Rational Design of Protein C Activators.

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

Inhibition of thrombin formation by active site mutated (S360A) activated protein C.

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K(D) of 0.11 +/- 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.

Manipulation of the affinity between protein and metal ions by imidazole and pH for metal affinity purification of protein C from Cohn fraction IV-1.

Protein C (PC) is an important anticoagulant in blood plasma. Cohn Fraction IV-1 (CFIV-1) is an inexpensive PC source but contains a large amount of factor II (FII). Immobilized metal affinity chromatography (IMAC) utilizes metal ions to adsorb proteins primarily via their surface histidine. Two major operation parameters for IMAC are imidazole concentration and pH: imidazole is a histidine analog and pH controls the protein surface protonation level. The effects of these two parameters on the adsorption and elution of PC and FII were studied for each protein individually and also together as a mixture. For the individual proteins, low FII (16%) and high PC (98%) adsorption were achieved at 8 mM imidazole, pH 8.0. At 11 mM imidazole, 92% of the adsorbed FII was eluted, with only a 3% PC loss. At 40 mM, 97% of the adsorbed PC was recovered. For the protein mixture, very similar adsorption and elution results were obtained, but slightly greater PC loss (16%) during elution at 11 mM imidazole. This result shows that there is a high potential for the PC purification from CFIV-1 by appropriately adjusting the imidazole concentration and pH in the IMAC process.

Anticoagulant blood factor deficiencies (protein C).

Anti-coagulant proteins are essential to maintain blood hemostatis for the supply of oxygen and nutrients to tissue cells and for the removal of toxic by-products from metabolism. Hereditary or acquired deficiencies of Protein C, Protein S, or Antithrombin III can lead to disease states such as deep vein thrombosis (DVT) with the possibility of producing lung emboli. Phenomena named Factor V Lieden can produce a similar pathologic condition. Anti-coagulant deficiencies, including Factor V Lieden, are HIDDEN blood conditions that can allow blood clot development, especially with trauma to the tissue and circulatory system. It is proposed that all children between ages twelve to fourteen be checked hereditary deficiencies and Factor V Lieden complications. This would require the development of inexpensive assay equipment12. The present research focuses on the low cost production of Zymogen Protein C via purification from blood plasma Cohn Fraction IV-1. This process is difficult due to the several Homologous Vitamin K dependent proteins in the blood coagulation cascade. Traditional chromatography (ion exchange) cannot achieve the desired separation. Some more exotic technologies are very expensive so our work proposes to use Immobilized Metal Affinity Chromatography (IMAC). It is hoped to produce a lower cost product that can be used prophylactic ally to treat Protein C deficiencies and possibly other coagulation problems.

Effect of pH and imidazole on protein C purification from Cohn fraction IV-1 by IMAC.

Cohn Fraction IV-1 (CFIV-1) is a by-product (often discarded) of a plasma fractionation process. It retains 90% of Protein C (PC) of plasma but contains several coagulants structurally homologous to PC. Of these coagulants, Factor II (FII) has the longest half-life (12 times of PC) and largest quantity (9 times of PC) in CFIV-1. Current purification process for PC is by immunoaffinity chromatography using monoclonal antibodies, which is very expensive. Immobilized metal affinity chromatography (IMAC) is an inexpensive process that uses metal ions to adsorb proteins via their surface histidines. Affinity of PC to the metal ions in IMAC is higher than that of FII because PC has 15 surface histidines and FII has 5. Two important factors in an IMAC process are pH and imidazole concentration. PH controls protonation of histidine, and imidazole, a histidine analog, competitively reacts with metal ions. The effects of pH and imidazole on adsorption and elution of PC and FII during IMAC process were studied. The effect of pH on PC and FII adsorption was similar within the range of 6.0 and 8.0. At concentrations below 15 mM imidazole, little PC or FII eluted. At 15 and 20 mM imidazole 2.5% of PC was eluted, while 20-30% of FII was eluted.

Separation of protein C from Cohn Fraction IV-1 by mini-antibody.

