Release of endogenous α-defs during blood coagulation. (A) α-Defs in plasma and serum. Blood collected from healthy volunteers in citrate to prepare plasma was allowed to sit at room temperature for 1 hour to prepare serum. Blood was centrifuged at 1500g for 10 minutes, and the concentration of α-defs 1-3 in the plasma and serum was measured by ELISA.⁵⁴  The results shown are the mean ± SD from 10 human healthy volunteers (*P < .05). (B) Role of contact activation vs thrombin. Whole blood from 11 healthy donors collected in citrate or with no anticoagulant was clotted along a glass tube or following the addition of thrombin and calcium chloride. The concentrations of α-defs measured by ELISA in sera prepared by each method were compared. The results shown are the mean ± SD in samples from 11 healthy volunteers (*P < .05). (C) Role of the intrinsic vs extrinsic pathway. Serum was generated from citrated whole blood following the addition of calcium chloride and tissue factor (TF) or kaolin, with or without aprotinin, and the concentration of α-defs was measured as in panel A. (D) Role of kallikrein. Isolated human neutrophils were added to normal plasma, prekallikrein-deficient plasma (PK (−)), or prekallikrein-deficient plasma supplemented with prekallikrein (PK (−) + PK). Clotting was initiated by adding kaolin and calcium chloride. The concentration of α-defs in the serum was measured as in panel A. The results shown are the mean ± SD of 3 experiments (*P < .05). (E) Isolated human neutrophils in phosphate-buffered saline (PBS) containing 1 mM calcium chloride were incubated with FXIIa, FXIa, FIXa, or FXa alone or together with prekallikrein (PreK) or with prekallikrein alone for 30 minutes, followed by centrifugation and separation of the supernatant fluids. In some experiments, colchicine or CTI was added along with the coagulation factors, where indicated. The concentration of α-defs in the supernatants was measured as in panel A. (F) Incorporation of α-defs into blood clots. An aliquot of ¹²⁵I-α-Def-1 was added to purified fibrinogen. Clotting was induced by adding thrombin, and radioactivity in the fibrin clot and supernatant was measured. The mean ± SD of 3 experiments in shown. (G) Release of α-defs from lysed blood clots. Blood clots were formed using blood collected from healthy human volunteers as in panel B by contact with glass or by adding thrombin, separated by centrifugation, lysed by addition tPA, and recentrifuged, and the concentration of α-defs in the supernatants was measured. The mean ± SD of 3 experiments is shown.