Von Willebrand Factor A1 Research Articles

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions

Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.

SLA Class III 영역의 돼지 Complement Factor B(CFB) 유전자의 Cloning, cSNP 동정 및 유전자형 분석

ABSTRACT The primers for RT-PCR and RACE-PCR were designed by aligning the pig genomic sequence and the human complement factor B (CFB) coding sequence (CDS) from the GenBank. Each PCR product was amplified in pig cDNA and sequencing was carried out. The CDS length of pig CFB gene was determined to be 2298 bp. In addition, the pig CDS was more longer than human and mouse orthologs because of insertion and deletion. The identities of porcine nucleotide sequences with those of human and mice were 84% and 80%, and the identities of amino acids were 79% to 77%, respectively. Three complement control protein (CCP) domains, one Von Willebrand factor A (VWFA) domain and a serine protease domain, that are revealed typically in mammals, were found in the pig CFB gene. Based on the CDSs determined, the primers were designed in intron regions for amplification of entire length of exons. In amplification and direct sequencing with genomic DNAs of six pig breeds, three cSNPs (coding single nucleotide polymorphisms) were identified and verified as missense mutations. Using the Multiplex-ARMS method, we genotyped and verified the mutations identified from direct sequencing. To demonstrate recrudescence, we performed both direct sequencing and Multiplex-ARMS with two randomly selected DNA samples. The genotype of each sample exhibited the same results using both methods. Therefore, three cSNPs were identified from pig CFB gene and that can be used for haplotype analysis of the swine leukocyte antigen(SLA) class III region. Moreover, the results indicate that the Multiplex-ARMS method should be powerful for genotyping of genes in the SLA region.(

Crystal Structure of Von Willebrand Factor (VWF) A1 Domain in Complex with Aptamer ARC1172, An Inhibitor of VWF-Platelet Binding

ARC1779 is a modified 40-mer oligonucleotide aptamer that binds with high affinity to the VWF A1 domain and blocks VWF binding to platelet GPIb-alpha. When given intravenously to volunteers and assayed ex vivo, ARC1779 dose-dependently inhibits VWF activity and prolongs the PFA-100 closure time with EC90 values of 2–3 μg/ml. ARC1779 also inhibits carotid artery thrombosis induced by electrical injury in nonhuman primates. These properties suggest that ARC1779 may be useful to prevent or treat platelet-dependent thrombosis in acute coronary syndromes, percutaneous coronary intervention procedures, or thrombotic thrombocytopenic purpura. To characterize this aptamer-VWF interaction, we determined the crystal structure of a complex between recombinant VWF domain A1 (residues 497–705, mature subunit numbering) expressed in E. coli and ARC1172, a 41-mer DNA oligonucleotide with the sequence 5′-GGC GTG CAG TGC CTT CGG CCG TGC GGT GCC TCC GTC ACG CC-invdT-3′. ARC1779 was derived from ARC1172 by adding modifications to prevent digestion by nucleases, and both aptamers bind VWF A1 with the same affinity. Crystals of the complex belonged to space group P212121 and diffracted to 2.4 angstroms. The A1 domain (Figure) has essentially the same structure as observed previously in isolation or bound to GPIb-alpha, botrocetin, bitiscetin, or Fab NMC4. Therefore, aptamer binding does not substantially alter the conformation of the VWF A1 domain. ARC1172 consists of three short helical segments and three connecting loops. Nucleotides C32-C40 interact with two segments G23-G26 and G2-G6 to form a 9-basepair helix with an interruption in one strand. The interruption comprises residues C7-T22, which protrude to form a 3-basepair helix between G11-C13 and G17-C19. The loop of ARC1172 composed of nucleotides G26-C32 makes extensive contacts with VWF A1 residues in helix alpha-4 (yellow) and alpha-5 (orange) and loop betaD-alpha4. For example, the side chains (shown in Figure) of Q625, Q628, R632 and Y637 participate in numerous hydrogen bonds and salt-bridges with ARC1172 (e.g., Q625 contacts C29), and R632 is buried within a deep pocket of the aptamer. Botrocetin occupies a similar interface on VWF A1, which accounts for the ability of ARC1172 or ARC1779 to block botrocetininduced platelet aggregation. In addition, nucleotide T10 extends toward helix alpha3 (lime green) and obstructs residue K599, which is required for binding to platelet GPIbalpha. The results provide a structural explanation for many of the functional properties of aptamers ARC1172 and ARC1779. [Display omitted]

