Binding ofStaphylococcus aureusProtein A to von Willebrand Factor Is Regulated by Mechanical Force

ABSTRACTBinding ofStaphylococcus aureusto the large plasma glycoprotein von Willebrand factor (vWF) is controlled by hydrodynamic flow conditions. Currently, we know little about the molecular details of this shear-stress-dependent interaction. Using single-molecule atomic force microscopy, we demonstrate that vWF binds to theS. aureussurface protein A (SpA) via a previously undescribed force-sensitive mechanism. We identify an extremely strong SpA-vWF interaction, capable of withstanding forces of ∼2 nN, both in laboratory and in clinically relevant methicillin-resistantS. aureus(MRSA) strains. Strong bonds are activated by mechanical stress, consistent with flow experiments revealing that bacteria adhere in larger amounts to vWF surfaces when the shear rate is increased. We suggest that force-enhanced adhesion may involve conformational changes in vWF. Under force, elongation of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site to which bacteria firmly attach. In addition, force-induced structural changes in the SpA domains may also promote strong, high-affinity binding. This force-regulated interaction might be of medical importance as it may play a role in bacterial adherence to platelets and to damaged blood vessels.IMPORTANCEStaphylococcus aureusprotein A (SpA) binds to von Willebrand factor (vWF) under flow. While vWF binding to SpA plays a role inS. aureusadherence to platelets and endothelial cells under shear stress, the molecular basis of this stress-dependent interaction has not yet been elucidated. Here we show that the SpA-vWF interaction is regulated by a new force-dependent mechanism. The results suggest that mechanical extension of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. Moreover, strong binding may be promoted by force-induced structural changes in the SpA domains. This study highlights the role of mechanoregulation in controlling the adhesion ofS. aureusand shows promise for the design of small inhibitors capable of blocking colonization under high shear stress.

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Mechanism and functional impact of CD40 ligand-induced von Willebrand factor release from endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th14-04-0336 ◽

2015 ◽

Vol 113 (05) ◽

pp. 1095-1108 ◽

Cited By ~ 10

Author(s):

Kerstin Möller ◽

Oliver Adolph ◽

Jennifer Grünow ◽

Julia Elrod ◽

Miruna Popa ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Von Willebrand Factor ◽

Cd40 Ligand ◽

Dependent Manner ◽

Vascular Remodelling ◽

String Formation ◽

Stress Dependent ◽

Von Willebrand ◽

Willebrand Factor

SummaryCo-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 tris-phosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimerplatelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

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Shear-stress-induced von Willebrand factor binding to platelets causes the activation of tyrosine kinase(s)

Biochemical Journal ◽

10.1042/bj3020681 ◽

1994 ◽

Vol 302 (3) ◽

pp. 681-686 ◽

Cited By ~ 55

Author(s):

K Razdan ◽

J D Hellums ◽

M H Kroll

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Tyrosine Phosphorylation ◽

Von Willebrand Factor ◽

Arterial Blood Flow ◽

Shear Stresses ◽

Arterial Blood ◽

Stress Dependent ◽

Von Willebrand ◽

Willebrand Factor

Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca2+ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29,000-31,000, 36,000, 50,000, 58,000, 64,000, 76,000, 85,000 and 105,000 was observed. PTP in response to a threshold shear stress of 0.3 mN/cm2 (30 dyn/cm2) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm2 (90 dyn/cm2) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca2+ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r) approximately 31,000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r) approximately 29,000 was shear-stress-dependent but independent of extracellular Ca2+. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r) approximately 76,000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.

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High Concentrations of Thrombospondin-1 Suppress Shear Stress Induced Cleavage of Von Willebrand Factor by ADAMTS13.

