Platelet Collagen Receptor Research Articles

GP VI-Mediated Platelet Activation and Procoagulant Activity Aggravate Inflammation and Aortic Wall Remodeling in Abdominal Aortic Aneurysm.

Platelets play an important role in cardiovascular and cerebrovascular diseases. Abdominal aortic aneurysm (AAA) is a highly lethal, atherosclerosis-related disease with characteristic features of progressive dilatation of the abdominal aorta and degradation of the vessel wall, accompanied by chronic inflammation. Platelet activation and procoagulant activity play a decisive role in the AAA pathology as they might trigger AAA development in both mice and humans. The present study investigated the impact of the major platelet collagen receptor GP (platelet glycoprotein) VI in pathophysiological processes underlying AAA initiation and progression. For experimental AAA induction in mice, PPE (porcine pancreatic elastase) and the external PPE model were used. Genetic deletion of GP VI offered protection of mice against aortic diameter expansion in experimental AAA. Mechanistically, GP VI deficiency resulted in decreased inflammation with reduced infiltration of neutrophils and platelets into the aortic wall. Furthermore, remodeling of the aortic wall was improved in the absence of GP VI, as indicated by reduced MMP (matrix metalloproteinase)-2/9 and OPN (osteopontin) plasma levels and an enhanced α-SMA (α-smooth muscle actin) content within the aortic wall, accompanied by reduced cell apoptosis. Consequently, an elevation in intima/media thickness and elastin content was observed in GP VI-deficient PPE mice, resulting in a significantly reduced aortic diameter expansion and reduced aneurysm incidence. In patients with AAA, enhanced plasma levels of soluble GP VI and fibrin, as well as fibrin accumulation within the intraluminal thrombus might serve as new biomarkers to detect AAA early. Moreover, we hypothesize that GP VI might play a role in procoagulant activity and thrombus stabilization via binding to fibrin. In conclusion, our results emphasize the potential need for a GP VI-targeted antiplatelet therapy to reduce AAA initiation and progression, as well as to protect patients with AAA from aortic rupture.

Antiplatelet effects of the CEACAM1-derived peptide QDTT

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and “inside-out” GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.

Platelets modulate cardiac remodeling via the collagen receptor GPVI after acute myocardial infarction.

Platelets play an important role in cardiovascular diseases. After acute myocardial infarction, platelets display enhanced activation and migrate into the infarct zone. Furthermore, platelets trigger acute inflammation and cardiac remodeling leading to alterations in scar formation and cardiac function as observed in thrombocytopenic mice. GPVI is the major collagen receptor in platelets and important for platelet activation and thrombus formation and stability. Antibody induced deletion of GPVI at the platelet surface or treatment of mice with recombinant GPVI-Fc results in reduced inflammation and decreased infarct size in a mouse model of AMI. However, the role of GPVI has not been fully clarified to date. In this study, we found that GPVI is not involved in the inflammatory response in experimental AMI using GPVI deficient mice that were analyzed in a closed-chest model. However, reduced platelet activation in response to GPVI and PAR4 receptor stimulation resulted in reduced pro-coagulant activity leading to improved cardiac remodeling. In detail, GPVI deficiency in mice led to reduced TGF-β plasma levels and decreased expression of genes involved in cardiac remodeling such as Col1a1, Col3a1, periostin and Cthrc1 7 days post AMI. Consequently, collagen quality of the scar shifted to more tight and less fine collagen leading to improved scar formation and cardiac function in GPVI deficient mice at 21d post AMI. Taken together, this study identifies GPVI as a major regulator of platelet-induced cardiac remodeling and supports the potential relevance of GPVI as therapeutic target to reduce ischemia reperfusion injury and to improve cardiac healing.

Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2

The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.