Intersection of Mechanisms of Type 2A Von Willebrand Disease through Defects in Von Willebrand Factor Multimerization, Secretion, ADAMTS-13 Susceptibility, and Regulated Storage

Type 2A von Willebrand disease (VWD) is characterized by the absence of large von Willebrand factor (VWF) multimers and decreased platelet binding function. These variants are classified as either group 1 (2A-1) with impaired assembly and secretion of VWF multimers, or group 2 (2A-2) with increased susceptibility to proteolysis by ADAMTS13. However, laboratory parameters in individual patients may not discriminate between 2A-1 and 2A-2; this must be established through expression studies. Type 2A VWD patients recruited through the TS Zimmerman Program For The Molecular And Clinical Biology Of VWD (ZPMCB VWD) were phenotypically identified based upon laboratory parameters. By sequencing genomic DNA we identified 13 potentially causative sequence variations in the VWF D2, D3, A1, and A2 domains of type 2A VWD patients. Expression studies were performed to examine the effect of these mutations onVWF processing and proteolysis, andVWF regulated storage. The VWF variants L1503R, S1506L, and V1607D demonstrated severely impaired secretion and lacked mid- and high-molecular weight multimers, consistent with a group 1 classification (2A – 1).Some variants including I1568N, G1579R, and G1631D were normally multimerized and secreted, but had an increased susceptibility to cleavage by ADAMTS13 cleavage, indicative of group 2 classification (2A – 2). Five variants involving cysteine residues (C1190S, C1099P, C1272R/S/Y) had multimer abnormalities, a modest reduction in secretion, and variably enhanced ADAMTS-13 cleavage. These variants did not easily fit the criteria for either 2A subgroup. We also identified sequence variations (M740I and I1380V) that had no effect on VWF secretion, structure, or proteolysis that are likely innocuous polymorphisms but are present in patients with a type 2A phenotype. VWF is stored for regulated release in endothelial cell Weibel-Palade bodies and in platelet alpha-granules. The effect of mutations on VWF regulated storage has been documented for few VWF variants. The 2A variants were expressed in HEK293 cells, immunostained, and examined by confocal microscopy. Several variants including C1272S/Y, L1503R, S1506L, and V1607D did not form storage granules. A loss or reduction in endothelial cell Weibel-Palade bodies may explain the diminished desmopressin response observed in many type 2A VWD patients. In sum, our data indicate that 2A-2 variants are associated with VWF A2 domain mutations and normal regulated storage, while 2A-1 mutations are not restricted to a particular domain and result in loss of regulated VWF storage. Mutations involving cysteines may not affect VWF secretion, but are likely to cause abnormalities in multimer structure. In summary, type 2A VWD appears to result from at least three intersecting mechanisms: intracellular retention, defective multimerization, or increased plasma proteolysis.

Thermodynamic Analysis of Von Willebrand Disease Type 2B and 2M Mutations on the Structural Stability of the Von Willebrand Factor A1 Domain.

