The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity

Plasma fibrinopeptide A (FPA) concentrations were measured in clinical blood samples incubated in the collecting syringe for different time periods before addition to heparin and Trasylol, and the rate of in vitro generation of FPA was calculated as the mean increment in FPA concentration per minute over the linear portion of the generation curve. 36 normal individuals had a mean plasma FPA level of 0.64 +/- 0.56 pmol/ml and an FPA generation rate of less than 0.5 pmol/ml per min. Clinical samples with elevated plasma FPA levels manifested slow (less than 1 pmol/ml per min) (28 patients) or rapid FPA generation (greater than 1 pmol/ml per min) (33 patients). Slow FPA generation was found in 10/10 patients with venous thrombosis, in 4/4 with aortic aneurysm, and in several patients with acquired hypofibrinogenemia. In one such patient, addition of fibrinogen resulted in rapid FPA generation whereas thrombin addition was without effect. Rapid FPA generation was generally linear, was usually associated with slower fibrinopeptide B generation and was inhibited by parenteral or in vitro heparin. It is thought to reflect increased thrombin activity and was seen in patients with pulmonary embolism, active systemic lupus erythematosus, renal transplant rejection, and after infusion of prothrombin concentrates. The initial rate of FPA cleavage by thrombin at fibrinogen concentrations from 0.05 to 4 mg/ml showed little change between 2 and 4 mg/ml with a Km of 2.99 muM. At a fibrinogen concentration of 2.5 mg/ml the FPA cleavage rate was 49.2 +/- 1.6 nmol/ml per min per U of thrombin. Exogenous thrombin added to normal blood generated 21.7 nmol/ml per U of thrombin FPA in the first minute with a nonlinear pattern reflecting inactivation of thrombin and the presence of alternative substrates. Hence, the thrombin concentration in the blood cannot be calculated from the FPA generation rate. The FPA generation rates in clinical samples with rapid generation (1-28 pmol/ml per min) could be produced by 2 X 10(-5) to 5.6 X 10(-4) thrombin U/ml acting on purified fibrinogen at physiological conditions of pH, ionic strength, and temperature.     

The effect of transmural pressure on pumping activity in isolated bovine lymphatic vessels

1. Isolated preparations of bovine mesenteric lymphatics containing about seven valved segments were cannulated and set up in a perfusion system so that, when the preparation was not contracting, the inflow and outflow pressures were exactly equal and there was no flow through the preparation. 2. Transmural pressure was varied by raising or lowering the inflow and outflow pressures simultaneously by the same amount. 3. The isolated vessels showed rhythmic spontaneous activity; it consisted of quick contractions which spread rapidly over the entire preparation, each followed by a rapid relaxation and a diastolic pause. 4. With each contraction, the preparation decreased in both length and diameter and generated an outflow pressure which pumped fluid in the direction determined by the orientation of the values. 5. Raising the transmural pressure in the preparation increased the output of the preparation; this was achieved by an increase in both the frequency and force of the individual contractions. 6. It was concluded that bovine lymphatics could propel fluid by their intrinsic activity at a rate which was related to the degree of distension of their walls.     

Identification and characterization of the thrombin binding sites on fibrin

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing gamma' chains, which are fibrinogen gamma chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 microM respectively). The presence of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on gamma' chains is a constitutive feature of fibrin as well as fibrinogen.     

Amidase activity and thermal stability of human thrombin

Previous studies of amidase activity of human alpha-thrombin have yielded variable results and the decrease of this activity as a function of time and temperature has never been quantified. As this protease is an efficient tool in biochemistry and biotechnology thanks to its extreme selectivity, amidase activity and stability of thrombin were investigated with the synthetic substrate Tos-Gly-Pro-Arg-pNa. Enzyme activity as a function of temperature showed an optimum peak at 45 degrees C. The pH dependence of the activity showed a maximum around 9.5. The addition of NaCl promoted an increase of the activity. Stability of thrombin decreased rapidly when increasing the temperature from 25-45 degrees C and when diluting the enzyme. The presence of glycerol and ethylene glycol promoted a small increase of thrombin half life, whereas polyethylene glycol had a more pronounced positive effect even at very low concentrations.     

