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Definition
Platelet factor 4 (Platelet heparin-neutralizing factor) is present in blood platelets as a proteoglycan and is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin.


Discovery
Human platelets contain a releasable low molecular weight protein which can neutralize heparin’s anticoagulant effect. This heparin-neutralizing protein, referred to as Platelet Factor Four, was first postulated by Conley et al., following clinical observations of heparin’s prolonged effect in thrombocytopenic patients 1.


Fragments
Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells. In a study, the effect of PF-4 on histamine release from rat peritoneal mast cells was investigated by employing its biologically-active carboxyl-terminal fragment, PF-4 (58-70). It was found that PF-4 (58-70) stimulated histamine release from mast cells in a dose-dependent manner (10(-8) to 10(-5) M). Histamine release induced by PF-4 (58-70) occurred rapidly (<30s) and was inhibited by extracellular Ca2+. These results suggest that PF-4 might play a crucial role at the site of inflammation and/or immune response 2.


Structural Characteristics
PF-4 belongs to the C-X-C chemokine family. This family also includes interleukin-8 (IL-8), ß-thromboglobulin, neutrophil-activating protein, interferon-inducing protein 10 (IP-10), and melanocyte growth-stimulating activity. PF-4 is a 7.8 kDaÅ protein of 70 amino acid length3. The crystal structure of human PF-4 has been solved to a resolution of 2.4 Å by molecular replacement4. The N-terminal residues form antiparallel ß-sheet-like structures. A positively charged ring of lysine and arginine side chains encircles the PF-4 molecule presenting multiple potential sites for heparin binding.


Mode of Action
The heparin-neutralizing activity in platelets is localized in the a granules. It is bound to a chondroitin 4-sulfate proteoglycan carrier with molecular weight of 5.9 kDa. PF4 is actively released during platelet aggregation induced by collagen, thrombin, adenosine diphosphate  and epinephrine. The COOH-terminal is unusual and seemed a likely binding site for glycosaminoglycans. In a study it was observed that the COOH-terminal tryptic peptide in high concentration partially reversed the prolonged thrombin time induced by heparin 5, suggesting heparin binding was localized to the COOH-terminal region of PF4. Support for the involvement of these lysines in the PF4-heparin binding activity was provided by the work of Handin and Cohen who showed that guanidination of lysines in PF4 decreased heparin neutralizing activity, but that modification of arginines was without effect 6.


Functions

PF-4 Modulates Fibroblast Growth Factor 2 Activity and Inhibits Fgf-2 Dimerization: A study investigated the mechanism of inhibition by examining the interaction of PF-4 and the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to high-affinity and low-affinity binding sites in murine microvascular endothelial cells (LEII cells) and proliferation. Maximum inhibition of binding to endothelial FGF receptors was observed at PF-4 concentrations between 5 and 10 µg/mL (half maximum inhibition at 0.6 µg/mL), and proliferation was completely inhibited at 2 µg/mL. Furthermore, to gain insight into the mechanism of inhibition, analysis of interaction of PF-4 with FGF-2/FGFR by using mutant heparan sulfate-deficient Chinese hamster ovary (CHO) cells transfected with the FGFR-1 cDNA (CHOm-FGFR-1) and the direct interaction with FGF-2 was conducted. It was found that in the absence of heparin, PF-4 inhibited binding of 125I-FGF-2 to CHOm-FGFR-1 cells in a concentration-dependent manner, although not completely. In the presence of heparin, PF-4 abolished totally the stimulatory effect of heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous or heparin-induced FGF-2 dimerization. These results indicate that PF-4 interacts with FGF-2 by complex formation, inhibiting FGF-2 dimerization, binding to FGF receptors, and internalization. This mechanism most likely contributes to the antiangiogenic properties 7of PF-4.


PF-4 Binds to Interleukin 8 Receptors and Activates Neutrophils When its N Terminus is Modified with Glu-Leu-Arg: Amino acid deletion and mutagenesis experiments have indicated that the sequences Glu-Leu-Arg (ELR) preceding the first cysteine at the N terminus of interleukin 8 (IL-8) is required for receptor binding and neutrophil activation. Platelet factor 4 (PF4) is structurally related to IL-8 (35% sequence identity) but lacks the N-terminal ELR sequence and comparable effects on neutrophils. In a study the ELR sequence was introduced at the N terminus of PF4 and found that the modified protein was a potent neutrophil activator and attractant. On the other hand, when the ELR sequence was introduced into the corresponding positions of two other proteins related to IL-8, gamma-interferon-inducible protein IP10 and monocyte chemoattractant protein 1, neither of them acquired neutrophil-activating properties, indicating that besides ELR additional structural determinants of IL-8 and PF4 are important for binding to IL-8 receptors. The conservation of these binding determinants suggests that PF4 may have evolved from a neutrophil activating protein 8.


References

1.     Conley CL, Hartmann RC & Lalley JS (1948). The relationship of heparin activity to platelet concentration. Proc. Soc. Exp. Biol. Med ., 69(2):284-287.

2.     Suzuki R, Kimura T, Kitaichi K, Tatsumi Y, Matsushima M, Zhao YL, Shibata E, Baba K, Hasegawa T, Takagi K (2002). Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells. Peptides, 23(10):1713-1717.

3.     Deuel TF, Keim PF, Farmer M, Heinrickson RL (1977). Amino acid sequence of human platelet factor 4. PNAS., 74(6):2256-2258.

4.     Zhang X, Chen L, Bancroft DL, Lai CK, Maione TM (1994). Crystal structure of recombinant human platelet factor 4. Biochemistry, 33 (27):8361-8316.

5.     Huang SS, Huang JS, Deuel TF (1982). Proteoglycan Carrier of Human Platelet Factor 4 – Isolation and characterization. J. Biol. Chem., 257 (19):11546-11550.

6.     Handin RI, Cohen HJ (1976). Purification and Binding Properties of Human Platelet Factor Four. J. Biol. Chem., 251(14): 4273-4282.

7.     Perollet C, Han ZC, Savona C, Caen JP, Bikfalvi A (1998). Platelet Factor 4 Modulates Fibroblast Growth Factor 2 (FGF-2) Activity and Inhibits FGF-2 Dimerization. Blood, 91(9):3289-3299.

8.     Clark-Lewis I, Dewald B, Geiser T, Moser B, Baggiolini M (1993). Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. PNAS., 90(8):3574-3577.

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