Von Willebrand Factor (VWF) initiates platelet adhesion at sites of vessel injury via the binding of platelet glycoprotein (GP) Ib-IX-V surface receptor to the VWF A1 domain. Congenital mutations occurring in the A1 domain result in the clinical bleeding diathesis of several subtypes of Type 2 Von Willebrand Disease. These mutations can either enhance VWF-platelet interactions (Type 2B) or cause a defect in this interaction (Type 2M). We hypothesize that these functional types of mutations could differentially alter VWF-platelet interactions in part by affecting the intrinsic stability of the A1 domain structure and consequently, a conformational transition induced by these interactions. In this study, we have characterized the thermodynamics of unfolding the A1 domain containing the Type 2B mutations (R1306Q, R1308L, and I1309V), the Type 2M mutation (G1324S) and a blocked disulfide variant of A1, (reduced and carboxyamidated, RCAM A1), that is representative of the Type 2M disulfide bond mutations, C1458Y and C1272(G,R,S). Using a combination of chemical and thermal denaturation monitored by circular dichroism spectroscopy and differential scanning calorimetry, we obtained a complete description of the thermodynamic stability of A1. Urea and guanidine-HCl denaturation show that the native conformation of A1 reversibly unfolds through a structurally stable intermediate, N·I·D, with two distinct conformational transitions that are widely separated in denaturant concentration. Thermal denaturation in the absence of denaturant shows that A1 unfolds via the two-step Lumry-Eyring mechanism, N·I→F, in which the reversible native to intermediate conformational transition is followed by the irreversible conversion of the intermediate to a final state that is unable to fold back to the native conformation. The clinically relevant mutations investigated affect only the thermodynamics of the first transition, N·I, suggesting that this conformational transition is important for the binding of A1 to the platelet GP1bα component of the GPIb-IX-V surface receptor. While the effects of Type 2B mutations on stability are moderate, the effects of the Type 2M mutations are extreme. G1324S increases the stability two-fold over wild type resulting in an increased concentration of urea required for unfolding and an increased melting temperature. Conversely, RCAM A1 completely destabilizes the native conformation resulting in a structure that resembles the intermediate conformation in the absence of denaturant. In conclusion, these thermodynamic observations parallel the clinical phenotypes associated with the mutations studied. The moderate effects of Type 2B mutations on A1 stability suggest that these mutations may impair quaternary interactions between A1 and neighboring VWF domains and enhance VWF-platelet interaction by exposing the GP1bα binding site. In contrast, Type 2M mutations cause extreme effects on stability. G1324S increases A1 stability and specifically reduces its conformational entropy, resulting in a more rigid structure with deficient capacity to interact with platelet GP1bα. RCAM-induced loss of the A1 disulfide bond dramatically destabilizes the native conformation, resulting in improper domain folding and defective interaction with platelet GP1bα.

Binding of ADAMTS13 to Von Willebrand Factor Under Static Conditions and Under Fluid Shear Stress

ADAMTS13 is a plasma metalloprotease that is essential for the normal proteolytic processing of von Willebrand factor (VWF). Dysfunctional ADAMTS13 may lead to thrombotic thrombocytopenic purpura, as uncleaved and unusually large VWF multimers accumulate in the blood and cause intravascular platelet aggregation. Many studies indicate that proteolysis of multimeric VWF involves conformational changes in the VWF A2 domain that expose the Y1605-M1606 scissile bond and also allow substrate binding to multiple exosites on ADAMTS13. For example, VWF is resistant to proteolysis by ADAMTS13 unless the VWF is subjected to fluid shear stress, mild denaturation with guanidine or urea, or adsorption onto a surface. However, the functional interactions between shear stress, various ADAMTS13 binding sites and VWF cleavage are not understood. Therefore, we investigated the effect of fluid shear stress and ADAMTS13 structure on ADAMTS13-VWF binding and VWF cleavage. Upon mixing recombinant VWF (rVWF) and ADAMTS13 in a physiological buffer (50 mM HEPES, 5 mM CaCl2, 1 μM ZnCl2, 150 mM NaCl, pH 7.4), we found that immunoprecipitation with anti-VWF also pulled down substantial amounts of ADAMTS13. Although less striking, a similar result was obtained with purified plasma VWF. Therefore, ADAMTS13 can bind VWF without gaining access to the cleavage site in VWF domain A2. When fluid shear stress was applied for 2 min with a bench-top vortexer, ADAMTS13 binding increased 3-fold and VWF was also cleaved. Lowering the ionic strength markedly increased the rate of VWF cleavage but did not affect ADAMTS13 binding, which suggests that cleavage and binding depend on distinct VWF-ADAMTS13 interactions. Shear-induced binding was reversible slowly upon removal of unbound ADAMTS13 or rapidly by addition of SDS. ADAMTS13-VWF binding was stable for at least 24 h after cessation of shear stress, indicating that the structural change in VWF that promotes binding was not readily reversible. Using a catalytically inactive ADAMTS13 variant to simplify the analysis of binding assays, 30 nM ADAMTS13(E231Q) bound to 30 μg/ml rVWF (120 nM subunits) with a stoichiometry of 0.012 ± 0.004 under static conditions and 0.098 ± 0.023 after shearing (mean ± SD, n = 3, P = 0.019). With 120 nM ADAMTS13(E231Q) the stoichiometry increased to 0.086 ± 0.036 under static conditions and 0.469 ± 0.033 after shearing for 2 min. Recombinant ADAMTS13 truncated after TSP-1 repeat 8 (lacking the C-terminal CUB domains, delCUB), or truncated after the Spacer domain (consisting of domains MDTCS), did not bind rVWF under static conditions, implicating the CUB domains in binding to VWF. In contrast, full-length ADAMTS13, delCUB and MDTCS bound similarly to rVWF after shearing. In a previous study, delCUB and MDTCS did not cleave VWF subjected to fluid shear stress (Zhang et al, Blood 2007; 110: 1887–1894). However, under the conditions employed in these experiments, MDTCS and delCUB displayed significant proteolytic activity, cleaving VWF at a rate comparable to that of full length ADAMTS13 when shear stress was applied over a time course of 0–160 sec. We conclude that ADAMTS13 CUB domains contribute to binding a few sites on multimeric VWF under static conditions, whereas ADAMTS13 MDTCS domains are sufficient to bind many sites in an altered conformation of VWF that is induced by fluid shear stress. Binding of ADAMTS13 to unsheared VWF multimers may facilitate the cleavage of VWF within a growing thrombus.

