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At least 703 records · Page 39Linked to original sources
PubMed1998

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule.

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

Animals↗
PubMed1982

Activated prothrombin complex concentrates: approaches to their preparation.

Locally produced prothrombin complex concentrate was activated by different methods and the resultant inhibitor bypassing activity compared with commercially produced activated concentrates using an assay based on the shortening of the clotting time of haemophilic plasma containing inhibitor to factor VIII. Analytical chromatography suggested that for most of these products the inhibitor bypassing activity could be explained in terms of classical activated coagulation factors. In addition, a preparation of Bridge Anticoagulant Neutralising Activity was examined. The results indicate this product merits in vivo investigation as a possible treatment for inhibitor patients.

Blood Coagulation Factors↗
PubMed1995

Comparative study on the use of anticoagulants heparin and recombinant hirudin in a rabbit traumatic anastomosis model.

Antithrombotic drugs, such as heparin, have been used in the clinics for a long time. Heparin acts by binding with antithrombin III to form a complex thereby enhancing the activity of antithrombin III to inactivate coagulation factors IIa, IXa, Xa, XIa and XIIa. Hirudin is a new antithrombotic agent and is reported to be much more powerful than heparin on a gravimetric basis. When both are administered systemically, one of the common complications seen is bleeding. Some previous studies have shown that local vascular endothelial concentrations of heparin are 30 to 7500 times greater than those found in the circulating blood. In order to avoid such complications, topical administration of antithrombotic drugs may be an ideal route of administration. The rabbit ear arterial crush-avulsion thrombosis model was used in this study. The animals were divided into five groups: one control group and four treatment groups which received varying concentrations of heparin and hirudin. In the saline control group, the patency rate was 19.23% at 24 hrs and 15.38% at 7 days. A higher patency rate at 7 days was obtained in groups treated with high concentration of heparin and hirudin. ACT, PT and APTT performed on samples drawn one hour after drug administration were within the normal range in both the control and the treatment groups. Scanning electron microscopy revealed the different extent of the clots on the injured intimal surfaces of the vessels in different groups. The results indicate that high concentrations of topically administered heparin or hirudin minimize the systemic complications and maximize the antithrombotic effects.

Administration, Topical↗
PubMed2000

Activation of factor IX by factor XIa.

Blood coagulation factor IX is activated during hemostasis by two distinct mechanisms. Activation through factor VIIa/tissue factor occurs early in the course of fibrin clot formation. Activation by factor XIa appears to be important for maintaining the integrity of the clot over time. In general, coagulation proteases are activated on a phospholipid surface in the presence of a protein cofactor. Until recently, activation of factor IX by factor XIa was thought to be the exception to this rule, as phospholipid has no effect on the reaction and no cofactor had been identified. These curious observations suggest that factor IX is activated by factor XIa in the fluid phase. A large amount of new evidence now indicates that factor IX activation by factor XIa occurs on the surface of activated platelets. The data suggest, however, that this reaction differs significantly from other protease-substrate interactions on the platelet surface. This is likely to be due, in part, to the unusual structure of the factor XI molecule.

Amino Acid Sequence↗
PubMed1994

Activation peptide of human factor IX has oligosaccharides O-glycosidically linked to threonine residues at 159 and 169.

O-Linked oligosaccharide chains were identified in the activation peptide (AP) of human blood coagulation factor IX. The peptide obtained from human factor IX was separated into three molecular species (AP alpha, AP beta, and AP gamma) by reversed-phase high-performance liquid chromatography. Amino acid analysis showed that AP alpha, but not AP beta and AP gamma, contained galactosamine in addition to glucosamine, thereby suggesting the presence of an O-linked sugar chain(s) in the molecule of AP alpha. A nonapeptide (AP alpha-D4, residues 157-165) and an undecapeptide (AP alpha-D5, 166-176) derived from AP alpha contained Thr-159 and Thr-169, neither of which could be identified using a gas-phase protein sequencer. All other serine and threonine residues present in AP alpha were identified by peptide sequencing. Component sugar and sialic acid analyses of AP alpha-D4 and AP alpha-D5 revealed that they contained 1 mol each of N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), and sialic acid. Fast atom bombardment tandem mass spectrometric analysis of AP alpha-D4 suggested the existence of Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures. On the basis of amino acid analysis after the isolation of AP alpha, it accounted for approximately 35% of the total activation peptide obtained. From these results, it was concluded that a part of the activation peptide of human factor IX in circulating blood has tri- and tetrasaccharides O-glycosidically linked to the threonine residues at 159 and 169.

