Gain- and loss-of-function mutants confirm the importance of apical residues to the primary interaction of human glycoprotein VI with collagen.

BACKGROUND: By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12. Lysine59 is proposed to lie on the apical surface of the receptor near the linker joining the two immunoglobulin (Ig)-like extracellular domains. Recently, others have postulated the involvement of a portion of the first domain distant from the interdomain hinge as being involved in an extended collagen-binding site. AIM AND METHODS: To extend our knowledge of the primary collagen-binding site of GPVI, a number of neighboring residues on the apical surface of recombinant soluble GPVI were mutated to alanine and binding of these mutants, as well as the lysine59 mutant, to fibrillar collagen was measured. RESULTS: Binding of recombinant GPVI to collagen, like CRP, was dramatically reduced by the mutation of residue lysine59 to glutamate. Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen. Mutation of the residue lysine41 to alanine dramatically increased binding to both CRP and collagen. This mutation abolished 10B12 binding, confirming its position in the epitope of our inhibitory phage antibody. CONCLUSIONS: Residues lysine59, arginine60, and arginine166, from both Ig-like domains of GPVI, are critical for collagen binding by the receptor. This provides additional evidence for a basic patch on the apical surface of the receptor as the primary collagen-binding site of GPVI.

Definition of novel GP6 polymorphisms and major difference in haplotype frequencies between populations by a combination of in-depth exon resequencing and genotyping with tag single nucleotide polymorphisms.

BACKGROUND: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. METHODS: To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. RESULTS: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. CONCLUSIONS: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.

Inhibition of MT1-MMP activity using functional antibody fragments selected against its hemopexin domain.

The membrane associated MMP, MT1-MMP, is a critical pericellular protease involved in tumour cell invasion and angiogenesis and is highly up-regulated in numerous human cancers. It therefore represents an exciting new therapeutic cancer-specific target. We have generated recombinant human scFv antibodies against the non-catalytic, hemopexin domain of MT1-MMP that modulate its interactions with collagen. One of these is an effective inhibitor of the invasive capacity of cancer cells and of angiogenesis in model systems. This demonstrates that targeting sites outside the catalytic domain presents a potential novel approach to proteinase inhibition that could have applications in cancer therapeutics.

A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder.

BACKGROUND: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. RESULTS: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. CONCLUSIONS: This study presents the first patient with a proven genetic GPVI defect.

Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

BACKGROUND: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). OBJECTIVE: The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. RESULTS: At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). CONCLUSIONS: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.

Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides.

Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine(biotin-x-cadaveri ne; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 muM CA2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 muM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting.

Threonine-145/methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbalpha express HPA-2 epitopes and show equal binding of von Willebrand factor.

Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIbalpha were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIbalpha in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIbalpha in vWF binding and indicate that recombinant GPIbalpha may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a.

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).

Rapid phenotyping of HPA-1a using either diabody-based hemagglutination or recombinant IgG1-based assays.

BACKGROUND: The HPA-1 system is carried on the beta3 integrin. HPA-1a (Zw(a), Pl(A1)) is immunogenic in an HPA-1b homozygote (HPA-1b1b). In pregnancy, 1 of 365 women forms anti-HPA-1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti-HPA-1a and the screening of donors to obtain HPA-1a-negative platelets for therapy need reliable, low-cost, automated assays. STUDY DESIGN AND METHODS: A diabody with dual specificity for HPA-1a x D and an IgG1 anti-HPA-1a have been constructed by the use of the genes encoding the first anti-HPA-1a fragment. With these reagents, two complementary HPA-1a phenotyping assays have been developed. RESULTS: This diabody was used in a simple hemagglutination technique to perform HPA-1a phenotyping on soluble glycoprotein IIb/IIIa from EDTA plasma samples. Over 1000 unselected donors have been correctly HPA-1a-phenotyped by use of the diabody. The human recombinant IgG1 anti-HPA-1a was produced in a rat myeloma cell line and was fluorescein labeled for use in a whole-blood flow cytometric HPA-1a phenotyping assay. This IgG1 anti-HPA-1a shows a clear differential between HPA-1a-positive and HPA-1a-negative platelets at nM antibody concentrations. CONCLUSIONS: The two recombinant reagents described are highly suitable for screening and confirmatory HPA-1a phenotyping. They permit rapid determination of the HPA-1a phenotype and are amenable to automation.

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence.

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.