Human platelet basic protein. Its relation to low affinity platelet factor 4 and beta-thromboglobulin.

Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein.

Human platelet basic protein associated with antiheparin and mitogenic activities: purification and partial characterization.

Platelet basic protein (PBP) (isoelectric point, 10.0-10.5; apparent Mr, 11,000-15,000) has been purified to homogeneity from material secreted by fresh human platelets after stimulation by thrombin. The purification, using preparative isoelectric focusing and chromatography on heparin-Sepharose, yielded two additional peptides with antiheparin activity that were immunologically identical with PBP: low-affinity platelet factor 4 and beta-thromboglubulin. The purity of the peptides was confirmed by immuoelectrophoresis and by NH2-terminal amino acid analysis. Dansyl chloride-treated PBP yielded a single dansylated amino acid residue (glycine). By using a specific radioimmunoassay it was shown that 10(9) human platelets contain 2-3 microgram of PBP which can be released in response to specific stimulation. PBP is associated with mitogenic activity as assayed in Swiss 3T3 mouse cells cultured in low-serum (0.4-1.5%) medium at levels of about 1 ng/ml and saturating at 10-40 ng/ml. The biological activity of different PBP preparations was variable, presumably due to inhibition by the varying amounts of ampholytes that interfered with the mitogenic activity of the peptide. Mitogenic activity was eluted from NaDodSO4/polyacrylamide gels and shown to comigrate with immunoreactive material and with conventional marker proteins of 14,000-17,000 daltons or with histones of 11,000-15,000 daltons. Evidence is presented that PBP is different from cationic platelet-derived growth factor which has an apparent Mr of 30,000.

Purification and characterization of lecithin:cholesterol acyltransferase.

The purification of lecithin:cholesterol acyltransferase (LCAT] from human plasma is reported. Hydroxylapatite fractions were approximately 16,000 fold purified over the starting plasma and were free of high-density lipoprotein (HDL) and albumin. The enzyme showed one band on polyacrylamide gel electrophoresis, SDS-urea polyacrylamide gel electrophoresis, isoelectric focusing, and one arc in immunodiffusion against a goat antiserum preparation. It was determined to be a glycoprotein with a molecular weight of approximately 70,000 and a pI of 3.7--4.0.

Immunological evaluation of LCAT deficiency.

Antibody towards a highly purified LCAT preparation was tested against LCAT-deficient sera by using the technic of immunodiffusion and immunoinhibition. Reaction of identity among normal serum, deficient sera, and purified LCAT was observed in immunodiffusion with two different antibodies obtained from two different goats. When the antibodies were mixed with deficient sera and tested for immunoinhibition of LCAT activity, suggesting that deficient sera contained an enzymatically inactive LCAT. The other antibody preparation showed inhibition of enzyme activity even in antigen excess. It appears that the precipitin lines observed in immunodiffusion do not represent LCAT in serum. In view of the higher titre of antibody in immunoinhibition experiments with this antibody, it remains to be determined whether at lower ratios of antibody to deficient serum, immunoinhibition by the antibody will be abolished in this case too.

Relationship between high density lipoproteins and the rate of in vitro serum cholesterol esterification.

The rate of in vitro esterification of serum cholesterol and the concentrations of serum high density lipoprotein cholesterol (HDL-C) were measured in 18 women and 9 men, 74--95 years of age. Subjects in this age group can have HDL-C levels below accepted limits of normal. The subjects were divided into two groups, one with HDL-C less than 40 mg/dl and the other with HDL-C 40 mg/dl or higher. The rate of in vitro serum cholesterol esterification in the former group was 2.00 n. moles/ml/min, significantly higher (p less than 0.02) than 1.62 n. moles/ml/min present in the latter group. There was no correlation between the cholesterol esterification and a protein (s), which can produce an immune gamma globulin that completely inhibits in vitro serum cholesterol esterification. These findings are discussed with reference to the putative relationships between HDL-C and atherosclerosis.

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.

A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase.

A method for processing 3 litres of human plasma for the purification of phosphatidylcholine-cholesterol acyltransferase is described. The method involves (NH4)2SO4 fractionation, citric acid treatment, and DEAE-cellulose and hydroxyapatite chromatography. At this stage the enzyme preparation is purified approx. 8000-fold. This preparation appears to be free of lipoproteins as determined by immunoelectrophoresis against anti-human serum and is minimally contaminated with albumin (less than 30 mug/mg of enzyme protein) as determined by immunodiffusion. The activity of the enzyme was stable for 4 days, but most of its activity was lost after 20 days On electrophoresis on 5% polyacrylamide gel, a fast-moving band with enzyme activity and a slow-moving band with no enzyme activity was observed. A faint band of albumin was also present. Extracts of enzymically active bands cut from ten gels and then pooled and extracted with 0.15 M-NaC1/4mM-sodium phosphate, pH 7.0, showed a single band on re-electrophoresis on 5% polyacrylamide gel.