Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2637-2645 ◽  
Author(s):  
Harish Shankaran ◽  
Paschalis Alexandridis ◽  
Sriram Neelamegham
Keyword(s):  
Shear Stress ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Fluid Shear ◽  
Fluid Forces ◽  
Platelet Receptor ◽  
Hydrodynamic Shear ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

The binding of plasma von Willebrand factor (VWF) to platelet receptor GpIb under high hydrodynamic shear leads to platelet activation and subsequent shear-induced platelet aggregation (SIPA). We quantitatively examined the aspects of fluid flow that regulate platelet activation by subjecting human blood and isolated platelets to well-defined shear conditions in a cone-plate viscometer. We made the following observations. First, Annexin V binding to phosphatidyl serine expressed on activated cells was detectable within 10 seconds of shear application. Second, fluid shear stress rather than shear rate controls platelet activation, and a threshold shear stress of approximately 80 dyn/cm2 is necessary to induce significant activation. Under these conditions, individual domains of soluble VWF and platelet GpIb are subjected to similar magnitudes of fluid forces on the order of 0.1 pN, whereas GpIb with bound VWF is subjected to 1 pN. Third, cell-cell collisions and time-varying stresses are not essential for platelet activation. Fourth, the mechanism of platelet activation can be resolved in 2 steps based on the contribution of VWF and fluid forces. Fluid shear and VWF are required during the first step, when GpIb-VWF binding likely occurs. Subsequently, high shear forces alone in the absence of VWF in suspension can induce platelet activation. In other experiments, purified VWF was subjected to shear in the viscometer, and VWF morphology was assessed using light scattering. These studies demonstrate, for the first time, the ability of hydrodynamic forces to induce VWF aggregation in suspension. This VWF self-association may be an additional feature involved in controlling cell adhesion rates in circulation.

scholarly journals von Willebrand factor self-association on platelet GpIbα under hydrodynamic shear: effect on shear-induced platelet activation

Blood ◽  
2010 ◽  
Vol 116 (19) ◽  
pp. 3990-3998 ◽  
Author(s):  
Kannayakanahalli M. Dayananda ◽  
Indrajeet Singh ◽  
Nandini Mondal ◽  
Sriram Neelamegham
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Cell Activation ◽  
Cell Surface Receptor ◽  
Platelet Glycoprotein ◽  
Fluid Shear ◽  
Hydrodynamic Shear ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488–conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ΔA1-488. Although ΔA1-488 alone did not bind platelets under shear, this protein bound GpIbα on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ∼ 60 to 70 dyne/cm2. ΔA1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ΔA1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-μm beads bearing either immobilized VWF or anti-GpIbα mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIbα via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

Binding of ADAMTS13 to Von Willebrand Factor Under Static Conditions and Under Fluid Shear Stress

