Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.

Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.

Cav 3.2 T-type calcium channel regulates mouse platelet activation and arterial thrombosis.

BACKGROUND: Cav 3.2 is a T-type calcium channel that causes low-threshold exocytosis. T-type calcium channel blockers reduce platelet granule exocytosis and aggregation. However, studies of the T-type calcium channel in platelets are lacking. OBJECTIVE: To examine the expression and role of Cav 3.2 in platelet function. METHODS: Global Cav 3.2-/- and platelet-specific Cav 3.2-/- mice and littermate controls were used for this study. Western blot analysis was used to detect the presence of Cav 3.2 and activation of the calcium-responsive protein extracellular signal-regulated kinase (ERK). Fura-2 dye was used to assess platelet calcium. Flow cytometry and light transmission aggregometry were used to evaluate platelet activation markers and aggregation, respectively. FeCl3 -induced thrombosis and a microfluidic flow device were used to assess in vivo and ex vivo thrombosis, respectively. RESULTS: Cav 3.2 was expressed in mouse platelets. As compared with wild-type controls, Cav 3.2-/- mouse platelets showed reduced calcium influx. Similarly, treatment with the T-type calcium channel inhibitor Ni2+ decreased the calcium influx in wild-type platelets. As compared with controls, both Cav 3.2-/- and Ni2+ -treated wild-type platelets showed reduced activation of ERK. ATP release, P-selectin exposure, and αIIb ß3 activation were reduced in Cav 3.2-/- and Ni2+ -treated wild-type platelets, as was platelet aggregation. On in vivo and ex vivo thrombosis assay, Cav3.2 deletion caused delayed thrombus formation. However, tail bleeding assay showed intact hemostasis. CONCLUSION: These results suggest that Cav 3.2 is required for the optimal activation of platelets.

Apo B/Apo A-I Ratio is Statistically A Better Predictor of Cardiovascular Disease (CVD) than Conventional Lipid Profile: A Study from Kathmandu Valley, Nepal.

BACKGROUND: Apo B and Apo A-I, are structural and functional components of lipoprotein particles that serve as transporters of cholesterol. The apo B/apo A-I ratio reflects the cholesterol transport and has been shown to be strongly related to risk of Myocardial infarction, stroke and other Cardiovascular manifestations. MATERIALS AND METHODS: Forty five participants with Cardiovascular Disease (CVD) and forty four healthy participants were included from different locations of Kathmandu valley, Nepal. Fasting blood samples were collected from ante-cubital vein and serum samples were used for lipid parameters, apo B and apo A-I levels measurement. RESULTS: Statistically significant differences were found for apo B/apo A-I ratio, HDL-c and apo B between the groups. The other lipid parameters and lipid ratios such as total cholesterol, triglyceride, low density lipoprotein, TC/HDL-c, TG/HDL-c and LDL-c/HDL-c were not found to be significant. CONCLUSION: Apo B/apo A-I ratio seems to have better predictive value than that of classical lipid parameters in cardiovascular risk assessment.