scholarly journals Anti-β2-GPI Antibodies Induce Von Willebrand Factor Release, Modulate Platelet Binding, and Affect VWF Proteolysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1444-1444
Author(s):  
Christopher J. Ng ◽  
Keith R. McCrae ◽  
Junmei Chen ◽  
Michael Wang ◽  
Marilyn J. Manco-Johnson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Syndrome ◽  
Von Willebrand Factor ◽  
Image Acquisition ◽  
Type I ◽  
Adamts13 Activity ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The antiphospholipid syndrome (APS) is characterized by predisposition to thrombosis. The cause for this pathology is poorly understood but is likely multifactorial, involving activation of blood cells and vasculature. The role that anti-β2-GPI antibodies play in von Willebrand factor (VWF) release from endothelial cells, VWF-platelet binding, and VWF cleavage by ADAMTS13 has not been well characterized in APS. We decided to study the effect of these antibodies on expressed ultra large VWF strings (ULVWF strings) that bind platelets (VWF-PLT strings) under flow to better understand platelet–VWF binding and ADAMTS13 regulation in APS. Hypothesis: We hypothesized that Anti-β2-GPI antibodies could induce VWF release from endothelial cells and modulate VWF’s prothombotic effect through alterations in VWF-Platelet binding and VWF cleavage by ADAMTS13. Methods: Human umbilical vein endothelial cells were seeded in 96-well plates/flow chambers prepared with a collagen Type I substrate for static/flow experiments, respectively. Static assays: Cells were incubated for 1 hr with Anti-β2-GPI or control antibodies and the conditioned media was assayed for VWF by ELISA, normalized to normal plasma. Flow Assay Analysis: After stimulation with agonist and perfusion with a platelet suspension, platelets bound to ULVWF in a string pattern were quantified via brightfield microscopy. Images of chambers were captured and VWF-PLT string-units (defined as a string length of 25μM) per slide were quantified. To minimize bias, image acquisition was standardized and the investigator was blinded at time of image acquisition/analysis. β2-GPI Flow assays: Endothelial cells in flow chambers were stimulated with 50ng/mL of phorbol myristate acetate (PMA), and a solution of fixed platelets with β2-GPI or β2-GPI+Anti-β2-GPI were perfused prior to image acquisition. ADAMTS13 assays: After stimulation with 25ng/mL PMA and perfusion with fixed platelets, images were acquired. Then control/patient plasma was perfused over formed strings. Images taken after plasma perfusion were quantified and compared to images prior to plasma perfusion. Data are shown as mean +/- SEM, and significance was determined as p<0.05 by student’s t-test or Mann-Whitney U Test, when appropriate. Results: Static Assays: Compared to control human IgG (8.28 +/- 3.34 mU/mL), VWF release was increased in the presence of two patient-derived Anti-β2-GPI antibodies, APS25-6 Anti-β2-GPI, 35.73 +/- 7.83 mU/mL (P = 0.008) and APS203-2 Anti-β2-GPI, 34.08 +/- 7.119 mU/mL (P = 0.039). As compared to control rabbit IgG (15.80 +/- 7.12 mU/mL), a rabbit polyclonal Anti-β2-GPI antibody, R24-6, also demonstrated increased soluble VWF (43.16 +/- 9.60 mU/mL, P = 0.013) release. β2GPI Flow Assays:The presence of β2GPI (2µM) reduced String-unit formation from 50.10 +/-5.57 Sting-units/image to 20.98 +/- 2.05 String Units/image (P < 0.0001) as compared to buffer. Addition of goat Anti-β2-GPI antibody (1µM) increased the VWF-PLT string observed as compared to β2GPI (2µM), 30.09 +/- 1.83 String Units to 20.98 +/- 2.05 String Units (P = 0.012) indicating that an Anti-β2-GPI antibody partially reverses the effect of β2GPI on reducing VWF-PLT string formation. ADAMTS13 Assay:Compared to pooled normal plasma (ADAMTS13 Activity 100%) (4.57 +/- 0.60 String Units/image cleaved), there was a significant decrease in the amount of string units/image cleaved in two APS plasmas with Anti-β2-GPI antibodies, APS232-9 (-0.23 +/- 0.98, P = 0.0003) and APS227-9 (2.23 +/- 0.73, P = 0.0009). ADAMTS13 Activity of patient plasma was 98.37% and 83.97%, respectively. These results suggest an inhibitory role of APS plasma on the cleavage of ULVWF strings. Conclusions: Anti-β2-GPI antibodies and antiphospholipid syndrome plasma may contribute to the prothrombotic phenotype observed in APS by three mechanisms: 1) the increased release of VWF from endothelial cells after incubation with Anti-β2-GPI, 2) increased platelet binding to ULVWF strings likely mediated by interfering with β2GPI’s known inhibition of Gp1bα VWF-platelet binding, and 3) a reduced ability to cleave VWF-PLT strings by APS plasma, suggestive of ADAMTS13 inhibition that does not correlate with ADAMTS13 activity. Taken together, our results suggest that VWF and its modulation may contribute to the prothrombotic phenotype observed in the antiphospholipid syndrome. Disclosures No relevant conflicts of interest to declare.

