Materials and Methods Materials. Human plasma factor XIII and plasma factor XIII b subunits were prepared according to procedures pre-viously described (Lorand et al., 1981). Recombinant human platelet factor XIII was a gift from Zymogenetics, Inc. The properties of recombinant human platelet factor XIII and human platelet-derived factor XIII have been previously re-ported to be similar (Hornyak et al., 1989). Fibrinogen was prepared by precipitating human plasma with/3-alanine as previously described (Lewis & Shafer, 1984). Generation of a2b2 and Appearance of the Active-Site Thiol Group. A 100-/iL aliquot of plasma factor XIII was mixed with a solution containing 840/iM [l-14C] iodoacetamide (NEN, 24.1 mCi/mmol) in 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% PEG, pH 7.5 at 37 C. At t= 0, a small volume of a-thrombin dilutedin 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% PEG, pH 7.5, was added such that the final composition of the reaction mixture was 1.21/tM plasma factor XIII, 45 mM [l-14C] iodoacetamide, and 19 nM a-thrombin. Incubation of plasma factor XIII with 19 nM a-thrombin under these conditions results in greater than 99% cleavage of AP after 30-min incubation (Januset al., 1983). The a-thrombin ac-tivity was quenched after 30 min of incubation by the addition of 11/* L of 30/iM PPACK (final molar ratio of PPACK to a-thrombin= 160). A 5-/tL sample of the incubate was pipetted onto a 1 cm2 piece of Whatman 3MM filter paper at this time and placed in a 10% trichloroacetic acid (TCA) bath to determine the fractionof active-site thiol group ex-posure prior to addition of calcium ion. A small volume of CaCl2 solution (0.04-0.4 M CaCl2 in 50 mM Tris-HCl, pH 7.5, depending upon the final concentration of CaCl2 desired) was added to the incubatecontaining a-thrombin-cleaved factor XIII, and 5-/iL samples were pipetted onto filter papers at various times thereafter. Immediately after being spotted, the filter papers were placed into a bath of 10% TCA and washed as previously described (Curtis et al., 1973, 1974). The amount of radioactively labeled factor XHIa precipitated on the filter papers was determined as noted previously (Hornyak et al., 1989).

In the experiment with fibrinogen, a 61.3-/tL incubate with a composition of 2.24/tM plasma factor XIII, 82/tM [1-14C] iodoacetamide, and 35 nM a-thrombin was used to cleave the factor XIII. After addition of the PPACK quench, 51.7/iL of 23.2/tM fibrinogen (previously dialyzed against 0.1 M Tris-HCl, 0.15 M NaCl, and 0.1% PEG, pH 7.5) was added to the cleaved factor XIII to standardize conditions to those of the experiments notcontaining fibrinogen. Calcium ion was added and the experiment completed as described above. No clotting of fibrinogen was observed during the time course of the experiment, consistent with complete activation of the a-thrombin by PPACK prior to addition of the fibrinogen. Rate of Exposure of the Active-Site Thiol Group of Platelet Factor XIII. Therate of exposure of the active-site thiol group