Evidence for the Interaction Between VWF Carboxyl-Terminal Domains and ADAMTS13 Domains Distal to Its Spacer Domain

ADAMTS13 plasma metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Y1605-M1606 bond in the A2 domain. Several studies have characterised high affinity binding sites in the A2 domain, probably interacting with the ADAMTS13 spacer and proximal domains. We have previously provided preliminary evidence for an additional ADAMTS13 binding site distal to the A domains, by measuring a ~7-fold increase in affinity for ADAMTS13 of plate-immobilised VWFA1CK (residues 1260-2813) (KD 11 nM) compared with VWFA1A2A3 (residues 1260-1874) (KD 78 nM) (Zanardelli et al, JTH, volume 5 Supplement I, XXI ISTH congress 2007, abstract number O-W-018). We subsequently expressed a series of VWF carboxyl-terminal fragments, VWFD4CK (residues 1874-2813), VWFA3CK (residues 1671-2813), VWFD4 (residues 1874-2299) and VWFB1CK (residues 2296-2813). Using an equilibrium binding assay, we assessed their binding to full-length ADAMTS13 and truncated ADAMTS13 proteins MDTCS (residues 34-685) and del(TSP5-CUB) (residues 34-894). KD values calculated for VWFD4CK and VWFA3CK binding to full-length ADAMTS13 were 134 and 108 nM, respectively. This confirmed the presence of an ADAMTS13 binding site outside VWF A2 domain, in the region D4CK. It also excluded a major role in the interaction for the VWF A3 domain. However, isolated VWFD4 and VWFB1CK did not bind significantly, suggesting that integrity of the VWF C-terminal region is also required for the binding. Furthermore, MDTCS bound to VWFA1CK and VWFA1A2A3 with similar affinity (KD 120 and 82 nM, respectively) and no significant interaction occurred with VWFD4CK. Similar results were found for the binding of del(TSP5-CUB) to the VWF fragments (VWFA1CK KD 85 nM, VWFA1A2A3 KD 65 nM and VWFD4CK no binding). Together these results suggested a multiple interaction between the carboxyl-terminal region of VWF (within D4CK) and the distal domains of ADAMTS13 (TSP5–8 and CUB domains). The VWF carboxyl-terminal binding site may be important for initial anchoring of ADAMTS13, allowing the subsequent binding between the spacer domain and VWF A2 domain.

