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Activation and inactivation of protein C during the clinical course of disseminated intravascular coagulation (DIC) was studied in three patients by qualitative (Western blotting) and quantitative (ELISA) analysis and the intensity of procoagulant activity monitored by the measurement of thrombin and factor Xa antithrombin III complexes. In one patient, inhibitor complexes of APC with protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1-AT) were observed and the latter predominated at presentation. Both disappeared during the development of remission but the loss of alpha 1-AT complexes preceded PCI complexes which on Western blotting appeared to increase in intensity prior to disappearance. The two other patients bled to death from uncontrollable haemorrhage. In both cases, APC/inhibitor complexes with alpha 2-macroglobulin (alpha 2-M) in addition to PCI and alpha 1-AT were detected and persisted until death. Although PCI appeared to be the primary inhibitor in all three cases, alpha 1-antitrypsin and particularly alpha 2-macroglobulin appeared to assume greater roles in the two fatal cases. These data are similar to previous findings in an experimental animal model of DIC that suggested that alpha 2-macroglobulin and alpha 1-antitrypsin become more important inhibitors of APC as the primary inhibitor PCI is consumed in the face of a sustained procoagulant challenge.

Activated protein C (APC), a serine protease, is regulated in plasma by protease inhibitors. This study was undertaken to determine the role of the major plasma inhibitors in regulating APC in plasma. Kinetic analysis and specific immunoassays for APC-inhibitor complexes were used to determine the inhibitors that form complexes with APC. Of the eight plasma inhibitors investigated, four interact with APC: protein C inhibitor (PCI), alpha 1-proteinase inhibitor (PI), alpha 2-antiplasmin (AP) and C1 esterase inhibitor (C1 Inh). The second order rate constants are: 1.3 x 10(4) M-1 s-1 (PCI); 15 M-1 s-1 (PI); 410 M-1 s-1 (AP); and < 6 M-1 s-1 (C1 Inh), with a relative effectiveness of each inhibitor to inactivate APC in plasma: 49:36:15: < 1, respectively. PCI, PI and AP are the major inhibitors of APC in plasma. Low concentrations of APC will be inhibited by PCI with PI and AP playing a secondary role. However, as increasing APC is generated, PI and AP begin to play more important roles as the PCI is consumed.

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein C inhibitor (PCI) concentration and other parameters of the protein C pathway were studied in 19 patients with symptomatic acute deep vein thrombosis before and during the first 5 days of heparin treatment. The mean levels of PCI antigen and activity decreased rapidly and significantly during the first day of heparin therapy from 83 and 84% to 60 and 59% of the pooled normal human plasma (p less than 0.01), respectively, and to 56 and 54% after 5 days of treatment (p less than 0.01). In contrast, antithrombin III decreased progressively 25% during 5 days of heparin treatment. Protein C antigen and activity and total protein S remained unchanged during heparin treatment. Free protein S was decreased before heparin treatment (83%, p less than 0.05) and increased to normal values after 5 days of treatment. C4b-binding protein was significantly increased before and during heparin treatment (p less than 0.01). Activated protein C (APC) complexed to its two major plasma inhibitors, PCI and alpha 1-antitrypsin (alpha 1AT) were measured by specific ELISA's. Before treatment, 18 of the 19 patients studied had increased levels of APC:alpha 1AT complexes with a mean value of 27 +/- 22 ng/ml (range, 6-86 ng/ml) compared to normal values (8 +/- 2 ng/ml) and 12 of the patients also had detectable APC:PCI complex levels with a mean value of 11 +/- 17 ng/ml (range, 5-68 ng/ml). Both APC:inhibitor complexes decreased significantly during heparin treatment.

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to less than 1 microliter of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations greater than or equal to 10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).

Inactivation of activated protein C (APC) in normal human plasma was studied in the absence and presence of heparin. In the absence of heparin APC inactivation followed pseudo-first order kinetics. In the presence of heparin the neutralization of APC was found to be biphasic. Up to 500 nM APC could be readily inactivated in normal plasma, indicating that the concentration of the APC inhibitor must be higher than previously assumed. Plasma deficient in the protein C inhibitor (PCI-I, as described by Suzuki and coworkers) and deficient in beta 2-glycoprotein I still possessed APC neutralizing capacity, presumably through the formation of complexes of APC with another plasma protein as was demonstrated by immunoblotting with anti-protein C antibodies. Together these data made us to conclude that a second inhibitor of APC (PCI-II) must be present in normal human plasma. This second inhibitor should be heparin independent, have a relatively high plasma concentration and form complexes with APC. Subsequently, we purified this PCI-II by isolating APC-PCI-II complexes from plasma deficient of vitamin K dependent proteins, PCI-I and beta 2-glycoprotein-I, to which purified human APC had been added. Purified PCI-II has a molecular weight of 50,000 daltons and aminoacid analysis revealed that PCI-II is identical with alpha 1-antitrypsin (alpha 1-AT). The second order rate constant for the reaction between purified alpha 1-AT and APC was found to be 269 M-1 min-1 in the absence of calcium and 602 M-1 min-1 in the presence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein C inhibitor (PCI) was purified from human plasma using immunoaffinity chromatography and heparin Sepharose chromatography, a method that allowed the purification of active and inactive inhibitor. PCI purified from outdated plasma was inactive and either in complex with plasma kallikrein or proteolytically degraded. Sequence analysis of cleaved PCI and of complexes between PCI and activated protein C or urokinase identified the previously recognized inhibitor cleavage site Arg354-Ser355. Two additional cleavage sites were observed in the modified inhibitor i.e. Arg357-Leu358 and Arg362-Leu363 which probably represent secondary cleavage of the inhibitor. Furthermore the sequence analysis of the inhibitor, whether purified from fresh or outdated plasma, revealed that it was microheterogeneous in the NH2-terminus as a result of cleavage by a trypsin like enzyme(s).