OC 10.1 The Impact of the Platelet Collagen Receptor GPVI on Inflammation and Extracellular Matrix Remodelling in Abdominal Aortic Aneurysm Formation

OC 10.1 The Impact of the Platelet Collagen Receptor GPVI on Inflammation and Extracellular Matrix Remodelling in Abdominal Aortic Aneurysm Formation

Reproducibility and Harmonization in Research using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide.

Metrology - the science of measure - is a subject few biological scientists are taught about in their training to their detriment; the application of simple standardization processes to everyday working practices provides confidence in data and reproducibility over distance and time. This method demonstrates how to standardize a core laboratory experiment used widely in hemostasis research and clinical practice, specifically, measuring responses to the platelet collagen receptor (glycoprotein [GP]VI) agonist collagen-related peptide, cross-linked (CRP-XL) by light transmission aggregometry (LTA). Using this approach will ensure intra-lab reproducibility and inter-lab harmonization, regardless of agonist stock or supplier. Importantly, this method is applicable to other platelet agonists and, indeed, many other biological molecules and bioassays. The process outlined below involves making a 6-8 point dilution series of the 'standard' and the 'test' (the material you are checking) and running them side by side in a chosen assay (in this case, LTA). CRP-XL is used at mass/volume concentrations, but not every material gives the same biological activity at a given concentration, so a dilution series is made to compare the standard and test material and determine what concentration is needed to give equivalent activity. The dilution series must span 0-100% aggregation. Data is plotted using non-linear regression, and the EC50 value of each sample (standard and test) is determined. To assign activity, divide the EC50 value of the standard by that of the test to determine how much more or less potent it is and adjust the concentration accordingly. This approach will ensure that the same biological 'activity' is added to the assay time and time again.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Evaluation of the effect of eltrombopag therapy on the platelet collagen receptor glycoprotein VI (GPVI) expression and soluble GPVI levels in young patients with immune thrombocytopenia

BackgroundPlatelet glycoprotein VI (GPVI) receptor is essential for platelet adhesion and aggregation. Eltrombopag is as an effective treatment for chronic immune thrombocytopenia (ITP); yet, its effect on platelet function is not fully characterized.AimThis prospective study investigated the effect of eltrombopag therapy on platelet function through assessment of GPVI receptor expression and soluble GPVI levels among pediatric patients with persistent or chronic ITP.MethodsThirty-six children and adolescents with persistent or chronic ITP were divided equally into two groups either to receive eltrombopag therapy or the standard of care. All patients were followed-up for 12 months with assessment of bleeding score and complete blood count (CBC). Evaluation of GPVI expression using flow cytometry and measurement of its soluble form by ELISA was done at baseline and at 6 months.ResultsITP patients on eltrombopag had significantly lower bleeding score after 6 months of therapy while the quality of life has significantly improved. Platelet count was significantly increased throughout the study. GPVI expression by flow cytometry and soluble GPVI levels were significantly increased after eltrombopag therapy. After 12 months, ITP patients on eltrombopag were able to maintain a good quality of life and low bleeding score.ConclusionOur data suggest that eltrombopag, through its effect on the GPVI receptor expression and its soluble form, might reduce bleeding manifestations and improve the quality of life of chronic and persistent ITP children independent of its effect on the platelet count.

HEMORRHAGIC PLEURAL EFFUSION IN A PATIENT WITH CHRONIC LYMPHOCYTIC LEUKEMIA ON IBRUTINIB THERAPY

HEMORRHAGIC PLEURAL EFFUSION IN A PATIENT WITH CHRONIC LYMPHOCYTIC LEUKEMIA ON IBRUTINIB THERAPY

Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking

BackgroundIn the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. ObjectivesWe determined if GPVI interacts with FXIII and how this might modulate clot formation. MethodsWe measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. ResultsGPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. ConclusionsThis is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors.

The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.

