Role of Gly197 in the structure and function of protein C.

We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved ~5-20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling.

Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.

Identification of novel small molecule inhibitors of activated protein C.

INTRODUCTION: Activated protein C (APC) is the central enzyme of the anticoagulant protein C pathway. Low concentrations of APC circulate in plasma and are believed to contribute to the maintenance of a normal haemostatic balance. MATERIALS AND METHODS: We have used a structure-based virtual screening approach to discover small drug-like molecules that inhibit the interaction between APC and its substrate FVa through inhibition of a predominant APC exosite, known to be involved in FVa substrate binding. We have combined in silico selection with functional screening and direct binding analysis to identify novel molecules and to ascertain and characterize the inhibition of the interaction between APC and FVa. RESULTS: We have identified a number of novel molecules that bind to APC and protein C with Kd values in the range of 10(-3)- 10(-5)M. Inhibition by these molecules is incomplete, which most likely reflects the extended surface that is involved in the interaction between APC and its substrates. Direct binding of hit molecules to variant APC molecules that were mutated in the targeted binding site revealed that several of the molecules presented a 100-500 fold lower affinity for the variant molecule, suggesting that these molecules indeed bind the exosite of APC. CONCLUSIONS: The protein-protein interaction inhibitors discovered here, could function as starting molecules for further development of small molecules with anti-APC properties. Such molecules may be of clinical interest, in particular in individuals where thrombin formation is compromised and the haemostatic balance is tipped towards bleeding tendencies, such as in haemophilia A.

Effect of Crocus sativus L. (saffron) on coagulation and anticoagulation systems in healthy volunteers.

Saffron showed some effects on blood coagulation and platelet aggregation in in vitro and in vivo studies. In a clinical trial with a limited number volunteers, saffron tablets influenced on bleeding time. In this study, the effect of saffron on plasma level of fibrinogen, factor VII (as coagulant agent), C and S protein (as anti-coagulant agent), PT and PTT in a larger sample size was evaluated. The study was a double-blind, placebo-controlled study consisting of 1 week treatment with 200 mg and 400 mg saffron tablets. Sixty healthy volunteers (age range 20-50 years) were selected for the study. The volunteers were divided into three groups of 20 each. Group 1 received placebo; Groups 2 and 3 received 200 mg and 400 mg saffron tablets, respectively, for 7 days (1 tablet per day). Before and after 7 days treatment and also 1 month after that, blood samples were taken. The plasma levels of fibrinogen, factor VII, C and S protein, PT and PTT were evaluated. Statistical analysis showed no difference between groups for any of evaluated factors. This study rejected any effect of saffron with dose of 200 and 400 mg for 1 week on coagulant and anticoagulant system.

Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry.

Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.

PAR1 cleavage and signaling in response to activated protein C and thrombin.

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Proteolysis of protein C in pooled normal plasma and purified protein C by activated protein C (APC).

Protein C is a vitamin-K dependent zymogen of the anti-coagulant serine protease activated protein C (APC). In this paper, we report four lines of evidence that APC can activate protein C in pooled normal plasma, and purified protein C. First, the addition of APC to protein C-deficient plasma supplemented with protein C produces a prolongation of the clotting time of plasma that is proportional to the amount of protein C. This behavior was observed with APC from the Chromogenix APC resistance kit (Dia Pharm, Franklin, OH, USA) and from APC derived from the thrombin activation of human protein C (Enzyme Research Laboratories, South Bend, IN, USA). Secondly, using immunoblotting after gel electrophoresis, the disappearance of epitopes for monoclonal antibodies that recognize protein C but not APC indicates a time course for the activation by APC of protein C in pooled normal plasma and protein C purified from plasma. Thirdly, the same time course for the disappearance of protein C specific epitope can be followed using ELISA. Finally, protein C can be activated by APC as indicated by the increase in APC specific synthetic substrate Tryp-Arg-Arg-p nitroaniline hydrolysis. Kinetic data indicate a value of 4.7+/-0.4 mM(-1) s(-1) for the activation of protein C by APC under physiological conditions and in the presence of calcium. These observations document that APC must function not only in the inactivation of activated factors V and VIII, but also in the activation of protein C. This additional action of APC may be important to consider more broadly because of APC in the treatment of sepsis.