Amino Acid Sequence↗
PubMed1997

Progress curve analysis of the kinetics with which blood coagulation factor XIa is inhibited by protease nexin-2.

Protease nexin-2 (PN-2), a soluble form of amyloid beta-protein precursor (APP) containing a Kunin protease inhibitor domain, has been shown to be a potent, reversible and competitive inhibitor of blood coagulation factor XIa (FXIa). We have analyzed progress curves of the hydrolysis of a sensitive fluorogenic substrate by FXIa in the presence of PN-2 to ascertain the kinetic rate constants governing the inhibition of FXIa by PN-2. The mechanism of this inhibition is best described as a slow equilibration between the free enzyme and inhibitor directly, without prior formation of a loosely-associated complex. The association rate constant (kon) and the dissociation rate constant (koff) were found to be 2.1 +/- 0.2 x 10(6) M-1 s-1 and 8.5 +/- 0.8 x 10(-4) s-1, respectively (n = 23). The inhibition constant calculated from these parameters (Ki) is 400 pM, in good agreement with previous reports. High molecular weight kininogen (HK) and Zn2+ ions exert opposite effects on the inhibition of FXIa by PN-2. HK protects FXIa from inactivation in a dose dependent and saturable manner (EC50 = 61 nM) whereas Zn2+ augments the ability of PN-2 to inhibit FXIa. When both Zn2+ ions and HK are present, only the accessory effect of Zn2+ is observed. PN-2 is known to be an abundant platelet alpha-granule protein (Van Nostrand et al., 1990a; Smith & Broze, 1992). We conducted sensitive measurements of FXIa activity in the presence of human platelets before and after their being activated with the thrombin receptor agonist peptide, SFLLRN-amide. We found that platelet activation, and ostensibly the release of PN-2, limits the lifetime of FXIa activity within the locus of activated platelets. As in the purified system, HK protects FXIa from inactivation and Zn2+ increases the inactivation of FXIa. However, when HK and Zn2+ are both present, it is the protective effect of HK which predominates and prolongs the lifetime of FXIa after platelet activation.

Amyloid beta-Protein Precursor↗
PubMed1985

Studies on the profibrinolytic actions of heparin and its fractions.

Utilizing modified immunochemical methods (ELISA and radioimmunoassay) tissue plasminogen activator, B beta 15-42 RPs and protein C antigen levels were measured in man and a subhuman primate model after subcutaneous and intravenous administration of various low molecular weight heparin fractions. A wide scatter in the data was observed in the t-PA and B beta 15-42 RP levels; however, statistical analysis of the data revealed that certain low molecular weight heparin fractions increased the levels of these endogenous markers of fibrinolysis. No significant alteration in the protein C levels was noted at any time; however, a wide scatter in these data was also evident. The profibrinolytic actions of low molecular weight heparin fractions may be related to the release of t-PA, which is easily measured in plasma. Since it has strong affinity for endogenous sites (thrombus, surface), wide scatter in the data may occur. Physical exercise may also increase the levels. Most of the results reported in our studies represent data on blood samples that were obtained using the simple venipuncture method. We also find that the intravenous administration of the low molecular weight heparin fractions also caused a shortening of the ELT. Since the low molecular weight heparin fractions are heterogeneous in nature, the profibrinolytic actions may be related to one or more of these constituent fragments. Thus, the molecular identity of the profibrinolytic component of low molecular weight fractions remains unknown at this time. Also unknown is if there is a relationship between this effect and anticoagulant activity.

Animals↗
PubMed1985

Polymorphism of normal factor IX detected by mouse monoclonal antibodies.

