Unit 217

Serpins are a class of proteins involved in the regulation of serine and other types of proteases (Huber and Carrell, 1989; Potempa et al., 1994; Church et al., 1997). To date, DNA and protein sequencing have identified >400 members of the serpin superfamily (Whisstock et al., 1999). Interestingly, not all serpins are of an inhibitory nature (Remold-O'Donnell, 1993). Noninhibitory serpins include ovalbumin, angiotensinogen, and pigment epithelium-derived factor. In addition to being found in humans, serpins have been found in other mammals, insects, plants, and viruses. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair (Huber and Carrell, 1989; Potempa et al., 1994; Church et al., 1997). Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a deficiency or a defect in functional protein. Examples of serpin-related pathologies include venous thromboembolic disease (an-tithrombin), breast cancer metastasis (maspin), emphysema (a1-protease inhibitor), and hereditary angioedema (C1 inhibitor) (Huber and Carrell, 1989; Potempa et al., 1994; Church et al., 1997). In this unit, the purification (see Basic Protocol 1) and assay (see Support Protocol) of antithrombin (AT; historically called antithrombin III) are first described. Then, protocols to determine the second-order rate constant of AT inhibition of thrombin in the absence and presence of heparin (see Basic Protocols 2 and 3) are presented. For a partial list of other serpins and their purification methods refer to Table 21.7.1. Antithrombin is a glycosaminoglycan-binding serpin that is found in human plasma at a concentration of 3 to 5 ^M. The target proteases of AT include the following blood-coagulation proteases: thrombin, Factors Xa, IXa, XIa, and Xlla.

CAUTION: Human blood products are a biohazard and must be handled and disposed of according to institutional guidelines. See appendix2a for general precautions.

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