Targeting of liposomes carrying recombinant fragments of platelet membrane glycoprotein Ibalpha to immobilized von Willebrand factor under flow conditions

Liposomes with covalently bound recombinant fragments of platelet membrane glycoprotein Ibalpha that retain the von Willebrand factor (vWf)-binding function (rGPIbalpha-liposomes) were prepared. Their interactions with an immobilized vWf surface under flow conditions were evaluated with a recirculating flow chamber, mounted on an epifluorescence microscope, which allows real-time visualization of fluorescence-labeled liposomes interacting with the surface. The interaction of rGPIbalpha-liposomes with the vWf surface was directly related to shear rate. At high densities of rGPIbalpha and vWf, rGPIbalpha-liposomes establishing contact with the vWf surface exhibited continuous displacement with decreased velocity relative to the hydrodynamic flow, depending on receptor density and matrix concentration. At lower densities of rGPIbalpha and vWf, rGPIbalpha-liposomes stopped only transiently, in the millisecond range, on the surface. This is the first study to demonstrate that the targeting of rGPIbalpha-liposomes is specific to the vWf surface under flow conditions.     

Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ibalpha under flow

We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibalpha. One fragment, rvWF508-704, represented the main portion of domain A1 (mature subunit residues 497-716) within the Cys509-Cys695 disulfide loop. The other, rvWF445-733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibalpha-binding site (residues 449-728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445-733 fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s-1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibalpha. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibalpha. Only in the presence of the exogenous modulator, botrocetin, did cyclic rvWF445-733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibalpha and the stability of binding.     

The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.     

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.     

Modeling and functional analysis of the interaction between von Willebrand factor A1 domain and glycoprotein Ibalpha

Binding of the von Willebrand factor (vWF) A1 domain to the glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to reactive substrates under high shear stress conditions, a key event in hemostasis and thrombosis. We have now used the known three-dimensional structure of the A1 domain to model the interaction with the GP Ibalpha sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand beta3 and preceding loop (residues 559-566) as well as in helix alpha3 (residues 594-603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ibalpha. To verify this model, 14 mutant A1 domain fragments containing single or multiple side chain substitutions were tested for their ability to mediate platelet adhesion under flow. Each of the vWF residues Tyr(565), Glu(596), and Lys(599) proved to be strictly required for A1 domain function, which, in agreement with previous findings, was also dependent on Gly(561). Moreover, an accessory functional role was apparent for a group of positively charged residues, including Arg at positions 629, 632, 636 and Lys at positions 643 and 645, possibly acting in concert. There was, however, no evidence from the model that these residues directly participate in forming the complex with GP Ibalpha. These results provide a partial model of the vWF-GP Ibalpha interaction linked to the manifestation of functional activity in platelet adhesion.     

The cytoplasmic domain of the platelet glycoprotein Ibalpha is phosphorylated at serine 609

The alpha chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbalpha is phosphorylated at Ser(609); this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbalpha C-terminal sequence S(606)GHSL(610) only when Ser(609) is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbalpha from platelet lysates, indicating a high proportion of GPIbalpha phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3zeta. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3zeta binding. A synthetic phosphopeptide corresponding to the GPIbalpha C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3zeta. Thus, phosphorylation at Ser(609) of GPIbalpha is important for 14-3-3zeta binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbalpha staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbalpha molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser(609) of GPIbalpha regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.     

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.     

Germ-line mosaicism for a valine-to-methionine substitution at residue 553 in the glycoprotein Ib-binding domain of von Willebrand factor, causing type IIB von Willebrand disease.

The origin of new single-gene mutations resulting in inherited disease is an issue which may be at least partially resolved by our enhanced ability to detect these changes. In this report we describe the identification of a missense mutation at codon 553 (guanine to adenine) in the von Willebrand factor (vWf) gene in affected members of a family with type IIB von Willebrand's disease (vWd). We found no evidence for this substitution in 190 normal vWf genes. The encoded substitution of a methionine for a valine at this residue is nonconservative in nature and has affected a vWf protein region which has been shown to facilitate binding to the platelet receptor glycoprotein Ib. In patients with type IIB vWd this interaction is characteristically increased in affinity. This mutation has also recently been recorded in four other type IIB vWd families. Thus, there is strong circumstantial evidence to incriminate this substitution as the disease causing mutation in this family. As further supporting evidence for this claim, we have shown by vWf polymorphism analysis that the mutation originated in a vWf gene transmitted from a phenotypically normal grandfather. Analysis of the sperm from this individual showed that approximately 5% of the germ line contained the mutant 553 sequence. These results confirm (1) that the candidate type IIB vWd mutation in this family occurred at some time during the development of the germ line of the grandfather and presumably was related to a mitotic cell division and (2) that, as a result, he is a low-level germ-line mosaic for the mutation.     

