Synthesis of an Arginine Analog of the Fragment 56-68 of Human Platelet Factor 4 Having Significant Antiheparin Activity

1979 ◽  
Author(s):  
S. Bajusz ◽  
I. Fauszt ◽  
É. Barabáa ◽  
D. Bagdy
Keyword(s):  
Solid Phase ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
Phase Method ◽  
Thrombin Time ◽  
Rich Region ◽  
Platelet Factor ◽  
Antiheparin Activity ◽  
Ionic Bonds ◽  
Solid Phase Method

Of the 70-residue polypeptide human platelet factor 4, which binds heparin stoichiometrlcally, the C-terminal fragment 50-70 /Tc-3/ showed a reduction of the heparin induced prolongation of thrombin time /Deuel et al., Proc. Natl. Acad. Sei. USA., 74:2256, 1977/.The arginine analog of the fragment 58-68 of Tc-3, which comprises the unique lysine rich region /Lys-Lys-Ile-Ile-Lys-Lys/, was prepared; Pro-Leu-Tyr-Arg-Arg-Ile-Ile-Arg-Arg-Leu-Leu-NH2. The aim of replacing of lyslne residues of the native molecule by arginine was to increase the interactions between the basic side chains of the peptide and SO3 - and/or COO- groups of heparin by introducing additional H-bridges and strengthening the ionic bonds. The new undecapeptide was synthesized by the standard solid phase method using Boc amino acids and dicyclo-hexylcarbodiimide condensation.1 nmole /1.8/ug/ of synthetic peptide completely inhibits the action of 0.3 units of heparin which corresponde a heparin/peptide molecular ration of about 1:6.

Critical Analysis of Platelet Factor 4 (Antiheparin Activity) Assays

1975 ◽  
Author(s):  
A. Hurlet ◽  
G. De Beys ◽  
M. Moriau ◽  
A. Monsieur ◽  
E. Schulzen ◽  
...  
Keyword(s):  
Calcium Chloride ◽  
Critical Analysis ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Platelet Factor 4 ◽  
Thrombin Time ◽  
Platelet Factor ◽  
Good Relationship ◽  
Antiheparin Activity ◽  
Set Up

Antiheparin activity is usually achieved by a heparin-thrombin time assay. Platelet free substrate plasma is adsorbed or not, calcium chloride used or not. Those technical conditions are analysed.Results are expressed by the amount of heparin units neutralized in the assay. With non adsorbed plasma substrate, calcium chloride generates thrombin, increasing antiheparin like activity. Lower antiheparin like activity is also observed with BaSO4 plasma substrate and with thrombin free of factor Xa. The amount of heparin units neutralized varies from 2.9 to 1.2 according to the system used.Tested without calcium, with Xa free thrombin and BaSO4 adsorbed substrate plasma, there exists a good relationship between the amount of heparin neutralized and various dilutions of serum, platelet rich plasma and platelet extract.An assay based on anti-Xa effect of heparin, set up according to the heparin assay of Yin, is compared to the method described above.

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.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Release of Platelet Factor 4 in Vivo during Intravascular Coagulation and in Thrombotic States

1968 ◽  
Vol 19 (03/04) ◽  
pp. 578-583 ◽  
Author(s):  
R Farbiszewski ◽  
S Niewiarowski ◽  
K Worowski ◽  
B Lipiński
Keyword(s):  
Coronary Thrombosis ◽  
Laboratory Diagnosis ◽  
Tolerance Test ◽  
Platelet Factor 4 ◽  
Thrombin Time ◽  
Significant Elevation ◽  
Platelet Factor ◽  
Thrombotic Diseases ◽  
Disseminated Intravascular Clotting

SummaryPlatelet factor 4 released from platelets into the circulating blood was determined using both the heparin thrombin time and paracoagulation methods. It has been found that thrombin injected intravenously into rabbits releases large amounts of this factor. Infusion of plasmin does not release this factor and this finding may be of importance for the differential diagnosis between disseminated intravascular clotting and primary fibrinolysis. PF4 is not released during the hyper coagulable condition induced by HgCl2 intoxication. Only small amounts of this factor are released after contact factor infusion.A significant elevation of extraplatelet PF4 was found in 23 patients with fresh coronary thrombosis and in 9 patients with thrombophlebitis and thromboembolic complications.The significance of the above findings for the pathogenesis, treatment and laboratory diagnosis of thrombotic diseases with particular reference to heparin tolerance test is discussed.

