Regular ArticleThe role of von Willebrand factor in thrombus formation
Introduction
Mature von Willebrand factor (VWF) is a multimeric protein composed of a variable number of identical subunits, each consisting of 2050 amino acid residues and up to 22 carbohydrate side chains [1]. Subunits are disulphide-bonded into dimers of approximately 500 kDa, which in turn are disulphide-linked into multimers of increasing size that can reach a molecular mass as high as 20 MDa. Our understanding of the structure and function of VWF and the mechanisms that underlie its involvement in haemostasis are developing constantly. The essential physiological functions of VWF have been elucidated, including binding and transportation of the pro-coagulant factor VIII (FVIII), mediating platelet adhesion to reactive surfaces, and mediating platelet aggregation and thrombus growth. Discrete domains within the VWF subunit exhibit specific functions that have been defined [2]. The major functional domains of VWF are A1, which contains the only binding site for the platelet receptor glycoprotein (GP) Ibα, A3, through which VWF binds to collagen, C1, which contains the RGD sequence recognized by the β3 integrins (αIIbβ3 and αvβ3) and D′–D3, which form the site that binds FVIII [2].
Many details regarding the biosynthesis and secretion of VWF are well established [3]. VWF is synthesised exclusively in endothelial cells and megakaryocytes. Following synthesis, the VWF produced in endothelial cells is secreted via one of two distinct pathways: a constitutive pathway directly linked to synthesis (i.e. molecules are released as soon as their synthesis is completed), and a regulated pathway involving storage of mature molecules for release after stimulation by secretagogues [3]. Because released VWF undergoes a process of regulated reduction of multimer size, the availability of a source of uncleaved larger multimers from cellular storage sites permits maximal function in areas where rapid platelet adhesion and aggregation is required. The storage organelles for VWF that are found in endothelial cells and megakaryocytes are the rod-shaped Weibel–Palade bodies and the α-granules, respectively [3], [4]. In megakaryocyte-derived circulating platelets, only the regulated pathway of VWF secretion is effectively operative in vivo. Therefore, the VWF circulating in plasma is essentially all of endothelial cell origin [5], as platelets release their α-granule content only when activated [5]. The VWF secreted from endothelial cells, through either the constitutive or regulated pathway, is directed towards both the lumen and the subendothelial matrix.
Von Willebrand factor is initially uncleaved, and the physiological reduction in the size of the multimers occurs through a controlled proteolytic cleavage event. The metalloproteinase, ADAMTS13 [6], cleaves specifically the Tyr1605-Met1606 peptide bond [7]. This cleavage has effects on the size of circulating multimers and consequently modulates proadhesive functions [8]. The largest VWF multimers display enhanced thrombogenic functions, possibly because multiple interactive sites for vessel wall components and platelets support more efficient adhesion. Thus, the controlled release of the largest multimers at the time of injury allows their presence at sites of tissue damage, and physiological regulatory mechanisms cause their disappearance from the circulation, possibly to prevent excessive thrombus formation [2].
In the present review article, recent advances in the role of VWF in the complex events of platelet thrombus formation are reviewed, with an emphasis on the processes influenced by shear rates occurring in pathological conditions.
Section snippets
The role of VWF in platelet adhesion and aggregation
VWF works to support thrombus formation not only with respect to maintaining platelet adhesion to sites of injury but also platelet–platelet cohesion or aggregation. Platelets respond rapidly to alterations of endothelial cells by attaching firmly to the site of lesion where exposure of subendothelial components may have occurred. The first layer of platelets is in contact with the thrombogenic surface (adhesion), whereas subsequent growth of the haemostatic plug depends on platelet-to-platelet
Mechanisms of platelet adhesion
In a flow field with shear rate above a threshold value (in human circulation around 1000 s− 1), only GP Ibα interaction with immobilised VWF multimers (for example bound to collagen) can initiate the tethering of circulating platelets to the vessel wall (Fig. 1) [9]. The binding of GP Ibα to the A1 domain of VWF occurs rapidly and is the essential adhesive interaction that can tether platelets to a surface when the shear rate is elevated. This interaction supports initially transient bonds, and
Mechanisms of platelet aggregation
The first layer of activated platelets that are firmly attached to a reactive surface becomes the substrate for accumulation of more platelets and thrombus growth (Fig. 2) [9]. Adhesive ligands, mainly fibrinogen and VWF, bind via activated αIIbβ3 on the membrane of the adherent platelets and become the substrate for the additional recruitment and attachment of incoming platelets. In this phase of thrombus growth (or platelet aggregation), the shear rate at the surface of the growing thrombus
Influence of shear rate on thrombus formation
The concept that VWF is required for thrombus formation on collagen when the shear rate is high, but not necessarily when it is low, can be demonstrated in real-time perfusion studies [9]. In one such experiment, thrombi formed when control blood was perfused over collagen type 1 fibrils at shear rates of 500 s− 1 and 1500 s− 1 (Fig. 3) [9]. However, when the same blood was treated before perfusion with an anti-VWF A3 antibody that blocks binding to collagen, thrombus formation was abolished when
Platelet adhesion and aggregation under elevated shear stress
Haemostasis and pathological arterial occlusion occur in distinct hydrodynamic environments. While platelet aggregation is conventionally thought to initiate after signalling-induced activation, it now appears that additional mechanisms can operate under blood-flow conditions comparable to those existing in stenotic coronary arteries.
Distinct platelet adhesion and aggregation mechanisms as a function of shear rate
A key function of VWF is to initiate platelet aggregation under elevated shear stress conditions, independent of activation [11]. This form of aggregation may precede, and is necessary for, stable adhesion to thrombogenic surfaces. We have recently demonstrated platelet aggregation independent of activation [11]. Thrombus formation was examined using model reactive surfaces composed of collagen type I fibrils or immobilised VWF in a flow chamber perfused with blood containing an α-thrombin
Evaluation of activation-independent platelet adhesion and aggregation
To enable a better visualisation of the process of activation-independent adhesion and aggregation, we prevented platelet activation, which would normally be nearly instantaneous following adhesion, by adding prostaglandin (PG) E1 to the blood, and prevented ligand binding to integrins by adding EDTA. Single platelets adhered to immobilised VWF at a shear rate of 3000 s− 1, but at 24,000 s− 1 rolling platelets aggregated within 100–200 μm from the boundary where immobilised VWF was exposed to
Activation-independent platelet aggregation in vivo
To evaluate whether VWF-mediated platelet aggregation occurs within the vasculature in vivo, we performed experiments in the mesenteric circulation of anaesthetised mice [11]. Platelets that had been treated with PGE1 were labelled with a red fluorochrome and non-treated platelets were labelled with a green fluorochrome. Platelets were then injected back concurrently into the mouse and their accumulation monitored at a site of injury. The green-labelled platelets formed thrombi and attached in
Conclusions
Pathological arterial blood flow generates fluid shear stresses that directly cause platelets to aggregate. At shear rates up to 10,000 s− 1, initial adhesion to a reactive substance and subsequent aggregation follow the generally accepted pattern of progressive accrual of single platelets. However, the studies described here have shown that at shear rates greater than 10,000 s− 1, activation-independent platelet aggregation mediated by soluble VWF facilitates adhesion and precedes stable
Acknowledgements
The author wishes to acknowledge the contribution of Dr Sandra Cox who provided writing assistance in the preparation of the manuscript, which is part of a proceedings supplement from a CSL-Behring-sponsored symposium in which the author received an honorarium.
Conflict of interest
None declared.
Role of the funding source
The original work referred to in this article was supported by grants from the National Institutes of Health of the United States (National Heart, Lung and Blood Institute grants
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