Addition Of Protamine Sulfate Research Articles

Complexation of Cell-Penetrating Peptide–Polymer Conjugates with Polyanions Controls Cells Uptake of HPMA Copolymers and Anti-tumor Activity

Cell penetrating peptides (CPPs) can mediate effective delivery of their associated drugs and drug carriers intracellularly, however their lack of cell specificity remains a major obstacle for their clinical development. We aimed at improving the cell specificity and therapeutic efficacy of HPMA copolymer-octaarginine (R8) conjugate (P-R8) in cells at the tumor micro-environment. To avoid premature cell-penetration, the positively charged R8 moieties were masked via electrostatic complexation with various polyanionic molecules (heparin sulfate, hyaluronic acid, fucoidan and poly-glutamic acid). We followed the kinetics of the FITC-labeled P-R8 penetration into endothelial and cancer cells over-time after its complexation in vitro and further tested whether the in situ addition of a stronger polycation can trigger the release of P-R8 from the complexes to resume cell penetration activity. A murine model of B16-F10 lung metastasis was then used as an in vivo model for assessing the therapeutic efficacy of the P-R8, loaded with doxorubicin (P-R8-DOX), after its complexation with PGA. The intracellular penetration of P-R8-FITC was reversibly inhibited by forming electrostatic interactions with counter polyanions, and can be restored either gradually over time by dissociation from the polyanions, or promptly following the addition of protamine sulfate. Mice injected with B16-F10 cells and treated with P-R8-DOX/PGA complexes, exhibited a significant prolonged survival times when compared with DOX-treated mice or relative to mice treated with either P-R8-DOX or P-DOX alone. The gradual release of P-R8 from P-R8-DOX/PGA may improve the therapeutic efficacy of water-soluble based nanomedicines for the treatment of solid lung tumors.

Anionic LPD complexes for gene delivery to macrophage: Preparation, characterization and transfection in vitro

In the present study, anionic lipid/peptide/DNA (LPD) complexes consisting of pH-sensitive liposome and protamine were introduced as the carriers targeting RAW 264.7 cell line, which had been reported to be difficult for transfection. The LPD complexes were physically characterized. The pH sensitivities and sizes of liposomes were investigated. The zeta potentials of LPD complexes altered significantly with the addition of protamine sulfate and anionic liposomes. It was demonstrated that the carriers produced an increase in the stability of plasmid DNA against DNase I. The TEM showed that the size distribution of LPD complexes was irregular. In the in vitro transfection, the efficiency of LPD complexes was higher than that of Lipofectamine™ 2000 and protamine/DNA complexes, but lower than that of electroporation. A possible mechanism for the internalization of plasmid DNA mediated by the anionic LPD complexes was also proposed. With a high safety certificated by MTT assay, LPD complexes prepared in this study might be potentially employed as a macrophage gene therapy.

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

Coagulation protein function VI: augmentation of anticoagulant function by acetaldehyde-treated heparin.

