Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Address correspondence to: Jerry Ware, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, Arkansas 72205, USA. Phone: 501.526.6096; Fax: 501.686.8167; E-mail: email@example.com.
First published November 25, 2013 - More info
von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbβ3 in platelets from mice expressing a vWD-type 2B–associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbβ3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin- or ADP-induced Ca2+ store release, which is required for αIIbβ3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.
Caterina Casari, Eliane Berrou, Marilyne Lebret, Frédéric Adam, Alexandre Kauskot, Régis Bobe, Céline Desconclois, Edith Fressinaud, Olivier D. Christophe, Peter J. Lenting, Jean-Philippe Rosa, Cécile V. Denis, Marijke Bryckaert
The knowledge gained from “experiments of nature” has always been paramount in identifying key players in pathophysiologic pathways. This is well characterized by naturally occurring bleeding and thrombotic disorders. In most cases, it is the absence of a particular protein that leads to recognition of its importance for normal physiology. On the other hand, gain-of-function mutations highlight not only the presence of the protein, but also how it regulates a particular physiologic response. In this issue of the JCI, Casari and colleagues define a previously unrecognized consequence of variant type 2B von Willebrand factor (vWF) binding to blood platelets. More than 30 years after an initial description of type 2B variant vWF, the consequence of this spontaneous variant vWF binding to platelets is viewed as a dysregulation of platelet signaling pathways contributing to the type 2B bleeding phenotype.
Among bleeding disorders, von Willebrand disease (vWD) is the most common. This inherited disorder is caused by production of a dysfunctional von Willebrand factor (vWF) (1). The current paradigm for the hemostasis regulation is that vWF circulates as a soluble multimeric protein with an indeterminate affinity for platelets or the intact vessel wall (2). Following vascular damage, surface-bound vWF is exposed in the subendothelium and captured from flowing blood to become a key bridging molecule, which supports platelet adhesion to an altered vascular surface. Thus, a simple explanation for the regulation of vWF function is that soluble vWF has an unmeasurable affinity for platelets, while surface-bound vWF interacts efficiently with platelets. The importance of vWF and its platelet receptor, glycoprotein Ib-IX (GP-Ib-IX), is highlighted by well-characterized mutations that lead to the absence of vWF or GP-Ib-IX, resulting in bleeding phenotypes (3, 4). vWD constitutes a complex spectrum of clinical phenotypes that can be further subclassified based on levels of circulating protein, degree of protein multimerization, and functional interactions with platelets and coagulation factor VIII (1, 3).
One of the more intriguing vWD subtypes is the gain-of-function mutation designated vWD-type 2B (5). In this case, single amino acid mutations within the GP-Ib-IX–binding domain of vWF lead to an altered conformation that supports the soluble interaction of vWF with platelets in the circulation (Figure 1A and refs. 6, 7). Because the consequence of type 2B vWF is a spontaneous interaction with circulating platelets, the phenotypic outcome might be expected to be a prothrombotic. Instead, vWD-type 2B leads to a counterintuitive phenotype — increased bleeding. The bleeding phenotype is thought to be promoted by the largest vWF multimers, which have the most hemostatic potential, being sequestered by platelets and unable to participate in hemostasis. The presumed consequences of soluble vWF binding to platelets are intravascular platelet clumping, platelet removal from the circulation, and varying degrees of thrombocytopenia. Thus, a combination of the absence of vWF with the most hemostatic potential and thrombocytopenia has historically been the explanation of the type 2B bleeding phenotype.
Regulation of the vWF/Platelet axis. (A) In response to endothelial cell damage, WT vWF binds to exposed extracellular matrix. This binding changes vWF affinity for the GP-Ib-IX, which promotes platelet adhesion. Platelet adhesion results in activation of integrins, including αIIbβ3, which in turn promotes platelet spreading, and aggregation. (B) A mutant vWF associated with vWD-type 2B has increased affinity for platelet GP-Ib-IX. Mutant vWF prevents activation of αIIbβ3, which reduces platelet adhesion, activation, and spreading in response to endothelial cell damage.
While thrombocytopenia and the absence of high-molecular-weight vWF are reasonable explanations for type 2B bleeding, the study by Casari et al. (8) in this issue of the JCI adds an additional explanation. Their current work suggests a third mechanism for type 2B bleeding; thrombocytopathy as a result of dysregulated platelet signaling is a consequence of variant vWF binding to platelets (Figure 1B). This dysregulated signaling impairs platelet aggregation, platelet secretion, and platelet spreading. For normal hemostasis, each of these platelet events is critical for the temporal sequence of events that support hemostasis. Specifically, Casari et al. provide evidence of decreased activation of the platelet fibrinogen integrin receptor, αIIbβ3, as a consequence of type 2B vWF binding to platelet GP-Ib-IX (8). Indeed, decreased activation of αIIbβ3 would significantly impact hemostasis. Thus, the work by Casari et al. provides an important mechanistic observation to explain bleeding associated with the type 2B phenotype more than 30 years after its recognition as a distinct subtype of vWD (5).
