Engagement of Fibrinogen and β3 Integrin Is Required for Maintenance of Platelet Intracellular and Cell Surface P-Selectin Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2868-2868
Author(s):  
Hong Yang ◽  
Sean Lang ◽  
Zhimin Zhai ◽  
Christopher M. Spring ◽  
Adili Reheman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Platelets ◽  
Human Patient ◽  
Platelet Factor ◽  
C Terminus ◽  
Significant Difference ◽  
Β3 Integrin ◽  
Von Willebrand ◽  
Selectin Binding ◽  
Deficient Mice

Abstract Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. While it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double deficient mice. Subsequently, we identified this was due to fibrinogen, but not VWF, deficiency. The impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in a human patient with severe hypofibrinogenemia and his heterozygous parents. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. This effect seems to be specific for P-selectin, as other alpha-granule proteins (e.g. thrombospondin-1, vitronectin, platelet factor 4) were not decreased in fibrinogen-deficient mouse or human platelets. We found that fibrinogen transfusion recovered the P-selectin expression in fibrinogen-deficient mice to >80% of the normal level within 48 hours, and four days post-transfusion, the P-selectin levels were nearly completely recovered. Furthermore, engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process since a similar impairment of P-selectin expression was also observed in fibrinogen γΔ5 mice and β3 integrin-deficient mice. To determine whether fibrinogen affects the biogenesis of platelet alpha-granules, platelets were examined via electron microscopy. No statistically significant difference in the number of platelet alpha-granules was observed in fibrinogen-deficient or β3 integrin-deficient mice compared to controls (P>0.05). These data suggest fibrinogen may play important roles in inflammation, including immune-mediated inflammation, thrombosis and hemostasis via enhancement of platelet P-selectin expression. Furthermore, there are 2–3 fold variations in human plasma fibrinogen levels in healthy populations, and there are patients with hypo- and afibrinogenemia, hyperfibrinogenemia, and Glanzmann’s Thrombasthenia (β3 integrin deficiency). Our data suggest that platelet P-selectin expression can be affected by all these conditions. Therefore, P-selectin as a platelet activation marker should be used with caution.

Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Hong Yang ◽  
Sean Lang ◽  
Zhimin Zhai ◽  
Ling Li ◽  
Walter H. A. Kahr ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Human Fibrinogen ◽  
C Terminus ◽  
Β3 Integrin ◽  
Von Willebrand ◽  
Selectin Binding ◽  
Willebrand Factor ◽  
Deficient Mice ◽  
Granule Release

Abstract Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/− platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Factor VIII Structure, Not Activity, Is the Primary Determinant of Immunogenicity in Hemophilia A and Combined Hemophilia A/Von Willebrand Disease Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1187-1187 ◽  
Author(s):  
Shannon L. Meeks ◽  
Courtney Cox ◽  
John F. Healey ◽  
Ernest T Parker ◽  
Bagirath Gangadharan ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Low Dose ◽  
Hemophilia A ◽  
Tail Vein ◽  
Fviii Inhibitor ◽  
Significant Difference ◽  
Von Willebrand ◽  
Inhibitor Titer ◽  
Deficient Mice