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 258-258
Author(s):  
Hendrik B Feys ◽  
Patricia J Anderson ◽  
J. Evan Sadler
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Time Course ◽  
Fluid Shear Stress ◽  
Proteolytic Processing ◽  
Full Length ◽  
Fluid Shear ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 is a plasma metalloprotease that is essential for the normal proteolytic processing of von Willebrand factor (VWF). Dysfunctional ADAMTS13 may lead to thrombotic thrombocytopenic purpura, as uncleaved and unusually large VWF multimers accumulate in the blood and cause intravascular platelet aggregation. Many studies indicate that proteolysis of multimeric VWF involves conformational changes in the VWF A2 domain that expose the Y1605-M1606 scissile bond and also allow substrate binding to multiple exosites on ADAMTS13. For example, VWF is resistant to proteolysis by ADAMTS13 unless the VWF is subjected to fluid shear stress, mild denaturation with guanidine or urea, or adsorption onto a surface. However, the functional interactions between shear stress, various ADAMTS13 binding sites and VWF cleavage are not understood. Therefore, we investigated the effect of fluid shear stress and ADAMTS13 structure on ADAMTS13-VWF binding and VWF cleavage. Upon mixing recombinant VWF (rVWF) and ADAMTS13 in a physiological buffer (50 mM HEPES, 5 mM CaCl2, 1 μM ZnCl2, 150 mM NaCl, pH 7.4), we found that immunoprecipitation with anti-VWF also pulled down substantial amounts of ADAMTS13. Although less striking, a similar result was obtained with purified plasma VWF. Therefore, ADAMTS13 can bind VWF without gaining access to the cleavage site in VWF domain A2. When fluid shear stress was applied for 2 min with a bench-top vortexer, ADAMTS13 binding increased 3-fold and VWF was also cleaved. Lowering the ionic strength markedly increased the rate of VWF cleavage but did not affect ADAMTS13 binding, which suggests that cleavage and binding depend on distinct VWF-ADAMTS13 interactions. Shear-induced binding was reversible slowly upon removal of unbound ADAMTS13 or rapidly by addition of SDS. ADAMTS13-VWF binding was stable for at least 24 h after cessation of shear stress, indicating that the structural change in VWF that promotes binding was not readily reversible. Using a catalytically inactive ADAMTS13 variant to simplify the analysis of binding assays, 30 nM ADAMTS13(E231Q) bound to 30 μg/ml rVWF (120 nM subunits) with a stoichiometry of 0.012 ± 0.004 under static conditions and 0.098 ± 0.023 after shearing (mean ± SD, n = 3, P = 0.019). With 120 nM ADAMTS13(E231Q) the stoichiometry increased to 0.086 ± 0.036 under static conditions and 0.469 ± 0.033 after shearing for 2 min. Recombinant ADAMTS13 truncated after TSP-1 repeat 8 (lacking the C-terminal CUB domains, delCUB), or truncated after the Spacer domain (consisting of domains MDTCS), did not bind rVWF under static conditions, implicating the CUB domains in binding to VWF. In contrast, full-length ADAMTS13, delCUB and MDTCS bound similarly to rVWF after shearing. In a previous study, delCUB and MDTCS did not cleave VWF subjected to fluid shear stress (Zhang et al, Blood2007; 110: 1887–1894). However, under the conditions employed in these experiments, MDTCS and delCUB displayed significant proteolytic activity, cleaving VWF at a rate comparable to that of full length ADAMTS13 when shear stress was applied over a time course of 0–160 sec. We conclude that ADAMTS13 CUB domains contribute to binding a few sites on multimeric VWF under static conditions, whereas ADAMTS13 MDTCS domains are sufficient to bind many sites in an altered conformation of VWF that is induced by fluid shear stress. Binding of ADAMTS13 to unsheared VWF multimers may facilitate the cleavage of VWF within a growing thrombus.

