Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin.

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.

Four states of tyrosine residues in the fibrinogen molecule.

The ionization of tyrosine residues in fibrinogen was studied by a spectrophotometric method. The total of 100 tyrosine residues in the fibrinogen molecule was classified into four states: (1) 28 tyrosine residues with pK 10.1 (m = 1.0). (2) tyrosine residues with pK 11.5 (m = 1.0), (3) 20 tyrosine residues with pK 12.2 (m = 3.0) and (4) 10 tyrosine residues non-ionizable. When fibrinogen was treated with 4 M guanidine . HCl, all of the tyrosine residues became ionizable with the ionization characteristics of pK 10.1 (m = 1.0). The ionization characteristics of tyrosine residues in plasmin-digested fibrinogen were similar to those of fibrinogen, while in CNBr-treated fibrinogen they were fairly different. The value, m, stands for the number of hydroxyl ions involved in the ionization of a tyrosine residue.

Fibrinogen and the early stages of polymerization to fibrin as studied by dynamic laser light scattering.

Human fibrinogen and the polymerization of fibrin after activation by the enzymes, thrombin and Batroxobin, were studied by means of dynamic laser light scattering (DLS). The apparent diffusion constant, D, for fibrinogen was measured and has a value of (1.80 +/- 0.42) X 10(-7) cm2 X s-1. D was found to contain contributions from the translational diffusion constant (Dt) as well as from the rotational diffusion constant (Dr). A comparison between experimental and calculated values of Dr and Dt suggests that fibrinogen in the absence of added Ca2+ expresses a certain degree of flexibility, while it is straightened in the presence of added Ca2+. The time dependence of D showed periodic oscillations, while the average D values decreased with time. Thrombin and Batroxobin caused similar behaviour of D. The period length was related to the enzyme concentration, clotting time (Ct) and the rate of release of fibrinopeptide A (FPA). No periodic oscillations were observed in experiments where the enzyme was replaced by saline, or in experiments using a dysfunctional fibrinogen (fibrinogen Aarhus) which displayed slow rates of FPA-release and polymerization. We propose that the periodic oscillations in a system far from equilibrium may be explained by conformational changes occurring in the fibrinogen molecule during enzyme activation and polymerization.

Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization.

Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.

Measurement of fibrinogen in dog liver cell fractions by electroimmunoassay and radioimmunoassay.

Dog liver was fractionated and the amount of fibrinogen, in the various cell fractions (organelles) involved in the secretory process, was determined both by the Laurell electroimmunoassay and by radioimmunoassay. An unequal distribution of fibrinogen was noted for each cell fraction after an appropriate correction was made. This involved the estimation of the level of contamination of each cell fraction by soluble (blood) fibrinogen. It was observed that significant amounts of added 125I-labeled fibrinogen could be found in each organelle following cell fractionation. This was especially true for the rough endoplasmic reticulum which contained as much as 31% of the added tracer. Both the smooth endoplasmic reticulum and the Golgi fractions contained much lower amounts of added tracer. Using this correction factor, it was found that the rough endoplasmic reticulum contains about 0.1-0.2 micrograms fibrinogen per mg organelle protein. On the other hand, the smooth endoplasmic reticulum and Golgi fractions contained much more fibrinogen. The fibrinogen concentration ranges found for the latter two organelles were 1.9-5.0 and 1.5-5.7 micrograms fibrinogen per mg organelle protein, respectively.

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity.

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.

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.

Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis.

