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Biomedical subjects

R H Wagner

Publications and source records attributed to R H Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1984

A quantitative method for studying platelet adhesion to collagen.

A simple turbidimetric method is described that permits quantitation of both the number and the rate at which human fixed washed platelets adhere to fibrillar collagen in suspension. Fixed washed platelets were mixed with buffer or test sample in an aggregometer cuvette. Collagen was added, and the change in light transmission was recorded at 37 degrees C. Percent adhesion was obtained from the maximum change in light transmission within 5 minutes, and the adhesion rate was calculated from the initial slope of the adhesion curve. In this system, the percent adhesion was optimal at ionic strengths of 0.1 to 0.15 in a pH range of 7.0 to 8.0. Percent adhesion could be increased either by lowering the platelet number or by increasing the collagen concentration. No adherence of fixed washed platelets to heat-denatured collagen or Cytodex 3 beads was observed. Adhesion rate increased with greater stirring speed, but decreased with increasing concentrations of bovine serum albumin or normal human plasma, but the percent adhesion remained relatively constant. The rate of adhesion in 20% normal human plasma was greater than that in 1% to 4% bovine serum albumin buffer. This suggests that normal plasma contains some factor(s) that can overcome the inhibitory effect of protein on the rate of adhesion of fixed washed platelets to fibrillar collagen.

Blood Platelets↗
PubMed1983

Botrocetin (venom coagglutinin): reaction with a broad spectrum of multimeric forms of factor VIII macromolecular complex.

Botrocetin, originally called venom coagglutinin, is a Bothrops factor that causes aggregation of blood platelets in the presence of the von Willebrand component of the factor VIII macromolecular complex. The complex consists of a series of multimers with a molecular weight of about 1-20 x 10(6). Ristocetin, another agent that causes platelet aggregation dependent on von Willebrand factor, reacts with only the higher molecular weight multimers. We report on the reactivity of botrocetin in relation to the multimeric structure of the factor VIII complex. Several plasmas or plasma fractions with abnormal distribution of the multimeric sizes were examined, including variant von Willebrand disease type IIA with lack of the higher molecular weight forms, commercial antihemophilic factor concentrates with a preponderance of lower molecular weight forms, cryoprecipitate-free plasma containing mainly the smaller multimers, and a chromatographic fraction of plasma containing only the highest molecular weight polymers. Factor VIII-related antigen content was adjusted to 25-100%. All of the preparations lacking the high molecular weight forms caused prompt platelet aggregation with botrocetin, but none of them caused aggregation in the ristocetin test made isochronal with the botrocetin test. The very high molecular weight polymers were equally effective with botrocetin and ristocetin. These findings indicate that the Bothrops factor is reactive with a broad spectrum of high to low molecular weight forms of the factor VIII complex, suggesting that bioassays of von Willebrand factor with botrocetin should correlate better with immunoassays for factor VIII-related antigen and could reflect better the full platelet-aggregating function of the complex than do ristocetin determinations.

Adolescent↗
PubMed1982

Hydrolysis of human platelet membrane glycoproteins with a Serratia marcescens metalloprotease: effect on response to thrombin and von Willebrand factor.

Metalloproteases from at least two Gram-negative organisms selectively hydrolyze the surface of human fixed washed platelets. The protease-treated platelets lose their ability to aggregate with bovine von Willebrand factor and human von Willebrand factor plus ristocetin. The present study reports the membrane glycoprotein alterations and loss of platelet function that occur after incubation of fresh washed human platelets with a purified protease produced by Serratia marcescens. The studies were undertaken in order to examine, using the Serratia protease as an enzymatic probe, the relationship between externally oriented glycoproteins and two known aggregating agents, bovine von Willebrand factor and thrombin. Platelet membrane glycoproteins were analyzed after discontinuous NaDodSO(4)/polyacrylamide gel electrophoresis using both staining with periodic acid-Schiff reagent and autoradiography of (3)H-labeled platelets. Both methods of detection demonstrated that at Serratia protease concentrations above 0.6 mug/ml there was hydrolysis within 3 min of a membrane glycoprotein (M(r) 185,000) corresponding to GPIb in control platelets. The loss of GPIb was accompanied by the appearance in the platelet supernatant of a glycopeptide (M(r) 156,000). Under reducing conditions, the hydrolyzed membrane glycopeptide in the supernatant had a consistently faster migration (M(r) 149,000). Platelets treated with Serratia protease at concentrations sufficient to give maximal cleavage of GPIb were unresponsive to bovine von Willebrand factor and did not aggregate or release [(14)C]serotonin at doses of von Willebrand factor up to 0.33 unit/ml. On the other hand, the response to bovine alpha-thrombin was only minimally impaired by treatment with Serratia protease. These results implicate GPIb in the response by platelets to bovine von Willebrand factor but suggest that surface components other than GPIb play a major role in the response by platelets to thrombin.

