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At least 19 recordsLinked to original sources
PubMed1998

Negative ion matrix-assisted laser desorption/ionization time-of-flight post-source decay calibration by using fibrinopeptide B.

Fibrinopeptide B (M(r) 1552.58) was employed as a calibration compound for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post-source decay (PSD) fragment ion analysis in the negative mode. Experiments were performed by using both continuous and delayed extraction, with the maximum reflectron voltages being 30 and 21 kV, respectively. For comparison, a common positive ion PSD calibrant, ACTH(18-39) (M(r) 2466.7), was also employed with positive ion calibration constants being applied to negative ion spectra. Using fibrinopeptide B as the calibrant, the negative ion PSD results for angiotensin II (M(r) 1046.2), renin substrate tetradecapeptide (horse) (M(r) 1759.0), and the custom-synthesized peptide (K2G4)2 (M(r) 987.1) showed a factor of 1.5-2 improvement in absolute mass accuracy. Typical absolute mass-to-charge ratio accuracies were within +/- 1 Thompson and were achieved even when the peptide being analyzed was more massive than fibrinopeptide B. In addition, both calibrants showed increased accuracy when experiments were conducted in the delayed extraction mode. Other advantages of using fibrinopeptide B are its moderate cost and the ability to perform calibration and sample analysis for negative ion PSD under the same instrumental conditions.

Angiotensin II↗
PubMed1991

The rate of fibrinopeptide B release modulates the rate of clot formation: a study with an acquired inhibitor to fibrinopeptide B release.

An asymptomatic 50-year-old male with a gamma globulin paraprotein was found to have prolonged prothrombin time, activated partial thromboplastin time, and thrombin time but a normal reptilase time. The prolonged clotting times were not the result of a factor deficiency because they were not corrected by the addition of normal plasma. Instead, this patient had an antibody that delayed thrombin-mediated fibrinopeptide B release thereby producing an apparent dysfibrinogenaemia. His isolated IgG prolonged the thrombin clotting time of both normal plasma and fibrinogen. Precincubation of his IgG with fibrinopeptide B, but not with fibrinopeptide A or thrombin, decreased its ability to prolong the thrombin clotting time. The patient's purified IgG but not control IgG delayed thrombin-mediated fibrinopeptide B release from fibrinogen without affecting the release of fibrinopeptide A. These studies define a novel, clinically silent dysfibrinogenaemia due to an antibody that delays thrombin-mediated fibrinopeptide B release from fibrinogen thereby markedly prolonging the clotting times.

Blood Coagulation Disorders↗
PubMed1981

Measurement of desarginine fibrinopeptide B in human blood.

Thrombin converts fibrinogen to fibrin in two steps. First fibrinopeptide A and fibrin I are formed and then fibrinopeptide B (B beta 1-14) and fibrin II. Since it is postulated that fibrin II is important in the genesis of thrombosis, it is of interest to measure fibrinopeptide B in peripheral blood samples. Previous difficulties in interpreting fibrinopeptide B immunoreactivity in plasma resulted from crossreaction of fibrinogen and of plasmin digest peptides B beta 1-42 and B beta 1-21 and from rapid loss of fibrinopeptide B immunoreactivity resulting from cleavage of arginine 14 by blood carboxypeptidase B. We have obviated these difficulties by removing fibrinogen from plasma by precipitation with ethanol and peptides B beta 1-21 and B beta 1-42 by adsorption on bentonite. Fibrinopeptide B is then converted to a desarginine fibrinopeptide B, which is measured in a new specific assay. Studies of the kinetics of fibrinopeptide cleavage showed that when whole blood was allowed to clot in vitro, fibrinopeptide A was cleaved more rapidly than fibrinopeptide B. In 18 patients on an acute care medical ward, desarginine fibrinopeptide B levels were lower than fibrinopeptide A levels and did not correlate with the levels of fibrinopeptide A or B beta 1-42. Desarginine fibrinopeptide B levels were less than 1 pmol/ml in all but two patients. In six patients receiving intraamniotic infusions of hypertonic saline to induce abortion, desarginine fibrinopeptide B levels increased 10-fold from the preinfusion mean level of 0.4 pmol/ml and then decreased. The pattern of changes resembled that of the fibrinopeptide A levels rather than of the B beta 1-42 levels. On the basis of these data it is suggested that plasma desarginine fibrinopeptide B levels reflect fibrin II formation in vivo.