The Role of N-Linked Glycosylation in Expression and Function of ADAMTS13

N-linked glycosylation is an important co-translational protein modification affecting protein folding, secretion, stability & function. We have previously demonstrated that N-linked glycosylation of Von Willebrand Factor (VWF) modulates its interaction with ADAMTS13, the plasma metalloprotease responsible for controlling VWF multimeric size. In the present study we have investigated the role of N-linked glycosylation in modulating ADAMTS13 expression, secretion & function.Lectin analysis of plasma derived & recombinant ADAMTS13 (rAD13) demonstrated that ADAMTS13 presents sialic acid & galactose residues, indicating the presence of complex glycans structures. Analysis with the lectin GNL, specific for high mannose structures showed that plasma derived ADAMTS13 also contained a small portion of basic high mannose glycans & this was increased in rAD13. Enzymatic modification of rAD13 with PNGase F to remove whole N-linked glycans & neuraminidase to remove terminal sialic residues were completely effective. However, neither modification affected the enzymatic activity of ADAMTS13 towards VWF, indicating that following secretion ADAMTS13 N-linked glycans are expendable.Using an ADAMTS13 ELISA and western blotting, we showed that expression of rAD13 in the presence of Tunicamycin prevented N-linked glycosylation & prevented secretion, indicating that N-linked glycosylation is essential for secretion of ADAMTS13. Expression in the presence of the glucosidase inhibitor castanospermine, that inhibits binding to the endoplasmic lectin chaperones calnexin & calreticulin, also prevented secretion of ADAMTS13 suggesting that glycan dependent interaction with these chaperones is essential for the correct cellular processing of ADAMTS13To further explore these observations, 10 individual point mutations encoding asparagine to glutamine changes were generated to prevent glycosylation at each of the 10 N-linked glycans sites in ADAMTS13: N462 & N46Q in the metalloprotease (MP) domain, N552Q in the cysteine rich domain, N579Q, N614Q & N667Q in the spacer domain, N707Q in the second Thrombospondin repeat (TSR), N828Q in the forth TSR and N1235Q and N1354Q in the CUB domains. Mutations N42Q, N579Q, N614Q, N707Q, N828Q and N1354Q caused only a slight to moderate reduction in ADAMTS13 secretion; 57%, 62%, 50%, 84%, 70% and 68% of wild type respectively. Mutations N552Q and N667Q significantly reduced ADAMTS13 secretion to 23% and 25% of wild type respectively indicating that glycosylation of the cysteine rich & spacer domains is required for efficient secretion. Significantly, mutation N46Q in the MP domain abolished secretion resulting in intracellular retention of ADAMTS13.All the secreted mutants with the exception of N828Q and N1235Q were able to effectively proteolyse the short VWF-115 substrate and full length VWF (FL-VWF). Interestingly, N828Q was demonstrated to have reduced activity towards VWF-115 and FL-VWF under both static and flow conditions. Although binding to immobilised VWF A2 domain and FL-VWF was not altered, a difference was observed in the binding of N828Q to globular FL-VWF. Conversely, mutation of N1235 increased activity of ADAMTS13 towards VWF.Together these data demonstrate that N-linked glycosylation of ADAMTS13, in particular at N46, is essential for efficient cellular processing & secretion of the enzyme. Following secretion the N-linked glycans are not required for proper function; however glycosylation of TSR 4 and the CUB domains are required for ADAMTS13 to assume a correct conformation for proper enzymatic activity towards VWF.

Force Dependent Structural Changes of the Von Willebrand Factor A2 Domain Are Required for VWF Cleavage by ADAMTS13

