Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays.

BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.

Plasma contains protein S monomers and multimers with similar direct anticoagulant activity.

BACKGROUND: Protein S (PS) has activated protein C-independent, direct anticoagulant activity (PS-direct). We reported that both multimers and monomers of affinity-purified PS have PS-direct similar to that in plasma, in contrast to another report. OBJECTIVE: We extended our studies to establish the molecular forms and activity of plasma PS. METHODS: Novel ELISAs were developed that could detect only multimeric, not monomeric, PS because they employed the same monoclonal antibody for capture and detection. PS forms were also examined on native PAGE immunoblots. A new activity assay for PS-direct was applied to plasma and gel-filtered plasma fractions. RESULTS: Plasma PS multimers were clearly demonstrated using the ELISAs; 30-60% of free plasma PS appeared to be multimeric, a proportion similar to that of affinity-purified PS. On immunoblots, plasma PS multimers were more easily detected after gel filtration; plasma PS monomers and several apparent multimers comigrated with respective forms of affinity-purified PS. Antigen elution profiles after gel filtration of plasma revealed at least one major peak of apparent PS multimers (40-55% of free PS appeared multimeric). Biotin-factor Xa could bind to both plasma PS monomers and multimers. Strong plasma PS-direct was demonstrated, and plasma PS monomers, multimers, and PS-C4b-binding protein complexes each reconstituted PS-depleted plasma to similar levels of PS-direct. CONCLUSION: Our data are in disagreement with a report that monomeric purified PS has little PS-direct and that only monomeric PS exists in plasma. We find that both affinity-purified and plasma PS exist as monomers and multimers with similar PS-direct.

Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.

BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

Molecular models of the procoagulant factor VIIIa-factor IXa complex.

BACKGROUND: Formation of the intrinsic tenase complex is an essential event in the procoagulant reactions that lead to clot formation. The tenase complex is formed when the activated serine protease, Factor IXa (FIXa), and its cofactor Factor VIIIa (FVIIIa) assemble on a phospholipid surface to proteolytically convert the zymogen Factor X (FX) into its active form FXa. The physiological relevance of the tenase complex is evident in hemophilia A or B patients who present with bleeding disorders. OBJECTIVES: The purpose of this study was to establish three-dimensional (3D) models of the FVIIIa-FIXa complex. METHODS: First, we built two new theoretical models of FVIIIa via homology modeling, inter-domain docking and loop simulation algorithms as well as a model for FIXa. This was followed by pseudo-Brownian protein-protein docking in internal coordinates with the ICM (Internal Coordinates Mechanics) program between the two FVIIIa and the FIXa structures. RESULTS: Ten representative models of this complex are presented based on agreements with known experimental data and according to structural criteria. CONCLUSIONS: These novel 3D models will help guide future site directed mutagenesis aimed at improving the functionality of FVIIIa and/or FIXa and will contribute to a better understanding of the role of this macromolecular complex in the blood coagulation cascade.

Potent blood coagulant activity of human semen due to prostasome-bound tissue factor.

Human semen contains very potent blood clotting activity; for example, seminal serum diluted up to 10,000-fold significantly decreased the recalcification clotting time of blood plasma. This seminal coagulant activity was dependent on factor X and calcium ions, suggesting the presence of a facto X activator. Immunoblotting analysis and immunoadsorption studies confirmed the presence of tissue factor antigen (45 kDa) in semen. Centrifugation studies suggested that tissue factor was membrane associated, and fractionation of seminal serum by gel filtration followed by immunoelectron microscopy revealed that tissue factor antigen was on the prostasome vesicle surface. Tissue factor originated from prostatic fluid and not from seminal vesicle secretions. Tissue factor antigen averaged 21 ng/ml in seminal serum. Hypothetical roles for very high levels of tissue factor in semen include several possibilities. In the event of abrasion and bleeding during intercourse, rapid blood clotting at lesion sites would prevent sperm and seminal components, including infectious agents such as human immunodeficiency virus, from entering the blood stream, generating antibodies, or promoting infectious disease. This could imply that development of infection from semen-borne agents or development of antisperm antibodies in some patients could result from impairment or absence of seminal tissue factor.

A two-allele polymorphism in protein C inhibitor with varying frequencies in different ethnic populations.

cDNAs for protein C inhibitor (PCI), prepared from human liver RNA, contained two forms of PCI, designated PCI*A and PCI*B. While PCI*A is identical to the published PCI sequence, PCI*B differs in 4 of 1221 bp and two amino acids, A36V and K86E. Frequencies for the PCI*B allele, determined from genomic DNA, differed among ethnic groups. Frequency distribution and historical migration of modern man suggest that PCI*A arose from the PCI*B allele. Antigen levels in plasma homozygous for PCI*A or PCI*B equalled that of pooled normal plasma. K86E in PCI*B causes a charge alteration in helix D which is likely involved in heparin binding in antithrombin III but not likely involved in glycosaminoglycan binding in PCI. Kinetic studies showed that plasmas homozygous for PCI*A and PCI*B are similar in their APC inhibiting properties and in their heparin sensitivity, consistent with the idea that helix D in PCI is not involved in heparin binding.