Compromised Proteolytic Cleavage of Von Willebrand Factor Type 2N Variants by ADAMTS13 in the Presence of Factor VIII (and Platelets) Under Fluid Shear Stress.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 28-28
Author(s):  
Christopher G. Skipwith ◽  
Sandra L. Haberichter ◽  
Wenjing Cao ◽  
Ashley L. Gehrand ◽  
X. Long Zheng
Keyword(s):  
Shear Stress ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Proteolytic Cleavage ◽  
Binding Activity ◽  
Clotting Factor ◽  
Fluid Shear ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 28 von Willebrand Factor (VWF) is a large, multimeric adhesive glycoprotein that is involved in the formation of a platelet plug after vascular injury. In addition, VWF functions as a carrier protein for clotting factor VIII (FVIII) which prevents rapid clearance of plasma FVIII. Decreased levels of VWF or defects in VWF function are found in patients with von Willebrand disease (VWD). Quantitative defects of VWF protein in type 1 and 3 VWD affect plasma levels of both VWF and FVIII, whereas qualitative defects in type 2 VWD result in abnormal binding of VWF to platelets such as in type 2A, 2B, and 2M, or to FVIII such as in type 2N. Previous studies have demonstrated that FVIII binds VWF, which dramatically accelerates the proteolytic cleavage of multimeric VWF by ADAMTS13 under mechanically-induced shear stresses. This rate-enhancing effect of FVIII on VWF proteolysis appears to depend on the ability of FVIII to bind VWF, as a FVIII variant lacking the A3 acidic region fails to exhibit the cofactor activity that accelerates VWF proteolysis. To determine whether reduced FVIII binding in VWF type 2N variants affects VWF proteolysis, we examined the proteolytic cleavage of recombinant VWF type 2N variants in the presence of FVIII (and lyophilized platelets) for variants with a moderate VWD phenotype (Arg854Gln and His817Gln) or severe VWD phenotype (Arg763Gly, Arg782Trp, Thr791Met, and Arg782Trp + His817Gln). Recombinant VWF type 2N variants (37.5 μg/ml or 150 nM) were incubated with ADAMTS13 (25 nM) in the absence and the presence of various concentrations of FVIII (0-40 nM) with or without lyophilized platelets (0-600×103/μl) under fluid shear stress. The proteolytic cleavage products (350 kDa) were determined by 5% SDS-PAGE and Western blot under denaturing but non-reducing conditions. We show that the proteolytic cleavage of VWF type 2N variants by ADAMTS13 under these conditions was variably reduced as compared that of wild type VWF. The reduction in the cleavage rate was proportional to the degree of reduction in VWF FVIII binding activity, which was assessed with a microtiter assay, with the least cleavage by ADAMTS13 of the variants with the lowest FVIII binding activity. This reduced cleavage of the type 2N variants was not correlated with the binding affinity between the type 2N variants and ADAMTS13 protease. These results provide further evidence that binding of FVIII to VWF, which may alter VWF conformation, is necessary to accelerate VWF proteolysis by ADAMTS13 under fluid shear stress. This variability in ADAMTS13 cleavage may contribute to the heterogeneity of bleeding phenotype of type 2N VWD variants. The bleeding phenotype may be modulated not only by plasma FVIII levels, but also the extent of VWF proteolysis by ADAMTS13 under physiological fluid shear stress. Disclosures: No relevant conflicts of interest to declare.