Neutralization of Antithrombin VI (Fibrinogen Breakdown Products) with Platelet Antiheparin Factor (Platelet Factor 4)*

1965 ◽  
Vol 14 (03/04) ◽  
pp. 490-499 ◽  
Author(s):  
S Niewiarowski ◽  
R Farbiszewski ◽  
A Popławski
Keyword(s):  
Zinc Acetate ◽  
Antithrombin Iii ◽  
Platelet Factor 4 ◽  
Breakdown Product ◽  
Platelet Factor ◽  
Chromatography Column ◽  
Antiheparin Activity

SummaryIt has been found that fibrinogen breakdown product – antithrombin VI – is neutralized by the purified preparation of platelet factor 4, obtained by means of zinc acetate precipitation and DEAE chromatography column. It has been suggested that antiheparin activity of platelet factor 4 and its ability to neutralize antithrombin VI may be related to the same protein.The purified preparation of platelet factor 4 does not influence the fibrinogen – fibrin conversion by thrombin. This means that platelet factor 2 and platelet factor 4 are not the same substance.Crude platelet extracts neutralize antithrombin III and V. However, the purified product did not interferes with the action of these antithrombins.

Coagulation Disorders in Thrombocythaemia, a Study of Seven Cases

1962 ◽  
Vol 07 (01) ◽  
pp. 114-128 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Halina Zywicka ◽  
Zbigniew Latałło
Keyword(s):  
Liver Parenchyma ◽  
Platelet Factor 4 ◽  
Coagulation System ◽  
Severe Bleeding ◽  
Clotting Factors ◽  
Platelet Factor ◽  
Antiheparin Activity ◽  
Thromboplastic Activity ◽  
Bleeding Episodes ◽  
Routine Coagulation

SummaryThe blood coagulation system has been studied in 7 patients with thrombocythaemia. 4 of these patients had thrombocythaemia after splenectomy, 2 of them had thrombocythaemia associated with myeloid leukemia, and 1 thrombocythaemia associated with polycythaemia. Severe bleeding episodes were noted in 5 cases, 2 patients had only mild bleeding symptoms.Each patient was examined several times. The period of observations varied from 2 months to 3 years. Platelet count varied from 350 000 to 3 800 000 per mm3.Bleeding time and tourniquet test were normal in all cases. Routine coagulation and fibrinolysis studies did not reveale characteristic abnormalities in plasma clotting factors. A decrease of prothrombin complex components was observed in 4 cases. This disturbance was due to the coexisting injury of liver parenchyma or myeloid changes but not to an increase of platelets or to the abnormalities in the platelet system.An increase of antiheparin activity was found in the plasma of 4 patients. This activity is probably due to the escape of platelet factor 4 from destroyed or qualitatively changed platelets into plasma.Platelet clotting factors were investigated in isolated platelet suspensions, A significant decrease of platelet factor 1 was observed in all patients and a decrease of platelet factor 4 in 5 patients. In 2 cases platelet factor 4 increased. Platelet thromboplastic activity showed a great variety of disturbances in conformity with other workers observations.Recent views on the pathogenesis of bleedings in thrombocythaemia are discussed. On the basis of their own investigations the authors suggest that the significant disturbances of platelet function may contribute to the development of bleeding, and that the increase of antiheparin activity in plasma may produce hypercoagulability and favorize the formation of thrombi.

Complete Covalent Structure of Human Platelet Factor 4

1979 ◽  
Vol 42 (05) ◽  
pp. 1652-1660 ◽  
Author(s):  
Francis J Morgan ◽  
Geoffrey S Begg ◽  
Colin N Chesterman
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Disulphide Bonds ◽  
Covalent Structure

SummaryThe amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.

Human Platelet Factor 4 (PF 4) Disappearance in Rabbits Fed an Atherogenic Diet

1986 ◽  
Vol 55 (01) ◽  
pp. 146-146 ◽  
Author(s):  
Marco Prosdocimi ◽  
Alberto Zatta ◽  
Fabrizio Fabris ◽  
Giuseppe Cella
Keyword(s):  
Human Platelet ◽  
Atherogenic Diet ◽  
Platelet Factor 4 ◽  
Platelet Factor

Clearance and In Vivo Release by Heparin of Human Platelet Factor 4 (PF4) in the Rabbit

1984 ◽  
Vol 52 (02) ◽  
pp. 157-159 ◽  
Author(s):  
M Prosdocimi ◽  
N Scattolo ◽  
A Zatta ◽  
F Fabris ◽  
F Stevanato ◽  
...  
Keyword(s):  
Half Life ◽  
Human Platelet ◽  
Slow Component ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
In Vivo Release ◽  
Partial Release ◽  
Different Doses ◽  
New Zealand Rabbits

Summary13 male New Zealand rabbits were injected with two different doses (25 μg/Kg and 100 μg/Kg) of human platelet factor 4 antigen (PF4). The disappearance of the protein was extremely fast with an half-life for the fast component of 1.07 ± 0.16 and 1.76 ± 0.11 min respectively. The half-life for the slow component, detectable only with the highest dosage, was 18.8 min.The administration of 2500 I.U. of heparin 30 min after PF4 administration induced a partial release of the injected protein and its clearance from plasma was slow, with half-life of 23.3 ± 5.9 min and 30.9 ± 2.19 min respectively.

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