One of the central features of the coagulation cascade is that each successive step in the process involves assembly of proteases with regulatory proteins (cofactors) and their substrate (~) on membrane surfaces. Despite the fact that these cofactors are essential to rapid activation of the target zymogens. relativell-little is known about how the cofactors function. The protein C activation complex offers some unique experimental advantages for investigating the functions of the cofactors in the coagulation cascade. The key features of the coinplex that aid in analysis follow. Chemically, protein C activation sinlply involves the release of a dodecapeptide from the amino terminus of the heavy chain of protein C (1). Rapid protein C activation, ho\vever. requires formation of a high affinity complex between thrombin and thrombomodulin (Kd= 0.2 nM). Complex formation enhances protein C activation approximately 1000-fold, but inhibits activation of normal thrombin substrates (2-4)(except for factor XI (5)). The reaction requires Ca2+. occurs in the absence of membrane surfaces. and requires the presence of the Gla domain of protein C for Ca2+ to facilitate activation. Phospholipid inembranes accelerate protein C activation. and since thrombin does not interact with membranes directly, and throinbomodulin is an integral membrane protein. the analysis of meinbraile participation ill substrate (protein C) presentation is greatly simplified. Finally. although thrombo~ nodulin is a very cornplex molecule. it can be fractionated into small domains with discrete functions. Relatively high affinity thrombin binding is maintained with fragments that are 80 residues or less in length (6), but good activity can be obtained with the addition of a single additional EGF domain (7-9). Overall, the properties of the activation complex are amenable to a variety of biochemical inanipulations that are not feasible with other activation complexes involved in coagulation. Since several reviews have appeared recently (3; 10-18). I will place the emphasis in this paper primarily on our o\vn studies and how we believe they contribute to an overall understanding of protease specificity in general and the function of the regulatory proteins in particular. A further advantage of the protein C activation system is that. although thrombin is a very specific protease. it nonetheless serves a variety of biological functions and hence has many substrates. Thus. the primary sequences adjacent to the