Marie N. O'Connor

Structural Basis for the Platelet-Collagen Interaction THE SMALLEST MOTIF WITHIN COLLAGEN THAT RECOGNIZES AND ACTIVATES PLATELET GLYCOPROTEIN VI CONTAINS TWO GLYCINE-PROLINE-HYDROXYPROLINE TRIPLETS

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

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

Summary.  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.

Selective Blockade of Glycoprotein VI Clustering on Collagen Helices

Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an “inert” scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)2 content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.

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

Summary.  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.

YS02 Screening and Characterisation of Novel Platelet Candidate Genes Derived from Genome‐Wide Association Studies for Function in Relation to Haemostasis and Thrombosis in the Model Organism Danio Rerio

Complementary approaches of microarray, proteomics, genome wide association and platelet functional studies have identified novel platelet proteins which are candidates for playing a role in thrombosis or conferring risk of myocardial infarction. The function of many of the proteins encoded by these candidate genes is unknown, and will need to be determined. To address the need for high‐throughput screening of candidate genes in a model organism, we have investigated the suitability of the zebrafish (ZF; Danio rerio), a genetically tractable vertebrate with nucleated thrombocytes. It is known that mouse knockouts of critical pro‐thrombotic genes [e.g. coagulation factor (F) VIII, integrin αIIb (ITGA2B), or glycoprotein VI] have subtle phenotypes and do not bleed spontaneously. We hypothesised that the sensitivity of the ZF model to screen for loss of platelet function could be enhanced by concurrent knock down of the FVIII gene and a pro‐thrombotic platelet gene. To test this, the genes for FVIII, ITGA2B or both were silenced by antisense oligonucleotides (morpholinos). Embryos were then assessed for body plan and bleeding at 3 days post‐fertilization (dpf). In parallel, a thrombosis model using laser injury to initiate thrombus formation was developed. This permits time to adhesion (TTA), time to occlusion (TTO) and time to thrombolysis (TTL) to be determined in morpholino‐injected and wild type ZF. As expected, individual knockdown of the FVIII or ITGA2B genes did not lead to spontaneous bleeding. Knockdown of the ITGA2B gene did, however, result in morphological malformations. Simultaneous knockdown of both genes resulted in a clear and reproducible bleeding phenotype visible in ∼20% of embryos. Using the laser injury model, venous thrombosis could be reproducibly induced in 3 and 6 dpf wild‐type ZF larvae with partial but not complete thrombolysis occurring within 5–10 min. In conclusion, ZF are a fast and informative way to screen for platelet function modifying genes.