PF4 Bovine
Description
Coagulation and Heparin Neutralization
Bovine PF4 inhibits heparin-like molecules on endothelial surfaces, promoting coagulation by neutralizing antithrombin III activity . This interaction underlies its role in heparin-induced thrombocytopenia (HIT), where PF4-heparin complexes trigger pathogenic antibody formation .
Immune Regulation
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Neutrophil Recruitment: Enhances neutrophil exocytosis of myeloperoxidase and lysozyme .
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Antiviral Activity: Stimulates NK cell cytotoxicity and antigen-presenting cell activation .
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Bacterial Binding: Binds to E. coli, S. aureus, and other pathogens, facilitating immune clearance .
Megakaryopoiesis Regulation
PF4 acts as a negative autocrine regulator of platelet production in vivo, suppressing megakaryocyte colony formation in bone marrow .
Heparin-Induced Thrombocytopenia (HIT)
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Antibody Formation: Bovine heparin induces PF4 antibodies at a higher rate (44.4%) than porcine heparin (30.6%) in cardiac surgery patients .
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Pathogenic Mechanism: PF4-heparin complexes form ultralarge complexes (ULCs) that bind HIT antibodies, activating platelets via FcγRIIA receptors .
Table 1: PF4-Dependent Cellular Responses in HIT
| Parameter | SRA-Positive Samples | SRA-Negative Samples |
|---|---|---|
| IgG Binding (Fold Change) | 3.9 ± 1.2 | 1.3 ± 0.4 |
| P-Selectin Expression | 3.78 ± 1.5 | 1.07 ± 0.3 |
| Heparin Inhibition | Complete at 10:1 GAG:PF4 | No effect |
Therapeutic Applications
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HIT Diagnostics: Used in ELISA assays to detect anti-PF4-heparin antibodies .
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Angiogenesis Inhibition: Explored in cancer therapy for blocking tumor vascularization .
Comparative Analysis with Human PF4
Challenges and Future Directions
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Aggregation Issues: Recombinant PF4 requires glycerol and DTT for solubility, complicating long-term storage .
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Therapeutic Targeting: Small-molecule PF4 antagonists (e.g., KKO inhibitors) are in development to disrupt ULC formation in HIT .
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Bacterial Sepsis: PF4’s role in pathogen binding warrants exploration as an antimicrobial adjuvant .
Product Specs
Platelet factor-4 (PF4) is a protein found in the granules of platelets. When platelets are activated, they release PF4. This protein plays a role in blood clotting by binding to a molecule called heparin. PF4 is also involved in inflammation and wound healing. Oncostatin-A, another protein, is similar in structure to PF4. This protein has been investigated for its potential to inhibit the formation of new blood vessels, a process that is important in tumor growth.
This product is a recombinant version of bovine Platelet Factor-4 (CXCL4) produced in E. coli bacteria. It is a single, non-glycosylated polypeptide chain composed of 88 amino acids with a molecular weight of approximately 9.5 kDa. The protein has been purified using proprietary chromatographic techniques.
The protein was lyophilized from a concentrated solution (0.2 μm filtered) in 20 mM phosphate buffer (PB) and 500 mM sodium chloride (NaCl) at pH 7.0.
To reconstitute the lyophilized Platelet Factor-4, it is recommended to dissolve it in sterile 18 MΩ-cm H2O to a concentration of at least 100 μg/ml. The reconstituted solution can be further diluted in other aqueous solutions.
Lyophilized CXCL4 can be stored at room temperature for up to 3 weeks, but for long-term storage, it is recommended to store it in a desiccated state below -18°C. Once reconstituted, Platelet Factor-4 should be stored at 4°C for a maximum of 2-7 days. For long-term storage, it can be stored below -18°C. Avoid repeated freeze-thaw cycles to maintain protein stability.
The purity of the protein is greater than 95.0% as determined by:
(a) High-Performance Liquid Chromatography (RP-HPLC) analysis.
(b) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis.
The biological activity of the protein was determined using a chemotaxis assay with human neutrophils. The effective concentration range for inducing chemotaxis in this assay is between 10-100 ng/ml.
CXCL4, PF-4, PF4, Iroplact, Oncostatin-A, SCYB4, MGC138298.
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Q&A
What is the structural difference between bovine PF4 and human PF4?