Blood Coagulation Factors↗
PubMed1981

A naturally occurring antibody that inhibits fibrin polymerization.

A 13-year-old girl with chronic aggressive hepatitis, postnecrotic cirrhosis, ulcerative colitis, and a coagulation defect acquired an antibody that specifically interfered with fibrin formation. We sought to characterize the antibody and determine the mechanism of its inhibitory activity. The patient's purified fibrinogen was functionally normal; however, the antibody inhibited the self-assembly of fibrin and prolonged the clotting times of the patient's plasma. This antibody, which belonged to the IgG class of immunoglobulins, acted early in the polymerization process to inhibit the association of fibrin monomers, as indicated by a prolonged lag time and a decreased slope in the polymerization curves. It did not inhibit fibrinopeptide cleavage or fibrin cross-linking. Affinity chromatography indicated that the antibody bound strongly to both fibrinogen and fibrin monomer.

Adolescent↗
PubMed1981

Proteolysis of human fixed, washed platelets by gram-negative bacterial metalloproteases: effect on von Willebrand factor-human platelet interactions.

Fixed, washed platelets (FWP) are usually stable to aggregation with von Willebrand factor (vWF) from human and certain animal plasmas over several months of storage. When one lot of FWP lost its stability in less than 1 week, studies demonstrated contamination with Serratia marcescens. Extracellular proteases produced by S. marcescens, as well as by Pseudomonas aeruginosa and Escherichia coli, were found to cause loss of FWP aggregability. Purified proteases were prepared from cell-free culture filtrates of S. marcescens and two different strains of P. aeruginosa. They were used to study the effect on the interaction of FWP and vWF. All three purified proteases destroyed FWP aggregability in a time- and concentration-dependent fashion. The protease produced by S. marcescens (SP) was found to be at least eight times more potent against FWP as a substrate than either of the two P. aeruginosa enzymes. The ability of SP to destroy FWP aggregability was prevented by EDTA and could be restored by the addition of Zn2+ in slight molar excess. When compared with trypsin and chymotrypsin, SP was found to be highly selective in digesting the FWP membrane, even at concentrations greater than that established to give a similar loss of FWP aggregability. SP does not induce aggregation of fresh, washed platelets or PRP, but renders them unaggregable with vWF. These proteases may be useful research tools for studying membranes and vWF-platelet interactions.

Blood Coagulation Factors↗
PubMed1981

The effect of extracellular proteases from gran-negative bacteria on the interaction of von Willebrand factor with human platelets.

FWPs are usually stable for several months. However, in less than a week, one lot of FWPs lost its ability to aggregate with bovine PAF or human vWF plus ristocetin. Initial experiments suggested that the loss of aggregability was caused by contamination of the FWPs with an extracellular protease of Serratia marcescens. Highly purified protease preparations from the culture filtrates of S. marcescens (SP), as well as from two strains of Pseudomonas aeruginosa, destroyed the aggregability of FWPs as a function of time and concentration. On the basis of azocasein units, the SP was found to be at least eight times more potent against FWPs as a substrate than either of the P. aeruginosa proteases. The effect of SP on FWP aggregability was inhibited by prior EDTA treatment and was restored by addition of Zn2+ in slight molar excess. Purified PAF, but not dilutions of bovine plasma, lost all PAF activity when incubated with SP. SP-treated FWPs would still aggregate with 10 microgram/ml polylysine. SP digestion of FWPs was more selective than digestion with trypsin or chymotrypsin, on the basis of both the polyacrylamide gel electrophoresis pattern and the amount of protein in the platelet digest supernatant. SP does not aggregate fresh washed platelets or initiate the release reaction but renders them unaggregable with vWF. SP and related proteases may be useful in the study of platelet membranes.