Comparative study of biological glues: cryoprecipitate glue, two-component fibrin sealant, and "French" glue

BACKGROUND: Although biological glues have been used clinically in cardiovascular operations, there are no comprehensive comparative studies to help clinicians select one glue over another. In this study we determined the efficacy in controlling suture line and surface bleeding and the biophysical properties of cryoprecipitate glue, two-component fibrin sealant, and "French" glue containing gelatin-resorcinol-formaldehyde-glutaraldehyde (GRFG). METHODS: Twenty-four dogs underwent a standardized atriotomy and aortotomy; the incisions were closed with interrupted 3-0 polypropylene sutures placed 3 mm apart. All dogs had a 3- by 3-cm area of the anterior wall of the right ventricle abraded until bleeding occurred. The animals were randomly allocated into four groups: in group 1 (n = 6) bleeding from the suture lines and from the epicardium was treated with cryoprecipitate glue; in group 2 (n = 6) bleeding was treated with two-component fibrin sealant; group 3 (n = 6) was treated with GRFG glue; group 4 (n = 6) was the untreated control group. The glues were also evaluated with regard to histomorphology, tensile strength, and virology. RESULTS: The cryoprecipitate glue and the two-component fibrin sealant glue were equally effective in controlling bleeding from the aortic and atrial suture lines. Although the GRFG glue slowed bleeding significantly at both sites compared to baseline, it did not provide total control. The control group required additional sutures to control bleeding. The cryoprecipitate glue and the two-component fibrin sealant provided a satisfactory clot in 3 to 4 seconds on the epicardium, whereas the GRFG glue generated a poor clot. There were minimal adhesions in the subpericardial space in the cryoprecipitate and the two-component fibrin sealant groups, whereas moderate-to-dense adhesions were present in the GRFG glue group at 6 weeks. The two-component fibrin sealant was completely reabsorbed by 10 days, but cryoprecipitate and GRFG glues were still present. On histologic examination, both fibrin glues exhibited minimal tissue reaction; in contrast, extensive fibroblastic proliferation was caused by the GRFG glue. The two-component and GRFG glues had outstanding adhesive property; in contrast, the cryoprecipitate glue did not show any adhesive power. The GRFG glue had a significantly greater tensile strength than the two-component fibrin sealant. Random samples from both cryoprecipitate and the two-component fibrin glue were free of hepatitis and retrovirus. CONCLUSIONS: The GRFG glue should be used as a tissue reinforcer; the two-component fibrin sealer is preferable when hemostatic action must be accompanied with mechanical barrier; and finally, the cryoprecipitate glue can be used when hemostatic action is the only requirement.     

Collagen proteins in electrorefining: Rate constants for glue hydrolysis and effects of molar mass on glue activity

Animal glue (collagen proteins) degradation was studied in water and in a simulated copper electrolyte (150 g/L H₂SO₄, 46 g/L Cu²⁺ as CuSO₄) by size-exclusion chromatography. The rate of degradation was relatively slow in pure aqueous solutions, and depending on the temperature and glue concentration, some association to larger molar mass species was observed. For simulated electrolyte in a temperature range of 42 °C to 70 °C and a glue concentration range of 100 to 3000 mg/L, the degradation rate constant was described with the following relation:k′ = 1.5· 10⁷exp (-9951/T), min^-1 The degradation rate was zero order with respect to initial concentration of the protein and first order with respect to acid concentration. The results show that glue degradation under normal tankhouse operation should be rapid, with degradation to number-average molar mass (M _n ) < 10,000 units occurring in about 40 to 80 minutes depending on the mass transfer rate (or mixing) of the electrolyte solution. Samples of glue from three different sources showed almost no difference in degradation rates. Results calculated from the rate equation for glue degradation have been correlated with cathode polarization data from the literature, and the results suggest that critical glueM _n below which the glue loses most of its activity is 3700. Formerly Postdoctoral Fellow with the Department of Chemistry, University of Saskatchewan     

Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels of the human testis

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor L-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.     

Nerve repair using freezing and fibrin glue: immediate histologic improvement of axonal coaptation

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify alpha-subunits of chicken rod and cone transducins (Tr alpha and Tc alpha, respectively), we produced two site-directed antibodies which discriminate between bovine Tr alpha and Tc alpha. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine Tr alpha specifically stained the rod outer segments. On the other hand, the antibody against bovine Tc alpha uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against Tc alpha recognized a single band of chicken photoreceptor protein, whose molecular weight (42,000) was in good agreement with that of bovine Tc alpha (41,000). The antibody against Tr alpha recognized a protein having the same molecular weight as that of bovine Tr alpha (39,000). These observations strongly suggested that all types of chicken cone cells have a single common Tc alpha (42 kDa) structurally related to bovine Tc alpha, though each cone cell type has a distinct visual pigment.     

