White Paper Knowledge Library
Coagulation Testing
Inflammation, endothelial damage, and platelet activation trigger secondary hemostasis, comprising initiation and propagation of the coagulation cascade and the formation of insoluble fibrin to consolidate and stabilize the thrombus.
MLM offers a panel of commercial and proprietary tests as well as nearly three decades of expertise in the area of coagulation to support your translational and clinical research needs.
Functional and Phenotypic Microparticle Analysis
Circulating stimuli, including inflammatory cytokines and some therapeutics, activate platelets and endothelial cells. Measurement of circulating mciroparticles serves as an indicator of in vivo vascular function, and in clinical studies is used to identify risk of thrombosis and bleeding.
Microparticle Procoagulant Activity-ELISA Detection
Activated platelets and endothelial cells release microparticles with exposed phosphatidyl-serine (PS), a procoagulant stimulus for throm-bin generation. Procoagulant plasma microparticles are quantitated using an ELISA platform in which PS-presenting microparticles are captured on an annexin V-coated microplate. In vitro thrombin gen-eration and catalytic activity are then measured as a readout for total microparticle concentration.
Microparticle Phenotyping-Flow Cytometry Detection
Cell origin and microparticle phenotype are established utilizing anti-body labeling of cell-type specific proteins on the microparticle sur-face. In normal donors, endothelial microparticle (EMP) levels are low (<5%), and are differentiated from other circulating microparticles, the majority of which are platelet-derived, by labeling E-Selectin (CD62e), a cell adhesion molecule expressed exclusively by endothelial cells.
Platelet-Leukocyte Agrregate Function Assay
Increasing evidence implicates platelets as key cellular mediators of inflammation and vascular disease. Platelets express and secrete a variety of molecules and inflammatory mediators, such as platelet factor 4, P-selectin, CD40L, vWF, CD63, thrombospondins, MMPs, polyphosphates, ADP, thromboxane, Platelets nitric oxide, IL-1b and GPIb. Many of these mediators promote recruitment of leukocytes to sites of endothelial damage or tissue inflammation and this pathway likely contributes to pathogenesis of inflammatory and cardiovascular diseases, such as atherosclerosis, diabetes, and rheumatoid arthritis.
Oxidative Burst Analysis by Flow Cytometry
The growing number of monoclonal antibody therapies have provided better, more targeted treatment for the different types of cancers. With these exciting new therapies, the side effects of immune sup-pression have been declining over the years. However, even these new therapies can be associated with increased opportunistic infections. The complex network of pathways and cells that encompass immune response regulation make the assessments of immune suppression one of the most challenging aspects to monitor in recovering patients. Understand-ing the mechanisms by which new therapies contribute to immune suppression and increase the incidence of opportunistic infections is a key aspect for immunooncology.
Platelet Function Analyzed by Flow Cytometry
Flow cytometry is a sensitive technique for assessing specific components of platelet function, including degranulation, receptor signaling and surface protein activation. Platelet activation results in changes in platelet shape, degranulation, and signaling, including cal-cium flux, and protein phosphorylation. Agonists such as thrombin or ADP bind protease-activated receptors (in human platelets, PAR-1 and PAR-4) or purinergic receptors (P2Y12), respectively, to in-duce platelet activation.
Receptor Occupancy Assay for Assessment of Therapeutic Agents
Receptor occupancy (RO), the measurement of the bind-ing of a therapeutic to its cellular target, is increasing-ly important in the development of biologically based therapeutic agents. RO assay measurements can be used to determine minimal drug thresholds and guide dosing decisions during the clinical development of a drug. RO assays using flow cytometry describe the qualitative or quantitative measurement of the binding of a therapeutic agent to its cell surface target. Therefore, RO assays can measure the number of cell surface receptors bound by a therapeutic agent or can be designed to address more complicated scenarios such as receptor signaling, inter-nalization or shedding events once a receptor engages a therapeutic agent. Data generated from RO assays can also be used to assess whether doses of an experimental therapeutic agent and its administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent.
Thrombin Generation Assay
Thrombin is the final product of the intrinsic and extrinsic pathways, converting soluble fibrinogen into fibrin. It also serves as a potent activator of platelet function. As a key enzyme in hemostasis and thrombosis, it is imperative that reliable moni-toring of thrombin be performed in study subjects during pharmacologic safety trials, and in patients dosed with hemostasis-modulating medication.
Established methods such as partial thromboplastin time (PT) or activated partial thromboplastin time (aPTT) are routinely used to monitor coagulation, and are simple, reliable, and internationally standard-ized. They are, however, a single end-point analysis of the coagulation cascade. The elegance and chal-lenge of hemostasis is the complexity of the system. Novel molecular techniques allow us to study not only an individual factor, like thrombin, but its “life-time” activity before activation, during activation, and down-regulation. Current instrumentation allows monitoring of the coagulation cascade in a cost-effec-tive manner and is readily customizable.