The snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation

Botrocetin is a snake venom protein that enhances the affinity of the A1 domain of plasma von Willebrand factor (vWF) for the platelet receptor glycoprotein Ibalpha (GPIbalpha), an event that contributes to bleeding and host death. Here we describe a kinetic and crystallographic analysis of this interaction that reveals a novel mechanism of affinity enhancement. Using high-temporal-resolution microscopy, we show that botrocetin decreases the GPIbalpha off-rate two-fold in both human and mouse complexes without affecting the on-rate. The key to this behavior is that, upon binding of GPIbalpha to vWF-A1, botrocetin prebound to vWF-A1 makes no contacts initially with GPIbalpha, but subsequently slides around the A1 surface to form a new interface. This two-step mechanism and flexible coupling may prevent adverse alterations in on-rate of GPIbalpha for vWF-A1, and permit adaptation to structural differences in GPIbalpha and vWF in several prey species.     

GPIbα-vWF rolling under shear stress shows differences between type 2B and 2M von Willebrand disease

Both type 2B and type 2M von Willebrand disease result in bleeding disorders; however, whereas type 2B has increased binding affinity between platelet glycoprotein Ibα and von Willebrand factor (vWF), type 2M has decreased binding affinity between these two molecules. We used R687E type 2B and G561S type 2M vWF-A1 mutations to study binding between flowing platelets and insolubilized vWF mutants. We measured rolling velocities, mean stop times, and mean go times at 37°C using high-speed video microscopy. The rolling velocities for wt-wt interactions first decrease, reach a minimum, and then increase with increasing shear stress, indicating a catch-slip transition. By changing the viscosity, we were able to quantify the effects of force versus shear rate for rolling velocities and mean stop times. Platelet interactions with loss-of-function vWF-A1 retain the catch-slip bond transition seen in wt-wt interactions, but at a higher shear stress compared with the wt-wt transition. The mean stop time for all vWF-A1 molecules reveals catch-slip transitions at different shear stresses (gain-of-function vWF-A1 < wt vWF-A1< loss-of-function vWF-A1). The shift in the catch-slip transition may indicate changes in how the different mutants become conformationally active, indicating different mechanisms leading to similar bleeding characteristics.     

GPIbα-vWF Rolling under Shear Stress Shows Differences between Type 2B and 2M von Willebrand Disease

Both type 2B and type 2M von Willebrand disease result in bleeding disorders; however, whereas type 2B has increased binding affinity between platelet glycoprotein Ibα and von Willebrand factor (vWF), type 2M has decreased binding affinity between these two molecules. We used R687E type 2B and G561S type 2M vWF-A1 mutations to study binding between flowing platelets and insolubilized vWF mutants. We measured rolling velocities, mean stop times, and mean go times at 37°C using high-speed video microscopy. The rolling velocities for wt-wt interactions first decrease, reach a minimum, and then increase with increasing shear stress, indicating a catch-slip transition. By changing the viscosity, we were able to quantify the effects of force versus shear rate for rolling velocities and mean stop times. Platelet interactions with loss-of-function vWF-A1 retain the catch-slip bond transition seen in wt-wt interactions, but at a higher shear stress compared with the wt-wt transition. The mean stop time for all vWF-A1 molecules reveals catch-slip transitions at different shear stresses (gain-of-function vWF-A1 < wt vWF-A1< loss-of-function vWF-A1). The shift in the catch-slip transition may indicate changes in how the different mutants become conformationally active, indicating different mechanisms leading to similar bleeding characteristics.     

Identification of peptide antagonists to glycoprotein Ibalpha that selectively inhibit von Willebrand factor dependent platelet aggregation

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.     

Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain

The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.     