scholarly journals von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 290-290 ◽  
Author(s):  
Anil K. Chauhan ◽  
Janka Kiucka ◽  
Alexander Brill ◽  
Meghan T. Walsh ◽  
Denisa D. Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Leukocyte Rolling ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adherent Leukocytes

Abstract von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

scholarly journals Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease [published erratum appears in Blood 1990 Nov 1;76(9):1901]

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1466-1472 ◽  
Author(s):  
BM Ewenstein ◽  
A Inbal ◽  
JS Pober ◽  
RI Handin
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Umbilical Vein ◽  
Von Willebrand Disease ◽  
Type I ◽  
Molecular Defects ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12- myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

scholarly journals Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 5010-5018 ◽  
Author(s):  
Thomas D. Nightingale ◽  
Krupa Pattni ◽  
Alistair N. Hume ◽  
Miguel C. Seabra ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Crucial Role ◽  
Shear Force ◽  
Small Gtpases ◽  
Cargo Protein ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Rab Effector

Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.

Von Willebrand Factor (VWF) Regulates Angiogenesis: Studies in Blood-Derived Endothelial Progenitor Cells From Von Willebrand's Disease Patients and VWF-Deficient Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5311-5311
Author(s):  
Richard D Starke ◽  
Koralia Paschalaki ◽  
Francesco Ferraro ◽  
Thomas A J McKinnon ◽  
Nicola H Dryden ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Network ◽  
Vascular Density ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
In Vitro Angiogenesis ◽  
Deficient Mice

Abstract Abstract 5311 Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesized by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, whilst deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis which are responsible for intractable bleeding. We recently showed that VWF is involved in angiogenesis. Inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA resulted in increased in vitro angiogenesis on Matrigel, proliferation and migration. Mechanism studies implicated the endothelial VWF receptor, integrin αvβ3 and the angiogenesis regulator angiopoietin-2. The findings were confirmed in EC from VWD patients and in VWF-deficient mice. Blood outgrowth endothelial cells (BOEC) isolated from peripheral blood of patients with VWD showed decreased VWF release, compared to control BOEC, and increased in vitro angiogenesis, migration and proliferation, similar to what observed with VWF siRNA-treated HUVEC. In vivo studies using the matrigel model and imaging of blood vessels in the ear showed increased angiogenesis and vascular network in VWF-deficient mice compared to controls. Recent studies in patients BOEC and in VWF deficient mice provide new insight into the complexity of this phenotype. We have used the mouse model of post-natal angiogenesis in the retina to carry out detailed analysis of angiogenic networks in the VWF-deficient mouse, and found increased vascular density and defective vascular network. Moreover, using BOEC from patients with type I and type II VWD, we have studied VWF intracellular distribution by immunofluorescence confocal analysis and found patterns of expression that point to a variety of defects in synthesis, storage and secretion. These studies define a new function for VWF, which may have clinical implications for VWD and for patients at risk of CV disease. Moreover, studies with BOEC from VWF patients provide a novel understanding of the physiopathology of this disease. Disclosures: No relevant conflicts of interest to declare.

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