SIGNALING ROLE OF REACTIVE OXYGEN SPECIES IN PLATELETS

Abstract: Review. Reactive oxygen species are formed as a result of redox reactions and are involved in signaling associated with cell growth, differentiation, and death. Platelets are both a source and a target of reactive oxygen species. The review regards the molecular mechanisms of the reactive oxygen species formation under platelet activation conditions and effects of altered redox balance on platelet function. The main platelet sources of reactive oxygen species are isoforms of NADPH oxidases, cyclooxygenase, xanthine oxidase, and mitochondrial electron transport chain. Reactive oxygen species produced during platelet activation by agonists act as secondary messengers and can interfere with signaling pathways that enhance agonist-induced platelet activation. The signaling pathway mediated by glycoprotein VI, the main platelet receptor for collagen, leading mainly to the generation of intracellular reactive oxygen species, as well as the signaling pathway activated by thrombin, leading to the production of extracellular reactive oxygen species are considered. Extracellular reactive oxygen species enhance initial signaling and support platelet recruitment and activation. Activated platelets are involved in the coupling of thrombosis, inflammation, and innate immune responses. The platelet antioxidant systems play an important role in preventing the cytotoxic effects of reactive oxygen species, as well as in the regulation of platelet signaling pathways. The authors of the review and other researchers have shown that the redox status of platelets can serve as a biomarker in the studies on prevention and treatment of diseases involving oxidative stress and can be used for objective assessments of the patients' condition severity (including depression and schizophrenia, in which the authors found a decrease in the activity of platelet glutathione -dependent antioxidant enzymes).

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia

The aimof the study was to assess the clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia (SS).Materials and methods.A total of 100 patients with SS were examined and followed-up for 15.3±8.3 months. The patients were divided into subgroups (SG): SG1 (n=51) – persons without events, SG2 (n=49) persons with recurrent ischemic events (hospitalization due to the development of pain syndrome, re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes), SGB (n=11) – persons with «major» recurrent ischemic events (re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes) , SGG (n=89) – persons without «major» events. The obtained survey data (general clinical, aggregometry, polymor phism of genes of platelet fibrinogen receptor ITGB3 (T1565C), platelet collagen receptor ITGA2 (C807T), ADP platelet receptor P2RY12, H1/H2 (T744C)) were analyzed using the STATISTICA 10.0 software.Results.In SG2, men predominated (χ2=9.2; p<0.01), past MI was more common (χ2=4.8; p<0.05), more stents were implanted (2.4±1.9 versus 1.7±1.1, p<0.05), TRAP-test values were higher (p<0.05) compared to SG1. In SGB, greater number of stents were implanted (3.1±2.2 versus 1.61±1.57, p<0.05), the carriage of the TC genotype of the ITGB3 gene was more common, (p<0.05), a combination of gene mutations ITGB3 and P2RY12 was more common, (p<0.05) compared to SGG. A logistic regression equation was constructed, including the presence of diabetes mellitus, the number of platelets in the blood test, the ASPI-test values, the carriage of the 1565C allele of the ITGB3 gene, the number of stents implanted, which makes it possible to determine the likelihood of developing «major» recurrent ischemic events with a cut-off threshold LP₀=0.0965, with sensitivity – 81.82 %, specificity – 78.48 %, overall accuracy – 78.89 %.Conclusions.The factors associated with the development of recurrent ischemic events are: male sex, previous MI, a greater number of implanted stents, and high TRAP-test values. The factors associated with the development of recurrent «major» ischemic events are: a greater number of implanted stents, carriage of the TC genotype of the ITGB3 gene, carriage of a combination of mutations of the H1/H2 polymorphic locus of the P2RY12 gene and the T1565C polymorphic locus of the ITGB3 gene, diabetes mellitus, the number of platelets in blood test, ASPI-test values.

Snake Venoms in Diagnostic Hemostasis and Thrombosis.

