Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Systems Biology Approach for Personalized Hemostasis Correction.

The correction of blood coagulation impairments of a bleeding or thrombotic nature employs standard protocols where the type of drug, its dose and the administration regime are stated. However, for a group of patients, such an approach may be ineffective, and personalized therapy adjustment is needed. Laboratory hemostasis tests are used to control the efficacy of therapy, which is expensive and time-consuming. Computer simulations may become an inexpensive and fast alternative to real blood tests. In this work, we propose a procedure to numerically define the individual hemostasis profile of a patient and estimate the anticoagulant efficacy of low-molecular-weight heparin (LMWH) based on the computer simulation of global hemostasis assays. We enrolled a group of 12 patients receiving LMWH therapy and performed routine coagulation assays (activated partial thromboplastin time and prothrombin time) and global hemostasis assays (thrombodynamics and thrombodynamics-4d) and measured anti-Xa activity, fibrinogen, prothrombin and antithrombin levels, creatinine clearance, lipid profiles and clinical blood counts. Blood samples were acquired 3, 6 and 12 h after LMWH administration. We developed a personalized pharmacokinetic model of LMWH and coupled it with the mechanism-driven blood coagulation model, which described the spatial dynamics of fibrin and thrombin propagation. We found that LMWH clearance was significantly lower in the group with high total cholesterol levels. We generated an individual patient's hemostasis profile based on the results of routine coagulation assays. We propose a method to simulate the results of global hemostasis assays in the case of an individual response to LMWH therapy, which can potentially help with hemostasis corrections based on the output of global tests.

Hypercoagulation detected by routine and global laboratory hemostasis assays in patients with infective endocarditis.

BACKGROUND: Coagulation system is heavily involved into the process of infective endocarditis (IE) vegetation formation and can facilitate further embolization. In this study we aimed to assess the coagulation and platelet state in IE implementing a wide range of standard and global laboratory assays. We also aim to determine whether prothrombotic genetic polymorphisms play any role in embolization and mortality in IE patients. METHODS: 37 patients with IE were enrolled into the study. Coagulation was assessed using standard coagulation assays (activated partial thromboplastin time (APTT), prothrombin, fibrinogen, D-dimer concentrations) and integral assays (thromboelastography (TEG) and thrombodynamics (TD)). Platelet functional activity was estimated by flow cytometry. Single nuclear polymorphisms of coagulation system genes were studied. RESULTS: Fibrinogen concentration and fibrinogen-dependent parameters of TEG and TD were increased in patients indicating systemic inflammation. In majority of patients clot growth rate in thrombodynamics was significantly shifted towards hypercoagulation in consistency with D-dimers elevation. However, in some patients prothrombin, thromboelastography and thrombodynamics were shifted towards hypocoagulation. Resting platelets were characterized by glycoprotein IIb-IIIa activation and degranulation. In patients with fatal IE, we observed a significant decrease in fibrinogen and thrombodynamics. In patients with embolism, we observed a significant decrease in the TEG R parameter. No association of embolism or mortality with genetic polymorphisms was found in our cohort. CONCLUSIONS: Our findings suggest that coagulation in patients with infective endocarditis is characterized by general hypercoagulability and platelet pre-activation. Some patients, however, have hypocoagulant coagulation profile, which presumably can indicate progressing of hypercoagulation into consumption coagulopathy.