Inhibition of plasminogen activation by polymerized ampicillin

The polymerized β-lactam antibiotic ampicillin inhibits the proteolytic activity of human plasmin upon ¹²⁵I-labeled fibrin clots. The inhibition is dose-dependent, with half-maximal inhibition occurring at 1.25 mM of the polymerized antibiotic. Polymerized ampicillin also inhibits binding of plasmin to fibrin, and 38% inhibition of binding occurs at 10 mM of the antibiotic. Furthermore, polymerized ampicillin inhibits the activation of plasminogen by either urokinase-like plasminogen activator (uPA) or tissue type-plasminogen activator (tPA). At 7.5 mM of polymerized ampicillin, the uPA-mediated plasminogen activation is suppressed by 94%, and half-maximal inhibition is obtained at 0.66 mM. The direct activity of uPA on the chromogenic substrate L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride (S-2444) is unaffected by polymerized ampicillin levels of up to 10 mM. The inhibitory effects of the polymerized antibiotic on the activation of plasminogen by both uPA and tPA is totally abolished in presence of fibrin. These interactions may serve as a novel model for ligands that enhance the clot-specificity of thrombolytic agents.     

Phosphorylation of coagulation factor XI by a casein kinase released by activated human platelets increases its susceptibility to activation by factor XIIa and thrombin.

Previous studies suggest that activated platelets facilitate the cleavage of factor XI by both factor XIIa and thrombin. Extracellular phosphorylation is a mechanism by which the function of plasma proteins can be regulated. Phosphorylation is mediated by a casein kinase which is released by activated platelets concomitant with large amounts of ATP and Ca2+. The purpose of this study was to investigate if factor XI is phosphorylated by a platelet casein kinase and whether phosphorylation may affect its activation properties. It was shown that supernatants from platelets which contain platelet casein kinase phosphorylated factor XI. By Western blot analysis it was shown that phosphorylation of factor XI substantially increased its susceptibility to cleavage by factor XIIa, and, to a lesser extent, by thrombin. The generated factor XIa was functionally active in that it cleaved the chromogenic substrate S2366, and in that factor XIa-antithrombin and thrombin-antithrombin complexes were generated when phosphorylated factor XI was added to blood plasma. The present study indicates that platelet-mediated phosphorylation of factor XI enhances the cleavage of factor XI into XIa and that the generated XIa possesses functional activity. Phosphorylation of factor XI might be an essential regulatory mechanism by which platelets mediate amplification of the coagulation cascade.     

A monoclonal antibody raised against human beta-factor XIIa which also recognizes alpha-factor XIIa but not factor XII or complexes of factor XIIa with C1 esterase inhibitor

A monoclonal antibody (mAb 2/215) against human beta-factor XIIa (beta-FXIIa), was shown by equilibrium binding studies to have a high affinity for alpha-factor XIIa (alpha-FXIIa) (Kd 1.8 nM) and beta-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or alpha-FXIIa:C1 esterase inhibitor (C1-INH) complex. Surface plasmon resonance studies showed that the mAb 2/215 bound to immobilized alpha-FXIIa with high affinity (KD 3.93 +/- 1.46 x 10(-11) M). Western blots employing mAb 2/215 indicated that human plasma contained small amounts of alpha-FXIIa but no beta-FXIIa. mAb 2/215 did not inhibit the amidolytic activity of beta-FXIIa and protected beta-FXIIa from inhibition by C1-INH. The recovery by ELISA,employing mAb 2/215 as the capture antibody, of alpha-FXIIa added to plasma was 11.3%, 42% after inhibition of alpha-FXIIa with 3:4dichloroisocoumarin, and 82% when 0.5% Triton-X100 was added to the assay. Gel filtration showed that the majority of plasma alpha-FXIIa existed as a complex (Mr approximately 170,000). This distinctive mAb increases the capacity to study the contact system in health and disease.     

High affinity binding of factor XIIa to an electronegative surface controls the rates of factor XII and prekallikrein activation in vitro

The incubation of normal human plasma in the presence of sulphatide vesicles results in the generation of amidolytic activity due to factor XIIa (FXIIa) and to kallikrein (KRN). The progress of the generation of the enzymes distinguished a high initial rate of enzyme generation, a decline of this rate to maximum amidolytic activity ([FXIIa]m and [KRN]m) and a negative pseudo-first-order rate attributed to enzyme inactivation by plasma C1-inhibitor (C1INH). [FXIIa]m and [KRN]m were determined after the treatment of various dilutions of plasma in the presence of 4, 15, or 40 microM sulphatide vesicles. At all levels of sulphatides, [FXIIa]m and [KRN]m initially increased with the concentration of plasma, to reach a plateau at higher concentration of plasma. The plateau activities of the generated enzymes and the optimal concentration of plasma both increased with the level of sulphatide vesicles. The pseudo-first-order inactivation rate for KRN increased progressively with the concentration of plasma but the respective rate for FXIIa was independent of the plasma concentration. The data suggest that contiguous binding of plasma FXIIa, factor XII (FXII), and the complexes of high molecular weight kininogen (HK) with prekallikrein (HK-PKRN) and factor XI (HK-FXI) to an electronegative surface induces a rapid generation of FXIIa and KRN. The concentration of the electronegative surface controls the levels of generated FXIIa and KRN and their release to the bulk phase. The released FXIIa and KRN are both inactivated by C1INH.     

