Biomedical subjects
Publications and source records attributed to H A Cooper.
Classical light-scattering measurements made on bovine Factor VIII preparations, which contain a series of multimers ranging from 1 greater than 12 X 10(6) molecular weight, gave a weight average molecular weight of about 8 X 10(6) and a z-average radius of gyration of about 3000 A. Characteristic dimensions for variously shaped models were calculated.
A simple technique using an aggregometer and fixed washed human platelets (FWP) and fibrillar collagen has been used to evaluate the contribution of the two components of the factor VIII (FVIII) complex to platelet-collagen interactions. FWP bound individually to collagen fibrils in suspension, and both the total number of FWP bound and the rate of adhesion increased with increasing collagen concentration. Von Willebrand's disease (vWD) type I or normal plasma immunoadsorbed with anti-factor VIII-related antigen (anti-FVIIIR:Ag) antiserum gave 20% and vWD type IIa gave 50% of the rate of adhesion obtained with normal, hemophilia A, or hemophilia A with inhibitor plasma, but the same percent adhesion was found with all plasmas. The rate of adhesion of both vWD type I and type IIa was corrected by the addition of purified FVIII complex. These results indicated that the FVIIIR:Ag and not the factor VIII coagulant activity (FVIII:C) in normal plasma or purified FVIII complex caused an accelerating effect on the rate at which FWP bound to collagen. Collagen fibrils not only bound FWP, but also adsorbed the FVIII complex with preferential adsorption of the forms of FVIIIR:Ag with the greatest ristocetin cofactor (FVIIIR:RCoF) activity. Saturation of collagen with FWP did not change the adsorption pattern of the FVIII complex. Also anti-FVIIIR:Ag blocked the accelerating effect of the FVIII complex but not the adhesion of FWP. Thus, FWP and FVIIIR:Ag appeared to bind to separate sites on 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.
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.
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.
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.
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.
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.
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.
The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property of a soluble glycoprotein, glycocalicin, that is loosely attached to the platelet surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose-response curve obtained with Brij 99-solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or Lens culinaris lectin. By using wheat germ agglutinin-Sepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4 degrees C in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents.
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.
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A retrospective study of 116 children with Hodgkin's disease diagnosed in the period 1935-1970 was undertaken to assess the prognostic role of histopathologic classiciation and clinical extent of the disease. The ages of the 80 boys and 36 girls ranged from 2.5 years to 15.0 years (mean, 10.0 years). The histopathologic diagnosis by lymph node biopsy revealed lymphocyte predominance in 22, nodular sclerosis in 67, mixed cellularity in 24, and lymphocyte depletion in 3. Within the subgroup of nodular sclerosis, 47 biopsies had classic well-developed collagenous bands, whereas 20 were in the cellular phase (10 without collagenous bands and 10 with minimal collagen). The clinical extent of disease was determined. There were 33 patients with Stage I disease, 38 with Stage IIA, 12 with Stage IIB, 24 with Stage III, and 9 with Stage IV. Survival correlated with histopathologic type and clinical stage, but not with age or sex. Survival was not dependent on the degree of collagenization in nodular sclerosis. There were 28 patients who survived for more than 10 years. Four of these 29 subsequently died owing to acute myelomonocytic leukemia, carcinoma of the breast, sepsis, and progression of Hodgkin's disease, respectively. Neoplasms developed in two other long-term survivors (thyroid carcinoma in one, and multiple basal cell carcinomas in the other).
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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.
Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.
Aggregation of human platelets by bovine plasma was recently recognized as a marker for the study of the antihemophilic and von Willebrand factors. A similar marker in porcine plasma is shown to be specific for the platelet-active von Willebrand factor of plasma, but not for the antihemophilic factor (factor VIII). A new quantitative assay for the von Willebrand factor is based on the dose-response relationship observed between the concentration of von Willebrand factor and platelet aggregation times determined macroscopically. Bleeder swine, homozygous for von Willebrand disease, had no detectable platelet aggregating activity, while heterozygotes, carriers of the disease, had reduced levels of approximately 50% of normal. Exact detection of non-obligate carriers, hitherto impossible, becomes readily feasible because of the gene dosage relationship to the von Willebrand factor plasma levels. Transfusion of bleeder swine with normal plasma results in an immediate post-transfusion rise of the von Willebrand factor, followed by a rapid falloff. At 24 hr post-transfusion, no von Willebrand factor is detectable at a time when the factor VIII response is at its maximum. The discordance in plasma levels of this platelet-active von Willebrand factor and factor VIII in carrier animals and in the bleeder animals after transfusion suggests a different molecular and genetic basis for these two biological activities.