Stabilization and accurate trimming of nerve ends: practical use of fibrin glue: technical note

OBJECTIVE: Peripheral nerve transection usually results in protrusion of the endoneurial contents ("mushrooming"). Trimming of the nerve ends before repair is often necessary to achieve cut nerve end planes. In this technical report, we describe a technique for stabilization and accurate trimming of nerve ends using fibrin glue. SURGICAL TECHNIQUE: The nerve ends of divided peripheral nerves are coated with fibrin glue and subsequently trimmed using a razor blade before repair. RESULTS: When fibrin glue is applied, a firm layer with a rubbery consistency is formed around the nerve. This layer stabilizes the nerve ends during trimming, and a clear-cut plane of the nerve can be achieved. Moreover, the fibrin glue stabilizes the nerve ends during manipulations caused by suturing of the nerve. CONCLUSION: The technique results in easier handling of the nerve during trimming and manipulation, minimal tissue damage to the nerve, and a clear-cut plane of the nerve.     

Microangioarchitectonics and microtopography of the blood vessels of the human stomach

The data of previous investigations on general principles of microarchitectonics of the human intraorganic gastric vessels have been checked. Peculiarities of branching and course of the stomach intraorganic vessels, arterio-venous anastomoses, twisted arterioles, sinusoid venules and veins confirm activity and variability of the organ's circulation at microcirculatory level. Certain slight differences in diameters and number of microvessels have been revealed in some anatomical parts of the serous membrane and submucous layer of the stomach. Maximal differences in vessel diameters of the microcirculatory bed and in number of capillaries per 1 mm2 have been revealed in the most active layers of the gastric wall--in muscular and mucous membranes. In the muscular membrane, large vessels and greater number of capillaries have been revealed in the area of the greater curvature and the pylorus. In the serous membrane in the pyloric area and in the area of the smaller curvature, microvessels have smaller diameters and the number of capillaries per square unit is less.     

Localization and expression of tissue kallikrein and kallistatin in human blood vessels

Human lysosomal acid lipase/cholesteryl ester hydrolase (hLAL) is essential for the intralysosomal metabolism of cholesteryl esters and triglycerides taken up by receptor-mediated endocytosis of lipoprotein particles. The key role of the enzyme in intracellular lipid homeostasis is illustrated by two lysosomal storage diseases inherited as autosomal recessive traits. Wolman disease, associated with deficient hLAL activity, leads to massive intracellular substrate accumulation and is always fatal in early infancy. Cholesteryl ester storage disease (CESD), in contrast, is characterized by very low levels of enzymic activity sufficient to allow survival of the affected patients into adulthood. In order to elucidate the underlying molecular defects in Wolman disease, we have characterized the hLAL gene in two female Wolman patients of German and Turkish origin by SSCP and DNA sequence analysis. Our results demonstrate that the German proband was compound heterozygous for an 8-bp deletion in exon 3 and a 2-bp deletion in exon 4 of the hLAL gene. These frameshift mutations lead to protein truncation at amino acid positions 24 and 116 and to complete loss of hydrolytic activity. The Turkish proband, in contrast, was homozygous for a G(1064)-->T substitution in exon 10 of the hLAL gene which converts the completely conserved glycine (GGG) residue at position 321 of the mature enzyme to tryptophan (TGG). In vitro expression of the hLAL(Gly(321)-->Trp) cDNA construct revealed that the amino acid replacement results in a more than 99% reduction of neutral lipid hydrolysis. The mutations provide new insights into the molecular basis of Wolman disease which is apparently more heterogeneous at the genetic level than cholesteryl ester storage disease.-Lohse, P., S. Maas, P. Lohse, A. C. Sewell, O. P. van Diggelen, and D. Seidel. Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease.     

Use of autologous fibrin glue in dermatologic surgery: application of skin graft and second intention healing

OBJECTIVE: To evaluate the efficiency of biological sealant, an autologous fibrin glue, in dermatological surgery. DESIGN: Randomized clinical trial. SETTING: The Dermatology Service of Hospital das Clinicas, Universidade de Campinas (UNICAMP), referral center. PATIENTS: 14 patients with malign epithelial cutaneous tumors participated in the evaluation, each having two tumors, generally facial and symmetrical, in order to perform a comparative evaluation on the same individual. PROCEDURES: The glue was prepared beforehand with a sample of autologous blood. Surgical extirpation of the tumor was followed by grafts or second intention healing. OUTCOMES: The efficiency of the sealant was then evaluated in relation to hemostasis, adhesion, surgical time and evolution of the granulation tissue, clinically and histologically. RESULTS: Immediate hemostasis and graft adhesion, with a significant reduction of surgical time, and in the open wounds there was immediate hemostasis and a clinical increase in granulation tissue, but with no histological differences among the groups on the 7th day. CONCLUSION: It is an adjuvant resource in skin cancer surgery.     