Cross Reactions↗
PubMed1975

Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes.

Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A (alha) and B (beta) chains of fibrinogen by various enzymes. Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was couple to desaminotyrosine and radiolabeled with 125I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slowere cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.

Alpha-Globulins↗
PubMed1988

Cardiovascular effects of fibrinopeptide B.

The effect of fibrinopeptide B (FpB) on isolated blood vessels and in the intact rat has been investigated. FpB contracted the rabbit superfused aorta preparation (EC50 = 7.5 nmol) and was some 20 times less potent than noradrenaline. Similarly, FpB (0.2-10 nmol) injected into the rat perfused kidney caused dose related, short-lived increases in perfusion pressure and potentiated the vasoconstrictor effect of injected noradrenaline. These effects were associated with increased efflux of PGE2 (but not TxB2) and were reduced but not abolished by indomethacin (10 microM). FpB injected intravenously into the urethane-anaesthetized rat exhibited vasoconstrictor activity (EC50 = 2.5 micrograms kg-1) but was less potent than noradrenaline (EC50 = 0.9 micrograms kg-1). The doses of FpB required to contract the rabbit isolated aorta and to constrict the vasculature of the rat kidney occur naturally in the bloodstream of patients with thrombotic disease. FpB released at the site of thrombus formation may play a part in regulating local blood vessel calibre.

Animals↗
PubMed1989

Conversion of fibrinogen to fibrin induced by preferential release of fibrinopeptide B.

Fibrin clot-promoting enzyme preferentially releasing fibrinopeptide B from fibrinogen was isolated from the crude venom of Agkistrodon contortrix and its mode of action was studied in detail. A purification procedure involving affinity chromatographies on immobilized lectin and arginine removed plasmin-like and kallikrein-like activities towards low-molecular-weight chromogenic substrates. Fibrin-promoting enzyme cleaved off only fibrinopeptides A and B from fibrinogen. The initial relative rate of release of fibrinopeptide B/fibrinopeptide A depended strongly on the presence of Ca2+. In the absence of Ca2+ the amount of fibrinopeptides released by fibrin-promoting enzyme at the gel point was greater. Fibrinopeptide B was no longer released before fibrinopeptide A from the non-polymerizing N-terminal disulphide knot of fibrinogen. Catalyzed by activated factor XIII, complete gamma-dimer and alpha-polymer formation was observed in fibrin from which only 23% of fibrinopeptide A, but 100% of fibrinopeptide B was released by fibrin-promoting enzyme. gamma-dimer formation markedly preceded alpha-polymer formation. These results strongly imply a similar overall arrangement of monomer molecules in this fibrin when compared with a thrombin-induced fibrin in which fibrinopeptide A is released before fibrinopeptide B. These observations support the concept that on fibrinopeptide B release from fibrinogen polymerization sites are exposed which reinforce the sites exposed on the release of fibrinopeptide A.

Calcium↗
PubMed2001

Fibrin monomer and fibrinopeptide B act additively to increase DNA synthesis in smooth muscle cells cultured from human saphenous vein.

PURPOSE: We investigated the hypothesis that fibrinogen increased DNA synthesis (and cell proliferation) of smooth muscle cells (SMCs) cultured from human saphenous vein and that the increased DNA synthesis was attenuated when cells were cultured on polymeric collagen. METHODS: SMCs were cultured from human saphenous vein on plastic, fibronectin, monomeric, and polymeric collagen. Fibrinogen products were prepared by proteolytic digestion. DNA synthesis was measured by bromodeoxyuridine incorporation into DNA, cell proliferation by cell counting, cyclic adenosine monophosphate by enzyme-linked immunosorbent assay, and fibrinopeptide B labeled with iodine 125 used for binding studies. RESULTS: Fibrin monomer (0.003-0.1 micromol/L) stimulated a concentration-dependent increase in DNA synthesis of up to 10-fold, which could be inhibited by the peptide Bbeta15-42. The stimulation of DNA synthesis was highest for cells cultured on plastic and lowest for cells cultured on type I collagen polymer. Much higher concentrations of fibrinogen (0.3-1 micromol/L) were required to effect similar increases in DNA synthesis. Fibrinogen had a particular effect to augment DNA synthesis, up to 14-fold, when cells were cultured on monomeric type I collagen. This augmented DNA synthesis was inhibited by a neutralizing antibody to urokinase-type plasminogen activator. Incubation of cells cultured on collagen monomer with fibrinogen resulted in production of fibrinopeptide B. Fibrinopeptide B (5 micromol/L) increased DNA synthesis by fourfold and had additive effects with fibrin monomer to increase DNA synthesis. Iodinated tyrosine fibrinopeptide B bound to SMCs (dissociation constant 0.6 micromol/L). CONCLUSION: Cultured human saphenous vein SMCs appear to have high-affinity receptors for fibrin monomer and fibrinopeptide B, the engagement of which stimulates DNA synthesis. These mechanisms may be pertinent to the association between fibrinogen and vein graft stenosis in vivo.