Background: An important regulatory mechanism of VWF functional activity is the proteolysis of the VWF high molecular weight multimers by ADAMTS13. The ADAMTS13 specific proteolytic site is located in the VWF A2 domain between Y1605 and M1606. Homology models by Sutherland et al. (1) predict the ADAMTS13 cleavage site of A2 to be covered by the helices of the domain. Thus an unfolding or opening of the domain is necessary in order to ensure cleavage by ADAMTS13. The main difference between the three neighboring VWF A domains is a disulfide bond connecting the respective N and C termini of the A1 and A3 domain, which is lacking in A2. An obvious assumption is that the disulfide bonds lock A1 and A3 in the folded state, while A2 can unfold in response to shear stress in blood vessels. To test this, we examined if the mutation N1493C in A2 enables the formation of a disulfide bond analogous to A1 and A3, thereby preventing unfolding of the A2 domain and stabilizing VWF against ADAMTS13.Methods: By in vitro mutagenesis of full length VWF we exchanged N1493 at the N-terminal site to Cysteine and C1670, one of two neighboring cysteines at the C-terminal site of the A2 domain, to Serine to allow creation of a Cysteine bonded loop (177 aa) in the A2 domain. Mutant and wt VWF were then digested by recombinant ADAMTS13 under denaturing non-reducing and reducing conditions, respectively, and analyzed by electrophoresis. Based on multiple sequence and structural alignments we created a homology model of the VWF A2 domain (residues 1488 to 1676 of human VWF) and the mutant A2 domain (N1493C and C1670S) from a human VWF A1 X-ray structure (PDB: 1AUQ). Our models cover a longer sequence (residues 1488 to 1676 of human VWF) than the model proposed by Sutherland et al.(1) The models were validated by molecular dynamics (MD) simulations. Force-probe MD simulations were applied to wildtype (wt) VWF to investigate unfolding.Results and Discussion: Successful introduction of a disulfide bond into A2 was validated by VWF multimer analysis. An additional loop due to a disulfide bond resulted in faster migration of VWF, suggesting a more compact structure. Persistence of the large VWF multimers and the lack of proteolytic products after treatment with recombinant ADAMTS13 indicated resistance of the A2 mutant to ADAMTS13. Electrophoresis of ADAMTS13 treated A2 domain mutant VWF under reducing conditions demonstrated a majority of undigested VWF monomers suggesting that proteolysis is not only prevented by creation of a disulfide bond spanning the proteolytic site but indeed by structural changes of the A2 domain, making the proteolytic site less accessible for ADAMTS13 in general. Physiological blood flow exerts forces onto VWF, stretching the multimeric protein to an elongated conformation. Force-induced unfolding of A2 is observed in force probe MD simulations by subjecting the termini of the A2 domain to a pulling force mimicking the tension in stretched VWF. The ADAMTS13 cleavage site is located at β-strand 4 (β4), which is after stepwise unfolding of α6, β6, α5, β5, and α4 exposed and therefore accessible for ADAMTS13. This explains the size regulation of VWF by ADAMTS13: larger multimers involve higher pulling forces and thereby a higher unfolding ratio at a given shear flow. As a result, larger VWF is cleaved more readily.Conclusion: Our combined experimental and theoretical approach suggests that locking of the VWF A2 domain by an artificial disulfide bond prevents a shear response and emphasizes the requirement of a force sensitive A2 domain structure for ADAMTS13 proteolysis. Respective experiments under shear conditions should confirm our hypothesis.

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.

Mechanistic Studies on ADAMTS13 Catalysis

The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I repeats (ADAMTS13) cleaves the Tyr 1605-Met 1606 peptide bond of von Willebrand factor (VWF), avoiding the accumulation of ultra large VWF multimers. Hydrolysis by ADAMTS13 of a VWF analog (Asp 1596-Arg 1668 peptide, fluorescence energy transfer substrate [FRETS]-VWF73) was investigated by a fluorescence quenching method (FRETS method) from 15°C to 45°C and pH values from 4.5 to 10.5. The catalysis was influenced by two ionizable groups, whose pK a values were equal to 6.41 ± 0.08 (ionization enthalpy = 32.6 ± 1.7 kJ/mol) and 4 ± 0.1 (ionization enthalpy = 3.8 ± 0.4 kJ/mol), whereas these values were equal to 6 ± 0.1 and 4.1 ± 0.1, respectively, in Co 2+-substituted ADAMTS13. The catalytic process of FRETS-VWF73 hydrolysis showed negative activation entropy (−144 kJ/mol), suggesting that the transition state becomes more ordered than the ground state of the reactants. The k cat/ K m values were not linearly correlated with temperature, as expression of change of the kinetic “stickiness” of the substrate. The Met 1606-Arg 1668 peptide product acted as hyperbolic mixed-type inhibitor of FRETS-VWF73 hydrolysis. Asp 1653, Glu 1655, Glu 1660, Asp 1663, together with the hydrophilic side chain of Thr 1656 were shown to form a “hot spot” in the VWF A2 sequence, which drives the molecular recognition and allosteric regulation of binding to ADAMTS13. The interaction of the Met 1606-Arg 1668 region of VWF with ADAMTS13 involves basic residues of the protease and is thus progressively inhibited at pH values >8.50. A molecular model of the FRETS-VWF73 showed that the substrate can fit into the active site only if ADAMTS13 assumes a C-like shape and, interacting with the acidic 1653–1668 region of VWF, properly orients the Tyr 1605-Met 1606 peptide bond for the cleavage by the zinc-aquo complex in the active site.

L1503R is a member of group I mutation and has dominant‐negative effect on secretion of full‐length VWF multimers: an analysis of two patients with type 2A von Willebrand disease

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T-->G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.

