Detection of an endogenous serine protease, inhibition of exterior serine proteases and a novel microdetection technique in platelet aggregation

Abstract

Serine Proteases are a class of proteolytic enzymes whose catalytic mechanism is based on an active-serine reside. 4-(2-Aminoethyl)-benzene sulfonyl fluoride (AEBSF) forms a covalent bond with the active serine residue in serine proteases. Alpha-Thrombin, a serine protease, activated platelet aggregation was completely inhibited by AEBSF, as expected. Platelet aggregations that were activated by non-proteolytic agonists, activating agents that do not contain an active serine residue for AEBSF to bind to, were also inhibited by AEBSF. This indicates that an endogenous platelet serine protease was required for aggregation to proceed independent of the activating agent. Inhibition of platelet aggregation is not usually associated with elimination of platelet shape change; however, shape change was not seen with AEBSF inhibition. This result suggests the serine protease may be involved with the platelet cytoskeleton. Western Blot studies indicate that there is at least one triton-insoluble protein whose ability to bind a serine protease inhibitor is lost with the addition of AEBSF and possibly another which gains binding ability. -Thrombin auto-catalyzes into - and - Thrombin. Inhibition of all of these agonists is essential for a complete anti-thrombotic response in instance where clotting is unwanted, due to each thrombin derivative having a unique pathway. Histone-1 effects on platelet aggregation initiated by various agonists were analyzed and significant in inhibition was seen in - and -thrombin. This research demonstrates the ability of Histone to inhibit -Thrombin induced platelet aggregation, which does not respond to commonly used anti-thrombotics Hirudin and Heparin. Platelet aggregation detection by impedance in whole blood has several advantages to turbidometric measurement of platelet aggregation by light transmission in Platelet Rich Plasma (PRP). In some instances it is not possible to separate nucleated platelets from red blood cells, such as in reptile and avian species. Having platelet aggregations being studied in whole blood replicates and in vivo environment much more effectively than PRP. Whole Blood aggregations and PRP both require relatively large amounts of blood per test, 0.5-1ml of whole blood is required for both. Therefore, a new method was required to examine platelet aggregation for instances where there are not large amounts of blood, such as with small animals, elderly patients, or children while still replicating an in vivo conditions. A microelectrode was developed in conjunction with Chronolog Co. that measures platelet aggregation via impedance while simultaneously measuring ATP release and only requires 150 l of whole blood per experiment. These experiments examined the role of both intrinsic and extrinsic serine proteases in platelet aggregation along with the development of a new methodology to examine these effects.%%%%M.S., Biomedical Science – Drexel University, 2001