The native fibrin gel structure formed in vitro from plasma samples was examined by liquid permeation of the hydrated fibrin gel networks in 18 men who had suffered a myocardial infarction before the age of 45 years and in 20 control subjects. Patients with an elevated plasma fibrinogen concentration had a considerably lower fibrin gel porosity (permeability coefficient, Ks) compared with patients with a normal plasma fibrinogen level and with controls. The calculated fiber mass-length ratio of the fibrin gel networks was decreased in both patient groups. Gel porosity differed markedly between individuals at a given plasma fibrinogen concentration. Fairly strong inverse correlations were found between plasma orosomucoid level on the one hand and Ks (r = -0.617, p less than 0.01) or fiber mass-length ratio (r = -0.499, p less than 0.05) on the other. The low density lipoprotein (LDL) cholesterol concentration also correlated inversely with Ks (r = -0.471, p less than 0.05) and fiber mass-length ratio (r = -0.522, p less than 0.05). Significant inverse relations, which were independent of plasma fibrinogen and lipoprotein concentrations, were detected between Ks (r = -0.519, p less than 0.05) and calculated fiber mass-length ratio (r = -0.723, p less than 0.001) and number and severity of coronary artery stenoses determined by angiography. A proneness to formation of tight, rigid and space-filling fibrin network structures with small pores thus appears to be associated with premature coronary artery disease.

Association between bleeding time and platelet adherence to artery subendothelium.

The efficacy of five different factor VIII-von Willebrand factor (FVIII-VWF) preparations in mediating adherence of blood platelets to damaged vessel walls was tested in an annular perfusion chamber utilizing human arteries and reconstituted blood. FVIII-VWF-purified by Sepharose CL-4B chromatography and von Willebrand factor prepared from this preparation by dissociation with 0.25 M CaCl2 followed by Sepharose CL-6B chromatography were equally effective in mediating platelet adherence as FVIII-VWF in cryoprecipitate and in plasma from normal subjects. A commercial concentrate of FVIII-VWF (Hemofil, Hyland) used for the treatment of haemophiliacs did not mediate platelet adherence at normal levels of FVIII-VWF related properties. A recently developed high-purity FVIII-VWF preparation (Concentrate II) containing multimers of high molecular weight normalized the platelet adherence. Platelet adherence in plasma obtained from two patients with von Willebrand's disease (VWD) was impaired, but plasma samples obtained following treatment with Concentrate II mediated normal platelet adherence. The normalization of platelet adherence paralleled the normalization of the bleeding time. This platelet adherence assay offers an inexpensive and efficient in vitro tool to test the efficacy of FVIII-VWF preparations designed for VWD patients. Preparations such as cryoprecipitate and Concentrate II mediated the platelet adherence and normalized the bleeding time. The commercial preparation did not mediate platelet adherence and had no effect on the bleeding time.

Application of thrombin based fibrin glue and non-thrombin based batroxobin glue on intact human blood vessels: evidence for transmural thrombin activity.

An alternative method of uniting small diameter vessels to obtain tissue union while limiting the thrombogenic effect of suture placement at a vessel anastomosis involves the use of a thrombin based fibrin glue as a surgical sealant. This investigation addresses whether the in vitro application of a thrombin based glue (TG), or batroxobin glue (BG), a non-thrombin based glue made with the snake venom enzyme batroxobin, alters intravascular platelet deposition (PD) or cleaves blood fibrinogen, as measured by fibrinopeptide A (FPA) production, when the respective glue is applied to the external surface of an intact human placental artery or an artery with an anastomosis. When TG was applied to the adventitial surface of an intact vessel or an anastomosis (n = 7) of control and experimental vessels, there was a significant increase in intraluminal platelet deposition, an effect not realized with BG (n = 12, intact vessel TG p = 0.01, BG p = 0.66, anastomosis TG p <0.01, BG p <0.01). Both TG and BG significantly increased FPA levels when human whole blood was perfused through both intact vessels or vessels containing an anastomosis when compared to control vessels (intact vessel TG and BG p <0.01, anastomosis TG and BG p <0.01). Labelled thrombin studies document the rapid passage of thrombin through an intact vessel wall or vessels with an anastomosis when TG was applied to the adventitial surface of the vessel. The data suggest that TG and BG are drug delivery systems for their respective enzymes that either pass through or transfer a message across not only a surgically created anastomosis, but also an intact vessel wall.

Fibrinogen: evolution of the structure-function concept. Keynote address at fibrinogen 2000 congress.