Effects of tannins from Geum japonicum on the catalytic activity of thrombin and factor Xa of blood coagulation cascade

Bioassay-guided fractionation of the MeOH extract of the whole plant of Geum japonicum led to the isolation of seven known tannins. They were identified by spectroscopic methods as penta-O-galloyl-beta-glucoside (1), pedunculagin (2), 2, 3-(S)-hexahydroxydiphenoyl-D-glucose (3), tellimagrandin II (4), 2, 6-di-O-galloyl-D-glucose (5), casuariin (6), and 5-desgalloylstachyurin (7). Compounds 1, 2, 4, 6, and 7 showed potent anticoagulant activity by significantly prolonging the clotting of rabbit plasma. The inhibitory effect of 2 was competitively directed against thrombin. Its IC50 values for inhibition of the enzymatic activity of thrombin on synthetic substrate and fibrinogen were 0.18 and 0.15 microM, respectively. On the other hand, compounds 1, 4, 6, and 7 are mixed noncompetitive inhibitors of thrombin. Their IC50 values for inhibition of fibrinogen hydrolysis were twofold to sevenfold lower than those for the inhibition of synthetic substrate hydrolysis. Factor Xa was competitively inhibited by compounds 1, 2, 4, 6, and 7. The phenolic hydroxyl groups of the active tannins appear to play an important role in their inhibitory effect on the enzymes.     

X-ray and neutron scattering density profiles of the intact human red blood cell membrane

Hemoglobin-free human red blood cell membranes have been prepared with glutaraldehyde to maintain an intact structure on partial dehydration. Treatment of resealed ghosts with poly(L-lysine) produced an essentially constant structural unit and permitted correlation of electron microscopy results with x-ray and neutron diffraction profiles. These profiles provide detailed information, for the intact membrane, on the location and relative distribution of lipids and proteins.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

The effect of batroxobin on coronary segmental resistance and coronary blood flow in acute myocardial ischemic dogs

To study the effects of batroxobin on coronary circulation and cardiac performance in acute myocardial ischemia, Batroxobin was given intravenously to dogs with experimental coronary stenosis. A dose-dependent increase of coronary blood flow (CBF) was observed. Forty minutes after batroxobin (2 BU.kg-1 at infusion rate 0.1 BU.kg-1.min-1) administration, CBF increased by 12% (P < 0.05), small coronary resistance(RS) decreased from 4.1 +/- 0.5 to 3.2 +/- 0.5 mmHg.min.ml-1 (P < 0.01), while large coronary resistance(RL) changed insignificantly from 3.9 +/- 0.8 to 3.8 +/- 0.7 mmHg.min.ml-1 (P > 0.05). Two hours following drug administration, the changes in CBF, RS and RL still remained and RT decreased by 13% (P < 0.05). The + LV(dp/dt)max and -LV(dp/dt)max increased by 14% and 16% (P < 0.05) respectively compared with those in control group. It is concluded that batroxobin improves the ischemic canine coronary circulation and cardiac performance by way of lowering the small coronary resistance and thus increasing CBF. The data also suggest the benificial effect of batroxobin in acute myocardial ischemia.     

Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin gel from plasma cryoprecipitate containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on the gel at different seeding ratios. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained within 10-15 days in culture. Early expression of basal membrane proteins was detected by immunostaining with laminin and type IV collagen antibodies. Cell proliferation was detected both in the epidermal layer and in the fibroblast embedded in the gel as assessed by BrdU incorporation. Detachment of composite cultures from dishes or flasks is a simple and quick procedure without the need for dispase treatment. Grafting of composite cultures to nude mice gave rise to an orderly stratified, orthokeratinized epithelium resembling human epidermis. A number of advantages including a large expansion factor without the need of 3T3 feeder layer, the availability of fibrin/plasma cryoprecipitate from blood banks and the versatile manipulation of composite cultures suggest that this system could be suitable for the definitive coverage of severely burned patients.     

The ability of thrombin inhibitors to reduce the thrombin activity generated in plasma on extrinsic and intrinsic activation

In a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action of influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced. By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki < or = 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1-0.6 microM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.