Abstract Abstract 1187 A major complication in the treatment of severe hemophilia A is the development of anti-fVIII antibodies in approximately 30% of patients. In these patients and in animal models injection of fVIII by an intravenous route, which traditionally is considered a tolerogenic route of protein delivery, not only fails to induce tolerance but induces a brisk T- and B- cell immune response. The role of fVIII structure and function in the immunogenicity of fVIII remains unclear. In this study we tested four interrelated hypotheses: (i) FVIII is immunogenic due to its role in promoting production of thrombin, leading to downstream events that provide an immunogenic milieu. (ii) FVIII is immunogenic because it is exposed to the immune system in the context of active inflammation (i.e. at the site of a clot). (iii) Structural determinants intrinsic to the fVIII molecule are immunogenic. (iv) FVIII is protected from the immune system until it is released from its large carrier protein von Willebrand factor (VWF). To address these hypotheses we constructed wild-type B domain deleted fVIII (wt fVIII) and 2 structurally intact inactive fVIII molecules, R372A/R1689A fVIII and an A2 domain point mutant, V634M fVIII. R372A/R1689A fVIII is inactive due to substitutions at thrombin and factor X proteolytic activation sites. It is not released from VWF, and thus may not be present at the site of a clot. V634M fVIII undergoes normal thrombin cleavage but has specific procoagulant activity that is less than 1% of wt fVIII. The immunogenicity of the fVIII molecules was compared in 3 protocols. In a low dose protocol, fVIII deficient mice were injected with 6 weekly tail vein injections of 0.2 μg followed by 2 injections of 0.5 μg wt fVIII or R372A/R1689A fVIII. In a varying dose protocol, the immunogenicity of wt fVIII, R372A/R1689A fVIII, and V634M fVIII was determined in fVIII deficient mice following 4 weekly tail vein injections of 0.5 μg, 1.0 μg, 1.5 μg, or 2.0 μg fVIII per dose followed by 1 boost at twice the dose. Finally, the immunogenicity of wt fVIII, R372A/R1689A fVIII, and V634M fVIII was compared in fVIII/VWF deficient mice following 6 weekly injections at 0.6 μg followed by 2 boost injections at 1.5 μg. In the low dose protocol 68% of fVIII deficient mice injected with wt fVIII had positive ELISA titers with a median titer of 400 compared with 40% of those injected with R372A/R1689A fVIII with a median titer of 0. Mice injected with wt fVIII had a median inhibitor titer of 10 BU/ml compared with a median titer of 0. Although R372A/R1689A fVIII was statistically less immunogenic with lower ELISA and inhibitor titers (p=0.027 and 0.018, respectively, Mann-Whitney test) this may not be clinically relevant as 40% of the mice mounted an immune response. In the varying dose protocol, there was no difference in median ELISA fVIII inhibitor titers at any dosing level. At the 2.0 μg dose all mice except for 1 in the V634M fVIII cohort mounted an immune response. The median ELISA titers at 2.0 μg were1760 for wt fVIII, 447 for R372A/R1689A fVIII, and 1480 for V634M fVIII. The median inhibitor titers at the 2.0 μg dose were 310 BU/ml for wt fVIII, 103 BU/ml for R372A/R1689A fVIII, and 288 BU/ml for V634M fVIII. There was no significant difference between wt fVIII, R372A/R1689A fVIII and V634M fVIII in either ELISA or inhibitor titers (p=0.2 and p=0.35, respectively, Kruskal-Wallis test). In the fVIII/VWF deficient mouse protocol, 85% of mice had positive ELISA titers in the wt fVIII cohort compared with 79% for R372A/R1689A fVIII and 85% for V634M fVIII. The median ELISA titers were similar for each group at 354 for wt fVIII, 179 for R372A/R1689A fVIII, and 363 for V634M fVIII. Inhibitor titers were similar for each group with a median inhibitor titer of 107 BU/ml for wt fVIII, 46 BU/ml for R372A/R1689A fVIII, and 198 BU/ml for V634M fVIII. There was no significant difference between wt fVIII, R372A/R1689A fVIII and V634M fVIII in either ELISA or inhibitor titers (p=0.46 and p=0.32, respectively, Kruskal-Wallis test). In conclusion, there was no significant difference in the immunogenicity of wt fVIII and V634M fVIII in fVIII deficient mice. R372A/R1689A fVIII was slightly less immunogenic in fVIII deficient mice in 1 of 2 protocols tested. In the absence of VWF, wt fVIII, R372A/R1689A fVIII, and V634M fVIII were equally immunogenic. This suggests that the immunogenicity of fVIII is intrinsic to fVIII structure and not its cofactor activity, while VWF may have a small protective effect. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Subcellular platelet factor VIII antigen and von Willebrand factor.

1975 ◽  
Vol 141 (5) ◽  
pp. 1101-1113 ◽  
Author(s):  
R L Nachman ◽  
E A Jaffe
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Platelets ◽  
Factor Vii ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Factor Viii Antigen ◽  
Von Willebrand ◽  
Willebrand Factor

Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.

Thrombin Stimulated Platelet Accumulation on Protein Coated Glass Capillaries: Role of Adhesive Platelet α-Granule Proteins

1989 ◽  
Vol 62 (03) ◽  
pp. 989-995 ◽  
Author(s):  
Juliette N Mulvihill ◽  
J Andrew Davies ◽  
Florence Toti ◽  
Jean-Marie Freyssinet ◽  
Jean-Pierre Cazenave
Keyword(s):  
Human Platelets ◽  
Capillary Perfusion ◽  
Coated Glass ◽  
Bovine Collagen ◽  
Glass Capillaries ◽  
Human Thrombin ◽  
Platelet Accumulation ◽  
Granule Proteins ◽  
Von Willebrand

SummaryThe generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22° C from solutions of thrombin 100 NIH units (33 μg)/ml gave surface concentrations in the range 0.019-0.101 μg/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37° C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s−1 or 2,000 s−1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet α-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s−1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by antithrombospondin antibodies.

Interactions of Purified Rat Factor VIII/von Willebrand Factor with Rat and Human Platelets – Effect of Albumin and Ristocetin

1984 ◽  
Vol 52 (01) ◽  
pp. 057-059 ◽  
Author(s):  
E Dejana ◽  
M Furlan ◽  
B Barbieri ◽  
M B Donati ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rat Plasma ◽  
Human Platelets ◽  
High Concentrations ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Rat 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.

