Bovine Activated Protein C Research Articles

Effects of activated protein C on coagulation and fibrinolysis in rabbits with endotoxin induced acute lung injury

Sepsis induced acute lung injury (ALI) as a common syndrome in clinical practice has a high mortality. Recombinant human activated protein C (APC) can significantly reduce the mortality of patients with severe sepsis. Several studies have implicated that APC may be protective in ALI. Twenty-one rabbits were operatively prepared and randomly divided into sham, control, or APC groups (n = 7 in each group). After a tracheotomy had been performed, ALI was produced in the control and APC groups by infusion of Escherichia coli endotoxin 100 microg/kg per hour intravenously for 1 hour. The sham group received only the vehicle, infusion of 20 ml of 0.9% saline. The rabbits were studied under anesthesia for 6 hours and were ventilated with 40% oxygen. Bovine APC (25 microg x kg(-1) x h(-1)) was intravenously administered. The infusion was initiated half an hour post-injury and lasted for 4 hours. The animals were resuscitated with Ringer's lactate solution. In comparison with nontreatment in the control group, the infusion of APC significantly reduced the increase of thrombomodulin level (TM; control group was (0.68 +/- 0.06) ng/ml, vs APC group of (0.62 +/- 0.07) ng/ml at 6 hours, P < 0.05), and significantly attenuated the fall in protein S (PS; control group was (2.32 +/- 0.03) microg/ml at 2 hours, (2.24 +/- 0.06) microg/ml at 4 hours and (2.21 +/- 0.09) microg/ml at 6 hours, vs APC group (2.46 +/- 0.04) microg/ml at 2 hours, (2.40 +/- 0.05) microg/ml at 4 hours and (2.39 +/- 0.07) microg/ml at 6 hours, P < 0.01). In addition, APC limited the increase in plasminogen activator inhibitor-1 (PAI-1) both in plasma (control group was (0.68 +/- 0.12) ng/ml at 1 hour, (0.84 +/- 0.06) ng/ml at 2 hours, (0.87 +/- 0.08) ng/ml at 4 hours and (0.91 +/- 0.05) ng/ml at 6 hours, vs APC group (0.42 +/- 0.16) ng/ml at 1 hour, (0.43 +/- 0.04) ng/ml at 2 hours, (0.45 +/- 0.09) ng/ml at 4 hours and (0.45 +/- 0.14) ng/ml at 6 hours, P < 0.01) and in bronchoalveolar lavage fluid (at 6 hours: sham, (1.05 +/- 0.05) ng/ml; control, (1.13 +/- 0.06) ng/ml; APC, (1.06 +/- 0.06) ng/ml; P < 0.05). However, APC failed to prevent the decrease in PaO(2)/FiO(2) ratio. APC-treated rabbits showed no significant difference in platelet count and antithrombin but exhibited less D-dimer production than did the controls. Moreover, APC limited the histopathological score of lung injury (2.6 +/- 0.8 in control, vs 1.4 +/- 0.6 in APC group, P < 0.01). Anti-coagulation and pro-fibrinolysis activity may be two of the possible mechanisms by which activated protein C attenuated endotoxin-induced ALI.

Amino Acid Residues in Thrombin-sensitive Region and First Epidermal Growth Factor Domain of Vitamin K-dependent Protein S Determining Specificity of the Activated Protein C Cofactor Function

Human protein S (PS) potentiates the anticoagulant activity of human but not bovine activated protein C (APC), whereas bovine PS is a cofactor to APC from both species. The structural requirements for the specificity of the APC cofactor function of human PS are located in its thrombin-sensitive region (TSR) and the first epidermal growth factor (EGF1)-like module. To elucidate which residues in these two modules determine the specificity of the APC cofactor activity, 41 human PS mutants were expressed. All mutants were cofactors to human APC and some also to bovine APC. Residues in TSR (positions 49 and 52) and EGF1 (residues 97 and 106) together determined the specificity of the APC cofactor function, whereas substitution of individual residues did not change specificity. Bovine PS, and mutants expressing cofactor activity to bovine APC, stimulated phospholipid binding of bovine APC. In contrast, human PS and mutants lacking cofactor activity to bovine APC failed to support binding of bovine APC to phospholipids. These data indicate that residues in TSR and EGF1 cause the specificity of the APC cofactor activity and support the concept that key residues in these two modules interact with APC on the phospholipid surface.

Synergistic Cofactor Function of Factor V and Protein S to Activated Protein C in the Inactivation of the Factor Villa – Factor IXa Complex

Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.

