Factor V is a substrate for the transamidase factor XIIIa

Coagulation Factor V (Mr = 330,000), upon cleavage by thrombin, produces Factor Va, which is composed of two subunits with Mr values of 94,000 and 74,000, along with two activation fragments possessing no known function. Studies were undertaken to assess the ability of the transamidase Factor XIIIa to covalently incorporate the lysine analogs [3H]putrescine and dansylcadaverine into the thrombin-cleaved (activated) and unactivated forms of human and bovine Factor V. The incorporation of either probe into thrombin-activated Factor V proceeded at an initial rate approximately twice that for unactivated Factor V. The extent of the incorporation of [3H]putrescine or dansylcadaverine into activated or unactivated human Factor V was identical; 4 mol of either probe per mol of Factor V. In the case of bovine Factor V, however, while 4 mol of probe were bound per mol of the unactivated pro-cofactor, 5 mol of either lysine analog were covalently linked to 1 mol of thrombin-cleaved Factor V. Polyacrylamide gel fluorography, immunoaffinity chromatography, and immunoprecipitation identified the largest activation fragment of human Factor V (Mr = 150,000) and bovine Factor V (Mr = 120,000) to contain the sites of incorporation of the covalently bound probes. High molecular weight, apparently covalent polymers of Factor V were produced by the action of Factor XIIIa on activated and unactivated human or bovine Factor V. The absence of either probe in the reaction mixtures did not appear to allow an enhancement of protein polymerization.     

Inactivation of factor Va by plasmin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.     

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.     

New fluorogenic peptide substrates for plasmin

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.     

Purification of six human vitamin K-dependent proteins in a single chromatographic step using immunoaffinity columns

The major human vitamin K-dependent proteins were purified from plasma using immunoadsorbents made with antibodies specific for each protein. Monoclonal antibodies to Factor VII, Factor IX, Factor X, Protein C, and Protein S were prepared from mice immunized with isolated vitamin K-dependent antigens. Purified monoclonal antibodies and a purified burro polyclonal anti-prothrombin immunoglobulin were individually coupled to Sepharose and used in a tandem series of columns to purify each of the vitamin K-dependent proteins from eluates of barium citrate precipitates of plasma. The proteins were eluted from the columns by sodium thiocyanate and retained functional activity following dialysis. Prothrombin, Factor VII, Factor IX, Factor X and Protein C were essentially homogeneous as judged by NaDodSO4-PAGE; Protein S was isolated as a Protein S-C4b binding protein complex. These results indicate the utility of monoclonal antibody immunoadsorbents for purifying the human vitamin K-dependent proteins and represent a considerable simplification over other purification schemes.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.     

Isolation of rabbit C3, Factor B, and Factor H and comparison of their properties with those of the human analog

The rabbit complement components C3, Factor B, and Factor H were isolated and characterized and were compared to the corresponding proteins of human serum. Chromatographic behavior, chemical properties, and functional interactions show great similarities between the components in both species. By SDS polyacrylamide gel electrophoresis, the m.w. were estimated to be 195,000 for C3, 86,000 for Factor B, and 155,000 for Factor H. The amino acid compositions of the rabbit proteins resembled those of the human analog. The total carbohydrate content of rabbit C3 and Factor H was approximately one-half that of the human proteins. In addition, a qualitative difference in the carbohydrate moieties of the C3 proteins was observed. The serum concentration of the rabbit proteins was markedly lower than that of the human proteins. The rabbit C3b,Bb enzyme resembled the human analog with respect to half-life, control by Factor H, and stabilization by nickel ions.     

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor VIII coagulant activity and factor VIII-related antigen released from isolated perfused human spleens.

Eight human spleens were perfused for up to 65 h at normothermia and the coagulant Factor VIII activity measured in the perfusate. In addition, in three experiments Factor VIII-related antigen was determined in the perfusate. Although the spleens were pathologically enlarged and the normal structure involved by different diseases, all spleens released Factor VIII coagulant activity and Factor VIII-related antigen. On average the total amount of Factor VIII coagulant activity released was equivalent to that of 3.5 l of human plasma.     

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.     

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.     

Coagulation Factor V contains copper ion

Preparations of bovine and human coagulation Factor V were analyzed for copper using both atomic absorption and atomic emission spectroscopy. All preparations of the bovine and human protein were found to contain copper ion at a ratio of 1 copper ion bound per mol (Mr = 330,000) of Factor V. As a result of copper binding and sequence homology between ceruloplasmin and Factor V, bovine Factor V and thrombin-activated Factor V (Va) were assessed with respect to their visible and near ultraviolet absorption spectra and to their ability to oxidize N,N-dimethyl-p-phenylenediamine (a substrate for ceruloplasmin). Factor V and Factor Va exhibited absorption spectra with no maxima at either 310 or 610 nm, indicating that the copper is not bound in a site analogous to Type I or Type III copper sites in ceruloplasmin. Further, Factor V and Factor Va are not capable of serving as catalysts for the oxidation of N,N-dimethyl-p-phenylenediamine under solution conditions that are optimum for ceruloplasmin oxidase activity. These data suggest that the copper ion bound to Factor V may be functionally and structurally distinct from the Type I and Type III copper ion bound to ceruloplasmin.     

Species specificity of recognition by the alternative pathway of complement

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.     

Purification and structural studies on the complement-system control protein beta 1H (Factor H).

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.     

Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZalphaA vector under the control of AOX1 promoter, which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native N- and C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.