Standardisation of von Willebrand Factor in Therapeutic Concentrates: Calibration of the 1st International Standard for von Willebrand Factor Concentrate (00/514)

2002 ◽  
Vol 88 (09) ◽  
pp. 380-386 ◽  
Author(s):  
Dawn Sands ◽  
Andrew Chang ◽  
Claudine Mazurier ◽  
Anthony Hubbard
Keyword(s):  
Von Willebrand Factor ◽  
International Study ◽  
Expert Committee ◽  
International Standard ◽  
A Value ◽  
Significant Difference ◽  
Factor Concentrate ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Good Agreement

SummaryAn international study involving 26 laboratories assayed two candidate von Willebrand Factor (VWF) concentrates (B and C) for VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCo) and VWF:Collagen binding (VWF:CB) relative to the 4th International Standard Factor VIII/VWF Plasma (4th IS Plasma) (97/586). Estimates of VWF:Ag showed good agreement between different methods, for both candidates, and the overall combined means were 11.01 IU/ml with inter-laboratory variability (GCV) of 10.9% for candidate B and 14.01 IU/ml (GCV 11.8%) for candidate C. Estimates of VWF:RCo showed no significant difference between methods for both candidates and gave overall means of 9.38 IU/ml (GCV 23.7%) for candidate B and 10.19 IU/ml (GCV 24.4%) for candidate C. Prior to the calibration of the candidates for VWF:CB it was necessary to calibrate the 4th IS Plasma relative to local frozen normal plasma pools; there was good agreement between different collagen reagents and an overall mean of 0.83 IU per ampoule (GCV 11.8%) was assigned. In contrast, estimates of VWF:CB in both candidates showed large differences between collagen reagents with inter-laboratory GCV’s of 40%. Candidate B (00/514) was established as the 1st International Standard von Willebrand Factor Concentrate by the WHO Expert Committee on Biological Standardisation in November 2001 with assigned values for VWF:Ag (11.0 IU/ampoule) and VWF:RCo (9.4 IU/ampoule). Large inter-laboratory variability of estimates precluded the assignment of a value for VWF:CB.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

The Relationship Between The Multimeric Structure Of F VIII-VWF And The Facilitation Of Platelet Adhesion To Human Subendothelium (Von Willebrand Factor Activity VWF-A)

1981 ◽  
Author(s):  
Jan J Sixma ◽  
Kjell S Sakariassen ◽  
Piet A Bolhuis
Keyword(s):  
High Purity ◽  
Human Platelets ◽  
Gel Chromatography ◽  
Absolute Requirement ◽  
Mild Reduction ◽  
Agarose Electrophoresis ◽  
Von Willebrand ◽  
The Relationship ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

The relation between the VWF-A of F VIII-VWF and its multimeric structure was studied in an annular perfusion chamber according to Baumgartner, with a steady flow system. 51Cr-labelled aspirin treated human platelets and human post mortem renal arteries were employed. F VIII-VWF was purified from cryoprecipitate by agarose gel chromatography and radiolabelled by the lactoperoxydase method. The multimeric distribution was determined by SDS-agarose electrophoresis. Five different commercial high purity concentrates contained multimers between 0.5 and 3.5 x 106 mol wt. None of these concentrates had VWF-A at ristocetin cofactor activities (RIC0F-A) of 1.0 u/ml. A low potency concentrate with mul- timers in the same mol wt range had normal VWF-A. Mild reduction (dithioerythritol-DTE ≤ 2mM) caused a shift in , mol wt range from 3.0 - 20.0 × 106 towards 0.5 - 2.0 × 106 with little decrease in RIC0F-A and VWF-A. Reduction with 10 mM DTE produced multimers of 1.5 and 0.5 × 106 without RICOF-A and VWF-A, but binding normally to the vessel wall. The void volume peak of 125I-VIII-VWF was rechromatographed on Sepharose-2B and arbitrarily divided in fractions A: mol wt 8,0 - 18.0 × 106 ;B: mol wt 4.5 - 11.0 × 106 ; and C: mol wt 2.5 - 6.5 × 106 . Binding of 125I-VIII-VWF to the subendothelium was highest for A, intermediate for B and lowest for C. Correction for mean mol wt showed that almost equal numbers of molecules bound from all three fractions. When the quantity of bound VIII-VWF was thus expressed, all fractions had a similar relative VWF-A.These data indicate that high purity concentrates do not correct the bleeding time at normal RICOF-levels, because they lack VWF-A. Multimers of high mol wt normally carry both RICOF-A and VWF-A, but the high mol wt is no absolute requirement. These data are in agreement with the notion that VWF-A resides on a specific polypeptide chain in the molecule.

scholarly journals Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway

2001 ◽  
Vol 21 (9) ◽  
pp. 3192-3205 ◽  
Author(s):  
Douglas Woods ◽  
Holly Cherwinski ◽  
Eleni Venetsanakos ◽  
Arun Bhat ◽  
Stephan Gysin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signaling Pathway ◽  
Erk Signaling ◽  
Integrin Expression ◽  
3T3 Cells ◽  
Extracellular Signal ◽  
Integrin Gene ◽  
Β3 Integrin ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

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