Role of Platelets, Thrombin, Integrin llb-llla, Fibronectin and Von Willebrand Factor on Tumor Adhesion in Vitro and Metastasis in Vivo

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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Unwinding the von Willebrand factor strings puzzle

Blood ◽

10.1182/blood-2012-07-442285 ◽

2013 ◽

Vol 121 (2) ◽

pp. 270-277 ◽

Cited By ~ 84

Author(s):

Karen De Ceunynck ◽

Simon F. De Meyer ◽

Karen Vanhoorelbeke

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Cleavage Sites ◽

Normal Functional ◽

Von Willebrand ◽

Willebrand Factor

Abstract von Willebrand factor (VWF) is amongst others synthesized by endothelial cells and stored as ultra-large (UL) VWF multimers in Weibel-Palade bodies. Although UL-VWF is proteolysed by ADAMTS13 (a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13) on secretion from endothelial cells, in vitro experiments in the absence of ADAMTS13 have demonstrated that a proportion of these UL-VWF multimers remain anchored to the activated endothelium. These multimers unravel, bind platelets, and wave in the direction of the flow. These so-called VWF “strings” have also been visualized in vivo, lining the lumen of activated mesenteric veins of Adamts13−/− mice. Various studies have demonstrated the extraordinary length of these VWF strings, the availability of their platelet binding and ADAMTS13 cleavage sites, and the possible nature of their endothelial attachment. VWF strings are also capable of tethering leukocytes and parasite-infected red blood cells. However, the majority of studies have been performed in the absence of ADAMTS13, a condition only experienced in thrombotic thrombocytopenic purpura. A normal functional role of VWF strings in healthy persons or in other disease pathologies remains unclear. In this review, we discuss some of the puzzling characteristics of VWF strings, and we debate whether the properties of VWF strings in the absence of ADAMTS13 might be relevant for understanding (patho)physiologic mechanisms.

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Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645964 ◽

1987 ◽

Vol 58 (02) ◽

pp. 753-757 ◽

Cited By ~ 33

Author(s):

M F López-Fernández ◽

C López-Berges ◽

R Martín ◽

A Pardo ◽

F J Ramos ◽

...

Keyword(s):

Von Willebrand Factor ◽

Proteinase Inhibitors ◽

Relative Proportion ◽

Variable Degree ◽

Bleeding Diathesis ◽

Myeloproliferative Syndrome ◽

Von Willebrand ◽

Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

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Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656135 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1182-1188 ◽

Cited By ~ 74

Author(s):

Ulrich M Vischer ◽

Claes B Wollheinn

Keyword(s):

Endothelial Cells ◽

Adenylate Cyclase ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Free Calcium ◽

Camp Content ◽

Von Willebrand ◽

Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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Mouse models to study von Willebrand factor structure-function relationships in vivo

Hämostaseologie ◽

10.1055/s-0037-1616933 ◽

2009 ◽

Vol 29 (01) ◽

pp. 17-20 ◽

Cited By ~ 2

Author(s):

I. Marx ◽

I. Badirou ◽

R. Pendu ◽

O. Christophe ◽

C. V. Denis

Keyword(s):

Structure Function ◽

Transient Expression ◽

Von Willebrand Factor ◽

Large Size ◽

Mouse Hepatocytes ◽

Function Relationship ◽

Von Willebrand ◽

Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

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Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

10.1055/s-0038-1652578 ◽

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.

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Covalent crosslinking of von Willebrand factor to fibrin

Blood ◽

10.1182/blood.v68.1.95.95 ◽

1986 ◽

Vol 68 (1) ◽

pp. 95-101 ◽

Cited By ~ 50

Author(s):

M Hada ◽

M Kaminski ◽

P Bockenstedt ◽

J McDonagh

Keyword(s):

Von Willebrand Factor ◽

Initial Rate ◽

Factor Xiiia ◽

Covalent Bonds ◽

Covalent Crosslinking ◽

Von Willebrand ◽

Willebrand Factor ◽

Plasmin Inhibitor

Abstract Factor XIIIa crosslinks a limited number of substrates via epsilon(gamma-glutamyl)-lysyl bond formation. It crosslinks fibrin to itself, alpha 2-plasmin inhibitor and fibronectin to fibrin, and fibronectin to collagen. Results presented here show that plasma von Willebrand factor (vWF) is a substrate for factor XIIIa and can be crosslinked to fibrin during gel formation. vWF-fibrin crosslinking was studied in purified systems and in plasma with 125I-vWF and 131I- fibrinogen. vWF incorporation into fibrin increased with time or increasing factor XIIIa. After electrophoresis of dissolved clots, distribution of 125I and 131I was measured and showed that vWF was crosslinked to the alpha chain of fibrin and entered the high-mol-wt alpha polymer. vWF-fibrin crosslinking decreased the initial rate of alpha polymer formation. Crosslinking of vWF polymer to itself could not be demonstrated under physiologic conditions but occurred if vWF was reduced first. Factor XIIIa catalyzed incorporation of putrescine into both monomeric and polymeric vWF. Altogether, these studies indicate that factor XIIIa can readily form covalent bonds between glutamine in vWF and lysine in fibrin alpha chains. This reaction occurs readily in vitro when plasma clotting is slow and may occur in vivo under similar conditions.

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Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽

10.1182/blood.v99.12.4486 ◽

2002 ◽

Vol 99 (12) ◽

pp. 4486-4493 ◽

Cited By ~ 109

Author(s):

Gregor Theilmeier ◽

Carine Michiels ◽

Erik Spaepen ◽

Ingrid Vreys ◽

Désiré Collen ◽

...

Keyword(s):

Von Willebrand Factor ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Perfusion Studies ◽

Von Willebrand ◽

Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

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

Blood ◽

10.1182/blood.v108.11.1535.1535 ◽

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.

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Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.3919.3919 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3919-3919

Author(s):

Zhou Zhou ◽

Han Hyojeong ◽

Miguel A. Cruz ◽

Jose A. Lopez ◽

Jing-fei Dong ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Von Willebrand Factor ◽

Elevated Level ◽

Dependent Manner ◽

Cell Disease ◽

Von Willebrand ◽

Willebrand Factor

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

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