scholarly journals Antigenic and antiheparin properties of human platelet factor 4 (PF4)

Blood ◽  
1975 ◽  
Vol 45 (4) ◽  
pp. 537-550
Author(s):  
N Nath ◽  
CT Lowery ◽  
S Niewiarowski
Keyword(s):  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Polyacrylamide Gel ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heat Stable ◽  
Antiheparin Activity ◽  
Heat Stable Protein

Platelet factor 4 (PF4, a heparin-neutralizing protein) was isolated from washed human platelets. It was found to be homogenous by SDS- polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophoresis, when tested with monospecific antibody produced in rabbits. PF4 is a heat-stable protein, but its antiheparin activity and antigenicity are destroyed by trypsin. The molecular weight of PF4 as calculated by amino acid analysis is approximately 8000 and by SDS- polyacrylamide gel electrophoresis with beta-mercaptoethanol, 7100 daltons. PF4 migrated to the cathode at pH 8.6. The interaction of PF4 with heparin resulted in the formation of a complex which migrated to the anode, as tested by immunoelectrophoresis. Incubation of purified PF4 with its antibody at 37 degrees C resulted in a loss of antiheparin activity. The presence of antiheparin activity and of PG4 antigen in material released during platelet aggregation by various agents and at various stages of the preparative procedure closely correlated. It has been concluded that PF4 antigen and antiheparin activity are two properties of the same protein. Comparison of human and pig PF4 revealed significant biochemical and antigenic differences.

Antigenic and antiheparin properties of human platelet factor 4 (PF4)

Blood ◽  
1975 ◽  
Vol 45 (4) ◽  
pp. 537-550 ◽  
Author(s):  
N Nath ◽  
CT Lowery ◽  
S Niewiarowski
Keyword(s):  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Polyacrylamide Gel ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heat Stable ◽  
Antiheparin Activity ◽  
Heat Stable Protein

Abstract Platelet factor 4 (PF4, a heparin-neutralizing protein) was isolated from washed human platelets. It was found to be homogenous by SDS- polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophoresis, when tested with monospecific antibody produced in rabbits. PF4 is a heat-stable protein, but its antiheparin activity and antigenicity are destroyed by trypsin. The molecular weight of PF4 as calculated by amino acid analysis is approximately 8000 and by SDS- polyacrylamide gel electrophoresis with beta-mercaptoethanol, 7100 daltons. PF4 migrated to the cathode at pH 8.6. The interaction of PF4 with heparin resulted in the formation of a complex which migrated to the anode, as tested by immunoelectrophoresis. Incubation of purified PF4 with its antibody at 37 degrees C resulted in a loss of antiheparin activity. The presence of antiheparin activity and of PG4 antigen in material released during platelet aggregation by various agents and at various stages of the preparative procedure closely correlated. It has been concluded that PF4 antigen and antiheparin activity are two properties of the same protein. Comparison of human and pig PF4 revealed significant biochemical and antigenic differences.

Release Of Platelet Factor-4 In Vivo After Heparin Injection

1981 ◽  
Author(s):  
A Koneti Rao ◽  
John C Holt ◽  
Pranee James ◽  
Stefan Niewiarowski
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Plasma Samples ◽  
Platelet Factor ◽  
Immunoreactive Material ◽  
Elution Pattern

A single bolus of heparin administered to 8 normal volunteers resulted in a significant increase in levels in platelet poor plasma (PPP) of platelet factor-4 (PF4) but not low-affinity platelet factor-4/β-thromboglobulin (LA-PF4/βTG). However, the presence of heparin interfered with the binding of 125I-PF4 to antibody in radioimmunoassay (RIA). This effect was overcome by increasing the concentration of NaCl from 0.15 to 0.5 M in the buffer used for RIA. In order to establish that the increased amount of immunoreactive material present in PPP was indeed PF4, the protein was isolated from postheparin plasma. A bolus of 5000 units of porcine lung heparin (Upjohn) was administered intravenously to 2 volunteers and plasma samples obtained before and 5 minutes after the injection. The levels of PF4 in PPP rose from 18 and 10 ng/ml before to 185 and 454 ng/ml at 5 minutes after injection in the two volunteers, respectively. The 5 minute samples were adsorbed to heparin agarose columns and PF4 levels decreased to 16 and 10 ng/ml respectively. The immunoreactive material was eluted with 1.2 M NaCl from the heparin agarose columns, showing typical elution pattern for PF4. This material was applied to SDS-polyacrylamide gel electrophoresis in parallel with purified PF4 obtained from human platelets. RIA carried out on eluates from gel slices revealed a species of the same molecular weight as standard PF4. Thus, heparin injection results in appearance in the circulation of a material identical to PF4. LA-PF4/βTG and PF4 are located in same granules and released in parallel during platelet stimulation. Further, LA-PF4 is cleared from plasma 4 times slower than PF4. Therefore, the elevation of PF4alone suggests release from sites other than platelets.

