scholarly journals Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451 ◽  
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

Identification and characterization of PF4varl, a human gene variant of platelet factor 4

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

scholarly journals Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 219-223 ◽  
Author(s):  
M Poncz ◽  
S Surrey ◽  
P LaRocco ◽  
MJ Weiss ◽  
EF Rappaport ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Genomic Dna ◽  
Cdna Probe ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Coding Region ◽  
Platelet Factor ◽  
Amino Acid Homology

Abstract We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

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.

Nomenclature of Secreted Platelet Proteins – Report of the Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets

1985 ◽  
Vol 53 (02) ◽  
pp. 282-284 ◽  
Author(s):  
Karen L Kaplan ◽  
Stefan Niewiarowski
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Cellulose Acetate ◽  
Working Party ◽  
Platelet Factor 4 ◽  
Cellulose Acetate Electrophoresis ◽  
Platelet Factor ◽  
Amino Terminal ◽  
Platelet Proteins

SummaryStandard nomenclature for a number of secreted platelet proteins was agreed upon by The Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets. Platelet factor 4 will continue to be used for the molecule with high heparin affinity, subunit molecular weight of 7780, and the described amino acid sequence. β-Thromboglobulin will be used to designate β-Thromboglobulin (81 amino acids/subunit, β-mobility on cellulose-acetate electrophoresis, pI 7), low-affinity platelet factor 4 (85 amino acids/subunit, y-mobility on cellulose-acetate electrophoresis, pI 8), and platelet basic protein (94 amino acids/ subunit, pI 10) when these are measured immunologically in plasma, but that thromboglobulin with a superscript designation of the pI should be used when assays are conducted on samples after isoelectric focusing, and a subscript amino-terminal amino acid can be added when a purified protein is described. Thrombospondin will continue to be the designation for the high molecular weight trimer that has previously been called thrombospondin or glycoprotein G. Platelet derived growth factor will be used for the group of closely related proteins of molecular weight about 30,000 and isoelectric point about 10.

scholarly journals Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding

1995 ◽  
Vol 312 (2) ◽  
pp. 357-365 ◽  
Author(s):  
K H Mayo ◽  
E Ilyina ◽  
V Roongta ◽  
M Dundas ◽  
J Joseph ◽  
...  
Keyword(s):  
Alpha Helix ◽  
Platelet Factor 4 ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Heparin Binding ◽  
Nmr Studies ◽  
Platelet Factor ◽  
Binding Data ◽  
Arginine Residues ◽  
Proton Resonances

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.

scholarly journals Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 219-223 ◽  
Author(s):  
M Poncz ◽  
S Surrey ◽  
P LaRocco ◽  
MJ Weiss ◽  
EF Rappaport ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Genomic Dna ◽  
Cdna Probe ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Coding Region ◽  
Platelet Factor ◽  
Amino Acid Homology

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

High- And Low-Affinity Heparin Binding Proteins From Bovine Platelets

1981 ◽  
Author(s):  
Raymond E Ciaglowski ◽  
Daniel A Walz
Keyword(s):  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Cellulose Acetate Electrophoresis ◽  
Platelet Factor ◽  
Amino Terminal ◽  
Binding Domains ◽  
Specific Proteins ◽  
Related Variant ◽  
Function Relationship ◽  
Relationship Of

The class of platelet-specific proteins which displays anti heparin and/or growth stimulating activities includes: high affinity platelet factor 4 (HA-PF-4), low affinity platelet factor 4 (LA-PF-4) and its closely related variant β-thromboglobulin (β-TG), and platelet derived growth factors) (PDGF). In an effort to determine to what extent these proteins and their activities might be broad-based, a comparative study using bovine platelets has been undertaken. Fresh, washed bovine platelets were either thrombin stimulated or freeze-fractured and the ensuing supernatants chromatographed on either heparin-Sepharose (HS) or dextran- sulfate-Sepharose (DSS). Bovine protein which eluted with either 1.0M NaCl (HS) or 1.5M NaCl (DSS) was subsequently gel filtered to homogeneity. Using S2222 chromogenic assays, the bovine protein (9000 dal tons) and human HA-PF-4 (7800 dal tons) each had similar heparin neutral ization equivalence, about 40 U/mg. Using 3T3 cells, growth promoting activity was not found to be associated with the purified bovine HA- PF-4. Although bovine HA-PF-4 has 15 additional residues from the amino terminal end of human HA-PF-4, the acidic, neutral and basic regions have remained similar. HSand DSS fractions from thrombin released platelets eluted with 0.5M NaCl and gel filtered yielded a homogeneous protein of molecular weight 12,000 dal tons. This product has a heparin neutralization activity of approximately 2 U/mg; cellulose acetate electrophoresis of the bovine protein migrated into the gammaglobulin region, and we have consequently referred to it as bovine LA-PF-4. The bovine LA-PF-4 protein had 3T3 growth stimulating activity at least comparable to that of a DSS intermediate salt eluted fraction. The amino terminal 12 residues of bovine LA-PF-4 are not similar to human LA-PF-4 or β-TG. We conclude that these bovine anti heparin proteins are functionally homologous to their human counterparts. We are currently completing the sequence of bovine HA-PF-4 in order to establish the structure-function relationship of the heparin binding domains of these proteins.