Thrombocytopathy associated with vWD-type 2B could also explain 2 additional features of the type 2B phenotype. The first is a better explanation for why type 2B does not result in some potential for microthrombi formation. Dysregulation of platelet signaling and reduced αIIbβ3 activation would support a type 2B–dependent antithrombotic mechanism. Second, a diagnositic feature of vWD-type 2B is platelet agglutination at low doses of ristocetin, even though the overall aggregation may be reduced. Again, thrombocytopathy might be expected to reveal itself with an overall diminished platelet aggregation response, as observed using type 2B plasma.
The quest to understand the molecular basis of the vWF/platelet GP-Ib-IX regulation has been a cornerstone of basic hemostasis research. Studies of vWD-type 2B molecules have suggested that conformational changes induced by mutations in vWF increase the platelet-binding affinity of vWF. For normal vWF, the surface-bound form is assumed to undergo key conformational changes that facilitate an increased affinity for platelet GP-Ib-IX. In the case of vWD-type 2B, the intrinsic mutation itself leads to an increased affinity for platelets (6). A similar increased affinity between vWF and platelet GP-Ib-IX can be caused by mutations in GP-Ib-IX that result in a phenotype described as “platelet-type vWD (Pt-vWD)” or “pseudo-vWD” (9, 10). Whether it is a gain-of-function mutation in the ligand (vWD-type 2B) or a mutation in the receptor (Pt-vWD), the net result is an increased bleeding risk. In support of the findings of Casari et al., earlier work on the molecular basis of Pt-vWD described a disruption of signaling pathways as a consequence of normal vWF binding to the mutant GP-Ib-IX receptor (11). Both cases pose the question of whether gain-of-function mutations in the ligand or receptor mimic the molecular events that occur during normal hemostasis or thrombosis. It is well documented that the vWF/GP-Ib-IX interaction between normal molecules precedes platelet activation and facilitates hemostasis. How the interaction of a mutant ligand or receptor in the vWF/GP-Ib-IX axis leads to signaling dysregulation awaits more detailed investigation.
Whether or not the interaction between vWF and platelets requires structural changes in both vWF and the platelet receptor, GP-Ib-IX, remains a long-standing debate. Exposure of vWF and platelets to a high-shear environment leads to a spontaneous interaction; however, it is less than clear whether this shear-induced interaction mimics those events occurring following vascular damage and surface-bound vWF. Solving the crystal structures of vWF in complex with the ligand-binding domain of GP-Ib-IX has used the type 2B mutations in vWF or the Pt-vWD mutation in GP-Ib-IX to facilitate complex cocrystallization (12). Considering the evidence that mutations in either the ligand or receptor are dysregulating platelet-dependent signaling events, we wonder whether these structures are telling us everything we should know about the molecular interactions in normal hemostasis. This is difficult to answer at this point and further confounded when it is recognized that all these structures have been determined using purified domains of vWF and GP-Ib-IX. The current structural analysis disregards any complementation that might occur from other domains of vWF or GP-Ib-IX.
The work by Casari and colleagues (8) may have implications for antithrombotic targeting of the vWF/GP-Ib-IX axis. As with any antithrombotic approach, the difficulty is finding efficacy without tipping the hemostatic balance to increased bleeding risk. Indeed, as a player in primary hemostasis, the vWF/platelet GP-Ib-IX axis has, to date, been a challenge to target without considerable risk. The findings by Casari et al. suggest an antithrombotic pathway mechanistic approach that has yet to be considered. While antagonizing the ligand/receptor interaction could be justified for interrupting primary hemostasis, a type 2B mimetic has potential to bind to normal platelet GP-Ib-IX and alter signaling pathways. Could such an approach lead to an antithrombotic effect beyond just blocking a ligand/receptor interaction? Could such a strategy lead to inhibition of activation pathways that mirror aspirin and/or be complementary? While exploring this strategy is beyond the scope of the study by Casari et al., the identification of altered signaling as a consequence of mutant vWF binding to platelets suggests such an approach might be feasible. Casari and colleagues are to be commended for providing important data to explain molecular consequences for a gain-of-function mutation in vWF many years after the recognition of type 2B as a distinct subtype of vWD.
I would like to thank Ginell Post and K.I. Varughese (University of Arkansas for Medical Sciences) for critical comments and helpful discussions.
Conflict of interest: The author has declared that no conflict of interest exists.
Reference information: J Clin Invest. 2013;123(12):5004–5006. doi:10.1172/JCI73169.
See the related article at von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbβ3.