Inhibition of the cerebroside sulphate (sulphatide)-induced contact activation reactions by platelet factor four

Cerebroside sulphate or sulphatides, present in erythrocyte and leucocyte cell membranes, have recently been shown to be a potent activator of the contact phase of coagulation. Platelet factor-four (PF4), purified from human washed platelets, was found to inhibit the sulphatide induced APTT and plasma prekallikrein activation in a dose dependent manner. A four-fold increase in the sulphatide-APTT was observed at 156pM PF4 in the reaction mixture and 30% inhibition of prekallikrein activation was observed at 1.6nM. PF4 was found to be an approximately 1000-fold more potent inhibitor than protamine sulphate in inhibiting activation of clotting but was not as potent in inhibiting activation of prekallikrein. The physiological form of PF4 as released from platelets, was also found to cause inhibition of plasma prekallikrein activation, with 50% inhibition observed when the concentration of PF4 in plasma was 2.25 g/ml (0.29 M). The ability of PF4 to inhibit the contact activation reactions induced by sulphatides may be of some physiological significance in that when released during platelet activation and vessel wall injury, it may have a protective effect against the development of thrombosis.     

Interaction of trypsin, beta-factor XIIa, and plasma kallikrein with a trypsin inhibitor isolated from barley seeds: a comparison with the corn inhibitor of activated Hageman factor

A trypsin inhibitor was purified from barley seeds by a modification of published procedures. We determined the dissociation constant, Ki, for the complexes of the barley inhibitor with trypsin, beta-Factor XIIa, and plasma kallikrein. We compared these constants for those of the same enzymes with the corn Hageman Factor inhibitor, which is a homolog of the barley inhibitor. The strength of interaction of the barley inhibitor with the three enzymes was: trypsin greater than beta-Factor XIIa greater than plasma kallikrein. In contrast, the corn inhibitor inhibits beta-Factor XIIa most strongly and does not inhibit plasma kallikrein at all. A possible structural basis for the difference in inhibition specificity is discussed.     

Constitutive plasminogen activator inhibitor 1 (PAI-1) biosynthesis in human Hep G2 hepatoma cells is maintained by an autocrine factor.

We studied the production of PAI-1 by human Hep G2 hepatoma cells. When culturing these cells in fresh medium (FM), PAI-1 accumulation rate was not linear: PAI-1 antigen after 24 h was 200 ng/10(6) cells while in subsequent 24 h periods, it was on average 1,000 ng/10(6) cells. Culture of Hep G2 cells in regular changes of a 12 h conditioned medium (CM) obtained from other Hep G2, instead of in FM, resulted in a 2-fold higher PAI-1 accumulation. In cells incubated in FM, PAI-1 mRNA declined rapidly after medium change and returned to basal levels after 24 h. In contrast, PAI-1 mRNA remained relatively stable when CM was used. The acute phase mediator interleukin 6 (IL-6) was not responsible for the autocrine stimulation of PAI-1: neither IL-6 nor antibodies to IL-6 altered the observed variations in PAI-1 expression. Our studies suggest the presence of an unknown PAI-1 stimulating factor.     

Stability of plasminogen activator inhibitor 1 (PAI-1)

The stability of PAI-activity has been studied at different conditions. The inactivation followed first order kinetics. Lowering the temperature and decreasing the pH both, increased the stability of PAI-1 dramatically. Addition of the PAI-1 binding protein, vitronectin, to reactivated PAI-1, about doubled the half-life of PAI-1 at all conditions studied. In the presence of chloramine T, the inactivation of reactivated PAI-1 was very rapid. In this case the protective effect of purified vitronectin, human plasma or fetal calf serum, but not of bovine serum albumin, was pronounced. The stability of the spontaneously active high Mr form of PAI-1 (partially purified or in plasma), constituting a complex between PAI-1 and vitronectin, was quite similar to reactivated PAI-1 in the presence of vitronectin. Addition of pure vitronectin, human plasma or fetal calf serum to such material had no further stabilizing effect. Reactivated PAI-1, which was inactivated by incubation at physiological conditions could again be fully reactivated, in contrast to chloramine T-oxidized PAI-1, which was irreversibly inactivated.     