Acetaldehyde (AcH) at preincubation concentrations of 447, 89.4, and 17.9 mM potentiates the effects of heparin on the clotting time of plasma. While control plasma clotted in the range of 12.6+/-0.1 to 13.8+/-0.1 sec, and heparin-treated plasma clotted in a range from 131.5+/-2.5 to 168.2+/-1.2 sec, heparin that was preincubated at room temperature for 30 min with 89.4 or 447 mM AcH did not clot plasma in 300 sec. Heparin exposed to 17.9 mM AcH clotted plasma in 193+/-1.1 sec. Ethanol at a 404 mM concentration also prolonged the clotting time of heparin-treated plasma >300 sec, while 202 mM ethanol prolonged the clotting time of heparin-treated plasma from 149.0+/-2.0 sec to 219.5+/-1.7 sec. It is suggested that AcH alters the tertiary structure of heparin by adduct formation, possibly by formation of cyclic acetals with iduronic and glucuronic acids, thereby more readily affecting binding of the glycosaminoglycan to antithrombin III and/or thrombin, prolonging clotting time. Ethanol, which does not react covalently with heparin, might affect its conformation as a consequence of an organic solvent effect. Protamine sulfate prolonged the clotting time of plasma from 13.6+/-0.1 sec to 17.9+/-0.2 sec. Protamine sulfate-treated heparin clotted plasma in 21.0+/-0.4 sec relative to heparin-treated plasma (160.4+/-1.7 sec). In subsequent experiments, AcH-treated protamine sulfate extended the clotting time of protamine sulfate from 17.9+/-0 sec to 33.7+/-0.6 sec. Prior addition of protamine sulfate to AcH-heparin mixtures or heparin to protamine sulfate-AcH mixtures before addition to plasma resulted in clotting times of 22.0+/-0.4 sec and 24.1+/-0.5 sec, respectively, relative to control clotting times of 162.3+/-2.6 sec for plasma-heparin mixtures. These results confirm both the reduction in coagulation time of heparin-treated plasma by protamine sulfate and the prolongation of clotting time of plasma by protamine sulfate. Furthermore, and importantly, they indicate that acetaldehyde-treated protamine sulfate is a more effective anticoagulant than protamine sulfate. It is suggested that reversible adduct formation between acetaldehyde, heparin, and protamine sulfate may occur as a means explaining the essentially identical coagulation time of these mixtures when added to plasma regardless of the order of premixing. Ethanol (404 mM) did not influence protamine sulfate effects. Lastly, the potentiation of the anticoagulant function of heparin by acetaldehyde suggests that a structural modification of the glycosaminoglycan may occur in alcoholics.

Heparin binding growth factors and the control of teratoma cell proliferation.

The role of basic fibroblast growth factor (bFGF) in the control of teratoma cell proliferation was examined. It was found that bFGF stimulates proliferation at low concentrations but induces cell migration at higher doses. These effects could be efficiently counteracted by addition of protamine sulphate. Moreover the bFGF gene is actively transcribed in primary human testicular embryonal carcinomas but was not expressed in any other embryonic tumour examined. The biological implications of these findings will be discussed.

Heparin and heparan sulphate are inhibitors of human leucocyte elastase

1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(L-alanine)3-p-nitroanilide (50% inhibition of 250 microliters of 10 micrograms of human leucocyte elastase/ml was obtained with 80 microliters of 2.8 micrograms of heparin/ml and 8 micrograms of heparan sulphate/ml). Less significant inhibition at the same concentrations was seen with the other glycosaminoglycans tested: hyaluronic acid and chondroitin sulphates A-C. 2. Heparin and heparan sulphate also strongly inhibited human leucocyte elastase activity towards insoluble human lung elastin, as determined by an e.l.i.s.a. for soluble elastin-derived peptides released by elastolytic activity on the elastin. This inhibition was shown not to be due to a direct interference of the glycosaminoglycans in the e.l.i.s.a. nor to the inhibition causing a change in the size of the elastin-derived peptides. However, unlike the chromogenic assay with n-succinyl-(L-alanine)3-p-nitroanilide as substrate, where heparin was the more effective inhibitor, in this assay system heparan sulphate was the more effective inhibitor (50% inhibition of 100 microliters of 50 ng of human leucocyte elastase/ml was obtained with 100 microliters of 4.5 micrograms of heparin/ml and 0.8 microgram of heparan sulphate/ml). These results suggest that heparin and heparan sulphate, as components of cellular and basement membranes, are likely to have a role in protecting structural proteins, such as elastin, from the proteolytic activity of human leucocyte elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis.

We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.

Protamine sulfate causes pulmonary hypertension and edema in isolated rat lungs.