Coagulation of blood is such an evident phenomenon that its observation can be traced back to earliest historical times. The great philosophers and physicians of antiquity discussed and provided interesting explanations. However, it was not until the end of the seventeenth century that the structural component of the blood clot was described by Malpighi as a white fibrous substance. In the middle of the nineteenth century this was identified as a constituent of pathological thrombi and given the name fibrin. At about that time its precursor in blood, fibrinogen, was isolated in a highly purified form by Hammarsten who suggested that, preceding fibrin formation, activation of fibrinogen by thrombin occurred by limited proteolysis. The activation mechanism was eventually clarified in the 1950s. It was shown to proceed in two discrete steps, by removal of low molecular weight activation peptides. Ferry postulated, based on physicochemical observations, that the activated molecules aligned in a half-staggered fashion to form polymers. The rapid post-war development of biochemical technology permitted evaluation of the primary structure of fibrinogen. With that followed identification of molecular domains in the activated firbinogen molecules that participate in polymer formation, crosslinking of polymeric structures, and domains for cellular attachment. Crystallization of fragments and, recently, of the entire molecule has confirmed and extended this knowledge. Lately, it has also been possible to obtain detailed information on the architecture of the fiber network in the fibrin gel. The gel structure is primarily determined by the initial rate of fibrinogen activation, but without infringement of this primary rule, several factors in blood may modulate the structure. Fibrinogen and fibrin play important roles in normal hemostasis, wound healing, and pathological processes, such as thrombosis and atherosclerosis.

Characterization of a fibrin glue-GDNF slow-release preparation.

One novel method to deliver trophic factor locally in the CNS is to mix it into fibrin glue. In the present studies, [125I]-labeled GDNF-containing fibrin glue balls were used to determine binding and spread of the trophic factor. First, the binding of different concentrations of [125I]-labeled GDNF in fibrin glue was determined in vitro. Within the six concentrations used (from 200 nM to 0.004 nM, 0 M as control), there was a strong linear correlation between the [125I]-GDNF concentration and the recovered radioactivity (r = 0.992). The mean bound radioactivity in 16 samples with 4 nM [125I]-GDNF was 71262 +/- 2710 CPM, and accounted for 89.8% of the mean initial count of free [125I]-GDNF (79369 +/- 3499 CPM). Second, [125I]-GDNF-containing glue balls were implanted into the anterior chamber of adult rats. The implanted fibrin glue balls decreased in size with time, but could still be identified on the irises 2 wk after implantation. Radioactivity was concentrated at the implantation sites in the early stages with a distribution in the surrounding iris tissue, which became separated into focal radioactive spots at the third week. Counts of radioactivity were significantly higher in the [125I]-GDNF glue ball-implanted irises than controls until 14 days after implantation. A study of the [125I] decay over time using least-squares linear regression demonstrated first-order kinetics (r = -0.98, p <0.02) with k = 0.0091 and T 1/2 = 76 h. Finally, [125I]-GDNF-containing glue balls were implanted in the spinal cord of adult rats. Radioactivity was concentrated at the implantation sites in the early stages and was later distributed more widely in the surrounding thoracic cord. The [125I]-GDNF-containing glue degraded over time and became a porous meshwork with decreasing radioactivity at the later time points. Radioactivity in the spinal cords subjected to implantation of [125I]-GDNF-containing glue balls was higher than in controls for 14 days. Study of the [125I] decay by time with least-squares linear regression demonstrated first-order kinetics (r = -0.97, p = 0.001) with T 1/2 = 75.6 h. We conclude that the trophic factor GDNF becomes bound in the fibrin glue matrix from which it is gradually released. Our results suggest that fibrin glue is an effective substrate for keeping a trophic factor localized in situ for a finite period, protected from the circulation, surrounding aqueous humor or CSF.

Calcium and fibrin gel structure.