Antithrombotic Effects of Activated Protein C and Protein S in a Rabbit Model of Microarterial Thrombosis

The antithrombotic properties of activated protein C (APC) and protein S were investigated in a rabbit model of microarterial thrombosis. The study focused on the ability of intact and thrombin-cleaved bovine protein S to potentiate the biological effects of bovine APC in vivo. Segments of the central arteries of the ears were subjected to arteriotomy, deep-vessel wall trauma, and arteriotomy suture. Five minutes before vascular reperfusion, groups of rabbits were infused with boluses of 0.1 mg/kg bovine APC alone or combined with different doses of intact (0.5, 0.1, or 0.05 mg/kg) or thrombin-cleaved (0.5 mg/kg) protein S. APC in combination with the two higher doses of protein S produced a potent antithrombotic response, as judged by assessment of vessel patency rates, while only the group receiving APC+0.5 mg/kg protein S showed significant reduction of thrombus weights as well. The biological effect depended on the active site in APC, as the antithrombotic effect was lost on pretreatment of APC with the serine protease inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone. The potentiation of the APC response by protein S depended on the structural integrity of the protein, and cleavage of the thrombin-sensitive region in protein S by thrombin resulted in a loss of biological response. No hemorrhagic side effects were noted by the APC-protein S combination, and the anticoagulant response was mild, even to the highest doses of APC and protein S.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein S as an in vivo cofactor to activated protein C in prevention of microarterial thrombosis in rabbits.

The antithrombotic effects of bovine activated protein C (APC) and protein S were investigated in a rabbit model of microarterial thrombosis. Because of the species specificity of the APC-protein S interaction, bovine APC expresses potent anticoagulant activity in rabbit plasma only when bovine protein S is also present. This provided a way to assess the contribution of bovine protein S to the antithrombotic effect of bovine APC. Rabbits were infused with boluses of activated protein C (0.1, 0.2, 0.4, or 0.8 mg/kg), protein S (0.5 mg/kg), or activated protein C (0.1 or 0.01 mg/kg) plus protein S (0.5 mg/kg). APC alone produced a dose-dependent antithrombotic effect, but only the group receiving the highest dose differed significantly from controls. While a low dose of activated protein C (0.1 mg/kg) alone had no antithrombotic effect, together with protein S (0.5 mg/kg) it produced a potent response. The presented results demonstrate the in vivo significance of protein S as a cofactor to activated protein C. The data show that a potent antithrombotic effect, without hemorrhagic side effects or significant systemic anticoagulation, may be achieved by low doses of activated protein C when combined with protein S.

Fluorogenic Substrates for Activated Protein C: Substrate Structure-Efficiency Correlation

A series of 87 fluorescent peptide substrates have been synthesized and evaluated using human and bovine activated protein C (APC). These substrates contain various isomers of aminonaphthalenesulfonamides (ANSN) as the detecting group and were substituted at P-4, P-3, P-2, P′1, and P′2 positions. Substrates with 6,2-ANSN at P′1 had the highest fluorescence quantum yield, exceeding that of 6,1-ANSN 9.3-fold and 5,1-ANSN almost 1000-fold. Almost 50 substrates containing substituted ANSNs as leaving groups have KM values for APC below 100 μM, reaching as low as 4-10 μM for 8 of these substrates. These values are significantly lower than those reported for p-nitroanilide and 4-methylcoumaryl-7-amide substrates. Additionally, some of these substrates have relatively high kcat exceeding 50 s−1. These constants as well as kcat/KM are influenced by the nature of amino acid in the P3 and P2 positions, by the isomer of ANSN (P′1), and by the structure of substituent incorporated in the sulfonamide moiety (P′2). The highest kcat/KM were found for substrates with D-isomers of Leu, Phe, and Val in the P3 position when these amino acids were N-unblocked. For the P2 position Val, Phe, and Leu were preferable. Substrates containing n-butyl (bovine APC) and benzyl (human APC) substituents in the (P′2) structure have elevated kcat/KM. ANSN-containing substrates are hydrolyzed by both human and bovine APC at a similar rate.

Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity.

Vitamin-K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The APC-cofactor function of protein S is species specific, as human protein S potentiates the anticoagulant activity of human but not that of bovine APC, whereas bovine protein S is a cofactor to APC from both species. To elucidate which modules in protein S determine the species specificity, in vitro mutagenesis was used to construct six recombinant chimeric molecules between human and bovine protein S. Wild-type human and bovine protein S and the chimeras were expressed in 293 cells and the recombinant proteins purified by monoclonal antibody affinity chromatography. The recombinant proteins were found to be post-translationally modified, they bound C4b-binding protein and were functionally active as cofactors to APC. Chimeras having both the thrombin-sensitive region (TSR) and the first epidermal-growth-factor-(EGF)-like module of bovine origin expressed APC-cofactor activity similar to that of bovine protein S. Those chimeras, in which TSR or EGF1 derived from different species, manifested APC-cofactor activity similar to that of human protein S, i.e. they did not express cofactor activity to bovine APC. These data indicate that sequence differences in the TSR and EGF1 of human and bovine protein S cause the species specificity of the APC-cofactor activity. The data support the concept that these two modules of protein S interact with APC on the surface of negatively charged phospholipids.

Resistance to inhibition by alpha-1-anti-trypsin and species specificity of a chimeric human/bovine protein C.

