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.

The Effect of Unpaired Cysteine Residues and the C Domains On the Expression of Von Willebrand Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1078-1078
Author(s):  
Thomas A J Mckinnon ◽  
Susie Shaperio ◽  
Agata Anna Nowak ◽  
M. Laffan
Keyword(s):  
Von Willebrand Factor ◽  
Point Mutations ◽  
Full Length ◽  
Cysteine Residues ◽  
Wild Type ◽  
Free Thiol ◽  
Disulphide Bonds ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 1078 Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein that mediates platelet adhesion under high shear stress and is the carrier molecule for FVIII. VWF contains a large number of cysteine residues that were previously thought to all participate in either intra- or inter-molecular disulphide bonds. However, recently it has been shown that VWF contains a proportion of unpaired cysteine residues, or free thiols and these may be involved in lateral self association of VWF. Initially Choi et al concluded that two free thiols are present in the D3 domain C889 and C898) and seven in the C domains (C2448, C2451, C2453, C2490, C2491, C2528 and C2533). Moreover, Ganderton et al recently showed that expression of the isolated VWF C2 domain resulted in the formation of disulphide linked oligomers and suggested that lateral self-association of VWF involved the C2431-C2453 bond located in the VWF C2 domain. When they mutated C2453 to Alanine the extent of oligomerisaton was enhanced. However it is not clear how this relates to the full length VWF molecule. In the present study we investigated the effect of mutating the predicted unpaired cysteine residues on the expression of full length VWF. Initially we used the binding of MPB to VWF to compare the relative amount of free thiol on plasma derived and recombinant VWF (rVWF). Interestingly, rVWF presented an increased free thiol content compared to purified plasma derived VWF indicating that free thiols, at least in rVWF are formed without exposure to the circulation. Next we created nine individual point mutations, based the observations of Choi et al, changing the predicted unpaired cysteine residues to alanine in full length VWF and analysed their expression in HEK293T cells. Interestingly, all of the point mutations failed to secrete from HEK293T cells, with the protein being retained within the cell lysate. A double point mutant, C2431A-C2453A, similarly failed to secrete. Analysis of the pro-VWF:mature-VWF ratio and Endo-H digestion of intracellular VWF demonstrated that all the mutants were retained within the endoplasmic reticulum (ER). Co-expression experiments with wild type VWF partially restored expression of some mutants, however co-expression with a deletion A1/A3 construct, demonstrated that the molecules containing the cysteine point mutations were retained predominately in the ER. Together these data suggest that in full length VWF, correct disulphide bonding within the ER is required for protein secretion. Since the point mutations did not express we created a series of deletion mutants to remove portions of the C-terminus of VWF. While VWF with either its A1, A2, A3 or D4 domain was expressed at comparable levels to wild type, all of the created C-terminal deletion variants: ΔC1C6(2255–2720), ΔC1C2(2255–2428), ΔC3(2431–2494), ΔC3C4-(2400–2515) and ΔC3C6-(2400–2662) also failed to secrete at significant levels. This data suggest that in the full length VWF molecule an intact sequence of C-domains is required for proper expression. To establish if the cysteine mutants could be expressed in smaller VWF constructs we introduced the same mutations into VWF molecules spanning the A2-CK, A2C6 and C3-CK domains. Interestingly, the mutants failed to express in VWF-A2CK and A2C6, again being retained in the ER, but were secreted in VWF-C3CK although to a significantly less extent than wild type C3CK. Furthermore, the A2C6 construct only expressed as monomers with very few dimers. Together these data demonstrate that correct disulphide bonding and an intact series of C domains are required for passage through the ER into the Golgi and efficient VWF secretion. The location and mechanism by which certain disulphide bonds break, forming free thiols remains to be established. Disclosures: No relevant conflicts of interest to declare.

Excessively activated plasminogen in human plasma cleaves VWF multimers and reduces collagen-binding activity