Characterization of a cDNA for rhesus monkey protein C inhibitor--evidence for N-terminal involvement in heparin stimulation.

cDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Four of these differences (T352M, N359S, R362K, L363I) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C.

Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine protein S has a 10-fold higher Kd for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for the negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exception of Lys-429-Ile in porcine protein S. Differences between porcine and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contribute to the decreased affinity of porcine protein S for human C4BP. Moreover, the species specificity of cofactor activities of various species of protein S is determined for human versus bovine-activated protein C, and these results, combined with sequence comparisons, agree with previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C.

Protein C inhibitor is expressed in tubular cells of human kidney.

Protein C inhibitor (PCI) is a serpin that inhibits a number of proteases. PCI is found in urine and binds to kidney epithelial cells. To determine if kidney is a source of PCI, cDNA was produced from human kidney total RNA. Sequencing and restriction mapping showed identity between kidney and liver PCI cDNA sequences. Similar cDNAs were obtained from rhesus monkey kidney and liver RNAs. Conditioned medium from the rhesus monkey kidney cell line CCL7.1 was analyzed on immunoblots, showing a 57,000-D protein band that comigrated with human plasma PCI. Immunohistochemical staining and in situ hybridization of human kidney tissue sections showed that kidney PCI antigen and RNA were confined to tubular cells. The findings are consistent with the idea that PCI is synthesized and localized in kidney tissue where it may provide protease inhibitory activity and suggest that complexes of PCI with urokinase found in human urine may be produced locally in the kidney.

Activation of the host response in human Plasmodium falciparum malaria: relation of parasitemia to tumor necrosis factor/cachectin, thrombin-antithrombin III, and protein C levels.

PURPOSE: Hemostatic alterations and elevated tumor necrosis factor/cachectin (TNF alpha) serum levels may contribute to the pathogenesis of organ complications in human Plasmodium falciparum malaria. Therefore, we examined whether altered protein C (PC) and thrombin-antithrombin III (TAT) plasma levels correlated with TNF alpha serum concentrations, parasitemia, and the clinical course of human P. falciparum malaria. PATIENTS AND METHODS: Forty-seven patients with P. falciparum malaria were evaluated prospectively before and during antiparasitic therapy. TNF alpha serum levels were determined by immunoradiometric assay, PC and TAT plasma antigen by enzyme-linked immunosorbent assay, and PC and PC inhibitor-1 (PCI-1) activity levels by functional tests. Cultured endothelial cells were incubated with serum from four patients with malaria and from healthy control subjects and then assayed for procoagulant activity. Northern blot hybridization was used to detect tissue factor mRNA. RESULTS: In vivo, TNF alpha serum concentrations were elevated (median: 38.6 pg/mL; n = 47) while plasma levels of PC (antigen 55.4%; activity 39.0%; n = 47) and PCI-1 (0.56 U/L) were decreased in almost all patients before antiparasitic treatment. At the same time, TAT concentrations were high. These alterations correlated significantly (p less than 0.01) both with the severity of the disease (as defined by organ impairment) and with the number of circulating parasitized erythrocytes. Low PCI-1 activity correlated with low PC activity (p less than 0.001) and antigen (p less than 0.05) levels. The plasma level of coagulation factor IX, another vitamin K-dependent protein, was not significantly changed. In vitro, incubation of endothelial cells with patient serum (severe P. falciparum malaria) increased both endothelial cell procoagulant activity and cytoplasmic tissue factor mRNA levels. CONCLUSION: Elevated levels of TNF alpha and TAT, decreased plasma levels of anticoagulant PC, and the induction of procoagulant activity in endothelial cells by patient serum indicate a shift in the balance of hemostatic activity towards a procoagulant state in P. falciparum malaria. The alterations in TNF alpha, TAT, and PC levels may be a response to infection, since they correlate with parasitemia and are reversed during antiparasitic treatment.

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2.

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.

Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract.

Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.

[Malaria tropica with activation of blood coagulation and detection of tumor necrosis factor (NF-alpha) in serum]. / Malaria tropica mit Gerinnungsaktivierung und Nachweis von Tumornekrosefaktor (TNF-alpha) im serum.

A 38-year-old patient with cerebral P. falciparum malaria was admitted 12 days after a short trip to Kenya. The serum level of tumor necrosis factor (TNF-alpha) was elevated (251 pg/ml). In contrast, Protein C (plasma activity 36.1%; antigen concentration 31.7%) and protein C inhibitor 1 (activity 0.55 U/ml) levels were decreased. This suggested a state of functional activation of the clotting system which was confirmed by elevated levels (4.8 ng/ml) of circulating thrombin-antithrombin-III-complexes (TAT). Protein S (total and free) and coagulation factor IX levels were within normal range. Under successful antiparasitic therapy, TNF-alpha as well as protein C and protein C inhibitor 1 levels returned to baseline within one week. In the context of other studies that demonstrate procoagulant effects of TNF-alpha, it is remarkable that in the case of complicated P. falciparum malaria, an elevated concentration of TNF-alpha can be paralleled by a decreased plasma level of protein C and an increase in TAT suggesting a procoagulant state.