The polycation protamine sulfate increases microvascular permeability in the kidney by reducing glomerular charge. We have exposed the pulmonary vasculature to protamine sulfate to determine whether electrical charges play a role in protein permeability in lung vascular beds. In anephric rats, protamine sulfate increased hematocrit approximately 25%. With protamine sulfate doses of 0.08 and 0.04 mg/g body wt, lung blood-free wet-to-dry weight ratios were increased (5.24 +/- 0.8 and 4.89 +/- 0.7) compared with control (3.85 +/- 0.3) (P less than 0.05). In isolated, ventilated, and perfused lungs 0.04 mg/g body wt protamine sulfate increased pulmonary arterial pressure from 5.2 +/- 1.4 to 16.3 +/- 3.9 mmHg (P less than 0.01). These lungs gained weight and lung wet-to-dry weight ratios were significantly increased (15.33 +/- 4.26 compared with 6.04 +/- 0.24 for control lungs). Poly-L-lysine, another polycation, also caused significant increases in pulmonary arterial pressure, lung weight, and lung wet-to-dry weight ratios. The addition of diphenhydramine to the perfusate 10 min before the addition of protamine sulfate did not prevent these changes. Heparin (90 U/mg protamine sulfate) reversed the abnormalities. Pulmonary arterial pressure (7.0 +/- 1.1 mmHg) was not significantly different from the control value, lung weight did not increase, and the lung wet-to-dry weight ratio was 6.24 +/- 0.23 (P greater than 0.05). We conclude that polycations have a significant effect on pulmonary vascular resistance and perhaps on permeability.

A simplified functional assay for protein C in plasma samples.

The important role of protein C (PC) in the regulation of hemostasis has been appreciated since the description of patients who were deficient in PC and presented with severe thromboembolic events. The potentially fatal complications associated with PC-deficiency require an early and reliable identification of those patients affected with this inherited disorder. The present study introduces a test procedure for the functional assessment of PC in plasma samples. The test utilizes the thrombin/thrombomodulin complex to achieve complete and rapid formation of activated PC whose proteolytic capacity is subsequently determined with a chromogenic substrate. Homogenate obtained from rabbit lung effectively substituted the purified component thrombomodulin in the assay system. This new approach simplifies the test procedure without losing specificity and accuracy. Proteases, such as plasmin, streptokinase and urokinase did not influence the assay and the inhibitory effect of heparin on the PC-activation could easily be overcome by the addition of protamine sulphate. The PC-activity in a group of unselected patients (n = 50), who did not reveal any abnormalities in global coagulation tests, amounted to 100 +/- 12% (mean +/- SD) with a range from 54 to 143% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Since the synthesis of PC depends on the availability of vitamin K, patients receiving phenprocoumon have also been analyzed. These patients (n = 103) presented 40 +/- 11% residual PC-activity accompanied by a concomitant decrease in PC-antigen levels to 43 +/- 10% (mean +/- SD). The test described is specific, sensitive, less time-consuming and can be performed on a routine basis.

Effect of anticoagulants on the chromogenic Limulus lysate assay for endotoxin.

We have examined the effects of anticoagulants on the chromogenic Limulus lysate assay for endotoxin. The results indicate that both heparin and CPD Adenine 1 have a striking dose related inhibitory effect on the assay. At concentrations of heparin as low as about 30 U/ml there was a 90% reduction in detectable endotoxin. The inhibitory effect of up to 100 U/ml of heparin could be neutralised by the addition of protamine sulphate. Anticoagulants appear to be an additional factor which can adversely affect the Limulus chromogenic assay for endotoxin. This further emphasises the need for alternative approaches to the measurement of endotoxin.

The effect of protamine on serum levels of placental protein 5 (PP5) in normal and abnormal pregnancy: a possible relation to coagulation abnormalities.

The levels of placental protein 5 (PP5) in pregnancy serum show an apparent increase after the addition of protamine sulphate. The difference of the apparent concentrations of serum PP5 in the presence and absence of protamine (delta PP5) was determined in 345 normal and abnormal pregnancies. A positive delta PP5 was less frequent in diabetic pregnancy (45 per cent) and pre-eclampsia (57 per cent) than in normal pregnancy (80 per cent). These findings provide further evidence that PP5 may be involved in the coagulation system, and in particular may relate to abnormal coagulation processes at the placental site.