2020 ◽  
Vol 168 (4) ◽  
pp. 355-363
Author(s):  
Kenshi Togashi ◽  
Satoshi Suzuki ◽  
Sae Morita ◽  
Yuki Ogasawara ◽  
Yasutada Imamura ◽  
...  
Keyword(s):  
Shear Stress ◽  
Human Plasma ◽  
Serine Protease ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Cleavage Sites ◽  
Binding Ability ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Plasmin (Pm) is a serine protease that can dissolve fibrin clots. Several possible functions of Pm in blood other than fibrinolysis have been proposed. To explore the effects of Pm on primary haemostasis, we evaluated the cleavage of von Willebrand factor multimers (VWFMs) in human plasma by streptokinase (SK)-activated plasminogen (Pg) and the binding ability of the digested VWFMs to collagen. SK-activated Pg and ADAMTS13 (a VWF-cleaving enzyme) in human plasma cleaved VWFMs in conformation-dependent manners through dialysis to the urea-containing buffer. However, VWFMs in human plasma under vortex-based shear stress were cleaved by SK-activated Pg but not by ADAMTS13. These results suggested that the VWFM-cleavage sites in human plasma are exposed to some extent by vortex-based shear stress for Pm but not for ADAMTS13. Additionally, we revealed that cleavage by SK-activated Pg reduced VWFMs’ binding ability to collagen, and VWFMs in human plasma were cleaved by Pm at several sites. These results suggest that SK-activated Pg degrades VWFMs, reduces their binding abilities to collagen and affects primary haemostasis. Because excessive Pg activation can degrade fibrinogen/fibrin, we propose that SK-activated Pg in blood may cause impaired primary and secondary haemostasis.

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.

The Mucin-like Macroglycopeptide Region of Glycoprotein Ibα is Required for Cell Adhesion to Immobilized von Willebrand Factor (VWF) Under Flow but not for Static VWF Binding

2002 ◽  
Vol 88 (10) ◽  
pp. 673-677 ◽  
Author(s):  
Chester Li ◽  
Jing-fei Dong ◽  
José López
Keyword(s):  
Shear Stress ◽  
Cell Adhesion ◽  
Ligand Binding ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Dominant Feature ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA dominant feature of the structure of platelet glycoprotein (GP) Ibα, the von Willebrand factor (VWF)-binding subunit to the GP IbIX-V complex, is the presence of an elongated, heavily glycosylated mucin-like stalk between the plasma membrane and the N-terminal 45-kDa ligand-binding domain. Here, we investigated the function of that region by expressing a mutant lacking residues 318-452 as part of a recombinant GP Ib-IX complex. We studied the VWF-binding function of this mutant under both static conditions and flow. The mutant GP Ibα was expressed normally on the surface of CHO bIX cells (stably expressing GP Ibβ and GP IX) and the proper conformation of the ligand-binding region was verified by the normal binding of 5 conformation-sensitive monoclonal antibodies. Under static conditions, cells expressing mutant GP Ibα bound VWF (binding induced by either botrocetin or ristocetin) in a manner indistinguishable from cells expressing wild-type GP Ibα. We also evaluated the ability of the mutant to mediate cell adhesion to immobilized VWF in the presence of fluid shear stress (at 2 and 10 dyn/cm2). When the mutant-expressing cells were incubated with immobilized VWF for 1 min before being exposed to shear, they rolled on the VWF surface in a manner similar to wild-type cells. However, if the cells were not first allowed to settle on the surface before the application of shear stress, the mutant GP Ibα was unable to capture the cells onto the VWF surface from the fluid stream, an indication that steric hindrance from other cell surface molecules may prevent access of the GP Ibα ligand-binding site to the surfaceimmobilized VWF.

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.

The R1306W Type 2B Natural Mutation of Von Willebrand Factor Dramatically Enhances the Multimer Sensitivity to Shear Stress

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3306-3306
Author(s):  
Raimondo De Cristofaro ◽  
Giovanni Luca Scaglione ◽  
Stefano Lancellotti ◽  
Maria Teresa Pagliari ◽  
Massimiliano Papi ◽  
...  
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Size Exclusion ◽  
Length Unit ◽  
Hydrodynamic Stress ◽  
Self Association ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Self Aggregation

Abstract Abstract 3306 Background. Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods. In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results. The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values <10dyn/cm2 the R1306W mutant, at variance with WT vWF, already showed an initial self-aggregation with formation of a network characterized by a roughness significantly higher than that of WT vWF. Mechanical stretching experiments, performed using the AFM cantilever, showed that R1306W vWF needs 30% less energy per length unit to be stretcted compared to WT protein (1.6 vs 2.2×10−11J/m) Conclusions. These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb. Disclosures: No relevant conflicts of interest to declare.

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

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
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.

A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
Author(s):  
Simon Allen ◽  
Adel M. Abuzenadah ◽  
Joanna Hinks ◽  
Joanna L. Blagg ◽  
Turkiz Gursel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Family Members ◽  
Amino Acid Position ◽  
Von Willebrand Disease ◽  
Molecular Defect ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

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

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
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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