Vimentin Exposed On Platelets Serves As an Adhesive Receptor for Von Willebrand Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 259-259
Author(s):  
Miguel A. Cruz ◽  
K. Vinod Vijayan
Keyword(s):  
Binding Site ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Platelet Receptor ◽  
Coated Surface ◽  
Initial Interaction ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 259 Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions. Disclosures: No relevant conflicts of interest to declare.

Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1144-1144
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Nola Fuller ◽  
J. Evan Sadler ◽  
Philip G de Groot ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
A Domains ◽  
Adhesive Proteins ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 1144 Introduction: Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding. Methods: Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control. Results: Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains. Conclusions: The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury. Disclosures: No relevant conflicts of interest to declare.

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1149-1155 ◽  
Author(s):  
Mitsuhiro Kuwahara ◽  
Mitsuhiko Sugimoto ◽  
Shizuko Tsuji ◽  
Shigeki Miyata ◽  
Akira Yoshioka
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Cytosolic Calcium ◽  
Flow Chamber ◽  
Flow Conditions ◽  
Color Changes ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Calcium Green

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Background It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. Results Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM) microscopy, here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the glycoprotein Ib and the A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. Conclusions These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Biomechanical Properties of Bonds Mediated by the von Willebrand Factor A1 Domain during Platelet Adhesion under Flow Conditions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3665-3665
Author(s):  
Marianna M. Machin ◽  
Jennifer N. Orje ◽  
Paolo Canu ◽  
Zaverio M. Ruggeri
Keyword(s):  
Residence Time ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Bond Formation ◽  
Biomechanical Properties ◽  
Hydrodynamic Forces ◽  
Flow Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The initial attachment of platelets to vascular lesions exposed to a high wall shear rate (γw) depends on the interaction between the membrane glycoprotein (GP) Ibα, a component of the GP Ib-IX-V receptor complex, and the A1 domain of surface-immobilized von Willebrand factor (VWFA1). We performed perfusion experiments under different flow conditions to measure transient platelet contacts onto immobilized recombinant VWFA1 and determine the probability of bond formation (capturing) and resistance to tensile stress (bond lifetime) of VWFA1-GP Ibα interactions. To define how molecular conformations influence the biomechanical properties of the bonds, we compared fragments exhibiting the native dimeric assembly of A1 domain with monomeric fragments obtained by selective purification of recombinant protein expressed in stable D. melanogaster cell lines. The minimum coating concentration of dimeric VWFA1 at which platelet adhesion events were statistically significantly different from nonspecific interactions on uncoated glass was 1 μg/ml. In the range of γw between 30 and 30,000 s−1, there was no threshold value for the initiation of adhesion, as seen for selectins. The number of adhering platelets first increased and then decreased with monotonically increasing γw, indicating the effect of transport phenomena as well as hydrodynamic forces on the VWFA1-GP Ibα interaction. Maximum event number was at 5,000 s−1 for dimeric and 1,500 s−1 for monomeric VWFA1. The platelet count had no statistically relevant influence on the efficiency of capturing and duration of adhesive contacts. As γw increased, a higher coating concentration of the VWF A1 domain was required to initiate platelet adhesion. The coating concentration determined the number of individual adhesion events that could occur over a defined period of time but did not affect the residence time, which is a measure of the strength of the bond between the receptor and the ligand. Hydrodynamic forces generated by blood flow shortened the duration of VWF-GP Ibα interactions. The percentage of platelets that had a residence time of less than 0.1 s increased almost linearly with increasing γw. Dimeric A1 domain was more efficient than the monomeric counterpart in promoting platelet adhesion as it displayed activity at lower coating concentrations. At permissible γw, a 10-fold higher monomer than dimer coating concentration (2 vs. 20 μg/ml) was required to obtain a similar capturing efficiency. Moreover, the upper limit of γw compatible with the initiation of adhesion was significantly higher for dimeric as compared to monomeric A1 domain. Doubling the dimer coating concentration resulted in a 5-fold increase in the γw limit for adhesion, but the same increase in the monomer coating concentration did not enhance the probability of bond formation at higher γw. In spite of the substantial difference in capturing efficiency, monomeric and dimeric VWFA1 supported platelet adhesion events of similar duration at any given γw, indicating that a different molecular conformation did not affect the lifetime of the interaction with GP Ibα. These results indicate that the dimeric assembly of A1 domains in VWF multimers may be crucial to support the initiation of platelet adhesion at high shear rates, but the duration of each adhesion event is limited by intrinsic properties of the individual VWFA1-GP Ibα bond.

KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS

1987 ◽  
Author(s):  
Hans H F I van Breugel ◽  
Philip G de Groot ◽  
Jan J Sixma
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Physical Parameters ◽  
Flow Conditions ◽  
Binding Studies ◽  
Platelet Suspension ◽  
Coated Surface ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.

scholarly journals Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4413-4424 ◽  
Author(s):  
Masaaki Moroi ◽  
Stephanie M. Jung ◽  
Shosaku Nomura ◽  
Sadayoshi Sekiguchi ◽  
Antonio Ordinas ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Initial Reaction ◽  
Adhesion Assay ◽  
Flow Conditions ◽  
Coated Surface ◽  
Von Willebrand ◽  
Willebrand Factor

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.

