A B S T R A C T Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surfacemediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen.

Lupus anticoagulants, commonly found in the immunoglobulin fraction of patients with the antiphospholipid syndrome (APS), and the normal plasma protein beta 2-glycoprotein 1 (beta 2GP1) may both contribute to the in vitro impairment of prothrombin activation associated with the APS. We examined the effects upon prothrombin activation supported by phospholipid vesicles of plasma IgG preparations from APS patients in the presence and absence of beta 2GP1. Using a purified system for measurement of prothrombin activation to thrombin, we demonstrated significant phospholipid concentration-dependent inhibition of prothrombin activation in the absence of beta 2GP1 by 11 consecutive patient IgG preparations. The degree of inhibition of prothrombin activation by equivalent concentrations of patient IgG correlated well with the extent of prolongation of the plasma clotting time in lupus anticoagulant assays of whole patient plasma. Additional studies with eight patient IgG preparations indicated that the addition of beta 2GP1 to patient IgG-phospholipid vesicle mixtures resulted in either independently additive inhibition by the two protein species (six cases) or potential inhibition of beta 2GP1 of the IgG inhibitory activity demonstrable in the absence of beta 2 GP1 (two cases). In addition, beta 2GP1-independent inhibition of prothrombin activation also occurred with three patient IgG preparations obtained by affinity binding to cardiolipin.

A B S T R A C T Two siblings with mild hemorrhagic symptoms had combined functional deficiencies of vitamin K-dependent clotting factors. Prothrombin (0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) were most severely affected. Antigenic amounts of affected coagulation factors were normal and normal generation of thrombin activity occurred in the patients' plasmas after treatment with nonophysiologic activators that do not require calcium for prothrombin activation. Hepatobilary disease, malabsorptive disorders, and plasma warfarin were not present. Both parents had normal levels of all coagulation factors. The patients' plasmas contained prothrombin that reacted both with antibody directed against des-y-carboxyprothrombin and native prothrombin. Crossed immunoelectrophoresis of patients' plasmas and studies of partially purified patient prothrombin suggested the presence of a relatively homogeneous species of dysfunctional prothrombin, distinct from the heterologous species found in the plasma of warfarin-treated persons. These studies are most consistent with a posttranslational defect in hepatic carboxylation of vitamin K-dependent factors. This kindred uniquely possesses an autosomal recessive disorder of vitamin K-dependent factor formation that causes production of an apparently homogeneous species of dysfunctional prothrombin; the functional deficiencies in clotting factors are totally corrected by oral or parenteral administration of vitamin KI.

ABSTRACTarPlasmin inhibitor (a2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified a2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 ;sg/ml), a2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. a2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). a2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of a2PI. These results suggest that, like other plasma protease inhibitors, a2PI possesses a broad in vitro spectrum of inhibitory properties.a2-Plasmin inhibitor (a2PI) has been recently identified as a fast-reacting inhibitor of plasmin in human plasma (1-3). a2PI has strong inhibitory activity against plasmin and is the first inhibitor that reacts with plasmin, whether plasminogen activation occurs in vitro or in vivo (4-6).Although the action of a2PI on plasmin is well characterized, relatively little information is available about the effect of this newly isolated inhibitor upon other plasma proteases (7,8). This paper reports the effect of physiologic concentrations of a2PI upon various proteases participating in blood coagulation and kinin generation and defines the in vitro spectrum of this inhibitor. We also compare the action of a2PI with that of some other plasma protease inhibitors in these systems. MATERIALS AND METHODSInhibitors. Human a2PI was prepared as described (1) and was judged greater than 95% homogeneous on disc gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, and immunoelectrophoresis. The concentration of purified a2PI was determined by using 7.03 as extinction coefficient El% at 280 um. a2-Macroglobulin (a2M), isolated from human plasma (9), gave a single band by sodium dodecyl sulfate gel electrophoresis after reduction with 2-mercaptoethanol (Mr, approximately 180,000). The concentration of a2M was determined by single radial immunodiffusion on Immuno-plates (Hyland, Costa Mesa, CA). Normal serum contains approximately 2 mg of a2M per ml. a2M at 2 mg/ml was essentially free of a2PI (<0.2 ,g/ml) when tested by a sensitive radioitnmunoassay for a2PI (unpublished data). a2M (8 mg/ml) contained no detectable C1 esterase inhibitor (Clinh), a1-antitrypsin, or antithrombin III as assayed by immunodiffusion using monospecific antiserum. Purified Clinh and monospecific antiserum against Clinh were generous gifts of J. Pensky

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