Determination of main inhibitor of activated factor XI

To study the contribution of alpha 1 antitrypsin (alpha 1AT) and antithrombin III (AT III) to the inactivation of F.XIa in plasma, we developed the assay system of F.XIa-AT III complex according to our method of F.XIa-alpha 1AT complex, and measured the levels of both complexes in the patients with disseminated intravascular coagulation (DIC) derived from various triggers. F.XIa-alpha 1AT complex level was always higher than F.XIa-AT III complex level in each patient, independently on the trigger of DIC, the administration of heparin and the levels of F.XI, alpha 1AT and AT III. These results indicate that alpha 1AT is the main inhibitor of F.XIa in plasma.     

Evidence for the participation of both activated factor XII and activated factor IX in cold-promoted activation of factor VII

The activation of ¹²⁵I-Factor XII and of ¹²⁵I-Factor IX in plasma during the cold-promoted activation of Factor VII was studied. Limited proteolysis of Factors XII and IX, a measure of activation, was determined from radioactivity profiles of reduced polyacrylamide gels following electrophoresis in the presence of sodium dodecyl sulfate. Significantly greater formation of Factor IX_a and Factor XII_a was found in citrated plasma incubated at 4° from four subjects whose Factor VII activity increased compared to plasma from five subjects whose Factor VII activity did not increase. Addition of an anti-Factor IX antiserum to citrated plasma inhibited 50% of the observed cold-promoted activation of Factor VII. The data are consistent with the hypothesis that both Factor XII_a and Factor IX_a are direct activators of Factor VII during cold-promoted activation of Factor VII in citrated plasma.     

Characterization of an inhibitor of neuronal plasminogen activator released by heart cells

A basic understanding of growth cone dynamics and developmental events involving growth cones requires an understanding of the function and regulation of molecules associated with and released by growth cones. Rat sympathetic neurons in culture release a urokinase-like plasminogen activator from their distal processes and/or growth cones (Pittman, 1985a). When sympathetic neurons are grown in cocultures with heart cells, however, plasminogen activator activity is not detected. The absence of plasminogen activator activity in cocultures of sympathetic neurons and heart cells appears to be due to the release of an inhibitor of plasminogen activator by heart cells. This inhibitor has a molecular weight of approximately 50 kDa in the presence of SDS and apparent molecular weights of approximately 50 and greater than 2000 kDa under native conditions. A significant fraction of the large-molecular-weight form of the inhibitor is converted to the smaller form following treatment with heparinase. Extremely stable complexes of 68 and 80 kDa are formed between the heart inhibitor and the plasminogen activator, urokinase, such that the complexes withstand boiling in SDS/mercaptoethanol. The data are consistent with the formation of an 80 kDa urokinase-inhibitor complex in the presence of heparan sulfate proteoglycan and a 68 kDa complex in the absence of heparan sulfate proteoglycan. A highly purified preparation of the heart inhibitor produces a 2- to 3-fold increase in neurite outgrowth from sympathetic neurons. These data indicate that the activity of the plasminogen activator released by sympathetic neurons can be regulated by a normal target tissue and that this regulation may result in increased neurite outgrowth from the neurons.     

Contact activation and factor VII after the use of an activated prothrombin complex concentrate (FEIBAR) in hemophiliacs with inhibitors

Administration of a brand of Activated Prothrombin Complex Concentrate (FEIBA) to Hemophiliacs with high-titre inhibitor to Factor VIII, induced a shortening of the Prothrombin Time and Thrombotest clotting time. This effect stems partly from the presence in the concentrate of high amounts of activated factor VII (α-VII_a) which indeed after the infusion, were detected in the plasma of Hemophiliacs. The attained levels of factor α-VII_a were shown to be proportional to the dose of concentrate administered. This form of activated factor VII was not generated as a result of activation of the contact activation mechanism, since after the infusion of the concentrate no changes were detected in the components of this system. The infused factor VII was indeed present in an activated form and this was further substantiated by the infusion of the concentrate in factor VII deficient patients. These observations suggest that factor α-VII_a is not generated $\text{in vivo}$ and thus is of exogenous origin. The clinical effect may well be due to the presence of this form of activated factor VII; in this context the alternative pathway involving factors IX and X should be implicated.     

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.     

Effect of fibrin-like stimulators on the activation of plasminogen by tissue-type plasminogen activator (t-PA)--studies with active site mutagenized plasminogen and plasmin resistant t-PA

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenized to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA-Glu275, a recombinant single chain t-PA in which the Arg of the plasmin sensitive Arg275-Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s-1) per 1 nM enzyme. In the absence of fibrin stimulation, the v0 for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s-1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s-1). In the presence of 1 microM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and 33 pM s-1 respectively), whereas with 1 microM CNBr-digested fibrinogen, the v0 for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s-1 respectively). In contrast, the v0 for nPlg and rPlg-Ala740 by single chain rt-PA-Glu275, two-chain rt-PA-Glu275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular activation of factor IX (christmas factor)

We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other.