Relation Of Heparin Binding And Conformation In PF4 AND LA-PF4 (βTG)

1981 ◽  
Author(s):  
John C Holt ◽  
Marek Kloczewiak ◽  
Daniel A Walz ◽  
Boguslaw Rucinski ◽  
Stefan Niewiarowski
Keyword(s):  
Amino Acid ◽  
Disulfide Bonds ◽  
Amino Acid Sequences ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Significant Difference ◽  
Lysine Residues ◽  
Α Helix ◽  
Β Sheet

Platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) are platelet-specific secreted proteins that bind to heparin. β-thromboglobulin (βTG) appears to be derived from LA-PF4 by proteolysis of four NH2-terminal residues. PF4 and LA-PF4 (βTG) show 50% sequence homology including four cysteine residues and two pairs of lysine residues near the C00H-terminus which are believed to be responsible for heparin binding. Despite these similarities, the two proteins have markedly different affinities for heparin. We have sought a structural interpretation of this difference by predicting the conformations of 0TG, LA-PF4 and PF4. First, the proportion of residues in α-helical, β-sheet and unordered conformations was estimated from circular dichroism measurements. The results for PF4 and LA-PF4 were experimentally identical, namely 16% α-helix and 20% β-sheet. These values were then applied as experimental constraints in the prediction of the secondary structure of PF4 and LA-PF4 based on their amino acid sequences. This was done by a computer program which compared local amino acid sequence (each residue and 8 residues on either side) with the conformation of similar sequences in 25 proteins of known structure. With the further constraint of the two disulfide bonds in each molecule, models were constructed representing the overall folding of the polypeptide chains. The only significant difference between the two proteins was in the COOH-terminal region of the chains. The models suggest that the lower affinity of LA-PF4 (and βTG) for heparin may result from steric hindrance by the longer and more negatively charged COOH-terminal segments of these molecules compared with PF4.

scholarly journals Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1290-1295 ◽  
Author(s):  
KS Park ◽  
S Rifat ◽  
H Eck ◽  
K Adachi ◽  
S Surrey ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Expression Vector ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Native Protein ◽  
Platelet Factor

Abstract Platelet factor 4 (PF4) is a 70 amino acid protein released from the alpha-granules of platelets after activation. The exact biologic function of this protein is unknown. We have constructed an expression vector for recombinant PF4 (rPF4) in the T7-based promoter vector pT7–7 to better study the relationship between PF4 structure and function. The protein was expressed in Escherichia coli and purified to homogeneity by heparin-agarose affinity chromatography and reverse- phase high-performance liquid chromatography. Purity of protein was confirmed by immunoblot analysis and sodium dodecyl sulfate- polyacrylamide gel electrophoresis, which resulted in a single component with a molecular weight of 8,000 daltons. The amino acid composition and sequence of the N-terminal 20 residues showed that rPF4 is identical to PF4 prepared from human platelets (hPF4), except for an N-terminal initiating methionine residue. Immunoblots revealed that rPF4 and hPF4 bound polyclonal anti-hPF4 equally well, while chemotaxis experiments demonstrated similar potencies as neutrophil attractants. Dose-dependent neutrophil chemotactic responses and competitive studies with polyclonal anti-hPF4 antiserum further demonstrate similar chemotactic properties of the two PF4 species. In conclusion, our data show that this recombinant protein and the native protein appears to have similar immunologic, heparin-binding, and chemotactic properties. The chemotactic properties of hPF4 appear to be entirely intrinsic to the protein and not due, in part, to any contaminating protein. Furthermore, our expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure/function relationships of this biologically important protein.

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

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.

Sign in / Sign up

Export Citation Format

Share Document