Inhibition of thrombin generation in plasma by inhibitors of factor Xa

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.     

Novel anticoagulants based on direct inhibition of thrombin and factor Xa

Decoquinate is an anticoccidial agent that inhibits respiration in the parasites mitochondrion. We examined human foreskin fibroblast cell cultures infected with the normally tissue cyst-less RH strain of Toxoplasma gondii and treated with decoquinate for evidence of tissue cyst induction and formation. Transmission electron microscopy observations demonstrated tissue cysts in decoquinate-treated cultures on days 3, 4, 5, and 6 after inoculation. Tissue cysts contained a tissue cyst wall that enclosed stages that resembled tachyzoites and stages that were structurally bradyzoites. Similar treatment of human foreskin fibroblast cells infected with tachyzoites of the TS-4 temperature-sensitive mutant of the RH strain did not result in production of tissue cysts.     

Regulation of thrombin formation by activated protein C: effect of the factor V Leiden mutation

Recent developments in arterial hemodynamics have indicated that the human arterial pressure waveform contains more information than is available from conventional sphygmomanometry. This information includes indices describing left ventricular systolic function and arterial properties. A cheap and reliable system was designed and implemented using readily available hardware for recording, analysis, and storage of arterial pressure waveforms. The system embodies an online technique for synthesizing ascending aortic pressure waveform from recordings made at different peripheral sites of the human arterial system. Eighteen indices are then derived from arterial pressure waveforms. This system can be used in an outpatient clinic for assisting in current pharmacological management of cardiovascular disease. It can also be extended to the critical care area, where the extra information provided aid in assessing the patient's condition.     

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.     

Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity

It is thought that only a limited number of residues in the extended binding pocket of coagulation proteases are critical for substrate and inhibitor specificity. A candidate residue from the crystal structures of thrombin and factor Xa (FXa) that may be critical for specificity at the S2 subsite is residue 99. Residue 99 is Tyr in FXa and Thr in activated protein C (APC). To determine the role of residue 99 in S2 specificity, a Gla-domainless mutant of protein C (GDPC) was prepared in which Thr99 was replaced with Tyr of FXa. GDPC T99Y bound Ca2+ and was activated by the thrombin-thrombomodulin complex normally. The T99Y mutant, similar to FXa, hydrolyzed the chromogenic substrates with a Gly at the P2 positions. This mutant was also inhibited by antithrombin (AT) (k2 = 4.2 +/- 0.2 x 10(1) M-1 s-1), and heparin accelerated the reaction >350-fold (k2 = 1.5 +/- 0.1 x 10(4) M-1 s-1). The T99Y mutant, however, did not activate prothrombin but inactivated factor Va approximately 2-fold better than wild type. To try to switch the specificity of FXa, both Tyr99 and Gln192 of FXa were replaced with those of APC in the Gla-domainless factor X (GDFX Y99T/Q192E). This mutant was folded correctly as it bound Ca2+ with a similar affinity as GDFX and was also activated by the Russell's viper venom at similar rate, but it cleaved the chromogenic substrates with a Gly at the P2 positions poorly. The mutant, instead, cleaved the APC-specific chromogenic substrates efficiently. The Y99T/Q192E mutant became resistant to inhibition by AT in the absence of heparin but was inhibited by AT almost normally in the presence of heparin (k2 = 3.4 +/- 0.5 x 10(5) M-1 s-1). The Y99T/Q192E mutant did not inactivate factor Va, and prothrombin activation by this mutant was impaired. These results indicate that 1) residue 99 is critical for enzyme specificity at the S2 subsite, 2) a role for heparin in acceleration of FXa inhibition by AT may involve the S2-P2 modulation, and 3) the exchange of residues 99 and 192 in FXa and APC may switch the enzyme specificity with the chromogenic substrates and inhibitors but not with the natural substrates.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Effects of tannins from Geum japonicum on the catalytic activity of thrombin and factor Xa of blood coagulation cascade

Bioassay-guided fractionation of the MeOH extract of the whole plant of Geum japonicum led to the isolation of seven known tannins. They were identified by spectroscopic methods as penta-O-galloyl-beta-glucoside (1), pedunculagin (2), 2, 3-(S)-hexahydroxydiphenoyl-D-glucose (3), tellimagrandin II (4), 2, 6-di-O-galloyl-D-glucose (5), casuariin (6), and 5-desgalloylstachyurin (7). Compounds 1, 2, 4, 6, and 7 showed potent anticoagulant activity by significantly prolonging the clotting of rabbit plasma. The inhibitory effect of 2 was competitively directed against thrombin. Its IC50 values for inhibition of the enzymatic activity of thrombin on synthetic substrate and fibrinogen were 0.18 and 0.15 microM, respectively. On the other hand, compounds 1, 4, 6, and 7 are mixed noncompetitive inhibitors of thrombin. Their IC50 values for inhibition of fibrinogen hydrolysis were twofold to sevenfold lower than those for the inhibition of synthetic substrate hydrolysis. Factor Xa was competitively inhibited by compounds 1, 2, 4, 6, and 7. The phenolic hydroxyl groups of the active tannins appear to play an important role in their inhibitory effect on the enzymes.     

Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.     

Rapid activation of the complement system by cuprophane depends on complement component C4

Hemodialysis with cuprophane dialyzer membranes promotes rapid activation of the complement system, which is thought to be mediated by the alternative pathway. Complete hereditary deficiency of complement C4, a classical pathway component, in two hemodialysis patients provided the opportunity to investigate a possible role of the classical pathway. In two hemodialysis patients with both C4 isotypes, C4A and C4B, and in one patient with C4B deficiency complement activation occurred immediately after the onset of hemodialysis, with peak levels of C3a and terminal complement complex (TCC) after ten to fifteen minutes. In patients with complete C4 deficiency, C3a and TCC remained unchanged for fifteen minutes and increased thereafter, reaching the highest level after thirty minutes. The leukocyte nadir was also delayed from fifteen to thirty minutes. In vitro incubation of normal, C4A- or C4B-deficient serum with cuprophane caused complement activation after fifteen minutes. In contrast, no activation was observed in sera of four C4-deficient patients. The addition of normal serum or purified human C4 restored the capacity for rapid complement activation. In one patient with severe immunoglobulin deficiency, C3a and TCC levels increased only moderately after 25 minutes of cuprophane dialysis. This patient's serum also exhibited delayed complement activation in vitro, which was normalized after pretreatment of cuprophane with immunoglobulins. Preincubation of normal serum with MgEGTA, a blocker of the classical pathway, inhibited rapid complement activation through cuprophane. As basal levels of C4a are markedly increased in hemodialysis patients (3450 +/- 850 ng/ml) compared to healthy controls (224 +/- 81 ng/ml), no further elevation of C4a was detectable during cuprophane hemodialysis. Incubation of normal serum with cuprophane, however, caused a slight increase in C4a after five minutes. These results indicate that the initial deposition of complement C3b on the cuprophane membrane, necessary for activation of the amplification loop of the alternative pathway, is mediated by the classical pathway C3-convertase C4b2a. We propose an extended concept of complement activation through cuprophane, which is based on four steps: (a) binding of anti-polysaccharide antibodies, (b) classical pathway activation, (c) alternative pathway activation and (d) terminal pathway activation.     

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro, and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage. DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-alpha, IL-1 beta, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.     

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.     

Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.     

Protein kinase C regulates macrophage tumor necrosis factor secretion: direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance

BACKGROUND: Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. METHODS: Murine macrophages were cultured +/- LPSp for 24 hours. Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator. Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay. RESULTS: LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA. PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells. Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages. PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells. CONCLUSIONS: Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages. Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.     

Contributions to maxima in protein kinase C activation

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.     

The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo

In response to fibroblast growth factor (FGF), FGF receptor-1 (FGFR-1) (flg) becomes tyrosine phosphorylated and associates with phospholipase C gamma (PLC gamma) and a 90 kDa protein. We report here that in cells transformed by v-Src, FGFR-1 becomes phosphorylated on tyrosine; however, neither PLC gamma nor p90 was found to be associated with tyrosine-phosphorylated FGFR-1. Instead, there was a strong constitutive association of FGFR-1 with the adaptor proteins Shc and Grb2 and the Ras guanine nucleotide exchange factor Sos. Association with Shc and Grb2 and Sos was not observed in response to FGF. Suramin did not prevent either tyrosine phosphorylation or Shc/Grb2/Sos association, indicating a non-autocrine mechanism. Thus, in cells transformed by v-Src, tyrosine phosphorylation of FGFR-1 results not in the expected association with PLC gamma and p90, but rather in the recruitment of the Ras activating Shc/Grb2/Sos complex. These data suggest a mechanism for Ras activation by v-Src involving phosphorylation of novel tyrosine(s) on FGFR-1.     

Dexamethasone abolishes the activation by nerve growth factor of protein kinase N: effects of nerve growth factor and dexamethasone on protein kinase N

Post-operative therapeutic rehabilitation in ligamentous-capsular injuries has a great importance and for the final result it is as the very operation. We begin it from making the patient realize that the good final result can be obtained only with patient, persistence and discipline. Early therapeutic rehabilitation after surgical treatment of ligamentous-capsular injuries is possible only when the ligament was reconstructed in a motor stable way. Painless, dosed, passive motion exercises with a limited range of movements did on a mechanical splint TELOS have a great importance for the final results.