L-ARGININE AND L-GLUTAMIC ACID INCREASE THE CONTENT OF PROTEIN C IN THE EARLY STAGES OF ISOLATION FROM DONOR PLASMA

Current large-scale production of blood-derived pharmacological preparations is aimed at expanding the list of products and deeper extraction of target proteins especially at the pre-purification stage. In particular, this problem becomes critical for the isolation of proteins like protein C (PC), which is present in plasma in trace amounts. Aim. We aimed to improve the buffer composition to minimize the interaction of PC with other proteins and lipids that are inevitably present in the stock material. Methods. The content of protein C in plasma and its derivatives was assessed by the amidolytic activity to the chromogenic substrate S2366. A decrease in homologous impurities and plasma enrichment with protein C was provided by selective bulk adsorption on DEAE-cellulose. Results. Here we describe that an equimolar mixture of two amino acids (L-arginine and L-glutamic acid) essentially increased the content of protein C at the stage of cryo-depleted plasma pre-purification, including initial dilution and subsequent enrichment of plasma with protein C due to selective bulk adsorption on DEAE- cellulose. Additionally, it was revealed that solutions of these amino acids, when combined, inhibit the induced amidolytic activity of protein C and increase its solubility (in contrast to other plasma proteases). Conclusion. Pre-adding of a mixture of amino acids L-arginine and L-glutamic acid to cryo-depleted plasma significantly optimizes the pre-purification stage of protein C, providing a 5-fold increase in its yield after elution from DEAE-cellulose.

The Fibronectin Type II Domain of Factor XII Ensures Zymogen Quiescence

Factor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation), plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation loop of FXII is shielded to regulate FXII activation and subsequent contact activation. In this study, we aimed to elucidate this mechanism by expressing and characterizing seven consecutive N-terminally truncated FXII variants as well as full-length wild-type (WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1–71), FXII cleavage products appear on Western blot. These fragments display spontaneous amidolytic activity, indicating that FXII without the fibronectin type II domain is susceptible to autoactivation. Additionally, truncated FXII Δ1–71 is more easily activated by PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation, we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A Δ1–71 is highly susceptible to cleavage by PKa, indicating exposure of the activation loop. In surface binding experiments, we found that the fibronectin type II domain is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation of full-length WT FXII by PKa increases approximately threefold in the presence of kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined, these findings suggest that the fibronectin type II domain shields the activation loop of FXII, ensuring zymogen quiescence.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition. Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%. We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation. The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays. Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. Disclosures Mast: Novo Nordisk: Research Funding.

Characterization of a Glycyl-Specific TET Aminopeptidase Complex from Pyrococcus horikoshii

ABSTRACT The TET peptidases are large self-compartmentalized complexes that form dodecameric particles. These metallopeptidases, members of the M42 family, are widely distributed in prokaryotes. Three different versions of TET complexes, with different substrate specificities, were found to coexist in the cytosol of the hyperthermophilic archaeon Pyrococcus horikoshii. In the present work, we identified a novel type of TET complex that we named PhTET4. The recombinant PhTET4 enzyme was found to self-assemble as a tetrahedral edifice similar to other TET complexes. We determined PhTET4 substrate specificity using a broad range of monoacyl chromogenic and fluorogenic compounds. High-performance liquid chromatographic peptide degradation assays were also performed. These experiments demonstrated that PhTET4 is a strict glycyl aminopeptidase, devoid of amidolytic activity toward other types of amino acids. The catalytic efficiency of PhTET4 was studied under various conditions. The protein was found to be a hyperthermophilic alkaline aminopeptidase. Interestingly, unlike other peptidases from the same family, it was activated only by nickel ions. IMPORTANCE We describe here the first known peptidase displaying exclusive activity toward N-terminal glycine residues. This work indicates a specific role for intracellular glycyl peptidases in deep sea hyperthermophilic archaeal metabolism. These observations also provide critical evidence for the use of these archaeal extremozymes for biotechnological applications.

