Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway.

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1-/- explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

Zenocutuzumab, a HER2xHER3 Bispecific Antibody, Is Effective Therapy for Tumors Driven by NRG1 Gene Rearrangements.

NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity-enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion-positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion-positive metastatic cancer were treated with Zeno. Two patients with ATP1B1-NRG1-positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74-NRG1-positive non-small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion-positive cancers. SIGNIFICANCE: NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion-positive cancers and is thus an effective therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1171.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency.

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

Prescriber Implementation of STOPP/START Recommendations for Hospitalised Older Adults: A Comparison of a Pharmacist Approach and a Physician Approach.

BACKGROUND: Two randomised controlled trials (RCTs) conducted simultaneously in the same Irish university teaching hospital have shown that provision of Screening Tool of Older Persons' Prescriptions (STOPP)/Screening Tool to Alert doctors to Right Treatment (START) recommendations to attending prescribers by a physician or a pharmacist can reduce in-hospital adverse drug reactions (ADRs) in older adults (≥ 65 years). The aims of this study were to compare the prescriber implementation rates of STOPP/START recommendations between the physician approach and the pharmacist approach in these two RCTs and to provide a narrative summary of the comparable clinical outcomes. METHODS: Data were extracted from the two RCT published papers and their associated computerised databases to calculate the percentage prescriber implementation rates for the STOPP/START recommendations. The Chi-square test was used to quantify the differences in prescriber implementation rates, with differences considered statistically significant where p < 0.05. RESULTS: Prescriber implementation rates of the STOPP and START recommendations made by the physician were 81.2% and 87.4% respectively, significantly higher than those made by the pharmacist (39.2% and 29.5% respectively), p < 0.0001. A greater absolute risk reduction in patients with ADRs was shown with the physician's intervention compared to the pharmacist's intervention (9.3% vs 6.8%). CONCLUSION: This study shows that the methods of communication and the medium through which the STOPP/START recommendations are delivered significantly affect their implementation. Non-implementation of some pharmacist-delivered recommendations may be contributing to preventable ADRs in older adults. Thus, future research should aim to identify the factors influencing prescriber implementation of pharmacist recommendations in order to inform the design of more effective pharmacist interventions in optimising older patients' pharmacotherapy.

Cost-Effectiveness Analysis of a Physician-Implemented Medication Screening Tool in Older Hospitalised Patients in Ireland.

BACKGROUND: A recent randomised controlled trial conducted in an Irish University teaching hospital that evaluated a physician-implemented medication screening tool, demonstrated positive outcomes in terms of a reduction in incident adverse drug reactions. OBJECTIVE: The present study objective was to evaluate the cost effectiveness of physicians applying this screening tool to older hospitalised patients compared with usual hospital care in the context of the earlier randomised controlled trial. METHOD: We used a cost-effectiveness analysis alongside a conventional outcome analysis in a cluster randomised controlled trial. Patients in the intervention arm (n = 360) received a multifactorial intervention consisting of medicines reconciliation, communication with patients' senior medical team, and generation of a pharmaceutical care plan in addition to usual medical and pharmaceutical care. Control arm patients (n = 372) received usual medical and pharmaceutical care only. Incremental cost effectiveness was examined in terms of costs to the healthcare system and an outcome measure of adverse drug reactions during inpatient hospital stay. Uncertainty in the analysis was explored using a cost-effectiveness acceptability curve. RESULTS: On average, the intervention arm was more costly but was also more effective. Compared with usual care (control), the intervention was associated with a non-statistically significant increase of €877 (95% confidence interval - €1807, €3561) in the mean healthcare cost, and a statistically significant decrease of - 0.164 (95% confidence interval - 0.257, - 0.070) in the mean number of adverse drug reaction events per patient. The associated incremental cost-effectiveness ratio per adverse drug reaction averted was €5358. The probability of the intervention being cost effective at threshold values of €0, €5000 and €10,000 was 0.236, 0.455 and 0.680, respectively. CONCLUSION: Based on the evidence presented, this physician-led intervention is not likely to be cost effective compared with usual hospital care. To inform future healthcare policy decisions in this field, more economic analyses of structured medication reviews by other healthcare professionals and by computerised clinical decision support software need to be conducted.

Prevention of Hospital-Acquired Adverse Drug Reactions in Older People Using Screening Tool of Older Persons' Prescriptions and Screening Tool to Alert to Right Treatment Criteria: A Cluster Randomized Controlled Trial.