A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma.

Human plasma contains an inhibitor of activated protein C (APC) which is termed according to its function protein C inhibitor (PCI). High purification of functionally active PCI with a yield of 18% is achieved by an improved procedure consisting of 4 steps: precipitation by rivanol, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE Sephacel and chromatography on dextran sulfate Sepharose. This purification results in the isolation of a homogeneous PCI which migrates in immunoelectrophoresis with the beta-globulins of human plasma and in SDS PAGE as one single band at Mr = 57,000 both under reducing and nonreducing conditions. The specific activity of the highly purified PCI was determined to be 226 units/mg, 1 unit being equivalent to the activity of 1 ml fresh human citrated plasma. PCI forms complexes with 1:1 stoichiometry (Ki: 1.4 x 10(-8) M) resulting in a loss of the amidolytic activity of APC as measured on Tos-Glu-Pro-Arg-pNA (S 2366). The inhibition rate of APC by PCI (k: 7.5 x 10(5) M-1 min-1) is significantly increased in the presence of 5 i.u./ml heparin (kH: 2.2 x 10(7) M-1 min-1). PCI also blocks the amidolytic activities of urokinase plasminogen activator (u-PA), thrombin and factor Xa on their chromogenic substrates in a heparin-dependent manner. According to the Ki-values measured for these reactions PCI is a noncompetitive inhibitor of these proteases. The Ki-values calculated do not differ significantly from those obtained for the inhibition of APC by PCI. Immunodepleted PCI-deficient plasma still contains an inhibitory activity against APC which, however, only slowly inactivates the amidolytic activity of APC and in a time and concentration-dependent manner. Addition of heparin has no influence on the inhibition rate. This finding suggests the existence of a second, heparin-independent PCI present in human plasma.

Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3.

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.

Participation and interactions of neutrophil elastase in haemostatic disorders of patients with severe infections.

The prognosis of septicaemia depends on the occurrence of complications such as shock and coagulation defects. The damage to haemostasis is usually explained by the action of the main coagulation and fibrinolysis enzymes, thrombin and plasmin. This paper presents data concerning the role of a third protease, granulocytic elastase. 82 patients who had been admitted to our hospital with suspected septicaemia were examined. Septicaemia was proven in 22 patients by the growth of microorganisms in blood cultures, and was clinically diagnosed in 9 patients. The plasma levels of neutrophil elastase-like protease complexed to a1antitrypsin (a1AT-ELP) were measured by zone immunoelectrophoresis assay (ZIA). The a1AT-ELP values were significantly increased in the 31 septic as compared to the 51 non-septic patients. In patients with complicated septicaemia, negative correlations of a1AT-ELP with factor XIII and the coagulation inhibitor antithrombin III were demonstrable. Among the patients with septic complications, the 3 who survived exhibited a dramatic decrease of a1AT-ELP, whereas in the other 16 patients who died the levels remained elevated. It might be of therapeutic significance that in 9 patients receiving fresh plasma and AT III-concentrate substitution for DIC the a1AT-ELP levels dropped, whereas they remained high in the other septicaemia patients. There were no correlations between a1AT-ELP and the a2antiplasmin-plasmin complexes (a2AP-P1), but strong correlations with signs of coagulation. The data suggest an interaction of coagulation and elastase release, probably involving the Hageman factor.

The clinical significance of alpha 1-antitrypsin-elastase (alpha 1AT-ELP) and alpha 2-antiplasmin-plasmin (alpha 2AP-PL) complexes for the differentiation of coagulation protein turnover: indications for plasma protein substitution in patients with septicaemia.

In inflammation, particularly in septicaemia, complex coagulation disorders may lead to a dangerous haemorrhagic diathesis. The conventional concept for this syndrome called DIC implicates the occurrence of active thrombin in the circulation, which may be followed by hyperfibrinolysis due to plasmin formation. In this study data are presented suggesting an important role for a third proteolytic system, granulocytic elastase. The complexes of plasmin and elastase with their specific inhibitors, alpha 2-antiplasmin-plasmin (alpha 2AP-PI) and alpha 1-antitrypsin-elastase (alpha 1AT-ELP) were determined immunologically. The alpha 1AT-ELP appears mainly in gram-negative septicaemia, particularly in meningococcal disease. The estimation of alpha 2AP-PI and alpha 1AT-ELP, together with a method for the detection of the antithrombin III--thrombin complex which remains to be established, is a suitable tool for for the differential diagnosis of the consumption of coagulation proteins. The assumption that at least three proteolytic systems participate in the development of the haemorrhagic diathesis during inflammation leads to the concept of a broad, comprehensive substitution therapy with e.g. concentrates of AT III, PPSB, or fresh frozen plasma. The aim of this treatment is to replace not only the consumed procoagulatory factors, but also the lacking inhibitors in order to control this "abnormal proteolysis syndrome".