Bovine PF4 is a single chain polypeptide with an apparent molecular weight of 12,300 Da and contains 107-109 amino acid residues. This is notably larger than human PF4, which has a molecular weight of approximately 8,000 Da and consists of only 70 residues . This structural difference contributes to their distinct biological activities, with bovine PF4 demonstrating higher antiheparin activity (558 units/mg) compared to human PF4 (489 units/mg) . When designing cross-species experiments, researchers should account for these differences in size and potency.
How is bovine PF4 typically isolated and purified for research applications?
Bovine PF4 can be efficiently isolated from freeze-thaw lysates of fresh bovine platelets using affinity chromatography . A single-step affinity chromatographic procedure typically yields approximately 142 μg of PF4 per mL of bovine platelets . For higher purity, researchers can employ a HiTrap Heparin high-performance affinity column followed by fast protein liquid chromatography (FPLC) using a Resource RPC FPLC column . Purity assessment should be conducted via SDS-PAGE with silver staining, and identity confirmation through immunoblotting with appropriate antibodies such as rabbit polyclonal antibodies against PF4 .
What are the primary biological functions of bovine PF4?
Bovine PF4 serves multiple biological functions:
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Neutralizes the anticoagulant effect of heparin by binding more strongly to heparin than to chondroitin-4-sulfate chains
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Releases during platelet aggregation, suggesting a role in hemostasis
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Functions as a negative regulator of megakaryopoiesis, affecting platelet production in vivo
These diverse functions make bovine PF4 relevant for research in hematology, immunology, and vascular biology.
What detection methods are commonly used to measure bovine PF4 levels?
Sandwich ELISA is the preferred method for quantifying bovine PF4 in research samples. Commercial kits provide detection ranges of 0.78-50 ng/mL with sensitivity levels of approximately 0.369 ng/mL . These assays are validated for multiple sample types, including:
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Serum
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Plasma
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Tissue homogenates
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Cell culture supernatants
When comparing results across studies, researchers should note the specific detection methodology and kit calibration standards used.
What methodological approaches can effectively study bovine PF4's role in megakaryopoiesis?
To investigate bovine PF4's regulatory role in megakaryopoiesis, researchers can employ several approaches:
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In vivo models: Study PF4 knockout mice and transgenic mice that overexpress PF4 to examine steady-state platelet counts and thrombocrit levels in relation to PF4 content
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Colony formation assays: Assess growth of megakaryocyte colonies in relation to PF4 content, including:
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Function-blocking experiments: Use anti-PF4 antibodies (typically at 25 μg/mL) to block endogenous PF4 and observe effects on megakaryopoiesis
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Recovery studies: Analyze the impact of PF4 on recovery from chemotherapy-induced thrombocytopenia using 5-fluorouracil (180 mg/kg) treatment models
These approaches provide complementary insights into PF4's regulatory mechanisms in platelet production.
What are the optimal parameters for using bovine PF4 as a replacement for BSA in embryo culture media?
Based on experimental data with porcine embryos, optimal parameters for using PF4 as a BSA replacement include:
When implementing PF4 as a BSA replacement, researchers should:
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Start with the 100-500 ng/mL concentration range
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Evaluate embryo cleavage rates at day 2
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Assess blastocyst formation rates at days 5 and 7
This methodology provides chemically defined conditions while maintaining developmental efficiency comparable to BSA-supplemented media.
How can researchers distinguish between effects of endogenous and exogenously added PF4?
To differentiate between effects of endogenous and exogenously added PF4, researchers can employ these methodological approaches:
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Neutralizing antibodies: Apply function-blocking anti-PF4 antibodies to neutralize endogenous PF4. Rabbit polyclonal anti-PF4 antibodies at concentrations of 25 μg/mL effectively block free PF4 activity
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Genetic models: Utilize PF4 knockout models (mPF4 −/−) to create systems lacking endogenous PF4, allowing clean assessment of exogenously added PF4
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Species-specific detection: When using human PF4 in bovine systems (or vice versa), employ species-specific antibodies or assays to distinguish the added protein from endogenous sources
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F(ab')2 fragments: For in vivo studies, F(ab')2 fragments prepared from anti-PF4 antibodies can be used (50-100 mg/kg) to block endogenous PF4 with reduced immunogenicity
These approaches enable researchers to parse the specific contributions of different PF4 sources in complex biological systems.
What methods can accurately assess the antiheparin activity of purified bovine PF4?
