggregation. Different size of vWF multimers were purified from human plasma through Sephacryl S-1000 column according to the method of Fowler et al. Fractions were analysed by SDS-agarose gel electrophoresis by the method of Ruggeri et al. When each fraction was examined for ristocetin cofactor activity (RCo), only larger multimers exhibited significant RCo. The maximum extent of ristocetin-induced platelet agglutination by larger multimers (10 μg/ml) was 80%, while that of intermediate and lower multimers at the same concentration was 20% and 0%, respectively. Each fraction was then added to washed platelet suspensions in the presence of 10 μM ADP and 0.3 mM CaCl2. Only larger multimers induced platelet aggregation, while intermediate and lower multimers failed to induce platelet aggregation. The maximum extent of aggregation in the presence of larger multimers (10 μg/ml) was 70% of that in the presence of fibrinogen instead. Similar experiments were peformed using platelet-rich plasma from a patient with afibrinogenemia in stead of washed normal platelets. ADP caused significant aggregation only when purified vWF larger multimers or fibrinogen was added. This vWF-mediated aggregation was completely inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM).Our results indicate that larger multimers of vWF play major roles in vWF interaction with GPIIb/IIIa.

scholarly journals Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin

2001 ◽  
Vol 134 (5) ◽  
pp. 1104-1112 ◽  
Author(s):  
Paul Jurasz ◽  
Michael W Stewart ◽  
Anna Radomski ◽  
Fadi Khadour ◽  
Marek Duszyk ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Tumour Cell ◽  
Differential Regulation ◽  
Von Willebrand ◽  
Willebrand Factor

β3 Tyrosine Phosphorylation in αIIbβ3 (Platelet Membrane GP IIb-IIIa) Outside-in Integrin Signaling

2001 ◽  
Vol 86 (07) ◽  
pp. 246-258 ◽  
Author(s):  
Lisa Nannizzi-Alaimo ◽  
K. S. Srinivasa Prasad ◽  
David Phillips
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Critical Role ◽  
Clot Retraction ◽  
Signaling Mechanism ◽  
Platelet Aggregate ◽  
Von Willebrand

SummaryThe platelet integrin αIIbβ3 not only binds fibrinogen and von Willebrand factor to mediate platelet aggregation and adhesion, it also serves as a signaling receptor. Platelet agonists such as ADP, thrombin and collagen induce “inside-out” signaling which activates the receptor function of αIIbβ3 for soluble fibrinogen. Subsequent platelet aggregation leads to “outside-in” signaling, inducing platelet aggregate stabilization and triggering a variety of functions important to platelet physiology. This review focuses on the role of β3 tyrosine phosphorylation in αIIbβ3 outside-in signaling. Tyrosine phosphorylation of β3 in platelets is a dynamic process which is initiated upon platelet aggregation and also by adhesion of platelets to immobilized fibrinogen. Tyrosine phosphorylation occurs on the β3 integrin cytoplasmic tyrosine (ICY) domain, a conserved motif found in thesubunits of several integrins. β3 ICY domain tyrosine phosphorylation induces the recruitment of two proteins to the cytoplasmic domains of αIIbβ3: the cytoskeletal protein myosin, important to clot retraction; and the signaling adapter protein Shc, important to platelet stimulation. The critical role of β3 tyrosine phosphorylation to platelet function was established by the diYF mouse, a novel strain which expresses an αIIbβ3 in which the two β3 ICY domain tyrosines have been mutated to phenylalanine. These mice are selectively impaired in outside-in αIIbβ3 signaling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. Taken together, the data suggest that the β3 tyrosine phosphorylation signaling mechanism is important to αIIbβ3 function and might be applicable to a wide variety of integrin-mediated events.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

2012 ◽  
Vol 107 (06) ◽  
pp. 1107-1121 ◽  
Author(s):  
Thomas Vanassche ◽  
Alexandre Kauskot ◽  
Jan Verhaegen ◽  
Willy Peetermans ◽  
Joanne van Ryn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Platelet Aggregation ◽  
Typical Feature ◽  
Human Platelets ◽  
Fibrin Scaffold ◽  
Immunoglobulin Binding ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryInteractions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

scholarly journals Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 996-103 ◽  
Author(s):  
KS Sakariassen ◽  
M Ottenhof-Rovers ◽  
JJ Sixma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Platelet Adherence ◽  
Human Arteries ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

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.

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