Blood ◽

10.1182/blood.v114.22.3063.3063 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3063-3063

Author(s):

Wenhua Zhou ◽

Han-Mou Tsai

Keyword(s):

Shear Stress ◽

Von Willebrand Factor ◽

Capillary Tube ◽

Suppressive Effect ◽

Dependent Manner ◽

Microvascular Injury ◽

High Concentrations ◽

Stress Dependent ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 3063 Poster Board II-1039 ADAMTS13 is a circulating metalloprotease that cleaves and down regulates the activity of von Willebrand factor (VWF) in the circulation. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP). VWF is critical for supporting platelet adhesion and aggregation at sites of microvascular injury. Nevertheless, the factors that protect VWF from cleavage by ADAMTS13 remain unknown. Thrombospondin-1 (Thbs-1), an adhesive protein that exhibit binding to multiple proteins such as collagen, fibronectin and fibrin, promotes thrombus adherence in animal models of vascular injury. To investigate the role of Thbs-1 in the regulation of VWF cleavage by ADAMTS13, we analyzed the cleavage of VWF flowing through a capillary tube. In the presence of physiological concentrations of ADAMTS13, VWF fragments were generated in a shear stress dependent manner. The cleavage of VWF in the capillary tube device was inhibited by Thbs-1 in a concentration dependent manner. At physiological concentrations (30 ng/mL) of Thbs-1, the cleavage of VWF was decreased insignificantly to 85% of control. The cleavage was decreased to 50% at 100 ng/mL Thbs-1, and to less than 10% at 1000 ng/mL Thbs-1 (P<0.0001). The suppressive effect of Thbs-1 was also observed with a truncated variant of ADAMTS13 that lacks the distal six thrombospondin type 1 repeats and the CUB domains, suggesting that the C-terminal part of ADAMTS13 is not necessary for the suppressive effect of Thbs-1. Furthermore, the presence of platelets did not negate the suppressive effect of Thbs-1 on VWF cleavage. Thbs-1 did not suppress the cleavage of FRET-VWF73 or guanidine hydrochloride treated VWF. It also did not affect the cleavage of VWF multimers induced by vortexing in the presence of ADAMTS13 and platelets. In summary, the suppressive effect of Thbs-1 on VWF cleavage is shear stress dependent. Thbs-1 is not expected to play a major role in regulating the size of circulating VWF multimers. Nevertheless, high concentrations of Thbs-1 released from platelets may protect sheared VWF from cleavage by ADAMTS13. This protective effect of Thbs-1 on VWF proteolysis may help stabilize the hemostatic plugs at sites of microvascular injury. Disclosures No relevant conflicts of interest to declare.

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Antibody-based prevention of von Willebrand factor degradation mediated by circulatory assist devices

Thrombosis and Haemostasis ◽

10.1160/th14-02-0148 ◽

2014 ◽

Vol 112 (11) ◽

pp. 1014-1023 ◽

Cited By ~ 25

Author(s):

Antoine Rauch ◽

Paulette Legendre ◽

Olivier Christophe ◽

Jenny Goudemand ◽

Eric van Belle ◽

...

Keyword(s):

Shear Stress ◽

Von Willebrand Factor ◽

Distal Portion ◽

Assist Devices ◽

Partial Inhibition ◽

Circulatory Assist Devices ◽

Bleeding Episodes ◽

Stress Dependent ◽

Von Willebrand ◽

Willebrand Factor

SummaryHaemorrhagic episodes in patients carrying circulatory assist devices represent a severe life-threatening clinical complication. These bleeding episodes may originate from a reduced functionality of von Willebrand factor (VWF), a multimeric protein pertinent to the formation of a haemostatic plug. It has been reported that the reduced functionality is due to increased proteolytic degradation by the enzyme ADAMTS13, a phenomenon that is facilitated by device-induced increases in shear stress to which VWF is exposed. Here, we have tested a series of VWF-derived protein fragments and monoclonal murine anti-VWF antibodies for their capacity to reduce shear stress-dependent degradation of VWF. Via direct binding experiments, we identified an anti-VWF antibody that partially blocked VWF-ADAMTS13 interactions (46 ± 14%). Epitope mapping experiments revealed that the antibody, designated mAb508, is directed against the distal portion of the VWF D4-domain (residues 2134–2301) and recognises a synthetic peptide encompassing residues 2158–2169. Consistent with its partial inhibition of VWF-ADAMTS13 interactions in binding assays, mAb508 reduced ADAMTS13-mediated VWF degradation in a vortex-based degradation assay by 48 ± 10%. In a HeartMateII-based whole bloodperfusion system, mAb508 was able to reduce degradation of highmolecular- weight (HMW)-VWF-multimers dose-dependently, with a maximal inhibition (83 ± 8%) being reached at concentrations of 10 μg/ml or higher. In conclusion, we report that partial inhibition of VWF-ADAMTS13 interactions using an anti-VWF antibody can prevent excessive degradation of HMW-VWF multimers. This strategy may be used for the development of therapeutic options to treat bleeding episodes due to shear stress-dependent VWF degradation, for instance in patients carrying circulatory assist devices.