Antiheparin activity assessment of bovine PF4 requires specific methodological approaches:
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Heparin neutralization assay: Measure the ability of purified PF4 to neutralize heparin's anticoagulant effect in standardized clotting assays
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Quantitative activity units: Express activity in units/mg, with bovine PF4 typically showing approximately 558 units/mg compared to human PF4's 489 units/mg
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Surface plasmon resonance: Determine binding kinetics and affinity between PF4 and heparin molecules of different lengths and sulfation patterns
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Competitive binding assays: Assess PF4's ability to displace labeled heparin from carrier molecules like chondroitin-4-sulfate
These methodologies provide comprehensive characterization of bovine PF4's heparin-binding properties, which are critical for understanding its biological functions and potential therapeutic applications.
How does bovine PF4 influence stem cell differentiation and viability?
Bovine PF4 enhances differentiation and viability of various stem cell lines in vitro . Concentration-dependent effects are observed:
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Concentration effects: Different concentrations (100-1000 ng/mL) produce varying effects, with 100-500 ng/mL typically showing optimal results for accelerating development
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Developmental markers: PF4 treatment influences expression of differentiation-specific markers in stem cell populations
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Survival enhancement: PF4 may protect stem cells from apoptosis under stress conditions, potentially through binding to cell surface glycosaminoglycans
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Microenvironment modification: PF4 can alter the cellular microenvironment, affecting paracrine signaling that influences stem cell fate decisions
Researchers working with stem cells should consider incorporating PF4 as a culture supplement to enhance differentiation protocols and improve cell viability.
What is the relationship between bovine PF4 and thrombocytopenia recovery?
Studies using genetic models with varying PF4 expression levels demonstrate that PF4 content inversely correlates with recovery from chemotherapy-induced thrombocytopenia:
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Recovery kinetics: Animals with higher PF4 expression show delayed recovery from 5-fluorouracil-induced thrombocytopenia
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Accelerated recovery: Treatment with anti-PF4 blocking antibodies speeds recovery, especially in animals with high endogenous PF4 levels
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Therapeutic potential: PF4 blockade represents a potential approach to limit the duration of chemotherapy-induced thrombocytopenia
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Individual variation: Approximately 8% of healthy adults have elevated platelet PF4 content (>2 times average), potentially predisposing them to more severe thrombocytopenia after bone marrow injury
These findings suggest that monitoring and potentially modulating PF4 levels could be clinically relevant for managing thrombocytopenia in certain patient populations.
How can bovine PF4 be effectively incorporated into defined culture systems?
For researchers developing chemically defined culture systems, bovine PF4 can be incorporated using these methodological considerations:
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Media formulation: Add PF4 to base media (e.g., NCSU-23 with 0.3 mg/mL PVA) at concentrations between 100-500 ng/mL for optimal effects
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Stability assessment: Evaluate PF4 stability in culture conditions by measuring residual activity at different time points
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Interaction effects: Test for potential interactions between PF4 and other media components through factorial experimental designs
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Schedule optimization: For extended cultures, determine whether single addition or regular replenishment of PF4 produces superior outcomes
This approach enables the creation of completely defined culture systems that eliminate variability associated with biological supplements like bovine serum albumin.
Product Science Overview
Platelet Factor-4 (PF4), also known as CXCL4, is a small cytokine belonging to the CXC chemokine family. It is released from the alpha granules of activated platelets during the process of platelet aggregation. PF4 plays a crucial role in various physiological and pathological processes, including coagulation, inflammation, and wound healing .
PF4 is a non-glycosylated polypeptide chain containing 88 amino acids and has a molecular mass of approximately 9.5 kDa . It forms a homotetramer with a high affinity for heparin, which allows it to neutralize heparin-like molecules on the endothelial surface of blood vessels, thereby promoting blood coagulation . Additionally, PF4 is chemotactic for neutrophils and monocytes and functions as an inhibitor of hematopoiesis, angiogenesis, and T-cell function .
Recombinant PF4 (rPF4) is produced using various expression systems, with Escherichia coli (E. coli) being one of the most commonly used hosts. The production process involves the insertion of the PF4 gene into a plasmid vector, which is then introduced into E. coli cells. The recombinant protein is expressed and subsequently purified using chromatographic techniques .
Recombinant PF4 has several applications in research and clinical settings. It is used in the diagnosis of heparin-induced thrombocytopenia (HIT), a condition where antibodies form against PF4-heparin complexes, leading to a decrease in platelet count . Additionally, rPF4 is utilized in studies investigating its role in various diseases, including liver fibrosis and cancer .