Screening and functional exploration of prothrombin Arg596 related mutations in Chinese venous thromboembolism patients

AimsDysfunctional prothrombin residue Arg596 associated mutation has been found to precipitate venous thromboembolism (VTE). In the current study we investigated the prevalence of Arg596 associated mutations in Chinese patients with VTE and explored the functional impact of Arg596Gln mutation on coagulation function in affected patients.MethodsProthrombin clotting activity was measured in 267 unrelated patients with unprovoked VTE. Patients with moderately decreased activities underwent further analysis of the F2 gene. Prothrombin amidolytic activity and antigen levels were detected in mutation carriers. Specific family members were investigated about their VTE histories and clinical phenotypes. The thrombin generation test (TGT) was used to evaluate thrombin function and antithrombin resistance assay was applied to assess the extent of impaired antithrombin inhibition of mutation carriers.ResultsTwo heterozygous mutation carriers of prothrombin Arg596Gln were identified, both of whom had moderately decreased clotting activities but normal amidolytic activities and antigen levels. Among the families of the two probands, nine out of 13 mutation carriers experienced episodes of VTE. TGTs showed that patients had elevated endogenous thrombin potential and prolonged start tail time. Thrombin generation could be inhibited in the presence of thrombomodulin. The thrombin Arg596Gln variant in patients’ plasma presented strong resistance to antithrombin inhibition.ConclusionProthrombin Arg596Gln mutation is a risk factor for Chinese patients with VTE due to its moderately decreased clotting activity but strong resistance to antithrombin inhibition. Prothrombin clotting activity screening and its encoding gene sequencing should be considered in patients with VTE when other established risk factors are absent.

Effect of IgG from multiple sclerosis patients on amidolytic activity of coagulation and anticoagulation factors of hemostasis

Background: Immunoglobulin G (IgG) is a major immunoglobulin (Ig) in blood that accumulates to a greater extent in the bloodstream of patients impacted by neuroimmunological disorders such as multiple sclerosis (MS). The aim of this study was to determine the effect of IgG obtained from MS patients on the amidolytic activity of coagulation and on anticoagulation factors, and to compare those effects to the effects of IgG from healthy donors. Methods: Spectrophotometric hydrolysis of specific chromogenic substrate by key haemostasis factors was examined. Results: Our study shows that unlike healthy individuals, patients suffering from MS express IgG which enhances the amidolytic activity of thrombin and protein C, but inhibits the activity of factor Xa. Conclusion: Our study shows that IgG and coagulation factors, indeed, interact with each other. IgG may be key mediators of neuroinflammation and, therefore, may serve as a potential target for therapeutic strategies for MS and other neuroimmunological diseases.

The Role of Sphingomyelin (SM) in Tissue Factor (TF) Encryption: ATP-Induced Activation of Acid Sphingomyelinase in Macrophages Decrypts Tissue Factor By Hydrolysis of SM in the Outer Leaflet