OBJECTIVES: To determine whether use of the Screening Tool of Older Persons' Prescriptions (STOPP) and Screening Tool to Alert to Right Treatment (START) criteria reduces incident hospital-acquired adverse drug reactions (ADRs), 28-day medication costs, and median length of hospital stay in older adults admitted with acute illness. DESIGN: Single-blind cluster randomized controlled trial (RCT) of unselected older adults hospitalized over a 13-month period. SETTING: Tertiary referral hospital in southern Ireland. PARTICIPANTS: Consecutively admitted individuals aged 65 and older (N = 732). INTERVENTION: Single time point presentation to attending physicians of potentially inappropriate medications according to the STOPP/START criteria. MEASUREMENTS: The primary outcome was the proportion of participants experiencing one or more ADRs during the index hospitalization. Secondary outcomes were median length of stay (LOS) and 28-day total medication cost. RESULTS: One or more ADRs occurred in 78 of the 372 control participants (21.0%; median age 78, interquartile range (IQR) 72-84) and in 42 of the 360 intervention participants (11.7%; median age 80, IQR 73-85) (absolute risk reduction = 9.3%, number needed to treat = 11). The median LOS in the hospital was 8 days (IQR 4-14 days) in both groups. At discharge, median medication cost was significantly lower in the intervention group (€73.16, IQR €38.68-121.72) than in the control group (€90.62, IQR €49.38-162.53) (Wilcoxon rank test Z statistic = -3.274, P < .001). CONCLUSION: Application of STOPP/START criteria resulted in significant reductions in ADR incidence and medication costs in acutely ill older adults but did not affect median LOS.

Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells.

Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIß in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

Prevention of Adverse Drug Reactions in Hospitalised Older Patients Using a Software-Supported Structured Pharmacist Intervention: A Cluster Randomised Controlled Trial.

BACKGROUND: Proven interventions to reduce adverse drug reactions (ADRs) in older hospitalised patients are lacking. Previous randomised controlled trial (RCT) data indicate that a structured pharmacist review of medication (SPRM) can reduce inappropriate prescribing in older hospitalised patients. However, no RCT data show that an SPRM reduces ADRs in this population. METHODS: We performed a cluster RCT comparing a clinical decision support software (CDSS)-supported SPRM intervention with standard pharmaceutical care in older patients hospitalised with an acute unselected illness. Over 13 months, we screened 1833 patients aged ≥65 years admitted to specialist services other than geriatric medicine for study inclusion. We randomised 361 patients to the trial intervention arm and 376 patients to the control arm, applying the intervention at a single timepoint within 48 h of admission. The primary endpoint (ADR incidence) was assessed at 7-10 days post-admission or at discharge (whichever came first). The secondary endpoints were the median hospital length of stay (LOS) and hospital mortality rate. RESULTS: Attending clinicians in the intervention group implemented 54.8% of SPRM/CDSS prescribing recommendations. Ninety-one ADRs occurred in 78 control patients (20.7%) compared with 61 ADRs in 50 intervention patients (13.9%), i.e., an absolute risk reduction of 6.8%. The number needed to treat (NNT) to prevent one patient having one ADR was 15; the total NNT to prevent one ADR was 14. The median LOS and hospital mortality were not significantly different. CONCLUSION: An SPRM delivered on a CDSS platform significantly reduces ADR incidence in acutely hospitalised older people.

Regulation of melanosome number, shape and movement in the zebrafish retinal pigment epithelium by OA1 and PMEL.

Analysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for the generation of cylindrical melanosomes in zebrafish, which in turn is required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together, our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localisation in the apical processes.

The impact of a structured pharmacist intervention on the appropriateness of prescribing in older hospitalized patients.