Heparan Sulfate Regulates Self-renewal and Pluripotency of Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure.

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.

Platelet-VWF complexes get the chop

In this issue of Blood, Shim and colleagues define a dual role for platelet glycoprotein (GP)Ibα (the major ligand-binding subunit of the GPIb-IX-V complex) in regulating ADAMTS13-mediated cleavage of von Willebrand factor (VWF) under shear: it alleviates an inhibitory effect of the VWF A1 domain on cleavage of the A2 domain,1 and it allows tensile force to be exerted on the A2 domain through at least 2 platelets binding per VWF multimer via the A1 domain (see figure).

Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

Clinical Facts & Curios

Human ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)–platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58–amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a “minimal” structure for allosterically regulated ADAMTS13.

Distinct Functional Conformations of von Willebrand Factor A1 Domain in Support of Platelet-Surface or Platelet-Platelet Interactions.

The adhesive protein, von Willebrand factor (VWF), is generally considered a key substrate for platelet adhesion to the vessel wall, yet its role in platelet cohesion (aggregation) may be equally important for normal thrombus formation. In either case, the function of VWF is mediated by the primary interaction of the VWF A1 domain (VWF-A1) with glycoprotein (GP) Ibα, a component of the GPIb-IX-V receptor complex on the platelet membrane. Because normal plasma VWF in solution and GPIb coexist in circulating blood without any appreciable interaction, it has been postulated that conformational changes occur when VWF becomes immobilized and/or under the effect of pathologically elevated shear stress, such that binding to the receptor becomes possible and resultis in platelet tethering to a surface and shear-induced aggregation. Changes of the molecular shape of VWF, from coiled to extended, have been shown under the effect of hemodynamic forces, but evidence for conformational changes within VWF-A1 has remained elusive. The crystal structure of VWF-A1 in complex with a GPIbα amino terminal fragment has revealed that the VWF-A1 residues involved in the interaction are comprised between positions 544–614 and, in particular, do not include several positively charged Arg and Lys residues located in helices α4 and 5 (residues 627–668). The latter appear as likely candidates to interact with negatively charged residues in GPIbα as a consequence of potential conformational changes induced by tensile stress on the bond following an initial ligand-receptor contact. We tested this hypothesis by evaluating the ability of selected VWF-A1 mutants to support platelet adhesion or aggregation, respectively, under controlled flow conditions.Methods. We expressed in insect cells and purified a series of VWF-A1 fragments comprising residues 445–733. One fragment had native sequence and 8 had single or multiple substitutions of positively charged amino acid residues in helices α4 and/or α5. None of the substituted residues contribute to contacts with GP Ibα in the known crystal structures of the corresponding complex, and all except one were between 8 and 20 angstroms away from the closest GPIbα residue. All the fragments were dimeric (d) owing to the presence of interchain disulfide bond(s).Results: Native dVWF-A1 in solution supported platelet aggregation in a laminar flow field. Of the 8 mutants, 5 had variably decreased function (up to 95% less aggregation) and 2 had increased function (up to 200% increase in aggregation). The same results were observed with platelet-rich plasma in suspension or by measuring platelet aggregate formation with blood cells perfused over immobilized VWF-A1 at wall shear rates as high as 10,000 1/s. In contrast, as judged by the number of tethered platelets and their rolling velocities, all mutants supported adhesion as well as or better that the native VWFA-1 at all shear rates tested (500–25,000 1/s).Conclusions: These results provide structural evidence for the existence of different VWF-A1 conformers that can modulate adhesive properties with distinct effects on platelet adhesion to a surface or platelet aggregation.

Cooperative Calcium Signaling Mediated by Glycoprotein (GP) Ib-IX-V, GPVI and Integrin α2β1 during Platelet Adhesion to Collagen under Flow.