Human protein C (PC) is a natural anticoagulant, antithrombotic, anti-inflammatory, and anti-apoptotic in the bloodstream. PC deficiency can lead to abnormal blood clot formation inside blood vessels, possibly causing heart attack, stroke, skin necrosis, or even death. PC can be, therefore, a valuable therapeutic with little side effect, unlike the currently used anti-coagulants. To reduce the cost involved in immuno purification of PC from blood plasma, single chain variable fragments (mini-Mab) are being produced by recombinant E. coli using phagemid technique. As an economic means of purifying the PC specific mini-Mab, metal affinity chromatography (IMAC) purification process was also investigated. Then using the purified mini-Mab, the feasibility of PC purification from the Cohn Fraction IV-1 was examined. Cohn Fraction IV-1 is usually a discarded side-stream from the blood plasma fractionation of human serum albumin. It holds 90% of PC in plasma, but is very cheap. Preliminary study of PC purification from the Cohn Fraction IV-1 showed 16% purification yield using mini-Mab immobilized NHS-activated Sepharose. The economic analysis for PC purification using mini-Mab showed that the overall process was found to be tens of times cheaper than that using Mab.

Treatment of adult patients with sepsis-induced coagulopathy and purpura fulminans using a plasma-derived protein C concentrate (Ceprotin).

BACKGROUND AND OBJECTIVES: The aim of this study was to document the effects of supplementation with a plasma-derived protein C concentrate in adult patients with infectious purpura fulminans. MATERIALS AND METHODS: We report the effect of the administration of a human protein C concentrate (Ceprotin, Baxter, Vienna, Austria) in eight adult patients with purpura fulminans. Five patients received the concentrate as level-adjusted continuous infusion (10 U/kg/h, target protein C activity 100%) and three patients received the concentrate as bolus infusions (100 U/kg every 6 h) in addition to standard sepsis therapy. Heparin, fresh-frozen plasma, antithrombin- and fibrinogen concentrates, low-dose rtPA, and platelet transfusions were given when appropriate. RESULTS: Six patients had overt disseminated intravascular coagulation: platelets, 19 g/l; fibrinogen, 60 mg/dl; antithrombin, 47%; prothrombin time, 32%; activated partial thromboplastin time (APTT), 88 s; d-dimer, 66 microg/ml; protein C activity, 29% (medians). Five patients had septic shock, six renal failure and four respiratory failure. Patients received between 5000 and 77,000 U of protein C concentrate over 2.5 days (median); the protein C activity increased to 184% (median) and coagulopathy resolved within 3 days in seven of the eight patients. Six patients survived, one died early from fulminant sepsis, and one died after 14 days from candida sepsis. CONCLUSIONS: Our data suggest that treatment with a plasma-derived protein C zymogen concentrate might be a useful support in adult patients with purpura fulminans.

Dual role for transactivator protein C in activation of mom promoter of bacteriophage Mu.

Transactivator C protein of bacteriophage Mu activates the mom gene of the phage by an unusual mechanism. DNA binding by C to its site results in unwinding of the neighboring sequences, realigning the out-of-phase promoter elements to facilitate RNA polymerase (RNAP) binding. High level stimulation of a C-independent constitutive promoter mutant (where RNAP is already bound) by the transactivator suggested an additional mechanism of transcription activation at a step after RNAP recruitment. In this study, we have investigated the various steps of promoter-polymerase interactions during transcription initiation by using both the promoter mutant and a positive control (pc) mutant of C protein. The transactivator does not influence formation of the open complex or its stability after facilitating the RNAP binding. However, at a subsequent step, the protein exerts an important role, enhancing the promoter clearance by increasing the productive RNAP.promoter complex. The pc mutant of the transactivator C is compromised at this step, supporting the additional downstream role for C in mom transcription activation. We suggest that this unusual multistep activation of Pmom has evolved to ensure irreversibility of the switch during the late lytic cycle of the phage.

Process scale-up studies for protein C separation using IMAC.

Protein C (PC) is the pivotal anticoagulant and antithrombotic in the human coagulation cascade. PC deficiency can disturb the blood hemostasis and cause thrombosis, inhibiting oxygen transport to tissue, and resulting in major medical problems such as deep vein thrombosis (DVT). The current treatment can cause bleeding and other major medical problems. PC circulates in the blood as a zymogen and is only activated when and where it is needed. PC is a safe anticoagulant without harmful side effects. A combination of ion-exchange chromatography and IMAC IDA-Cu was studied for the relatively large scaled PC separation from Cohn fraction IV-1. Almost half of the active PC was recovered by using this process. In future work, we will verify the linearity of the IMAC column scale-up. This process can be used to produce PC from Cohn fraction IV-1 at large quantities and low cost to treat PC-deficient patients.

Protein C production: metal ion/protein interfacial interaction in immobilized metal affinity chromatography.

