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Human Urokinase (uPA) Purified Two Chain High Molecular Weight, Human tPA 85% Single Chain Purified, and Human tPA 95% Two Chain Purified from Innovative Research were used in the following study:
PhD. Dissertation, University of Michigan
Hemostasis is a complex, regulated bodily process that repairs vascular damage following injury. Three major parts make up the hemostatic system: blood clotting (coagulation), platelet activation, and vascular repair. Of these parts, coagulation plays the largest role.
The coagulation cascade, established in the 1960s, describes a series of proteolytic cleavage reactions that transform inactive clotting factors (zymogens) into active enzymes, most of which are serine proteases. The cascade is divided into two pathways - the extrinsic pathway and the intrinsic pathway. In the extrinsic pathway, or tissue activation pathway, activation is induced by either tissue factor (TF) or a negatively charged contact surface. A series of proteolytic cleavages follow, and eventually the two pathways converge into what is known as the common pathway. The last step in the coagulation cascade is blood clot formation to seal the injured vasculature.
Compared to hemostasis, thrombosis describes the process in which an unwanted blood clot forms inside a blood vessel, which may cause an obstruction that can disrupt circulation. Interestingly, although thrombosis follows a separate activation process than hemostasis, the tissue factor pathway plays a role in activating blood clotting in both hemostasis and thrombosis.
Following vascular damage, TF is exposed and binds to FVIIa, a serine protease. This bound group then initiates the proteolytic cleavage of factor IX (FIX) and factor X (FX), whose activation is facilitated by TF residues remaining near or on the substrate binding site. In this study, it was hypothesized that there is an exosite-adjacent TF serine loop that selectively mediates binding site substrate via the TF-FVIIa complex. Mutagenesis testing and enzymatic assay observations suggested a correlation between the length of the TF serine loop and its effects on FIX and FX activation. For example, FX activation was observed to have decreased by up to 200-fold after changing the serine loop length by only one residue, however FIX activation was largely unaffected in this instance. This implies that the TF-FVIIa complex actively recognizes and selects between the two protein substrates when initiating and facilitating the formation of blood clots.
Under normal circumstances, serine proteases are typically synthesized in an inactive state and remain that way until reaching their target destination to avoid cellular damage and the potential for the development of various diseases. A secondary hypothesis was tested alongside the primary experiments to determine whether small concentrations of ATP and other nucleotides in the Endoplasmic Reticulum (ER) and Golgi would be able to prevent the premature activation of zymogens as they travel through these compartments towards their target destination. Their testing seemed to prove the hypothesis correct, suggesting that small amounts of ATP and other nucleotides may be able to serve as a safety mechanism to prevent cellular damage and the development of diseases caused by premature protease activation.
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