Human Platelet Factor 4

• Cat.No.: PF4-52H
• Product Overview: Human PF-4 a7.8 kDa protein consisting of 70 amino acid residues.

Specification

• Species: Human
• Source: Human Platelets
• Tag: Non
• Description: Platelet factor-4 is a 70-amino acid protein that is released from the alpha-granules of activated platelets and binds with high affinity to heparin. Its major physiologic role appears to be neutralization of heparin-like molecules on the endothelial surface of blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation. As a strong chemoattractant for neutrophils and fibroblasts, PF4 probably has a role in inflammation and wound repair. Oncostatin-A is a member of the CXC chemokine family. Human PF4 is used for the proof of heparin-induced thrombocytopenia. Furthermore it is used as an inhibitor in the angiogenesis during tumor therapy.
• Amino Acid Sequence: The sequence of the first four N-terminal amino acids was determined and was found to be Glu-Ala-Glu-Glu.
• Physical Appearance: Sterile Filtered white lyophilized powder.
• Purity: Greater than 95.0% as determined by: (a) Analysis by RP-HPLC. (b) Analysis by SDS-PAGE.
• Formulation: The protein was lyophilized in PBS buffer.
• Solubility: It is recommended to reconstitute the lyophilized CXCL4 in sterile 18 MΩ-cm H2O not less than 100 µg/ml, which can then be further diluted to other aqueous solutions.
• Activity Control: Binding of HIT-antibodies.
• Stability: Human CXCL4 although stable at 25℃ 1 week, should be stored desiccated below -18℃. Please prevent freeze-thaw cycles.
• Publications:
  Interactions of Spike-RBD of SARS-CoV-2 and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis (2021)

Gene Information

• Gene Name: PF4 platelet factor 4 [ Homo sapiens ]
• Synonyms: PF4; platelet factor 4; CXCL4; SCYB4; MGC138298; Iroplact; Oncostatin-A; C-X-C motif chemokine 4; chemokine (C-X-C motif) ligand 4
• Gene ID: 5196
• mRNA Refseq: NM_002619
• Protein Refseq: NP_002610
• MIM: 173460
• UniProt ID: P02776
• Chromosome Location: 4q12-q21
• Pathway: Chemokine signaling pathway; Cytokine-cytokine receptor interaction; Hemostasis; Signaling by GPCR
• Function: chemokine activity; heparin binding

Citation

Interactions of Spike-RBD of SARS-CoV-2 and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis

Journal: International Journal of Molecular Sciences    PubMed ID: 34445266    Data: 2021/8/9

Authors: Margherita Passariello, Cinzia Vetrei, Malgorzata Kloc

Article Snippet:The following human recombinant proteins and antibodies were used: human SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein (Sino Biological, 10108-H08H, Eschborn, Germany); human recombinant IgG1 Fc protein (R & D Systems, 110-HG, Minneapolis, MN, USA); human Platelet Factor 4 (Creative BioMart, PF4-52H, Shirley, NY, USA).. HRP conjugated anti-human Fc antibody (Sigma, AP113P, St. Louis, MO, USA); anti-human IgG (Fab’)2 goat monoclonal antibody (Abcam, ab98535, Cambridge, UK); rabbit polyclonal anti-PF4 antibody (Prodotti Gianni, ab9561, Milano, Italy).HRP conjugated anti-human Fc antibody (Sigma, AP113P, St. Louis, MO, USA); anti-human IgG (Fab’)2 goat monoclonal antibody (Abcam, ab98535, Cambridge, UK); rabbit polyclonal anti-PF4 antibody (Prodotti Gianni, ab9561, Milano, Italy).

Representative images of 3D structures of SARS-CoV-2 RBD, ACE-2 and PF4 proteins. ( A ) SARS-CoV-2 spike ectodomain structure (open state, 6vyb) . The RBD domain (319–541 aa) is highlighted in gold. ( B ) Crystal structure of SARS-CoV-2 Spike Receptor-Binding Domain bound with ACE2 (6vw1). ( C ) Crystal structure of platelet factor 4 1f9q. The different domains of the protein are indicated by different colors: the chemokine interleukin-8-like domain is shown by pink color, the interleukin 8-like chemokine domain in green; the CXC Chemokine domain in brown and the small cytokine C-X-C is shown in purple color . In the red circles are highlighted the domains of the Spike-RBD protein ( B ) and PF4 protein ( C ) which show a similarity in the structure with the common sequences indicated by blue arrows ( D ). All the structures were obtained by PDB IntrePro. The PF4 aa residues involved in VITT are highlighted in yellow ( D ). The Red color of the D indicates identical residues in the two sequences.

Cross-reactivity of the anti-PF4 or the anti-Spike-RBD antibodies for Spike-RBD and PF4, respectively. ( A , B ) Binding of anti-PF4 antibody to Spike-RBD of SARS-CoV-2. ( A ) ELISA assays were performed to test the binding of polyclonal anti-PF4 antibody to PF4 protein (black bars), to RBD/Fc chimeric protein (dark grey bars) or to Fc (light grey bars). ( B ) Binding curves of the polyclonal anti-PF4 antibody, tested at increasing concentrations on RBD/Fc (black curve) or Fc region (grey curve). Error bars depicted means ± SD. ( C , D ): Binding of the anti-Spike monoclonal and polyclonal antibodies to PF4. The novel human monoclonal D3 and S96 mAbs were tested by ELISA assays at the concentration of 100 nM on the immobilized PF4 protein (black bars), RBD/Fc protein (grey bars) or Fc region (white bars), used as positive or negative controls, respectively ( C ). The binding of the anti-Spike polyclonal antibody to the same proteins was tested at the concentration of 30 nM in a parallel assay ( D ). ( E ) ELISA assays to test the cross-reactivity for PF4 of the human anti-Spike polyclonal antibodies from vaccinated donors. The binding of aliquots of 15 μL (1×) or 30 μL (2×) of human plasma containing polyclonal antibodies of immunized donors was tested at increasing concentrations on RBD/Fc (grey bars) or PF4 (black bars) proteins immobilized on the plate at 5 μg/mL. The Fc domain (white bars) was used in parallel as a negative control. An anti-Spike polyclonal antibody was used as a positive control of specificity for RBD. Error bars depicted means ± SD.

Interference of the novel anti-Spike mAbs in the interaction of the anti-PF4 antibodies with the Spike-RBD protein. ( A ) The binding to RBD was analyzed by testing the anti-PF4 antibody (white bars) in the absence or in the presence of D3 or S96 mAbs (grey bars) used at increasing concentrations. The binding of the anti-PF4 antibody was detected by using the anti-rabbit-HRP secondary antibody. Error bars depict means ± SD * p < 0.05. ( B ) A model explaining the possible roles of RBD and anti-PF4 antibodies in the platelets aggregation. Spike-RBD protein could bind to ACE2 receptor expressed on platelets. The recognition of Spike-RBD protein by either anti-PF4 or anti-Spike antibodies and the parallel engagement of the Fc receptor on activated platelets could induce the release of PF4 and formation of immune complexes with consequent platelets aggregation and thrombotic events.