Animals↗
PubMed1981

The multimeric distribution of factor VIII-related antigen studied by an improved crossed-immunoelectrophoresis technique.

An improved XIEP technique has been described that is capable of resolving under nondenaturing conditions at least five populations of FVIIIR:Ag directly from microliter amounts of normal human plasma. Resolution has been achieved by increasing the concentration of agarose in the electrophoretic medium to 2% or 3% and extending the period of electrophoresis to take advantage of molecular sieving effects. Normal human plasma demonstrates an electrophoretic pattern indicating that the majority of FVIIIR:Ag is assembled as higher-molecular-weight multimers. The distribution of FVIIIR:Ag appears to be independent of antigen concentration and sample storage temperature. Plasmas from two individuals with variant VWD demonstrate a lack of the higher-molecular-weight multimers coupled with an absolute increase in the lower-molecular-weight antigenic populations, suggesting a defect in antigen assembly. Commercial FVIII preparations, reported to be ineffective in correcting the bleeding abnormality in VWD, have also been found to lack the higher-molecular-weight multimers. On the other hand, cryoprecipitate, preferred in the treatment for VWD, had essentially the same antigen distribution as normal plasma. The similarities in the distribution of FVIIIR:Ag in variant plasmas and commercial FVIII concentrates provide further evidence that it is primarily the higher-molecular-weight multimers that function in maintaining hemostasis.

Adolescent↗
PubMed1980

Structure-function of the factor VIII complex studied with an immobilized heteroantisera to VIII:C.

An antibody was raised in rabbits to the small active fragment of human factor VIII, obtained by Ca2+ dissociation of a human factor VIII preparation made from a multidonor plasma pool. After absorption, the antibody neutralized the factor VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions or neutralize von Willebrand factor (vwf) activity as measured by ristocetin aggregation of fixed washed platelets. Immune beads were prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were prepared with IgG fractions obtained from the rabbits before immunization and used throughout as a control. The amount of factor VIII coagulant activity (VII:C) removed from plasma by immune beads was time-dependent and proportional to the amount of beads used, but all of the VIII:C could not be readily removed. Removal of VIII:C by immune beads parallelled removal of factor VIII:antigen, but less VWF activity was removed. Immune beads could be blocked or saturated by treatment with large amounts of normal plasma, but not by von Willebrand disease plasma and only by some haemophilic plasma.

Absorption↗
PubMed1977

Platelet-aggregating factor and the aggregation of fixed washed platelets.

Platelet-aggregating factor (PAF) was removed from bovine plasma by human platelets fixed with 2% formaldehyde. The degree of adsorption was directly related to the platelet concentration and the length of incubation. Fixed washed platelets (FWP) aggregated with bovine plasma could be deaggregated by 1M KCl, Evans blue, and 8M urea but not by beta-galactosidase. Incubation with 1M KCl eluted some but not all of the PAF, as the deaggregated platelets spontaneously aggregated upon removal of the deaggregating conditions. Also, fixed platelets adsorbed PAF even in the presence of 1M salt or after treatment with Evans blue. Platelet aggregation was not affected by thrombin (20 micron/ml) but was abolished by trypsin at concentrations as low as 4 X 10(-1) microgram/ml. The data suggest that deaggregation is not the result of elution of the loosely bound aggregating factor from the platelet surface, but rather the disruption of noncovalent interplatelet bridging between one or more PAF molecules bound to a specific receptor.

Adsorption↗
PubMed1976

Purification and immunochemical characterization of human plasma factor XIII.