Specific interaction between placentae protein 5 and heparin

Levels of placental protein 5 (PP5) in paired serum and plasma samples obtained during the second half of pregnancy were measured by radioimmunoassay. The apparent level of PP5 was significantly higher in serum than in heparin-derived plasma. The addition of heparin to late pregnancy serum resulted in a dose-dependent decrease in the apparent PP5 concentrations, the maximal effect occurring at heparin concentrations greater than 4 U/ml. The same phenomenon was observed using purified PP5 and placental homogenate. The effect of heparin was readily reversed by the addition of protamine sulphate. The specificity of the interaction between heparin and PP5 was demonstrated by affinity chromatography with heparin as the ligand. The functional significance of these results are discussed in relation to observations on other placental proteins and proteins with affinity to heparin.

Dysfibrinogenaemia Characterized by Abnormal Fibrin Monomer Polymerization and Normal Fibrinopeptide A Release

Routine testing on plasma from a patient due to undergo a coronary artery bypass graft operation revealed a prolonged thrombin clotting time associated with a normal plasma fibrinogen level when this was determined by a method not dependent upon the rate of fibrin formation. Fibrinogen purified from the patient's plasma by precipitation with beta-alanine also gave a prolonged thrombin time and this confirmed the presence of a dysfibrinogenaemia. Increasing calcium chloride concentration, addition of protamine sulphate and decreasing ionic strength all produced a partial correction of the clotting defect. Addition of normal plasma to patient's plasma failed to correct the prolonged thrombin clotting time and a pH dependence of the defect was also observed. Kinetic studies of fibrinopeptide release, using a specific radioimmunoassay, demonstrated no delay in the release of patient fibrinopeptide A. The functional defect was localized as an abnormality in the polymerization of fibrin monomers by studying fibrin monomers prepared and isolated from plasma and from purified fibrinogen solution. An electrophoretic examination of the patient's fibrinogen using both agarose and polyacrylamide gels failed to demonstrate any alteration in mobility or any structural defect associated with the polypeptide chains A alpha, B beta and gamma. All seven of the living siblings of the propositus and also his daughter showed no abnormality in any clotting assay. However, because the propositus did not suffer from liver disease it has been assumed that the abnormality is genetic in origin.

Dysfibrinogenaemia Characterized by Abnormal Fibrin Monomer Polymerization and Normal Fibrinopeptide A Release

Summary. Routine testing on plasma from a patient due to undergo a coronary artery bypass graft operation revealed a prolonged thrombin clotting time associated with a normal plasma fibrinogen level when this was determined by a method not dependent upon the rate of fibrin formation. Fibrinogen purified from the patient's plasma by precipitation with β‐alanine also gave a prolonged thrombin time and this confirmed the presence of a dysfibrinogenaemia. Increasing calcium chloride concentration, addition of protamine sulphate and decreasing ionic strength all produced a partial correction of the clotting defect. Addition of normal plasma to patient's plasma failed to correct the prolonged thrombin clotting time and a pH dependence of the defect was also observed. Kinetic studies of fibrinopeptide release, using a specific radioimmunoassay, demonstrated no delay in the release of patient fibrinopeptide A. The functional defect was localized as an abnormality in the polymerization of fibrin monomers by studying fibrin monomers prepared and isolated from plasma and from purified fibrinogen solution. An electrophoretic examination of the patient's fibrinogen using both agarose and polyacrylamide gels failed to demonstrate any alteration in mobility or any structural defect associated with the polypeptide chains Aα, Bβ and γ. All seven of the living siblings of the propositus and also his daughter showed no abnormality in any clotting assay. However, because the propositus did not suffer from liver disease it has been assumed that the abnormality is genetic in origin.