scholarly journals Effect of heparin on the in vivo release and clearance of human platelet factor 4

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1208-1214 ◽  
Author(s):  
AK Rao ◽  
S Niewiarowski ◽  
P James ◽  
JC Holt ◽  
M Harris ◽  
...  
Keyword(s):  
Half Life ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Human Platelets ◽  
Single Component ◽  
Platelet Factor 4 ◽  
Component Model ◽  
Platelet Factor

Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet- poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from “postheparin plasma” by adsorption on heparin-agarose and elution with 2.0 M NaCl and “authentic PF4” isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

scholarly journals Effect of heparin on the in vivo release and clearance of human platelet factor 4

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1208-1214 ◽  
Author(s):  
AK Rao ◽  
S Niewiarowski ◽  
P James ◽  
JC Holt ◽  
M Harris ◽  
...  
Keyword(s):  
Half Life ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Human Platelets ◽  
Single Component ◽  
Platelet Factor 4 ◽  
Component Model ◽  
Platelet Factor

Abstract Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet- poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from “postheparin plasma” by adsorption on heparin-agarose and elution with 2.0 M NaCl and “authentic PF4” isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

scholarly journals Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals A set of surface proteins common to the circulating human platelet and lymphocyte

1974 ◽  
Vol 141 (3) ◽  
pp. 909-911 ◽  
Author(s):  
M. J. A. Tanner ◽  
D. H. Boxer ◽  
Jane Cumming ◽  
J. Verrier-Jones
Keyword(s):  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
Major Type ◽  
Cell Type ◽  
Peptide Maps

The surface proteins of the circulating human platelet and lymphocyte were labelled by using the lactoperoxidase iodination method. Polyacrylamide-gel electrophoresis showed that four corresponding labelled proteins are found on the surface of each cell type. The most intensely labelled protein contains little or no carbohydrate, but the remaining labelled proteins are all glycoproteins. The major labelled band from each cell was isolated and comparative peptide ‘maps’ showed that the two proteins are closely similar. The surface proteins of the lymphocyte and platelet are distinct from those on the erythrocyte, the remaining major type of circulating cell.

On the Plasmin Digestion of Factor XIII

1975 ◽  
Author(s):  
K. Mikami ◽  
T. Mikami ◽  
H. Suzuki ◽  
M. Fujimaki ◽  
K. Fukutake
Keyword(s):  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Platelet Factor ◽  
Subunit A ◽  
Plasma Factor ◽  
Congenital Afibrinogenemia

The plasmin digestion of factor XIII in the plasma with congenital afibrinogenemia has been reported by Suzuki et al. in 1967. The present investigation using polyacrylamide gel electrophoresis and crossimmunoelectrophoresis with anti-factor XIII (A & S) serum demonstrates that the process of plasmin digestion of factor XIII can be divided into three steps ; the subunit A of factor XIII such as placental or platelet factor XIII is ready to get plasmin digestion following the decrease of amount and activity of subunit A, and the subunit S in the A1 S complex like plasma factor XIII is more readily affectable on the plasmin digestion than the subunit A of the complex, and the A2 S complex coexisting with fibrinogen or plasma protein is fairly stable on exposure to plasmin.

Estimation of molecular weight by polyacrylamide gel electrophoresis using heat stable fluorophors

1976 ◽  
Vol 70 (2) ◽  
pp. 327-335 ◽  
Author(s):  
Bruce O. Barger ◽  
Fred C. White ◽  
Judith L. Pace ◽  
David L. Kemper ◽  
W.L. Ragland
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Heat Stable

Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody

1992 ◽  
Vol 67 (01) ◽  
pp. 137-143 ◽  
Author(s):  
Leopoldo Saggin ◽  
Flavia Cazzola ◽  
Giuseppe Corona ◽  
Emanuela Salvatico ◽  
Giuseppe Cella ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Factor Xa ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Molar Ratio ◽  
Rabbit Platelet ◽  
Platelet Factor ◽  
Chromogenic Assay ◽  
Antiheparin Activity ◽  
Human Molecule

SummaryWe have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

scholarly journals Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets

1986 ◽  
Vol 237 (1) ◽  
pp. 139-145 ◽  
Author(s):  
M G Low ◽  
R C Carroll ◽  
A C Cox
Keyword(s):  
Substrate Specificity ◽  
Phospholipase C ◽  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Platelets ◽  
Physiological Significance ◽  
Multiple Forms

The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.

Sign in / Sign up

Export Citation Format

Share Document