Bromodeoxyuridine↗
PubMed1979

Fibrinopeptide B and aggregation of fibrinogen.

Removal of fibrinopeptide B from human fibrinogen by reaction with the procoagulant enzyme from copperhead snake venom below 25 degrees C resulted in tight aggregation of the fibrinogen, which, in turn, progressively blocked a concomitant but sluggish release of fibrinopeptide A by the enzyme. When the clots obtained at less than 25 degrees C were warmed, they dissociated into soluble aggregates and monomers. Release of fibrinopeptide A then resumed, and a secondary coagulation followed. The aggregation induced by release of fibrinopeptide B itself involves a plasmin-susceptible segment located just distal to B in the B beta chain of fibrinogen, a segment previously shown to be of little importance in the aggregation induced by release of fibrinopeptide A.

Amino Acid Sequence↗
PubMed1987

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta-fibrin).

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.

Blood Coagulation↗
PubMed1984

Peptide heterogeneity in a preparation of synthetic fibrinopeptide B.

A commercially available preparation of synthetic human fibrinopeptide B (FpB) was shown by hplc to contain two chromatographically distinct peaks, one of which was identical to FpB. Our results suggest that the contaminant peptide (FpB-2), which represented approximately 43% of the total peptide composition, is FpB containing an alpha-aminosuccinimide (Asc) residue. This Asc residue probably arose as a result of the cyclization of 5Asp-6Asn during either the coupling or deprotection reactions. FpB-2 was rapidly hydrolysed by carboxypeptidase B to des-Arg-FpB-2. It was stable under acidic conditions but in dilute alkali was converted to equimolar amounts of FpB and FpB containing beta-Asp at residue 5. Since it has been suggested that 5Asp-6Asn is a major immunorecognition site in FpB, our observations emphasize the need to establish the purity of synthetic FpB preparations destined for use in the immunoassay of either FpB or des-Arg-FpB.

Amino Acids↗
PubMed1994

Endothelial desquamating activity of rat synthetic fibrinopeptide B and its analogues "in vivo": identification of responsible sequence.

The endothelial desquamating activity of the synthetic rat fibrinopeptide B (ATTDSDKVDLSIAR-OH), and its analogues was studied "in vivo" after intravenous administration to rats. Rat fibrinopeptide B (FPB) caused a significant increase in the count of circulating endothelial cell carcasses at the dose of 100 nmol/kg. Maximal effect reaching about 270% of the normal value was achieved with the dose of 600 nmol/kg in 30 min. after the injection. No significant thrombocytopenia, no hemolysis and no other life-threatening complications were observed. The same endothelial desquamating effect was achieved with N-terminal FPB(1-7) peptide ATTDSDK-OH, but very low activity exhibited the two FPB mutant peptides: ATDSDKVDLSIAR-OH and ATTNSNK-OH. Our results indicate that N-terminal sequence (1-7) consisting of N-terminal "pig tail" (ATT), acid region (DSD) and basic amino acid (K) is responsible for endothelial desquamating activity of rat FPB. Similar corresponding sequences may be recognized in FPB of different species. The conservation of this common "active site" sequence is less apparent in primates.

Amino Acid Sequence↗
PubMed1977

Role of fibrinopeptide B release: comparison of fibrins produced by thrombin and Ancrod.