Calcium ions (Ca), when present in the gel forming system, were shown to influence liquid permeation of the gels formed, as judged from the Ks-values (permeability coefficients) of the final gels. On increasing Ca concentration, the Ks-values for Fibrin I and Fibrin II gels increase and a maximum is reached at about 10-20 mM for gels formed at ionic strengths between 0.18 and 0.24. For both gels, clotting times (Ct) were shortened on increasing Ca concentration and the shortening was accompanied by increase in Ks. Magnesium also shortened Ct but had no appreciable effect on Ks. The rate of activation of fibrinogen (release of FPA and FPB) was not much affected by Ca, but the activation required for gelation at Ct, decreased with increasing Ca concentration. After the gels were formed, the removal of Ca by EDTA did not change the flow properties. Our results showed that Ca is of importance for formation of the fibrin gel structure, but it may be of minor importance for preservation of the gel structure after its formation. There is a difference between Fibrin I and Fibrin II gels with regard to Ca dependence. The role of calcium in gelation as well as its physiological implications is discussed.

Thrombin-induced platelet reactions: effect of substrates and inhibitors on binding of thrombin and serotonin release.

Binding of 125I-thrombin to platelets and subsequent serotonin release were confirmed. The binding of unaltered thrombin to platelets was also measured by a new technique using a chromogenic substrate (S-2238). As compared to 125I-thrombin, this method gave similar constants for binding to the high affinity binding site, but lower for binding to the low affinity binding site. Furthermore, the results suggest that the platelets have two classes of independent binding sites. Substrates and inhibitors of thrombin inhibited thrombin induced serotonin release, suggesting that the release reaction depends on the proteolytic activity of thrombin. The serotonin release was more inhibited than the binding of thrombin, suggesting that the platelet binding site and the active site of thrombin are located in different parts of the thrombin molecule.

Fibrin gel structure and clotting time.

Liquid permeation studies of fibrin gels, which were formed in the presence of thrombin (Fibrin II) and in the presence of Batroxobin (Fibrin I), showed that those gels have distinctly different flow properties. For both gels the permeability coefficient (Ks) and average pore size varied depending on the conditions for gel formation. Low pH and ionic strength favour high Ks and large pore sizes, whereas high pH and ionic strength produce gels with low Ks and small pore sizes. Parallel turbidity studies showed correlations between the optical properties of the gels and permeation data. Of particular importance was the finding that the clotting time (Ct) is directly related to Ks of the final gels. Thus events preceding gel formation determine the gel structure. It is proposed that the average lengths of polymers formed prior to gelatin varies directly with Ct. Shorter polymers give infinite net works (gels) which are tight and longer polymers provide for formation of infinite net works which are more porous. At the same Ct, the net work formed with thrombin is, over a wide range of Ct, tighter than that formed with Batroxobin. It was also found that Ks is inversely related to the fibrin concentration (C) in the gels. The ratio Ct/C (permeability index) is thus an important determinant for the gel structure. The applicability of the permeability index to whole blood as a predictive test for thromboembolic and bleeding conditions is discussed.

Purification of the factor VIII complex.

Two high purity factor VIII concentrates, type I and type II were developed for clinical trials in patients with hemophilia A and von Willebrand's disease. Fresh frozen plasma containing 1% polyethylene glycol 4000 was thawed to form cryoprecipitate, which was subsequently dissolved in citrate buffer. By addition of glycine buffer to a final concentration of 2.0 M at 26 degrees C, the bulk of fibrinogen was precipitated while factor VIII remained in solution. Factor VIII was precipitated from the glycine supernatant by addition of solid sodium chloride. The recovery of factor VIII procoagulant activity (VIII:C) per kg plasma was 271 +/- 23 units (n = 4) and 386 +/- 47 units (n = 7) for the type I and the type II preparations, respectively, while the recovery of von Willebrand factor related activity (ristocetin cofactor, VIIIR:RC) was 518 +/- 75 units and 718 +/- 90 units per kg plasma, respectively. The specific activity (units per mg protein) of VIII:C in the type I and type II preparations were 2.53 +/- 1.02 and 7.56 +/- 1.33, respectively. The specific activity (units per mg protein) of VIIIR:RC for the type I and type II preparations were 4.86 +/- 2.32 and 13.6 +/- 3.7, respectively. VIIIR:Ag was present as multimers in both preparations, and the multimeric pattern was similar to that of normal plasma. The preparations have the ability to correct the prolonged bleeding time in severe von Willebrand's disease. The factor VIII complex in the type II preparations was further purified by gel filtration on Sephacryl S-1000. This preparation was free of fibrinogen and fibronectin. Its specific activity in terms of VIII:C was 47 u/mg protein and 104 u/mg protein in terms of VIIIR:RC. The subunit of reduced factor VIIIR:Ag had Mr of 210 Kd on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

On the occurrence of thrombin-like enzymes in mosquitoes.