The hydrophobic nature of residue-5 of human protein C is a major determinant of its functional interactions with acidic phospholipid vesicles.

We have previously proposed that a cluster of surface-exposed hydrophobic amino acids, viz., F4, L5, and L8, present at the amino-terminus of the Ca(2+)-bound form of gamma-carboxyglutamic acid domain (GD) of human protein C (PC), contributes a substantial portion of the total functional binding energy of PC and its activated form, APC, to acidic phospholipid (PL) vesicles. A deeper understanding of the importance of the hydrophobic nature of sequence position 5, and the particular relevance of leucine at that location, was sought by examination of the properties of a series of mutant proteins containing A5, V5, I5, and W5 as replacements for L5 in recombinant (r)-PC and APC. The Ca(2+)- and PL-dependent plasma-based anticoagulant activities of [L5A]r-APC, [L5V]r-APC, [L5I]r-APC, and [L5W]r-APC were determined to be approximately 28%, 51%, 98%, and 105%, respectively, of that of wild-type r-APC. A similar trend in activities of the mutant enzymes was observed in in vitro factor V/Va and factor VIII/VIIIa inactivation assays. Apparently normal Ca(2+)-dependent conformations were adopted by each of the mutant proteins, but the Ca(2+)-bound form of [L5A]r-PC was relatively the most defective of the mutants in its binding to PL. These results confirm the importance of the hydrophobic character at sequence position 5 as critical to the functional binding of PC to PL.

Hydrophobic amino acid residues of human anticoagulation protein C that contribute to its functional binding to phospholipid Vesicles.

The contributions to functional phospholipid (PL) binding of the cluster of amino acid side chains of human protein C (PC) comprising F4, L5, and L8 have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain replaced the wild-type hydrophobic groups at these positions. The PL-dependent plasma-based anticoagulant activities of [F4Q]-r-APC and [L8Q]r-APC were severely reduced to 5% and < 2%, respectively, of wild-type r-APC. Activity losses of the mutants toward inactivation of coagulation factor VIII, measured in the complete in vitro tenase system, have also been observed. As evidenced through Ca(2+)-induced intrinsic fluorescence changes, both [F4Q]r-PC and [L8Q]r-PC were able to adopt Ca(2+)-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca(2+)-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L8Q]r-PC showed greatly defective macroscopic binding properties to PL vesicles, as did to a lesser extent [F4Q]r-PC. These findings were similar to those reported previously for [L5Q]r-PC/APC [Zhang, L., & Castellino, F. J. (1994) J. Biol. Chem. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutants are related to their suboptimal binding to PL or to their misorientation on the PL surface leading to poor alignment of the active sites of the r-APC mutants with the complementary cleavage sites on fVIII/fVIIIa and fV/fVa.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein C deficiency following hematopoietic stem cell transplantation: optimization of intravenous vitamin K dose.

Patients undergoing hematopoietic stem cell transplantation (HSCT) are dependent on i.v. vitamin K supplementation to prevent deficiency. Vitamin K deficiency may contribute to the development of a hypercoagulable state by limiting hepatic synthesis of fully functional carboxylated anticoagulant protein C (PC). The ratio of PC antigen (CAg) to PC measured in a clot-based functional assay (CFx) reflects the degree to which PC is carboxylated. The 133 patients undergoing HSCT received vitamin K 10 mg per week (low dose, 101 patients) or 5 mg per day (high dose, 32 patients) i.v. as their sole exogenous source of vitamin K. CAg and CFx were assayed before HSCT preparative regimen and again 14 days later. CAg and CFx fell significantly in both groups from day 0 to day 14 but there were no differences between the low-dose and high-dose vitamin K groups. For both groups, CAg correlated strongly with CFx at day 14 (p = 0.0001). At day 14, the CAg/CFx ratio for the low-dose group was significantly greater than for the high-dose group (1.26 +/- 0.4 vs 1.09 +/- 0.1, p < 0.0002), suggesting that low-dose patients had a higher proportion of incompletely carboxylated PC. The CAg/CFx ratio at day 14 correlated with serum albumin for the high-dose group (p = 0.05), but not the low-dose group (p = 0.09), suggesting that the change in ratio in the low-dose group was not simply due to a lack of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)