We have investigated signaling mediated by GPIb-IX-V, GPVI and α2β1 during platelet adhesion to collagen type I either in the presence or absence of von Willebrand factor (VWF) A1A2A3 domain under flow conditions (wall shear rate 600 or 3000 s−1). We used platelets labeled with FLUO3-AM and real-time videomicroscopy with high-speed image acquisition to analyze concurrently adhesion, translocation and calcium transients in single platelets interacting with the surface. All experiments were performed in the presence of integrillin (100 μg/ml) to block the possible involvement of αIIbβ3. Platelet adhesion and activation at 600 s−1 were similar in the absence or presence of VWF A1A2A3 domain. Calcium signaling consisted of both short lasting peaks (release from intracellular stores) and long sustained waves (transmembrane flux). In our experimental condition blockade of α2β1 or GPVI with monoclonal antibodies reduced platelet adhesion by 50% and the proportion of adherent platelets that became activated by 20–30%. Concomitant inhibition of both receptors decreased platelet adhesion by >90%. Thus, both α2β1 and GPVI participate in the initial stage of platelet adhesion to collagen type I. At the higher shear rate of 3000 s−1, platelet adhesion and activation was 40–50% less than at 600 s−1 in the absence of VWF A1A2A3 domain; addition of the latter enhanced adhesion and activation by 12 to 14-fold, and >80% of the platelets exhibited stable adhesion during the 30 s observation period. Intracellular Ca++ peaks in adherent platelets reached 2–3 μM. Blockade of GP Ib-IX-V inhibited both platelet adhesion and activation by at least 90%. Blockade of α2β1 markedly reduced the number of adhering platelets, which were mostly (>90%) translocating on the surface and exhibited only short lasting Ca++ transients. Blockade of GPVI greatly decreased the number of adherent platelets, most of which, however, were firmly attached to the surface (<15% exhibiting translocation). Calcium transients in the activated platelet consisted of short lasting peaks and rare long lasting waves reaching concentrations <1.5–2 μM. These results suggest that, on a surface of collagen type I, α2β1 is required for normal platelet arrest following tethering through the VWF A1 domain-GPIb-IX-V interaction at higher shear rates, and Ca++ signaling results from the concerted action of the two receptors reinforced by GPVI. The adhesion potential of platelets exposed to collagen, therefore, appears to depend on signaling triggered by different ligand-receptor interactions varying as a function of flow conditions.

G-Quartet Oligonucleotide T70923 Blocks GP Ib-VWF Mediated Platelet Aggregation under Static and Flow Conditions.

Hemostasis at site of the vessel injury is initiated by platelets being tethered to exposed subendothelium. The tethering events are mediated by interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor GP Ib-IX-V complex. If the ligand-receptor interaction, when it occurs in intact vascular lumen or at the site of ruptured atherosclerotic plaques, results in life-threatening thrombosis found in patients with thrombotic thrombocytopenic purpura and myocardial infarction. The development of these thrombotic events suggests that the VWF-GP Ib interaction may be a potential target for therapeutic intervention. To explore such potentials, we have developed and examined the effects of a G-quartet oligonucleotide (T30923, GGGTGGGTGGGTGGGT) on the VWF-GP Ib interaction. G-rich DNA sequences, from which the G-quartets derive, were originally identified in the telomeres DNA and have the ability to form rigid G-quartet structures that interact with proteins. The T30923 structure was designed and determined by nuclear magnetic resonance to form an intramolecular G-quartet that is composed of two identical G-quartets in the center and two GTGT loops on the top and bottom of stacked G-quartets, respectively. Platelet aggregation was induced by four agonists: ristocetin, botrocetin, collagen, and ADP in the presence of either 20 mM of T30923 or the control oligonucleotide. We found that T30923 specifically and dose-dependently blocked platelet aggregation induced by ristocetin, but not by other agonists, with a maximal inhibition achieved at 20 mM. Computer-simulation based on the crystal structure of the VWF A1 domain and the N-terminal VWF-binding domain of GP Iba suggested that the G-quartet binds to multiple sites on VWF-A1. Since the action of ristocetin mimics that of fluid shear stress, we tested the ability of T30923 to block VWF-GP Ib interaction under flow condition. Shear-induced platelet aggregation at 100 dyn/cm2 was blocked 59.3%±11.8% by 20 mM of T30923, but not a control oligonucleotide. Furthermore, the G-quartet T30923 blocked over 80% of platelet adhesion to immobilized VWF, but not collagen under a constant wall shear stress of 30 dyn/cm2. These results demonstrate the potential of T30923 to serve as a VWF antagonist. Furthermore, alterations of DNA sequences and tertiary structure provide a large number of G-quartet variants to be generated and then screened for specific phenotypes. The approach may lead to a new class of potential G-quartet based antithrombotic agents.