While tissue factor (TF)-mediated blood coagulation is essential for maintaining hemostasis, the aberrant activation of TF-mediated coagulation is a major determinant of thrombotic occlusions, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. Typically, TF on cell surfaces exists in inactive coagulant status (cryptic TF). Cell injury leads conversion of cryptic TF to coagulant active/prothrombotic TF. Molecular differences between cryptic and procoagulant TF and the mechanisms that are responsible for the conversion from one to the other form are poorly understood and often controversial. A majority of the evidence in the literature suggest that level of anionic phospholipids, such as phosphatidylserine (PS), in the outer leaflet of the plasma membrane plays a critical role in regulating TF procoagulant activity at the cell surface. However, other pathways, such as the thioredoxin system or thiol-disulfide exchange pathways involving protein-disulfide isomerase (PDI), were also shown to contribute to TF activation by inducing structural changes in TF. It is unknown at present whether TF on cell surfaces of naïve cells exists primarily in the cryptic state because of the limited availability of anionic phospholipids at the outer leaflet or phospholipids present in the outer leaflet play an active role in maintaining TF in the cryptic state. In the outer leaflet of mammalian plasma membrane, sphingomyelin (SM) constitutes up to 50% of the total phospholipids present on the cell surface. It is possible that a high SM content in the outer leaflet may be responsible for maintaining TF in its cryptic state at the cell surface in naïve cells, and the hydrolysis of SM on the outer leaflet mediated by factors released in cell injury contributes to TF activation. The present study was carried out to investigate this possibility. First, we tested the potential effect of SM on TF activity in a reconstituted system in which full-length TF was reconstituted into phospholipid vesicles composed of varying molar concentrations of SM with the remainder of the vesicle consisting of phosphatidylcholine (PC). SM, at 35 mol % or higher concentration in the proteoliposome, inhibited TF coagulant activity significantly as measured in factor X activation assay. Ceramide, having a similar sphingosine backbone as of SM, had no inhibitory effect on TF-FVIIa activation of FX. Measurement of FVIIa-TF amidolytic activity showed that SM does not inhibit the amidolytic activity of FVIIa-TF, indicating that SM neither affects FVIIa binding to TF nor TF-FVIIa cleavage of the small substrate peptide. SM also inhibited significantly TF activity of TF reconstituted in PC/PS (94%:6% mol/mol) vesicles. Next, human monocyte-derived macrophages (MDMs) were treated with varying concentrations of bacterial sphingomyelinase (b-SMase) to hydrolyze SM in the outer leaflet. b-SMase treatment increased cell surface TF activity in a dose-dependent manner. SMase treatment also enhanced the release of TF-bearing microparticles (MPs). SMase treatment had no significant effect on cell surface prothrombinase activity or annexin V binding to MDMs, indicating that b-SMase treatment did not increase PS availability at the cell surface under our experimental conditions. Similar to that observed in bone marrow-derived mouse macrophages, ATP (200 µM) stimulation of MDMs increased cell surface TF activity by about 3-fold and triggered the release of TF+ MPs. Immunofluorescence confocal microscopy revealed that ATP stimulation induced in the translocation of acid(a)-SMase from intracellular compartments to the outer leaflet of the plasma membrane. Treatment of MDMs with sphingomyelinase inhibitors, desipramine and imipramine (1 and 5 µM), or silencing a-SMase with siRNA markedly reduced the ATP-induced increased TF activity at the cell surface and TF+ MPs release. Finally, ATP stimulation was shown to increase the hydrolysis of SM in the outer leaflet of MDMs markedly. a-SMase inhibitors or silencing of a-SMase attenuated the ATP-induced SM hydrolysis. In summary, our data indicate that SM plays a critical role in maintaining TF in the cryptic state in resting cells. Activation/translocation of a-SMase to the outer leaflet following the activation of ATP receptor P2X7 leads to hydrolysis of SM and thus relieves the inhibitory effect of SM on TF, leading to TF decryption and the release of TF+ MPs. Disclosures No relevant conflicts of interest to declare.

COMBINED USE OF HAEMOSTATIC SYSTEM INDICES FOR EVALUATION OF UPPER RESPIRATORY TRACT CANCER PROGRESSION

Aim: To analyze whether comprehensive assessment of haemostatic system components, in particular, indices of coagulation and fibrinolytic systems along with functionally related proteins, could be indicative of upper respiratory tract (URT) cancer progression. Materials and Methods: Indices of coagulation and fibrinolytic systems along with functionally related proteins, in particular, trypsinlike amidolytic activity, trypsin-like proteolytic activity, thrombin-like amidolytic activity, elastase-like amidolytic activity, fibrinolytic activity, potential amidolytic plasmin activity, content of fibrinogen, antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin, and prothrombin time were evaluated in blood plasma of patients with URT cancer of II (n = 10) and III (n = 25) stages with the use of routine biochemical methods. Results: For both groups of patients with URT cancer there have been shown notable differences for the majority of the studied indices, especially the indexes of proteolytic activities, from these of healthy donors, and in the case of URT cancer of III stage they reached statistical significance. In contrary, the changes in the content of antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin were insignificant. In both groups of patients significant increase of fibrinogen content has been registered, while the content of soluble fibrinogen didn’t change. Also, in both groups of patients there a significant increase of potential activity of plasminogen was documented, while clot lysis time was significantly increased only in patients with III stage URT cancer. Multifactorial analysis of haemostatic system indices evidenced for efficacy of their combined use for evaluation of URT cancer progression risk. Conclusion: Combined use of fibrinogen and α2-macroglobulin content and the level of amidolytic thrombinlike activity could serve as an indicator of URT cancer progression.