Snake venoms have evolved primarily to immobilize and kill prey, and consequently, they contain some of the most potent natural toxins. Part of that armory is a range of hemotoxic components that affect every area of hemostasis, which we have harnessed to great effect in the study and diagnosis of hemostatic disorders. The most widely used are those that affect coagulation, such as thrombin-like enzymes unaffected by heparin and direct thrombin inhibitors, which can help confirm or dispute their presence in plasma. The liquid gold of coagulation activators is Russell's viper venom, since it contains activators of factor X and factor V. It is used in a range of clotting-based assays, such as assessment of factor X and factor V deficiencies, protein C and protein S deficiencies, activated protein C resistance, and probably the most important test for lupus anticoagulants, the dilute Russell's viper venom time. Activators of prothrombin, such as oscutarin C from Coastal Taipan venom and ecarin from saw-scaled viper venom, are employed in prothrombin activity assays and lupus anticoagulant detection, and ecarin has a valuable role in quantitative assays of direct thrombin inhibitors. Snake venoms affecting primary hemostasis include botrocetin from the jararaca, which can be used to assay von Willebrand factor activity, and convulxin from the cascavel, which can be used to detect deficiency of the platelet collagen receptor, glycoprotein VI. This article takes the reader to every area of the diagnostic hemostasis laboratory to appreciate the myriad applications of snake venoms available in diagnostic practice.

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen

ObjectiveThe platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI‐dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. Methods and resultsELISA using biotinyl‐fibrinogen immobilized on streptavidin‐coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near‐plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI‐Fc2 or Revacept) or monomer (GPVI‐His: single chain of Revacept GPVI domain, with His tag). Clot‐bound proteins were analyzed by SDS‐PAGE/immunoblotting. GPVI‐dimer bound to noncrosslinked fibrin clots with classical one‐site binding kinetics, with µM‐level KD, and to crosslinked clots with higher affinity. Anti‐GPVI‐dimer (mFab‐F) inhibited the binding. However, GPVI‐His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot‐bound platelet‐derived proteins were integrin αIIbβ3, present at high levels, and GPVI. ConclusionsWe conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D‐fragment and D‐dimer, suggesting that fibrin(ogen)'s GPVI‐binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

Discovery of Small Molecule Adhesion GPCR Modulators

GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs). These GPCRs are unique in their possession of an autoproteolytic extracellular domain that cleaves the receptors as a prerequisite to activation. GPR56 couples to G12/13 family G proteins and we recently discovered that it functions as a platelet collagen receptor that is important for hemostasis. We hypothesize that GPR56 is an early-acting collagen sensor for platelets – enabling them to first recognize blood vessel injury and to initiate the platelet program that leads to repair. Despite being biologically relevant in a number of cellular processes and diseases, there is a distinct lack of available small molecule tool compounds for AGPCRs, including GPR56. We conducted a large-scale high-throughput screen to find agonists and antagonists of GPR56. Over 150,000 small molecules and extracts were screened at UM's Center for Chemical Genomics (CCG) using a cell-based serum response element (SRE) luciferase gene reporter assay in HEK293T cells. Primary screening yielded 47 initial candidate agonists that activated GPR56 in secondary cell-based reporter and biochemical GPCR reconstitution assays. GPR56 reconstitution assays with pure G proteins further revealed that 19 of these compounds caused at least a 5-fold increase over control in G protein GTPγS binding. A notable structural cluster was identified among these molecules with some members exhibiting a 10-fold increase in potency (~30 μM to ~3 μM EC50) and equivalent efficacy compared to the full agonist positive control, a synthetic peptide mimetic of the GPR56 tethered-peptide-agonist. Follow-up GPR56 activation assays investigating the structure-activity relationship (SAR) of these pharmacophores are being pursued in conjunction with orthogonal platelet activity assays. Knowledge of AGPCR mechanisms and pathologies is rapidly emerging. As such, the discovery of high affinity agonists and antagonists will be useful probes to explore their activities in endogenous contexts. These compounds may also serve as leads for the development of GPR56-targeted pro- or anticoagulants.

Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs

AbstractPlatelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.