Interaction of von Willebrand factor domain A1 with platelet glycoprotein Ibalpha-(1-289). Slow intrinsic binding kinetics mediate rapid platelet adhesion

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibalpha. Recombinant VWF A1 domain (residues Glu(497)-Pro(705) of VWF) bound stoichiometrically to a GPIbalpha-calmodulin fusion protein (residues His(1)-Val(289) of GPIbalpha; GPIbalpha-CaM) immobilized on W-7-agarose with a K(d) of 3.3 microM. The variant VWF A1(R545A) bound to GPIbalpha-CaM 20-fold more tightly, mainly because the association rate constant k(on) increased from 1,100 to 8,800 M(-1) s(-1). The GPIbalpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbalpha(G233V)-CaM and GPIbalpha(M239V)-CaM with a K(d) of 1.0 and 0.63 microM, respectively. The increased affinity of VWF A1 for GPIbalpha(M239V)-CaM was explained by an increase in k(on) to 4,500 M(-1) s(-1). GPIbalpha-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu(480)/Val(481)-Gly(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbalpha-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbalpha-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbalpha, and the increased binding affinity caused by mutations in VWF or GPIbalpha, are reproduced by isolated structural domains. The substantial increase in k(on) caused by mutations in either A1 or GPIbalpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k(on) and k(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.     

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of the A1 domain of von Willebrand factor (vWF) to glycoprotein Ibα (GPIbα) results in platelet adhesion, activation, and aggregation that initiates primary hemostasis. Both the elevated shear stress and the mutations associated with type 2B von Willebrand disease enhance the interaction between A1 and GPIbα. Through molecular dynamics simulations for wild-type vWF-A1 and its eight gain of function mutants (R543Q, I546V, ΔSS, etc.), we found that the gain of function mutations destabilize the N-terminal arm, increase a clock pendulum-like movement of the α2-helix, and turn a closed A1 conformation into a partially open one favoring binding to GPIbα. The residue Arg⁵⁷⁸ at the α2-helix behaves as a pivot in the destabilization of the N-terminal arm and a consequent dynamic change of the α2-helix. These results suggest a localized dynamics-driven affinity regulation mechanism for vWF-GPIbα interaction. Allosteric drugs controlling this intrinsic protein dynamics may be effective in blocking the GPIb-vWF interaction.     

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.     

Platelet aggregation by membrane-expressed A1 domains of von Willebrand Factor is dependent on residues Asp 560 and Gly 561

We have previously demonstrated that the isolated von Willebrand Factor (vWF)-A1-domain can activate platelets in a GPIb-dependent manner. Here we evaluated the functional impact of targeted point mutations Gly561His (G1324(561)H), an analog of a previously described 2M von Willebrand disease variant, and Asp560Ser (D1323(560)S) in the model of the membrane expressed A1-domain. Platelet aggregation in response to COS-7-cells stably transfected with wild type A1-domain was abrogated by both substitutions. Ristocetin did not increase the aggregatory potential of mutant vWF-A1, in contrast to native forms. Botrocetin boosted the aggregatory responses of all A1-domains tested. These data suggest that G1324(561) and D1323(560) comprise part of the GPIb binding motif essential for subsequent platelet aggregation. Botrocetin seems to alter the potential of vWF for GPIb interaction independently of that motif. The experimental system tested here provides a rapid and reproducible approach for the functional analysis of isolated A1-domain interactions with platelet-GPIb.     

Recombinant von Willebrand factor Arg578-->Gln. A type IIB von Willebrand disease mutation affects binding to glycoprotein Ib but not to collagen or heparin.

