Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets.

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.

Platelets control blood clotting in health and disease. Small molecule drugs are often used to study human platelets. Here, describe how Cellular Thermal Shift Assay (CETSA) can be used in platelets to investigate the binding between these drugs and their targets inside platelets. This technique can be used to increase our understanding of how existing and future drugs work in platelets.

The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Proteomic profiling of platelet signalling.

Platelets play a crucial role in hemostasis; activating and aggregating to arrest bleeding following vascular injury. Platelet activation is a complex and dynamic process, involving the co-ordination of numerous receptors to initiate shape change and aggregation. Under pathological conditions, alterations in the normal platelet response can favor a prothrombotic state and increase the risk of acute coronary syndromes (ACS). Receptor stimulation and the tyrosine phosphorylation of key signaling molecules underpin platelet activation in both hemostasis and cardiovascular disease. A lack of nucleus and low mRNA levels makes protein function the primary focus of platelet research. Advancements in proteomic technologies now allow for comprehensive analysis of the platelet proteome and its associated post-translational modifications. In this review, recent applications of proteomics in platelet signaling studies are discussed with particular focus on the elucidation of novel phosphorylation events following receptor activation.

Cleavage by MMP-13 renders VWF unable to bind to collagen but increases its platelet reactivity.

BACKGROUND: Atherosclerotic plaque rupture and subsequent thrombosis underpin thrombotic syndromes. Under inflammatory conditions in the unstable plaque, perturbed endothelial cells secrete von Willebrand Factor (VWF) which, via its interaction with GpIbα, enables platelet rolling across and adherence to the damaged endothelium. Following plaque rupture, VWF and platelets are exposed to subendothelial collagen, which supports stable platelet adhesion, activation, and aggregation. Plaque-derived matrix metalloproteinase (MMP)-13 is also released into the surrounding lumen where it may interact with VWF, collagen, and platelets. OBJECTIVES: We sought to discover whether MMP-13 can cleave VWF and whether this might regulate its interaction with both collagen and platelets. METHODS: We have used platelet adhesion assays and whole blood flow experiments to assess the effects of VWF cleavage by MMP-13 on platelet adhesion and thrombus formation. RESULTS: Unlike the shear-dependent cleavage of VWF by a disintegrin and metalloprotease with thrombospondin motif member 13 (ADAMTS13), MMP-13 is able to cleave VWF under static conditions. Following cleavage by MMP-13, immobilized VWF cannot bind to collagen but interacts more strongly with platelets, supporting slower platelet rolling in whole blood under shear. Compared with intact VWF, the interaction of cleaved VWF with platelets results in greater GpIbα upregulation and P-selectin expression, and the thrombi formed on cleaved VWF-collagen co-coatings are larger and more contractile than platelet aggregates on intact VWF-collagen co-coatings or on collagen alone. CONCLUSIONS: Our data suggest a VWF-mediated role for MMP-13 in the recruitment of platelets to the site of vascular injury and may provide new insights into the association of MMP-13 in atherothrombotic and stroke pathologies.

The application of proteomics technology to thrombosis research: the identification of potential therapeutic targets in cardiovascular disease.

Thrombus formation underpins the development of cardiovascular diseases, including acute coronary syndromes and ischaemic stroke. A number of well-characterised cardiovascular risk factors which contribute to the development of the majority of cardiovascular events have been identified, including dyslipidaemia, hypertension and diabetes. Individuals with type 2 diabetes mellitus (T2DM) have a 3- to 5-fold increased risk for development of cardiovascular disease (CVD). They may have a cluster of haemostatic abnormalities, including elevated levels of plasminogen activator inhibitor-1 (PAI-1) and fibrinogen, which contribute to acute thrombotic events. It is clear that additional unidentified risk factors contribute to the pathogenesis of cardiovascular events, and so the search for novel biomarkers and effectors, particularly in individuals with T2DM, remains a major challenge of cardiovascular medicine. Plasma and cellular proteins which contribute to thrombus formation have the potential to confer a pro-thrombotic state and represent a link between genotype, environment and disease phenotype. The comprehensive analysis of these proteins is now increasingly facilitated through the continued development of proteomic technologies which provide multifaceted approaches to the identification of novel biomarkers and/or effectors of thrombus formation and on which future anticoagulant and thrombolytic therapies may be based. This review provides an overview of current proteomic technologies. It focuses on the recent studies in which these technologies have been applied in the search for novel proteins that may confer increased risk of acute cardiovascular diseases and therefore that may influence disease progression and therapy.

MMP-13 binds to platelet receptors αIIbß3 and GPVI and impairs aggregation and thrombus formation.

BACKGROUND: Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. OBJECTIVES: Here we sought to elucidate whether matrix metalloproteinase-13 (MMP-13), a collagenolytic metalloproteinase up-regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. RESULTS: We demonstrate that MMP-13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbß3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbß3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. CONCLUSIONS: Our data demonstrate a role for MMP-13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP-13 in atherothrombotic pathologies.

Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner.

Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbß3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2ß1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

Complement C3 is a novel plasma clot component with anti-fibrinolytic properties.

BACKGROUND AND METHOD: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. RESULTS: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. CONCLUSION: These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.