Human activated protein C (APC) has been shown to be physiologically susceptible to inhibition by the abundant serpin inhibitor alpha-1-anti-trypsin (AAT). Studies on the inactivation by AAT [Heeb, M. J., & Griffin, J. H. (1988) J. Biol. Chem. 263, 11613-11616] have shown that the calculated rate of this inactivation matches that of the observed half-life of APC in vivo [Wydro, R., Oppenheimer, C., Rodger, R., & Miemi, S. (1988) Clin. Res. 36, 329A] and complex formation therefore probably represents a physiologic regulation process for APC. In this study we observed that bovine APC, in contrast to human APC, is nearly completely resistant to inactivation by human AAT. An additional difference between human and bovine APC is that human APC is a potent anticoagulant in human plasma, whereas bovine APC is only minimally active in human plasma. These functional differences exist despite considerable structural similarity between the human and bovine molecules. In order to identify specific molecular regions responsible for function, a chimeric molecule consisting of the light chain of human protein C (PC) and the heavy chain of bovine PC was constructed, expressed, and characterized. The activated chimeric PC is similar to human APC in having potent anticoagulant activity in human plasma, but displays nearly identical resistance to AAT inhibition with the bovine molecule. The similarity between the chimeric and bovine molecules in resistance to AAT inhibition indicates that the structural determinants for inhibitor interactions reside within the heavy chain (serine protease) domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin‐K‐dependent protein S

Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.

Neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C is species-dependent

The interaction of bovine and human activated protein C (APC) with type-1 plasminogen activator inhibitor (PAI-1) was studied in a cell-free system. Human plasma and a preparation of PAI-1 obtained from human endothelial cell cultures were used as sources of PAI-1. Bovine APC was able to neutralize PAI-1 inhibitory activity present in both sources in a dose-dependent manner; the concentration of bovine APC required to produce 50% (C 50) neutralization of endothelial PAI-1 (0.5nM) was 4 μg/ml (64nM). Moreover, when complexes between tissue plasminogen activator (t-PA, 60ng/ml, 0.84nM) and PAI-1(18.8ng/ml, 0.35nM) were incubated with bovine APC, the amount of such complexes decreased as a function of the concentration of added APC (C 50 = 2 μ/ml, 32 nM), and a concomitant increase in the amount of residual t-PA activity was observed. This effect was due to the formation of APC⊎PAI-1 complexes as detected by immunoblotting with monoclonal antibodies directed against PAI-1. By this mechanism bovine APC prevented the initial reaction between PAI-1 and t-PA and interfered with the stability of complexes between t-PA and PAI-1. The latter observation suggests that complexes between t-PA and PAI-1 may dissociate in the presence of bovine APC. In contrast with these findings, when the experiments were performed in an entirely human homologous cell-free system, human APC did not form a complex with PAI-1 and no effect either on PAI-1 or on the stability of preformed t-PA ⊎ PAI-1 complexes was observed. The results indicate that the neutralization of PAI-1 by APC is a phenomenon induced by interspecies molecular interactions.

Species specificity of the fibrinolytic effects of activated protein C

Activated protein C (APC) has been shown to stimulate fibrinolysis in both in vitro and in vivo experimental systems. In order to test the importance of protein S in the fibrinolytic activity of APC we have compared the activity of APC, prepared from rabbit, bovine and human plasma, in the stimulation of whole blood clot lysis, the inactivation of plasminogen activator inhibitors and anticoagulant activity. When whole blood clot lysis was performed using tissue plasminogen activator in either human or rabbit blood, APC was found to enhance clot lysis in a species specific manner that paralleled the pattern observed for its anticoagulant activity. Bovine APC, was the poorest stimulator of fibrinolysis in human plasma. However, if bovine protein S was also added to human plasma, bovine APC was as effective in promoting fibrinolysis as human APC. In contrast, no species specific effects on the inactivation of plasminogen activator inhibitor activity was observed. Though substantial effects of APC on plasminogen activator inhibitor levels were made by rabbit, human and bovine activated protein C in human plasma, there was no effect of activated protein C on the rate of clot lysis of human plasma. These results suggest that protein S is important for the expression of the fibrinolytic activity of activated protein C and that the effect of protein S may be useful for the differentiation of fibrinolytic effects of activated protein C that may be related to the inactivation of plasminogen activator inhibitors and those that are not.

Purification and characterization of plasma protein C inhibitor

Plasma protein C inhibitor (PCI) was purified to homogeneity (>95%) with good recovery (>25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr-57,000 into two products with Mr-54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr=54,000. PCI did not detectably inhibit α-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 × 10 4, 0.22 × 10 4, 0.08 × 10 4, 0.61 × 10 4, 2.01 × 10 4, 6.50 × 10 4 and 9.03 × 10 4 M −1s −1 in the absence of heparin and 1.58 × 10 6, 0.43 × 10 6, 0.03 × 10 6, 0.52 × 10 6, 0.09 × 10 6, 0.18 × 10 6 and 0.74 × 10 6 M −1s −1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 × 10 4 and 0.26 × 10 4 M −1s −1 in the absence of heparin and 0.54 × 10 6 and 0.71 × 10 6 M −1s −1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PCI and is not essential for the heparin stimulation of this reaction.