Protein C (PC) is an essential blood factor in the human blood coagulation cascade. PC can help achieve blood hemostasis in many deadly disease conditions such as sepsis, cancer, HIV, etc.; reduced oxygen transport due to blood agglutination within the body can cause tissue death and organ failure as a result of low oxygen transport. Our goal is to produce large quantities of low cost zymogen PC for the treatment and prevention of blood clotting resulting from many disease states, as well as provide an effective therapy for PC deficiency. Current studies show that Immobilized Metal Affinity Chromatography (IMAC) has high specificity and can be used for difficult separations among homologous proteins at relatively low cost compared to current methods, such as Immunoaffinity Chromatography. Thus, we are investigating the optimization of IMAC for the separation and purification of PC from Cohn fraction IV-I. Molecular interactions within the chromatography column involve many parameters that include: the use and type of chromatographic gel and buffer solution, the pH, temperature, metal ion, chelator, and the sequence and structure of the protein itself. These parameters all influence the protein's interaction with the column. Experimental equilibrium isotherms show that PC has primary and secondary binding characteristics, demonstrating that the interaction is not just a simple process of one protein binding to one metal ion. Understanding the thermodynamics of interfacial interaction between proteins and surface-bound Cu2+ is essential to optimizing IMAC for PC purification, as well as for separation of other proteins in general. Hence we are undertaking theoretical and experimental studies of IDA-Cu/PC adsorption. The differences in structures of PC and other critical homologous blood factors are examined using the protein visualization program Cn3D. A better understanding of the interfacial phenomena will help determine the most effective conditions to achieve our goal.

The crystal structure of Helicobacter cysteine-rich protein C at 2.0 A resolution: similar peptide-binding sites in TPR and SEL1-like repeat proteins.

Helicobacter pylori is a Gram-negative human pathogen that infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. Bacterial cell-surface and secreted proteins often play an important role in pathogen-host interactions and are thought to be selective mediators for the pathology of the infection. The Helicobacter cysteine-rich proteins (Hcp) represent a large family of secreted proteins that seem to be specific for microorganisms from the epsilon-subfamily of proteobacteria. Although significantly elevated levels of anti-Hcp antibodies were observed in many patients infected with H.pylori, details on the biological functions of Hcp proteins are sparse. Hcps belong to a large family of Sel1-like multi-repeat proteins. The crystal structure of HcpC was refined at 2.0 A resolution and revealed a super-helical topology composed of seven disulfide bridged alpha/alpha-repeats, an N-terminal capping helix and an extended C-terminal coil consisting of alternating hydrophobic and hydrophilic residues. In the crystal packing, the C-terminal coil interacts with the concave surface of a symmetry-related HcpC super-helix. A hydrophobic pocket and a cluster of negatively charged residues recognize the side-chains of Val290 and Lys287 from the C-terminal coil, respectively. The peptide nitrogen atom of His291 forms a short hydrogen bond with the side-chain of Asn66. The interactions seen in this crystal contact are strikingly similar to the peptide-binding modes of the Hsp70/Hsp90 organizing protein and the PEX5 receptor. The conservation of the peptide-binding mode suggests that HcpC might recognize its binding partner in a similar way.

Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.

Protein C separation from human blood plasma derivatives using low cost chromatography.

Protein C (PC) deficiency can cause thrombosis, inhibiting oxygen transport to tissue thus resulting in many complications, including death. Present treatment can cause catastrophic bleeding and other major medical problems. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. The vitamin K dependent (VKD) proteins are homologous proteins, making the separation of PC from plasma extremely difficult. Immobilized metal affinity chromatography (IMAC) is investigated to separate the VKD proteins to replace immunoaffinity chromatography, because of the high cost of monoclonal antibodies. An IDA-Cu column was found effective for the separation of PC from prothrombin, the most harmful contaminant. For Cohn fraction IV-1 separation, a DEAE column was found an efficient initial step, with about 25-fold PC purity increase. Following this step, an IDA-Cu column could remove many contaminants including prothrombin. The combination of DEAE and IDA-Cu resulted in PC purity increase of about 100-fold.

Preparation of vitamin K-dependent proteins, such as clotting factors II, VII, IX and X and clotting inhibitor protein C.

A review is given of preparative methods for the isolation of the vitamin K-dependent clotting factors II, VII, IX, X and clotting inhibitor protein C, all derived from human plasma. Factor II, activated factor VII and activated protein C are also obtained from recombinant animal cells. The methods for their purification are described. The problem of difference in posttranslational modifications between plasma derived and recombinant protein is discussed with regard to therapeutic proteins.

Chromatographic purification and properties of a therapeutic human protein C concentrate.

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.