A thrombin-like enzyme has been identified in mosquitoes and partially purified. The enzyme preparation displays on SDS gel electrophoresis a major protein band of about 22 kDa and one minor of about 28 kDa. The enzyme preparation cleaves a synthetic substrate (S-2238) for thrombin with Km 113 mumol/L (as compared to 3 mumol/L for thrombin). The enzyme clots fibrinogen and plasma. During activation of fibrinogen, fibrinopeptide A (but scarcely fibrinopeptide B) is released. The enzyme is not inhibited by hirudin and activates (if at all) factor XIII differently from thrombin. Predominantly gamma-dimers are formed in the cross linking process. As compared to thrombin a larger extent of activation is required to induce gelation (clotting) by the mosquito enzyme. At a given clotting time the enzyme produces tighter gel structures than thrombin. In its action the enzyme resembles the snake venom enzyme, batroxobin.

An in vivo study of a new factor VIII high purity preparation.

The efficacy of high purity preparations of factor VIII complex are usually lacking effect on the bleeding time in von Willebrand's disease. In this report we have studied the efficacy of a new high purity factor VIII preparation in two von Willebrand and two hemophilia A patients. The prolonged bleeding time in the patients with von Willebrand's disease was corrected after infusion of the preparation and a secondary rise in factor VIII procoagulant activity (VIII:C) demonstrated. The biological half-life time of VIII:C, in the two hemophilia A patients, was about 15 hrs. The investigation shows that this particular preparation is capable of correcting the prolonged bleeding time in von Willebrand's disease.

The fibrinogenolytic and procoagulant activity of southern copperhead venom enzymes.

The effect of four isolated proteolytic fractions of Agkistrodon contortrix contortrix venom on fibrinogen and its polymerizing ability was investigated by means of radioimmunoassay for fibrinopeptides A and B (FPA, FPB), fibrinogen fragment B beta 15-42 and by polyacrylamide gel electrophoresis. One enzyme (MW approximately 68,000) predominantly released FPB and activated fibrin stabilizing factor. The relative rates of the FPA/FPB release markedly depended on temperature. Calcium ions caused the decrease of FPB cleavage rate without any noticeable change in cleavage rate of FPA. The enzymes of lower molecular weight degraded fibrinogen into a family of degradation products.

The action of prothrombin activated by Ecarin on fibrinogen.

Highly purified human prothrombin was activated by Ecarin, a prothrombin activating principle of Echis carinatus venom and the generated thrombin-like activity was investigated. Kinetics of the release of fibrino-peptides A and B (FPA, FPB) from human fibrinogen was estimated using radioimmunoassay technique. The direct proteolytic action of Ecarin on fibrinogen was studied by means of polyacrylamide gel electrophoresis and by radioimmunoassay for fragment B beta 15-42 of fibrinogen. In a system containing Ecarin, fibrinogen and prothrombin both fibrinopeptides were cleaved off at a rate that was essentially similar to that observed with thrombin, the cleavage off of FPB being in the initial stage always slower than the release of FPA. At physiological concentration of prothrombin and at low concentration of Ecarin all FPB were liberated while less than 1% of B beta 15-42 immunoreactivity was released. It was possible to accelerate the release of FPB when prothrombin was activated by Ecarin in the presence of Fibrin I gel (formed from fibrinogen by Batroxobin which liberates FPA only) instead of fibrinogen. Fibrin I thus constituted a more favourable substrate than fibrinogen with regard to release of FPB. The results indicate that the coagulant activity generated from prothrombin by Ecarin is thrombin-like. No intermediate product capable of marked cleavage off of FPA only was detected.