17 research outputs found
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. Plaqueâderived matrix metalloproteinase (MMP)â13 is also released into the surrounding lumen where it may interact with VWF, collagen and platelets.
Objectives
We sought to discover whether MMPâ13 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 assess the effects of VWF cleavage by MMPâ13 on platelet adhesion and thrombus formation.
Results
Unlike the shearâdependent cleavage of VWF by ADAMTS13, MMPâ13 is able to cleave VWF under static conditions. Following cleavage by MMPâ13, immobilised 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 upâregulation and Pâselectin expression, and the thrombi formed on cleaved VWFâcollagen coâcoatings are larger and more contractile than platelet aggregates on intact VWFâcollagen coâcoatings or on collagen alone.
Conclusions
Our data suggest a VWFâmediated role for MMPâ13 in the recruitment of platelets to the site of vascular injury and may provide new insights into the association of MMPâ13 in atherothrombotic and stroke pathologies
MMP-13 binds to platelet receptors αIIbÎČ3 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 (αIIbÎČ3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbÎČ3 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
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.This work was supported by a British Heart Foundation programme grant,
RG/009/003/27122, and peptide synthesis, by grants from Medical Research Council and Wellcome
Trust.This is the author accepted manuscript. The final version can be found on the publisher's website at: http://www.jbc.org/content/early/2014/07/09/jbc.M114.58344
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 αIIbÎČ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2ÎČ1. 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
The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study
Background:
Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy.
Methods:
Consecutive women undergoing mastectomyâ±âIBR for breast cancer JulyâDecember, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomyâ±âIBR were compared and risk factors associated with delays explored.
Results:
A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [nâ=â675, 26.6%]; pedicled flaps [nâ=â105,4.1%] and free-flaps [nâ=â228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays.
Conclusions:
IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients
The purification, characterization and inhibition of three metalloproteinases from Echis ocellatus venom
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Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets
Publication status: PublishedSmall 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 proteinâs 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
Complement C3 is a novel plasma clot component with anti-fibrinolytic properties
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
The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13
This is the author accepted manuscript. The final version can be found on the publisher's website at: http://www.jbc.org/content/early/2014/07/09/jbc.M114.583443Remodeling 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 ? chains sequentially, at Gly775-Leu776 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 (at helix residues 775-6). 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.This work was supported by a British Heart Foundation programme grant, \ud
RG/009/003/27122, and peptide synthesis, by grants from Medical Research Council and Wellcome \ud
Trust