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Factor VIII and Platelets Cooperatively Accelerate Proteolytic Cleavage of Von Willebrand Factor by ADAMTS13 Under Shear Stress

Blood ◽

10.1182/blood.v112.11.590.590 ◽

2008 ◽

Vol 112 (11) ◽

pp. 590-590

Author(s):

Christopher G. Skipwith ◽

Wenjing Cao ◽

X. Long Zheng

Keyword(s):

Shear Stress ◽

Factor Viii ◽

Von Willebrand Factor ◽

Coagulation Factor ◽

Proteolytic Cleavage ◽

Cleavage Product ◽

Shear Stresses ◽

High Affinity ◽

Von Willebrand ◽

Willebrand Factor

Abstract ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 bond of the central A2 domain. Inability to cleave vWF results in thrombotic thrombocytopenic purpura (TTP), which is characterized by profound thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ involvement. Previous studies have demonstrated that coagulation factor VIII (Cao et al, PNAS, 105:7416–21, 2008) or platelets (Shim et al, Blood, 111:651–7, 2008) can accelerate proteolytic cleavage of multimeric vWF in solution by ADAMTS13 under mechanic induced shear stresses. However, the concentrations of factor VIII or platelets required to achieve a detectable increase in the proteolytic cleavage product (350K) were beyond the physiological ranges. Therefore, the physiological significance of these findings remained to be determined. In this study, we assessed whether factor VIII and platelets, both of which bind vWF with high affinity, cooperatively affected the proteolytic cleavage of vWF by ADAMTS13 under high shear stress. All experiments were performed with a fixed concentration of vWF (150 nM) and ADAMTS13 (25 nM), and various concentrations of factor VIII and platelets in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM CaCl2. The reaction mixtures (total volume 20 μl in a 0.2 ml PCR tube) were subjected to vortexing at 2,500 rpm in MixMate (manufactured by Eppendorf), which generates laminar shear stress of approximately 30 dynes/cm2. We showed that in the absence of factor VIII, lyophilized platelets at the concentration of 1,000×109 per liter did not increase the proteolytic cleavage of plasma-derived vWF by ADAMTS13. An addition of factor VIII (1, 2, and 5 nM) to the reaction mixture markedly accelerated the rate enhancing effect of lyophilized platelets in a concentration-dependent manner. In the presence of physiological concentration of factor VIII (1–2 nM), the proteolytic cleavage product (350K) detected by Western blot under non-reducing conditions reached the maximal intensity at the platelet concentration of 100×109 per liter. B-domain deleted factor VIII (FVIII-SQ) showed the similar cooperativity with platelets accelerating proteolytic cleavage by ADAMTS13. However, a construct with the acidic region (a3) that binds vWF with high affinity removed (FVIII-2RKR) did not show any cooperativity with platelets in vWF proteolysis by ADAMTS13. We conclude that binding of factor VIII and platelets to vWF cooperatively accelerates the proteolytic cleavage of vWF by ADAMTS13 under high (arterial) shear stress. These findings provide novel insight into the regulation of ADAMTS13-mediated vWF proteolysis by a coagulation factor and platelets under physiological conditions. The data also suggest a potential role of factor VIII and platelets in the therapeutic regimen for TTP.

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Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650338 ◽

1996 ◽

Vol 75 (04) ◽

pp. 655-660 ◽

Cited By ~ 59

Author(s):

Mario Mazzucato ◽

Luigi De Marco ◽

Paola Pradella ◽

Adriana Masotti ◽

Francesco I Pareti

Keyword(s):

Monoclonal Antibody ◽

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Transmembrane Flux ◽

Von Willebrand ◽

Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

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Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽

10.1182/blood.v91.10.3792.3792_3792_3799 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3792-3799 ◽

Cited By ~ 3

Author(s):

Hilde Depraetere ◽

Nadine Ajzenberg ◽

Jean-Pierre Girma ◽

Catherine Lacombe ◽

Dominique Meyer ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Platelet Glycoprotein ◽

Rotational Viscometer ◽

Induce Platelet Aggregation ◽

Static Conditions ◽

Von Willebrand ◽

Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

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