ABSTRACT: Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.
Project description:The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3? has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3? in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser(609) of GPIb? with alanine (S609A) significantly prevented the association of 14-3-3? with GPIb? before and after the VWF binding to GPIb?. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca(2+) levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3? with the cytoplasmic domain of GPIb? plays an important role in GPIb-IX-VWF interaction-induced signaling.
Project description:A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca(2+), morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by ?(IIb)?(3) blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIb?. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.
Project description:The interaction of platelet glycoprotein Ib? (GPIb?) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIb? binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with <i>Vwf</i> exon 28-encoding domains A1 and A2 replaced by the human homolog (VWF<sup>h28</sup>). VWF<sup>h28</sup> mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIb? on platelets (mGPIb?<sup>null</sup>;hGPIb?<sup>Tg</sup>; H1MA) to generate a new strain (H1HA) with humanized GPIb?-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIb?-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIb?-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIb?-VWFA1 binding, which also inhibited FeCl<sub>3</sub>-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIb?-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.
Project description:Von Willebrand Factor (VWF) A1-domain binding to platelet receptor GpIb? is an important fluid-shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N-terminus of the A1-domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D'D3-domain, A1-domain N-terminal flanking peptide (NFP), and O-glycans on this peptide.Full-length dimeric VWF (?Pro-VWF), dimeric VWF lacking the D'D3 domain (?D'D3-VWF), and ?D'D3-VWF variants lacking either the NFP (?D'D3NFP(?)-VWF) or just O-glycans on this peptide (?D'D3OG(?)-VWF) were expressed. Monomeric VWF-A1 and D'D3-A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ?Pro-VWF binding to immobilized GpIb? (KD?100 nmol/L) was 50- to 100-fold higher than other proteins lacking the D'D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on-rate of D'D3-A1 binding to immobilized GpIb? (kon=1.8±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.7 ?mol/L) was reduced compared with the single VWF-A1 domain (kon=5.1±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.2 ?mol/L). Thus, VWF-D'D3 primarily controls soluble VWF binding to GpIb?. In contrast, upon VWF immobilization, all molecular features regulated A1-GpIb? binding. Here, in ELISA, the number of apparent A1-domain sites available for binding GpIb? on ?Pro-VWF was ?50% that of the ?D'D3-VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ?Pro-VWF<?D'D3-VWF<?D'D3NFP(?)-VWF<?D'D3OG(?)-VWF.Whereas VWF-D'D3 is the major regulator of soluble VWF binding to platelet GpIb?, both the D'D3-domain and N-terminal peptide regulate platelet translocation and thrombus formation.
Project description:OBJECTIVE:Apheresis platelets for transfusion treatment are currently stored at room temperature because after refrigeration platelets are rapidly cleared on transfusion. In this study, the role of von Willebrand factor (VWF) in the clearance of refrigerated platelets is addressed. APPROACH AND RESULTS:Human and murine platelets were refrigerated in gas-permeable bags at 4°C for 24 hours. VWF binding, platelet signaling events, and platelet post-transfusion recovery and survival were measured. After refrigeration, the binding of plasma VWF to platelets was drastically increased, confirming earlier studies. The binding was blocked by peptide OS1 that bound specifically to platelet glycoprotein (GP)Ib? and was absent in VWF-/- plasma. Although surface expression of GPIb? was reduced after refrigeration, refrigeration-induced VWF binding under physiological shear induced unfolding of the GPIb? mechanosensory domain on the platelet, as evidenced by increased exposure of a linear epitope therein. Refrigeration and shear treatment also induced small elevation of intracellular Ca2+, phosphatidylserine exposure, and desialylation of platelets, which were absent in VWF-/- platelets or inhibited by OS1, which is a monomeric 11-residue peptide (CTERMALHNLC). Furthermore, refrigerated VWF-/- platelets displayed increased post-transfusion recovery and survival than wild-type ones. Similarly, adding OS1 to transgenic murine platelets expressing only human GPIb? during refrigeration improved their post-transfusion recovery and survival. CONCLUSIONS:Refrigeration-induced binding of VWF to platelets facilitates their rapid clearance by inducing GPIb?-mediated signaling. Our results suggest that inhibition of the VWF-GPIb? interaction may be a potential strategy to enable refrigeration of platelets for transfusion treatment.
Project description:The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib ? (GpIb?). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIb?.
Project description: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 ?IIb?3 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, ?IIb?3 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 ?IIb?3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLC?2 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.
Project description:von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ib?. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln(1238)-Glu(1260), in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIb? binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln(1238)-Glu(1260) stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIb?. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIb?. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIb?, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin(ogen)-coated surface. At a high shear rate of 1,500 s(-1), platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln(1238)-Glu(1260)) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIb? in solution under high shear forces.
Project description: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 Ib? (GpIb?) 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 ?A1-488. Although ?A1-488 alone did not bind platelets under shear, this protein bound GpIb? 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/cm(2). ?A1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ?A1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-?m beads bearing either immobilized VWF or anti-GpIb? mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm(2). Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIb? via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.
Project description:von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF-sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.