Platelet membrane glycoprotein I: structure and function. The domain of glycoprotein I involved in the von Willebrand receptor

The basic structure of platelet membrane glycoprotein I (GPI) and its relation to glycocalicin are now well understood. Glycocalicin is a proteolytic fragment produced by the action of an endogenous Ca2+ activated protease. GPI consists of two glycopeptides, an alpha and a beta chain connected by a disulphide bridge. Glycocalicin is the major part of the GPI alpha chain and can be split by trypsin into a heavily glycosylated trypsin-resistant fragment and a peptide containing at least one intramolecular disulphide bridge and a thrombin binding site. Both the alpha and the beta chains of GPI show hydrophobic properties and are probably integral membrane proteins. The position of the von Willebrand factor binding site within the GPI molecule is still controversial but the bulk of the evidence points to it lying within the non-glycosylated part of the glycocalicin fragment. It is however evident that the GPI beta chain may influence the GPI alpha chain in maintaining the correct conformation of the binding site. The von Willebrand factor binding site and the thrombin binding site appear to be independent but may nevertheless influence one another.     

Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.     

Isolation and functional characterization of the von Willebrand factor-binding domain located between residues His1-Arg293 of the alpha-chain of glycoprotein Ib

In the present report we describe the isolation of a functional domain of platelet membrane glycoprotein (GP) Ib which retains von Willebrand factor (vWF)-binding activity. Glycocalicin, a proteolytic fragment of the alpha-chain of GP Ib generated by an endogenous calcium-activated protease, was submitted to digestion with trypsin. The two resulting fragments, one of 45 kDa extending between residues His1 and Arg293 and representing the amino terminus of the alpha-chain, the other of 84 kDa corresponding to the previously described macroglycopeptide, were purified to homogeneity. Glycocalicin, as well as the 45- and 84-kDa fragments, inhibited the ristocetin-dependent binding of native vWF to platelet GP Ib. The concentration inhibiting 50% of binding (IC50) was between 1 and 5 microM with all these molecules. In contrast, the binding of asialo-vWF to platelet GP Ib, measured directly in the absence of ristocetin, was blocked by glycocalicin and the 45-kDa fragment with a similar IC50, but not by the 84-kDa fragment. Both glycocalicin and the 45-kDa fragment bound to purified surface-bound vWF in a ristocetin-dependent manner and with similar affinities. Monoclonal antibodies against vWF or GP Ib inhibited this interaction in a way consistent with their inhibition of vWF binding to platelet GP Ib. These studies demonstrate that the amino-terminal extracytoplasmic region of the alpha-chain, extending between residues 1 and 293, contains a functional domain that interacts with vWF in the absence of any other structure of the GP Ib complex or any other platelet membrane component. Whereas the ristocetin-dependent binding of vWF may involve also other domains in the macroglycopeptide region, the direct vWF-GP Ib interaction appears to be mediated only by a domain in the amino-terminal region of GP Ib alpha.     

Crystal structure of von Willebrand factor A1 domain complexed with snake venom,bitiscetin: insight into glycoprotein Ibalpha binding mechanism induced by snake venom proteins

Bitiscetin, a platelet adhesion inducer isolated from venom of the snake Bitis arietans, activates the binding of the von Willebrand factor (VWF) A1 domain to glycoprotein Ib (GPIb) in vitro. This activation requires the formation of a bitiscetin-VWF A1 complex, suggesting an allosteric mechanism of action. Here, we report the crystal structure of bitiscetin-VWF A1 domain complex solved at 2.85 A. In the complex structure, helix alpha5 of VWF A1 domain lies on a concave depression on bitiscetin, and binding sites are located at both ends of the depression. The binding sites correspond well with those proposed previously based on alanine-scanning mutagenesis (Matsui, T., Hamako, J., Matsushita, T., Nakayama, T., Fujimura, Y., and Titani, K. (2002) Biochemistry 41, 7939-7946). Against our expectations, the structure of the VWF A1 domain bound to bitiscetin does not differ significantly from the structure of the free A1 domain. These results are similar to the case of botrocetin, another snake-derived inducer of platelet aggregation, although the binding modes of botrocetin and bitiscetin are different. The modeled structure of the ternary bitiscetin-VWF A1-GPIb complex suggests that an electropositive surface of bitiscetin may interact with a favorably positioned anionic region of GPIb. These results suggest that snake venom proteins induce VWF A1-GPIbalpha binding by interacting with both proteins, and not by causing conformational changes in VWF A1.     

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.     

Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg(369)-Ile(370) bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (K(d) = approximately 10 nM, B(max) = approximately 1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn(2+)) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI. Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbalpha, in a Zn(2+)-dependent fashion (K(d) = approximately 52 nM). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn(2+), whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn(2+), glycocalicin binds FXI at a heparin-binding site in A3 (Lys(252) and Lys(253)) and not by amino acids previously shown to be required for platelet binding (Ser(248), Arg(250), Lys(255), Phe(260), and Gln(263)). However, binding in the presence of HK and Zn(2+) requires Ser(248), Arg(250), Lys(255), Phe(260), and GLn(263) and not Lys(252) and Lys(253). Thus, binding of FXI to GPIbalpha is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.     

Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin. A model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib

We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury.     

Regulation of von Willebrand factor binding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.     

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

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.     

Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.