The gelation time, opacity, light scattering, and elastic moduli of human fibrin gels clotted in the presence of thrombin, Ancrod, and Reptilase have been compared. At low ionic strength lateral association to thick fibers is observed in all cases. At all ionic strengths thrombin fibrin forms thicker fibers than does Ancrod fibrin. We have demonstrated that an increase in the extent of lateral association is linked to an increase in its velocity and to a decrease in the gelation time. One may consider the removal of fibrinopeptide B to act as a switch: after it is removed fibrin assembles rapidly to thick fibers and gelation is fast; but when this peptide is still attached, there is a slow assembly of thin fibers, and gelation, especially of dilute fibrin, is delayed. We believe that this delay is critical for the complete digestion by plasmin of fibrin formed during in vivo defibrination with Ancrod and of fibrin produced by very small amounts of thrombin (which would still contain fibrinopeptide B), and that slow release of fibrinopeptide B is part of a control mechanism for the regulation of fibrin formation and the prevention of intravascular coagulation.

Ancrod↗
PubMed1992

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.

Batroxobin↗
PubMed1988

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B.

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.

Animals↗
PubMed2006

Identification of a new truncated form and deamidation products of fibrinopeptide B released by thrombin from human fibrinogen.

Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.

Blood Coagulation Factors↗
PubMed1982

Immunochemical studies of antisera to human fibrinopeptide-B.

The immunochemical specificity of rabbit antisera to human fibrinopeptide-B (FPB) has been studied by comparing the relative abilities of FPB and of various proteins and peptides containing the NH2-terminal segment of the B beta-chain of human fibrinogen to inhibit the binding of a radioiodinated FPB derivative by each of seven anti-FPB sera. Anti-FBP sera varied in the extent to which they cross-reacted with fibrinogen, the NH2-terminal disulfide knot of fibrinogen (N-DSK), B beta 1(Pyr)-118(Met), B beta 1(Pyr)-42(Arg), and desarginyl-FPB. Anti-FPB sera have been identified that discriminate effectively between FPB and larger FBP-containing peptides; such antisera can be used to measure FPB in the absence of the larger peptides or to demonstrate the presence of larger peptides such as B beta 1(Pyr)-42(Arg) in extracts of clinical plasma samples by means of an increase in FPB immunoreactivity following thrombin treatment. One anti-FPB serum has been identified that is capable of detecting desarginyl-FPB, and this antiserum has been used in the development of a radioimmunoassay for desarginyl-FPB. Thus, by precisely defining the specificity of anti-FPB sera, it has been possible to identify antisera that are useful, not only in the measurement of FPB, but also in the detection of other important related molecules, such as B beta 1(Pyr)-42(Arg) and desarginyl-FPB. The immunochemical detection of these FPB-related peptides should provide useful information concerning the action of proteolytic enzymes, such as plasmin on the NH2-terminal segment of the B beta-chain of fibrinogen, and of carboxypeptidase-B on free FPB, in human plasma.

Cross Reactions↗
PubMed1979

In vitro stability of human fibrinopeptide B.

In anticipation of a future clinical application of plasma fibrinopeptide B (FPB) measurement, we studied the stability of FPB in an ultrafiltrate of normal plasma, normal urine and alkaline buffer by measuring the immunoreactivity of the peptide by FPB radioimmunoassay using anti FPB serum (R-29). FPB was unstable in an ultrafiltrate of plasma and urine and demonstrated a temperature dependent loss of activity. In plasma ultrafiltrate the loss of immunoreactivity was not significant during the first 24 hours, however, 92% of the peptide activity was lost at the end of seven days at 25 degrees C and 37 degrees C. The rate of FPB degradation in urine was comparable. The peptide was stable in an alkaline buffer (pH 8.5) at temperatures ranging from -10 degrees C to 37 degrees C or in plasma ultrafiltrate or urine when incubated at -10 degrees C. Treatment with carboxypeptidase B or leucine aminopeptidase for two hours at 37 degrees C (enzyme/substrate molar ratio of up to 1:100) did not cause a loss of FPB immunoreactivity. EDTA (1.0 mM) and Trasylol (500 units/ml) completely stabilized the peptide in a plasma ultrafiltrate.

Carboxypeptidases↗