A detailed study of the conditions for purification of plasma factor XIII was undertaken and an optimal procedure developed. This procedure involves adsorption with aluminum hydroxide, repeated fractionation with ammonium sulfate, heat denaturation of fibrinogen, precipitation with polyethylene glycol, cryoprecipitation, and chromatography on DEAE-cellulose with gradient elution. The specific activity of the final product, as measured by the fluorescent amine incorporation assay for factor XIII, averages about 3,000-4,000 mumol dansylcadaverine incorporation/mg protein. The yield is about 20%. In SDS electrophoresis the final product has two bands under nonreducing conditions, characteristic of the a and b subunits.

Ammonium Sulfate↗
PubMed1975

Antibody nature of circulating inhibitor of plasma von Willebrand factor.

A circulating plasma inhibitor of the "von Willebrand factor" was observed in a multiply tranfused subject with severe von Willebrand's disease. The platelet-active von Willebrand factor is associated with a plasma protein macromolecular complex that is deficient in the disease. The inhibitor appears to be an IgG antibody, kappa type, based on neutralization tests with goat antisera to specific human immunoglobulins. The IgG and inhibitor separated out together in plasma fractions obtained by "salting-out" and chromatographic procedures. Two separate inhibitor neutralization tests for the platelet-active factor, one with human plasma and ristocetin, the other with bovine plasma, gave similar results, based on the macroscopic aggregation time test of fixed human platelets. With cryoprecipitate transfusions the inhibitor was transiently neutralized with the temporary appearance of von Willebrand factor, factor VIII, and factor VIII-like antigen in the plasma. The plasma inhibitor level increased after transfusion, suggesting an anamnestic response. Lower titer inhibitor plasmas neutralized only the platelet activity. Highest titer plasma also neutralized human factor VIII, but only in part; it did not neutralize either bovine factor VIII or the human small active factor VIII fragment. The anti-factor VIII activity of the von Willebrand factor inhibitor may be due to steric hindrance, dependent on the spatial relationship of factor VIII sites on the macromolecular complex.

Animals↗
PubMed1975

Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.

When human, canine, or bovine factor VIII preparations are chromatographed on 4% agarose at ionic strength 0.2, the factor VIII activity elutes as a single peak in the void volume with slight tailing. Incubation of such preparations with dilute (0.01 U/ml) highly purified thrombin results in some activation of factor VIII. Chromatography of such incubation mixtures, under the same conditions as before, results in elution of two peaks of factor VIII activity one in the void volume and one much later with marked tailing. The void volume peak has most of the protein and some factor VIII activity. These void volume fractions also contain all the von Willebrand factor activity of thrombin-treated bovine preparations. Longer treatment with thrombin, or treatment with stronger thrombin, appears to shift much more of the procoagulant activity to the later eluting peak. Also, when the peak of factor VIII activity, found in the void volume after thrombin treatment, was again incubated with dilute thrombin, an increase in factor VIII activity occurred. Chromatography of this incubation mixture demonstrated only a small amount of activity in the void volume, while the bulk of the activity was present in the second peak. On the other hand, thrombin treatment of factor VIII activity from peak 2 caused a rapid decline of activity instead of a further increase. It is proposed that the residual factor VIII activity found in the void volume represents unreacted factor VIII, while the late eluting peak represents thrombin-activated material that is of smaller apparent size. The late eluting peak differs from the small active factor VIII fragment obtained by Ca2+ dissociation, as the latter can be activated by thrombin. A similar set of experiments was performed using ultracentifugation of bovine factor VIII preparations on sucrose density gradients. Results of these experiments agreed completely with those obtained with get chromatography. Preparations made from human hemophilic plasma, by the procudure employed in the purification of human factor VIII, were also incubated with thrombin and chromatographed. von Willebrand factor was again found only in the void volume fractions, but there was no factor VIII activity in any fractions eluted. In other control experiments, activated and unactivated factor VIII fractions did not clot fibrinogen and contained no assayable factor IX or X. The thrombin-modified factor VIII of small size was inactivated by both a naturally occurring human inhibitor to factor VIII and the gamma globulin fraction of a rabbit antisera produced against the calcium-dissociated small active factor VIII fragment.

Animals↗