von Willebrand factor (vWF) is a multimeric plasma glycoprotein that mediates platelet adhesion to the subendothelium via binding to platelet glycoprotein Ib (GPIb) and to components of the vessel wall. Recently, missense mutations that cause type IIB von Willebrand disease (vWD) were described, clustered within a disulfide loop in the A1 domain of vWF that has binding sites for GPIb, collagen, and heparin. In type IIB vWD, plasma vWF exhibits increased affinity for platelet GPIb, but decreased binding to collagen and heparin. The effect was studied of a type IIB vWD mutation, Arg578-->Gln, on the interaction of vWF with GPIb, collagen, and heparin. Recombinant wild type rvWF and mutant rvWF(R578Q) were expressed in COS-7 cells. Ristocetin-induced binding of rvWF(R578Q) to GPIb was markedly increased compared with rvWF, confirming that the Arg578-->Gln mutation causes the characteristic gain-of-function abnormality of type IIB vWD; botrocetin-induced binding was only slightly increased. Binding to collagen type III and heparin-agarose was compared for rvWF(R578Q) and plasma vWF from patients with four different type IIB mutations: Arg543-->Trp, Arg545-->Cys, Val553-->Met, Arg578-->Gln. For all of the plasma samples, binding to collagen and to heparin was reduced compared with normal plasma. In contrast, binding of rvWF(R578Q) to collagen and heparin was normal compared with wild type rvWF. Therefore, the Arg578-->Gln mutation increases the affinity of vWF for GPIb but does not directly impair vWF interaction with collagen or heparin. Arg578 may therefore be necessary to prevent normal vWF from interacting with GPIb. In type IIB vWD, the defective binding of plasma vWF to collagen and heparin may be secondary to post-synthetic modifications that occur in vivo, such as the loss of high molecular weight vWF multimers.     

Dynamic force spectroscopy of glycoprotein Ib-IX and von Willebrand factor

The first stage in hemostasis is the binding of the platelet membrane receptor glycoprotein (GP) Ib-IX complex to the A1 domain of von Willebrand factor in the subendothelium. A bleeding disorder associated with this interaction is platelet-type von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Ibalpha subunit of GP Ib-IX. Using optical tweezers and a quadrant photodetector, we investigated the binding of A1 to GOF and loss-of-function mutants of GP Ibalpha with mutations in the region containing the two known naturally occurring mutations. By dynamically measuring unbinding force profiles at loading rates ranging from 200-20,000 pN/s, we found that the bond strengths between A1 and GP Ibalpha GOF mutants (233, 235, 237, and 239) were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined (p < 0.05). In addition, mutants 231 and 232 exhibited significantly lower bond strengths with A1 than the wild-type receptors (p < 0.05). We computed unloaded dissociation rate constant (k(off)(0)) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k(off)(0) value of 5.47 +/- 0.25 s(-1) to be significantly greater than the GOF k(off)(0) values and significantly less than the loss-of-function k(off)(0) values. Our data illustrate the importance of the bond kinetics associated with the VWF/GP Ib-IX interaction in hemostasis and also demonstrate the drastic changes in binding that can occur when only a single amino acid of GP Ibalpha is altered.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

Characterization of recombinant von Willebrand factor corresponding to mutations in type IIA and type IIB von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from defects in von Willebrand factor (vWF). Although both type IIA and IIB vWD are characterized by the absence of high molecular weight multimers in plasma, vWF from patients with type IIA vWD demonstrates a decreased affinity for the platelet receptor glycoprotein Ib (GPIb), whereas vWF from patients with type IIB vWD show an increased affinity for GPIb. To investigate how structural alterations in vWF affect its interaction with GPIb, we reproduced the reported potential mutations in type IIA and IIB vWD in vWF cDNA and expressed the recombinant proteins in COS-1 cells. The type IIA vWF potential mutation was represented by a G-->A transversion which results in the substitution of Lys for Glu at position 875 in the mature vWF subunit (rvWFLys875). The type IIB vWF mutation corresponds to a duplicated ATG codon, resulting in three contiguous methionines starting at position 540-541 in the normal vWF sequence (rv-WFduplMet540-541). The subunit composition and multimeric structure of both mutant proteins were similar to the wild-type rvWF. The rvWFLys875 bound to fixed platelets in the presence of ristocetin similar to wild-type rvWF. The rvWFduplMet540-541 bound to fixed platelets in the absence of agonist. The rvWFLys875 appears to interact normally with GPIb, and the decreased affinity for the platelet receptor observed in plasma is probably a consequence of prior reduction in multimeric size resulting from the defect. In contrast, the duplication of Met540-541 increases the reactivity of vWF for its platelet receptor.     

Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibalpha complex reveals conformation differences with a complex bearing von Willebrand disease mutations

The adhesion of platelets to the subendothelium of blood vessels at sites of vascular injury under high shear conditions is mediated by a direct interaction between the platelet receptor glycoprotein Ibalpha (GpIbalpha) and the A1 domain of the von Willebrand factor (VWF). Here we report the 2.6-A crystal structure of a complex comprised of the extracellular domain of GpIbalpha and the wild-type A1 domain of VWF. A direct comparison of this structure to a GpIbalpha-A1 complex containing "gain-of-function" mutations, A1-R543Q and GpIbalpha-M239V, reveals specific structural differences between these complexes at sites near the two GpIbalpha-A1 binding interfaces. At the smaller interface, differences in interaction show that the alpha1-beta2 loop of A1 serves as a conformational switch, alternating between an open alpha1-beta2 isomer that allows faster dissociation of GpIbalpha-A1, as observed in the wild-type complex, and an extended isomer that favors tight association as seen in the complex containing A1 with a type 2B von Willebrand Disease (VWD) mutation associated with spontaneous binding to GpIbalpha. At the larger interface, differences in interaction associated with the GpIbalpha-M239V platelet-type VWD mutation are minor and localized but feature discrete gamma-turn conformers at the loop end of the beta-hairpin structure. The beta-hairpin, stabilized by a strong classic gamma-turn as seen in the mutant complex, relates to the increased affinity of A1 binding, and the beta-hairpin with a weak inverse gamma-turn observed in the wild-type complex corresponds to the lower affinity state of GpIbalpha. These findings provide important details that add to our understanding of how both type 2B and platelet-type VWD mutations affect GpIbalpha-A1 binding affinity.     

Determination of interaction kinetic constants for HIV-1 protease inhibitors using optical biosensor technology

The interaction between HIV-1 protease and inhibitors has been studied with optical biosensor technology. Optimized experimental procedures and mathematical analysis permitted determination of association and dissociation rate constants. A sensor surface with native enzyme was unstable and exhibited a drift that was influenced by the binding of inhibitor. This was hypothesized to be due to a specific mechanism involving autoproteolysis and/or dimer dissociation. The use of a mutant predicted to be less susceptible to autoproteolysis (Q7K) than wild-type enzyme resulted in a minor effect on surface stability, while a completely stable surface was obtained by treatment of the immobilized enzyme with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide; the most stable surface was achieved by chemically modifying the Q7K enzyme. The stabilized surface was enzymatically active and the interaction with inhibitors was similar to that for native enzyme. Several of the inhibitors had very high association rates, and estimation of kinetic constants was therefore performed with a binding equation accounting for limited mass transport. Of the clinical inhibitors studied, saquinavir had the highest affinity for the enzyme, a result of the lowest dissociation rate. Although the dissociation rate for ritonavir was sixfold faster, the affinity was only twofold lower than that for saquinavir since the association rate was threefold faster. Nelfinavir and indinavir exhibited lower affinities relative to the other inhibitors, a consequence of a slower association for nelfinavir and a relatively fast dissociation for indinavir. These results show that biosensor-based interaction studies can resolve affinity into association and dissociation rates, and that these are characteristic parameters for the interaction between enzymes and inhibitors.     

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

Structural basis of von Willebrand factor activation by the snake toxin botrocetin

The A1 domain of von Willebrand factor (vWF) mediates platelet adhesion to sites of vascular injury by binding to the platelet receptor glycoprotein Ib (GpIb), an interaction that is regulated by hydrodynamic shear forces. The GpIb binding surface of A1 is distinct from a regulatory region, suggesting that ligand binding is controlled allosterically. Here we report the crystal structures of the "gain-of-function" mutant A1 domain (I546V) and its complex with the exogenous activator botrocetin. We show that botrocetin switches the mutant A1 back toward the wild-type conformation, suggesting that affinity is enhanced by augmenting the GpIb binding surface rather than through allosteric control. Functional studies of platelet adhesion under flow further suggest that the activation mechanism is distinct from that of the gain-of-function mutation.