A VWF Fragment Spanning the A1, A2, and A3 Domains Supports Platelet Adhesion Better Than the Isolated A1 Domain and Is Considerably More Resistant to Cleavage by Adamts-13 Than the Isolated A2 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3664-3664
Author(s):  
Arnoldo A. Padilla ◽  
Jose A. Lopez ◽  
Miguel A. Cruz
Keyword(s):  
Platelet Adhesion ◽  
Flow Chamber ◽  
Multimeric Protein ◽  
A Domains ◽  
The Individual ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Better Than

Abstract von Willebrand factor (VWF) is a large multimeric protein whose monomers are of the domain structure D’-D3-A1-A2-A3-D4-B1-B2-B3C1-C2. The three tandem A domains mediate several important functions of the protein including supporting platelet adhesion, through an interaction of A1 with the platelet GP Ib-IX-V complex, and binding collagen, through A3 and A1. Further, the A2 domain contains the cleavage site for the plasma metalloprotease ADAMTS-13, which processes the newly synthesized and extremely adhesive ultra-large forms of VWF (ULVWF) to the forms normally found in plasma. Our goal was to compare the functions of the individual A domains as isolated domains to their function in the context of the A1-A2-A3 fragment. All recombinant domains were produced in bacteria. The A1-A2-A3 fragment contained an N-terminal thioredoxin-tag and a C-terminal 6Xhis tag and was purified in 2 steps using nickel and heparin columns. The recombinant protein was correctly folded, as indicated by the fact that it was recognized by a number of conformation-specific antibodies. Using ELISA, we examined the interaction of the fragment with both glycocalicin (the soluble extracellular domain of GP Iba) and collagen. At equivalent concentrations, A1-A2-A3 bound collagen better than did the isolated A1 and A3 domains, but the binding to glycocalicin was approximately 7.5 times lower than the A1 domain. We then tested the ability of the fragment to support platelet adhesion under flow, by coating the fragment onto a coverslip that made up the bottom of a parallel-plate flow chamber. In contrast to the findings with immobilized glycocalicin, immobilized A1-A2-A3 supported the attachment, rolling, and firm adhesion of the platelets to the surface, whereas A1 did not support firm adhesion. Finally, we tested cleavage of the fragment by plasma ADAMTS-13, using both ELISA and western blotting to detect cleavage. The rate of cleavage of the A1-A2-A3 fragment was much slower than that of the recombinant A2 domain, needing a longer incubation time to achieve a similar percentage of cleavage in a 15 fold higher concentration of plasma. These results show clearly that within the triplicate A domains of VWF, adjacent A domains influence each other’s functions, possibly by binding each other or by conformational modulation. The fact that the fragment is able to support firm platelet adhesion in isolation suggests that it may contain a second, previously unidentified binding site for platelet receptors.

Mutations in the von Willebrand Factor A1 Domain That Increase the Affinity for Collagen Have Different Effects on the Binding to Glycoprotein Ib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3661-3661
Author(s):  
Miguel A. Cruz ◽  
Liza D. Morales
Keyword(s):  
Binding Site ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Type I ◽  
Fibrillar Collagen ◽  
Wild Type ◽  
Collagen Types ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The interaction of plasma von Willebrand factor (VWF) with collagen at the site of vascular injury plays a critical role in the initiation of thrombus formation under high shear stress. It does this by forming a bridge between the fibrils of collagen in the subendothelium and the platelet glycoprotein Ib-IX-V complex (GPIb). The A1 domain of VWF is the binding site for GPIb whereas the collagen-function of VWF is controlled by both A1 and A3 domains. The VWF-A3 domain is important to support binding to fibrils of collagen Types I and III while the A1 domain is involved in the binding to microfibrillar collagen Type VI. It is assumed that the interaction of VWF with fibrillar collagen (via the A3 domain) may regulate the expression of the GPIb-binding site in the A1 domain. However, there is no a definite data to substantiate that hypothesis. Our goal was to demonstrate that a direct interaction between the A1 domain and fibrillar collagen Types I or III exposes the GPIb binding site. Thus, we postulated that platelet GPIb is able to interact with isolated A1 domain that is bound to collagen. We have demonstrated that the VWF-A1 protein binds specifically to human placenta collagen Types I and III with a KD ~ 200 nM by using surface plasmon resonance (SPR). Using plasma-free blood, we have provided strong evidence that isolated VWF-A1 domain bound to either collagen Type I or III is able to support platelet adhesion under high flow conditions. This platelet interaction was effectively blocked with antibodies against either GPIb or A1 domain. These results clearly show the ability of the A1 domain to concurrently interact with both GPIb and collagen fibrils and they also suggest that the collagen-A1 binding may regulate the expression of the GPIb-binding site in the A1 domain. To test this hypothesis, we analyzed three residues that in a previous mutagenesis study they increased the binding of VWF to GPIb, reasoning that they may have an effect on the collagen binding activity as well. The three residues are located in the a7 helix (rear face) of the folded A1 domain and mutagenesis studies of other I(A)-domains have demonstrated that this helix plays a role in regulating the affinity of the ligand-binding. We introduced point mutations into the 3 residues and the recombinant mutant proteins were expressed in bacteria. The three mutants (R687E, D688R, and E689R) were purified as wild type and their structural integrity was confirmed with three conformation-specific antibodies. All the mutants bound to both collagens Type I or III with an affinity much higher than the wild type (WT) (KD~ 9 -1 nM). The mutants were assessed by their ability to mediate platelet adhesion to collagen, and their ability to inhibit both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. Interestingly, in the three assays the R687E mutant had an activity higher than WT while the D688R had a markedly decrease activity. The mutant E689R had an activity similar to that of WT for the three assays. Together our data indicate that a direct association between the VWF-A1 domain and collagen fibrils influences the expression of GPIb binding function in VWF. Further, these data indicate that residue R687 located in the a7 helix plays a novel and important role in modulating the collagen/A1/GPIb binding.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Background It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. Results Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. Conclusions These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

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