BACKGROUND: Throughout the literature, drug-related problems (DRPs), such as medication reconciliation issues and potentially inappropriate prescribing, have been reported to be associated with adverse outcomes in older individuals. Both structured pharmacist review of medication (SPRM) interventions and computerized decision support systems (CDSSs) have been shown to reduce DRPs. OBJECTIVE: The objectives of this study were to (i) evaluate the impact of a specially developed SPRM/CDSS intervention on the appropriateness of prescribing in older Irish hospital inpatients, and (ii) examine the acceptance rates of these recommendations. METHODS: We prospectively reviewed 361 patients, aged ≥65 years who were admitted to an Irish university teaching hospital over a 12-month period. At the point of admission, the patients received a SPRM/CDSS intervention, which screened for DRPs. Any DRPs that were identified were then communicated in writing to the attending medical team. The patient's medical records were reviewed again at 7-10 days, or at the point of discharge (whichever came first). RESULTS: Of the 361 patients reviewed, 181 (50.1 %) were female; the median age was 77 years [interquartile range (IQR) 71-83 years). A total of 3,163 (median 9, IQR 6-12) and 4,192 (median 12, IQR 8-15) medications were prescribed at admission and discharge, respectively. The SPRM generated 1,000 recommendations in 296 patients. Of the 1,000 recommendations, 548 (54.8 %) were implemented by the medical teams accordingly. The SPRM/CDSS intervention resulted in an improvement in the appropriateness of prescribing as defined by the medication appropriateness index (MAI), with a statistically significant difference in the median summated MAI at admission (15, IQR: 7-21) and follow-up (12, IQR: 6-18); p < 0.001. However, the SPRM did not result in an improvement in appropriateness of underprescribing as defined by a modified set assessment of care of vulnerable elders (ACOVE) criteria. CONCLUSION: This study indicated that DRPs are prevalent in older Irish hospitalized inpatients and that a specially developed SPRM intervention supported by a CDSS can improve both the appropriateness and accuracy of medication regimens of older hospitalized inpatients.

Inappropriate prescribing: criteria, detection and prevention.

Inappropriate prescribing is highly prevalent in older people and is a major healthcare concern because of its association with negative healthcare outcomes including adverse drug events, related morbidity and hospitalization. With changing population demographics resulting in increasing proportions of older people worldwide, improving the quality and safety of prescribing in older people poses a global challenge. To date a number of different strategies have been used to identify potentially inappropriate prescribing in older people. Over the last two decades, a number of criteria have been published to assist prescribers in detecting inappropriate prescribing, the majority of which have been explicit sets of criteria, though some are implicit. The majority of these prescribing indicators pertain to overprescribing and misprescribing, with only a minority focussing on the underprescribing of indicated medicines. Additional interventions to optimize prescribing in older people include comprehensive geriatric assessment, clinical pharmacist review, and education of prescribers as well as computerized prescribing with clinical decision support systems. In this review, we describe the inappropriate prescribing detection tools or criteria most frequently cited in the literature and examine their role in preventing inappropriate prescribing and other related healthcare outcomes. We also discuss other measures commonly used in the detection and prevention of inappropriate prescribing in older people and the evidence supporting their use and their application in everyday clinical practice.

Adverse drug reactions in older patients during hospitalisation: are they predictable?

BACKGROUND: adverse drug reactions (ADRs) are a major cause of morbidity and healthcare utilisation in older people. The GerontoNet ADR risk score aims to identify older people at risk of ADRs during hospitalisation. We aimed to assess the clinical applicability of this score and identify other variables that predict ADRs in hospitalised older people. METHODS: we prospectively studied 513 acutely ill patients aged ≥65 years. The GerontoNet ADR risk score was calculated for all patients. ADRs were identified through patient and physician consultation together with analysis of case notes. Receiver operator characteristic (ROC) curves were constructed to test the ability of the GerontoNet risk score to predict ADRs. Multivariate logistic regression examined the influence of individual variables on the presence of ADRs. RESULTS: in-hospital ADRs were identified in 135 patients (26%). The area under the ROC curve was 0.62 (95% CI: 0.57-0.68). Variables which increased ADR risk include (i) renal failure (OR: 1.81, 95% CI: 1.12-2.92), (ii) increasing number of medications (OR: 1.09, 95% CI: 1.02-1.17) (iii) inappropriate medications (OR: 2.40, 95% CI: 1.26-4.50) and (iv) age ≥75 years (OR: 2.12, 95% CI: 1.23-3.70). CONCLUSION: the GerontoNet ADR risk score incorrectly classified 38% of patients as low risk. Inappropriate medications and increasing age also contribute to ADR risk.

Pharmacotherapy at the end-of-life.

Older people reaching end-of-life status are particularly at risk from inter-related adverse effects of pharmacotherapy, including polypharmacy, inappropriate medications and adverse drug events. These adverse effects of pharmacotherapy may be highly detrimental, as well as highly expensive. End-of-life pharmacotherapy is sometimes perceived to be complex and challenging, probably unnecessarily. This relates in part to the poorly developed evidence base and lack of high-quality research in this area. In this article, we deal with some of the key issues relating to pharmacotherapy in end-of-life patients, namely (i) the guiding principles of drug selection, (ii) the main drugs and drug classes that are best avoided, (iii) the benefits of 'oligopharmacy' (i.e. deliberate avoidance of polypharmacy) in end-of-life patients.

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.

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.

Production of calmodulin-tagged proteins in Drosophila Schneider S2 cells: a novel system for antigen production and phage antibody isolation.

We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.

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.

Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.