Sciatic Nerve Compression During Anticoagulation Therapy

Severe hip pain developed in a patient who was receiving anticoagulation therapy of heparin sodium and warfarin sodium (Coumadin) for femoral vein phlebitis. The patient had exquisite tenderness but no ecchymoses along the sciatic nerve. A drop in the hemoglobin level showed a large volume loss of blood. Retroperitoneal hematoma with lumbar plexopathy was suspected, but a computerized tomographic scan revealed instead hemorrhage into the gluteal muscles. The anticoagulation therapy was corrected with the addition of protamine sulfate and vitamin K; the patient's pain lessened and the hemoglobin level stabilized. Computerized tomography allowed clear visualization of the hematomas in the buttock, and invasive studies were avoided. Hip pain and a falling hemoglobin level in a patient receiving anticoagulation therapy should alert one to the possibility of bleeding into the retroperitoneal space, thigh, or buttock.

The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex

Protamine sulfate was found to precipitate completely the nonactivated [ 3H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [ 3H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [ 3H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800 g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 m m CaCl 2 to the cytosol resulted in the appearance of activated [ 3H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by p I. Protamine sulfate could not affect the separation of activated [ 3H]dexamethasone-receptor complex at salt concentrations above 100 m m NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [ 3H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation.

Isolation of a Cytochrome b6-f Particle from Chloroplasts

System 1 particles were prepared from lettuce chloroplasts by the addition of digitonin and differential centrifugation as described previously (Nelson, N., Dreschler, Z., and Neumann, J. (1970) J. Biol. Chem. 245, 143), but higher detergent concentrations were used. A cytochrome b6-f particle was isolated from System 1 particles by the addition of protamine sulfate and centrifugation. The particle was purified by Bio-Gel and DEAE-column chromatography and ammonium sulfate precipitation. The presence of detergents during purification was required in order to prevent aggregation of the cytochromes. The ratio of cytochrome b6 to cytochrome f in the purified particle was close to 2. The particle contained non-heme iron and phospholipids. It was devoid of chlorophyll but contained carotenes. The calculated minimum molecular weight of the particle was 103,000 per 1 heme c. The molar extinction coefficient of the cytochromes in the particle was determined by measuring the absorbance of the pyridine hemochrome complexes. For cytochrome b6 the molar extinction coefficients obtained were as follows: reduced minus oxidized at 563 nm minus 575 nm, 24,400; and at 563 nm minus 542 nm, 20,700. For cytochrome f the molar extinction coefficients obtained were as follows: reduced minus oxidized at 554 nm minus 538 nm, 20,600; and at 554 nm minus 565 nm 23,000. The E0 of cytochrome f in the particle is +330 mvolts and that of cytochrome b6 is -100 mvolts. The cytochrome b6-f particle from chloroplasts shows similarities with the cytochrome b-c1 complex (Complex III) from mitochondria. Some implications of the present findings for the operation of photosynthetic electron transport pathways are discussed.

Role of Pyruvate and S -Adenosylmethionine in Activating the Pyruvate Formate-Lyase of Escherichia coli

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.

A medium free of agar, serum and peptone for plaque assay of herpes simplex virus.

SummaryMethods have been described for quantitative plaque assay of herpes simplex virus either in bottle culture or in Leighton tubes. The overlay employed was devoid of agar, peptones and serum, which are viewed as undesirable constituents. Use of agar overlay results in production of plaques from only a small fraction of the potential plaque-forming population. The inhibition of plaque formation by agar can be reversed by addition of protamine sulfate at a level of approximately 800 μg/ml. Possible uncertainties associated with the use of mixtures of agar and protamine can be avoided by substitution of methyl cellulose with which a further 10-fold increase in sensitivity of the assay was obtained.

Estimation of the Activation of the Thromboplastic System in Blood During Extracorporeal Circulation

A method is described by which it is possible to demonstrate an activation of the thromboplastic system during extracorporeal circulation.The principle is the same as used in the three-stage thromboplastin activation test where the thromboplastic activity in the plasma sample is estimated by its ability to induce thromboplastic activity in normal, platelet-rich plasma in the thromboplastin activation test. Heparin is neutralized by the combined addition of protamine sulphate and toluidine blue.