Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype.

OBJECTIVE: Reduced blood flow and tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We, therefore, sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions. APPROACH AND RESULTS: In a cohort of patients with metabolic and peripheral artery disease, platelet activity was enhanced, and inhibition with oral antiplatelet agents was impaired compared with platelets from control subjects, suggesting a difference in platelet phenotype caused by the disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O2) had increased expression of some proteins that augment platelet activation compared with platelets in normoxic conditions (21% O2). Using a murine model of critical limb ischemia, platelet activity was increased even 2 weeks postsurgery compared with sham surgery mice. This effect was partly inhibited in platelet-specific ERK5 (extracellular regulated protein kinase 5) knockout mice. CONCLUSIONS: These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses because of changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should, therefore, be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of antiplatelet therapy.

Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion.

BACKGROUND: Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes. In comparison with platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species-rich environments may not be the same as in normal healthy conditions. Extracellular regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase family member activated in hypoxic, reactive oxygen species-rich environments and in response to receptor-signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells after ischemia. We present evidence that platelets express ERK5 and that platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent reactive oxygen species-mediated mechanisms in ischemic myocardium. METHODS AND RESULTS: Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and that platelet-specific ERK5(-/-) mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5-regulated proteins is reduced in ERK5(-/-) platelets post-MI. CONCLUSIONS: ERK5 functions as a platelet activator in ischemic conditions, and platelet ERK5 maintains the expression of some platelet proteins after MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment.

Emerging roles for platelets as immune and inflammatory cells.

Despite their small size and anucleate status, platelets have diverse roles in vascular biology. Not only are platelets the cellular mediator of thrombosis, but platelets are also immune cells that initiate and accelerate many vascular inflammatory conditions. Platelets are linked to the pathogenesis of inflammatory diseases such as atherosclerosis, malaria infection, transplant rejection, and rheumatoid arthritis. In some contexts, platelet immune functions are protective, whereas in others platelets contribute to adverse inflammatory outcomes. In this review, we will discuss platelet and platelet-derived mediator interactions with the innate and acquired arms of the immune system and platelet-vessel wall interactions that drive inflammatory disease. There have been many recent publications indicating both important protective and adverse roles for platelets in infectious disease. Because of this new accumulating data, and the fact that infectious disease continues to be a leading cause of death globally, we will also focus on new and emerging concepts related to platelet immune and inflammatory functions in the context of infectious disease.

Small cells, big effects: the role of platelets in transplant vasculopathy.

Platelets are the cellular mediator of thrombosis, but also represent an important part of the immune system. Platelets store numerous pre-formed inflammatory molecules in their granules and produce immune mediators de novo upon stimulation. Activated platelets express adhesion molecules that can interact with endothelial cells and leukocytes both at the site of inflammatory insult and in the circulation. Platelets therefore drive and potentiate vascular inflammation by direct and indirect interactions with both endothelial cells and leukocytes. Platelet activation has been associated with the pathogenesis of inflammatory diseases such as atherosclerosis, arthritis, and cerebral malaria, in addition to transplant vasculopathy. In this review we will discuss immune roles for platelets in the context of transplant vascular inflammation.

A random mutation capture assay to detect genomic point mutations in mouse tissue.

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.

Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbß, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium.

Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion.

In humans, vWF levels predict the risk of myocardial infarction and thrombosis; however, the factors that influence vWF levels are not completely understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels, though the functional relationship between STXBP5 and vWF is unknown. We hypothesized that STXBP5 inhibits endothelial cell exocytosis. We found that STXBP5 is expressed in human endothelial cells and colocalizes with and interacts with syntaxin 4. In human endothelial cells reduction of STXBP5 increased exocytosis of vWF and P-selectin. Mice lacking Stxbp5 had higher levels of vWF in the plasma, increased P-selectin translocation, and more platelet-endothelial interactions, which suggests that STXBP5 inhibits endothelial exocytosis. However, Stxbp5 KO mice also displayed hemostasis defects, including prolonged tail bleeding times and impaired mesenteric arteriole and carotid artery thrombosis. Furthermore, platelets from Stxbp5 KO mice had defects in platelet secretion and activation; thus, STXBP5 inhibits endothelial exocytosis but promotes platelet secretion. Our study reveals a vascular function for STXBP5, validates the functional relevance of a candidate gene identified by GWAS, and suggests that variation within STXBP5 is a genetic risk for venous thromboembolic disease.