Filamin A binding to the cytoplasmic tail of glycoprotein Ibα regulates von Willebrand factor–induced platelet activation

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2122-2129 ◽  
Author(s):  
Shuju Feng ◽  
Julio C. Reséndiz ◽  
Xin Lu ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Fusion Protein ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Filamin A ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibα (GpIbα) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)–induced platelet activation. To begin, we examined filamin binding to GpIbα in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbα's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbα's filamin A–binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbα cytoplasmic domain fusion protein. One peptide (residues 557-575; designated “A4 peptide”) inhibited filamin A binding to the GST-GpIbα cytoplasmic domain fusion protein and competed with GpIbα for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbα and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbα regulates proaggregatory tyrosine kinase signaling.

scholarly journals ADAMTS13 Synergizes with HDL in Preventing Von Willebrand Factor Self-Association Under Shear Stress

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3451-3451
Author(s):  
Dominic W Chung ◽  
Junmei Chen ◽  
Minhua Ling ◽  
Taisha Doo ◽  
Teri Blevens ◽  
...  
Keyword(s):  
Shear Stress ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Knockout Mice ◽  
Tube Surface ◽  
Wild Type ◽  
Double Knockout ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand factor (VWF) is a plasma glycoprotein that mediates platelet adhesion at sites of vessel injury. It is synthesized in megakaryocytes and endothelial cells and is assembled in the endoplasmic reticulum and Golgi into an array of multimers. Upon secretion from microvascular endothelium, VWF multimers can further self-associate under shear stress and form surface-bound fibers of potentially enormous sizes capable of spanning the lumens of vessels up to 300 mm in diameter (Zheng et al. Nature Communications 2015 In press). These structures are normally removed by the plasma metalloprotease ADAMTS13. However, when ADAMTS13 is inactivated or when massive VWF secretion overwhelms the capacity of ADAMTS13 to process VWF, these structures persist in the microcirculation and bind platelets avidly to form occlusive thrombi, a process characteristic of the devastating disease thrombotic thrombocytopenic purpura (TTP). These microvascular VWF-platelet thrombi have also been implicated in the microvascular dysfunction that accompanies malaria, sickle cell disease, and sepsis. We recently identified high density lipoprotein particles (HDL) as being able to prevent VWF self-association into thick strands (Chung et al. Blood 2015 in revision). In these studies, we also studied VWF self-association in citrated human plasma under shear stress in a test tube in the presence of EDTA (to inhibit ADAMTS13). VWF self-associated and adsorbed to the tube surface, a phenomenon prevented by addition of HDL at concentrations above those already present in plasma. When EDTA was not added to the plasma, the majority of the VWF was not cleaved but was nevertheless stabilized in solution. This result suggests that when ADAMTS13 has been progressively inactivated by citrate at 37°C, it is able to prevent VWF self-association. It is not clear why EDTA-inhibited ADAMTS13 did not stabilize VWF to the same extent as citrate-inhibited ADAMTS13. It is possible that EDTA and citrate have different effects on the stabilization function of ADAMTS13. Further, addition of recombinant ADAMTS13 to citrated plasma (final ratio VWF monomer:ADAMTS13 = 1.6:1) did not enhance VWF cleavage under shear, but completely stabilized the VWF multimers. These results demonstrate a new function for ADAMTS13: it regulates VWF adhesive activity by preventing VWF self-association through direct binding instead of cleavage. Therefore, we hypothesize that the relative levels of VWF, HDL, and ADAMTS13 in plasma regulate the propensity of VWF multimers to self-associate under shear stress. While high VWF levels and high shear stress favor VWF self-association, high HDL and ADAMTS13 levels prevent self-association. We tested the hypothesis with plasma from wild-type or knockout mice on the C57BL6 background. In comparison to humans, wild-type C57BL6 mice have low VWF levels, high HDL levels (calculated from HDL-cholesterol levels), and express a truncated version of ADAMTS13. Further, ADAMTS13-deficient C57BL6 mice do not spontaneously develop microvascular occlusion. Unlike human citrated plasma, when citrated plasma from wild-type mice was sheared in the presence of EDTA, the VWF multimers did not self-associate. We attributed this difference from human plasma to the low VWF:HDL ratio in this mouse strain. When the plasma from apolipoprotein (Apo) A-I knockout mice was sheared in the presence of EDTA, the VWF multimers also did not self-associate, which we attributed to the low VWF level and the ability of EDTA-inhibited truncated ADAMTS13 to stabilize VWF. When the plasma of a double knockout of ApoA-I and ADAMTS13 was sheared, the VWF self-associated and adsorbed to the tube surface. Addition of HDL to this double knockout plasma stabilized the VWF. The VWF antigen levels in wild-type, single and double knockout mouse plasma were comparable. Double knockout mice challenged with a bolus injection of VWF developed more severe thrombocytopenia than did mice with either single ApoA-I or ADAMTS13 deficiency. Together, these results suggest that ADAMTS13 synergizes with HDL in stabilizing VWF and dampening its self-association into hyperadhesive forms under shear stress, and that interplay between concentrations of VWF, ADAMTS13, and HDL particles can determine the propensity for developing TTP and its severity once developed. Disclosures No relevant conflicts of interest to declare.

Inhibitory Effect of Prostacyclin and Nitroprusside on Type IIB von Willebrand Factor-promoted Platelet Activation

1996 ◽  
Vol 76 (03) ◽  
pp. 469-474 ◽  
Author(s):  
Mariangela Francesconi ◽  
Alessandra Casonato ◽  
Stefano Pagan ◽  
Arianna Donella-Deana ◽  
Elena Pontara ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Protein Tyrosine ◽  
Platelet Receptor ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand disease (vWD) of type IIB is a hereditary haemorragic disorder characterised by an excessive interaction of von Willebrand factor (vWF) with the platelet receptor GPIb which promotes platelet activation and aggregation through a phospholipase A2-mediated release of arachidonic acid.The present report shows that prostacyclin and nitroprusside, vaso-dilator-compounds that enhance the cAMP and cGMP concentration respectively, cause a drastic inhibition of the type IIB vWF-induced platelet responses including increase of cytosolic Ca2+ concentration, phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa), secretion of ATP and serotonin, and aggregation parallel to a decrease of arachidonic acid release. Type IIB vWF also elicits tyrosine phosphorylation of proteins with apparent molecular mass of 60,74,82 and 130 kDa. Prostacyclin, which induces per se tyrosine-phosphoryla-tion of proteins of about 38 and 45 kDa, inhibits drastically the type IIB vWF-promoted tyrosine-phosphorylation of the 74 kDa protein while inhibits slightly that of 60 kDa band. The protein tyrosine-kinase inhibitor genistein causes a little decrease in the type IIB vWF-induced release of arachidonic acid.It is concluded that the inhibition exerted by prostacyclin and nitroprusside on type IIB vWF-elicited platelet activation seems to be largely ascribable to prevention of the phospholipase A2 activation with the ensuing decrease of the subsequent protein tyrosine phosphorylation

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