Hemophilia B is an X-chromosomal recessive disease due to deficiency of coagulation factor IX. Three monoclonal antibodies against factor IX were prepared and used to develop immunoradiometric assays (IRMAs) of factor IX antigen (IX-Ag). IX-Ag was measured in 65 normal individuals with one IRMA based on polyclonal anti-IX antibodies and two IRMAs based on three monoclonal anti-IX antibodies. One of the monoclonal antibodies differed in specificity since it neutralized less than 50% of the clotting activity of factor IX (IX-C), whereas the other two monoclonal antibodies neutralized 80-95%. When the former antibody was used as the solid phase in IRMA, two groups of normal individuals were distinguished: group A with measurable IX-Ag, and group B without demonstrable IX-Ag. There were no differences between the groups either in IX-C or in IX-Ag measured with polyclonal antibodies. A subgroup comprising only women could be distinguished in group A, in whom intermediate IX-Ag concentrations were found. Family studies showed the group B variant of normal factor IX to be transmitted according to the pattern of X-linked recessive inheritance. The allelic frequency of group A was 0.66, and that of group B was 0.34.

Adolescent↗
PubMed1995

Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display.

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.

Amino Acid Sequence↗
PubMed1994

Large-scale preparation and biochemical characterization of a new high purity factor IX concentrate prepared by metal chelate affinity chromatography.

Metal chelate affinity chromatography on copper-charged Chelating Sepharose has been used to purify a factor IX concentrate from 4,000- to 5,000-kg pools of human plasma, with an overall yield of 194 IU/kg. Unwanted proteins and solvent-detergent reagents added to inactivate lipid-enveloped viruses were removed during the chromatographic step. The freeze-dried product was > 80% pure factor IX with a mean specific activity of > 160 IU/mg protein. The concentrate showed no evidence of clotting factor activation by in vitro tests for potential thrombogenicity or by direct assay for activated factor IX. The concentrate did not exhibit proteolytic activity against a range of synthetic peptide chromogenic substrates. Full functional factor IX activity was retained and there was no evidence of protein degradation. Metal chelate affinity chromatography therefore appears to present less physicochemical challenge to the protein than other factor IX purification methods, while allowing the preparation of a clinical factor IX concentrate at a large scale.

Chelating Agents↗
PubMed2004

Identification of prothrombin as a major thrombogenic agent in prothrombin complex concentrates.

Prothrombin complex concentrates (PCC) were compared in an in vitro test system for thrombogenicity (thrombin generation assay) employing plasma from coumarin-treated patients. Among these concentrates one had a proven history of thrombogenicity, whereas the remainder did not cause such fatal casualties in the past. Investigations into the thrombogenic component were performed by spiking experiments in which we biased a typical PCC without reported thromboembolic complications into one with a performance in the thrombin generation assay like that with a proven history of thrombogenicity. Hereby, it was possible to identify prothrombin as the most plausible thrombogenic component. Additional experiments performed with anticoagulant components (antithrombin together with heparin) resulted in a perfect reversal of the observed in vitro thrombogenicity. Our in vitro observations corroborate on an experimental basis the widespread medicinal usage of antithrombin administration as a regimen for the avoidance of thromboembolic complications during treatment with PCC and related products, and vice versa. Our observation casts doubts upon the widely accepted idea of activated factor IX as the thromboembolic agent in PCC. Also, our finding may be taken as an example for the feasibility of this test system as an in vitro model for thrombogenicity.

Blood Coagulation Factors↗
PubMed1989

Defective propeptide processing and abnormal activation underlie the molecular pathology of factor IX Troed-y-Rhiw.

Affected members of a South Wales haemophilia B family (from Troed-y-Rhiw) were shown by Western blotting and immunoperoxidase detection to have a factor IX molecule of higher than normal molecular weight which also shows impaired calcium binding. Gene cloning and DNA sequencing revealed the same arginine to glutamine mutation at position -4 of the propeptide that has been found in two previously described factor IX variants which circulate with the propeptide still attached. The mutation also abolishes a HaeIII restriction enzyme recognition site. A potential carrier was shown to be normal both by Western blotting and DNA studies. The way in which the attached propeptide interferes with normal factor IX function was investigated by activation studies with crude normal and patient factor IX Troed-y-Rhiw preparations using Western blotting and detection with iodinated immunopurified polyclonal antifactor IX serum. We demonstrate that the -4 mutation appears to block cleavage between the Arg145-Ala146 peptide bond in the activation peptide, thus preventing the normal activation of factor IX Troed-y-Rhiw. A small amount of normally processed factor IX is produced, implying that the -4 mutation does not completely prevent propeptide cleavage, thus accounting for the low levels of factor IX activity measured in the plasma of affected family members.

Base Sequence↗