The control of angiogenesis and tumor invasion by platelet factor-4 and platelet factor-4-derived molecules

Platelet factor-4 (PF-4) is an ELR-negative chemokine that exhibits antiangiogenesis properties. PF-4 inhibits endothelial cell proliferation and migration, angiogenesis in vitro and in vivo, and tumor growth. However, tumor cells are not directly inhibited by PF-4. To date, a cell surface receptor that would explain the biological effects mediated by PF-4 has not been identified. PF-4 is able to interact directly with angiogenesis growth factors such as fibroblast growth factors (FGFs) and vascular endothelial growth factors and inhibits their interaction with cell surface receptors. Furthermore, dimerization of fibroblast growth factors is abrogated by PF-4. Whether PF-4 plays a role as an endogenous angiogenesis regulator is at present not clear. Several PF-4 fragments and modified molecules have been made that exhibit antiangiogenesis properties. Among these is a C-terminal fragment that has a defined structure, retains all the antiangiogenesis properties of the parent molecule, inhibits growth factor receptor binding, associates with FGFs, and destabilizes their three-dimensional structure. The relevance of these observations for the treatment of malignant disease is discussed.     

Genetic variants in platelet factor 4 modulate inflammatory and platelet activation biomarkers

Background- African Americans suffer from higher prevalence and severity of atherosclerosis compared with whites, highlighting racial and ethnic disparities in cardiovascular disease. Previous studies have pointed to the role of vascular inflammation and platelet activation in the formation of atherosclerotic lesions. Methods and Results- We explored the role of genetic variation in 4 chemokine/chemokine receptor genes (CX3CR1, CX3CL1, CXCR3, and PF4) on systemic inflammation and platelet activation serum biomarkers (fractalkine, platelet P-selectin, platelet factor 4 [PF4], and tumor necrosis factor-α). In total, 110 single nucleotide polymorphisms were tested among 1042 African Americans and 763 whites. The strongest association with serum PF4 levels was observed for rs168449, which was significant in both racial groups (P value: African Americans=0.0017, whites=0.014, combined=1.2 × 10(-4)), and remained significant after permutation-based multiple corrections (P(c) value: combined=0.0013). After accounting for the effect of rs168449, we identified another significant single nucleotide polymorphism (rs1435520), suggesting a second independent signal regulating serum PF4 levels (conditional P value: African Americans=0.02, whites=0.02). Together, these single nucleotide polymorphisms explained 0.98% and 1.23% of serum PF4 variance in African Americans and whites, respectively. Additionally, in African Americans, we found an additional PF4 variant (rs8180167), uncorrelated with rs168449 and rs1435520, associated with serum tumor necrosis factor-α levels (P=0.008, P(c)=0.048). Conclusions- Our study highlights the importance of PF4 variants in the regulation of platelet activation (PF4) and systemic inflammation (tumor necrosis factor-α) serum biomarkers.     

Protease-induced immunoregulatory activity of platelet factor 4

Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity.     

Recombinant platelet factor 4 for heparin neutralization

Protamine sulfate has been used for many years to reverse the effects of unfractionated heparin, but it can cause hemodynamic changes and other serious side effects. Platelet factor 4 (PF4) is a naturally occurring protein synthesized in megakaryocytes and eventually stored in the alpha granules of platelets for later release. Although the complete physiologic role of PF4 is unknown, it is highly effective for the neutralization of heparin anticoagulation. Several preliminary animal studies and trials using blood obtained from cardiopulmonary bypass circuits suggested recombinant PF4 (rPF4) would be an effective alternative to protamine. In the first open-label, phase 1 human study, patients received rPF4 in doses of 0.5, 1.0, 2.5, or 5.0 mg/kg over 3 minutes to reverse heparin anticoagulation after diagnostic cardiac catheterization. There were no important hemodynamic changes and the rPF4 was highly effective in neutralizing heparin. Serial measurements of rPF4 levels showed a monophasic elimination pattern with a serum half-life of 25.5 +/- 13.5 minutes that was independent of dose administered. A randomized and blinded trial comparing rPF4 to protamine confirmed the safety and effectiveness of rPF4. Although rPF4 was initially being evaluated as a clinical alternative to protamine, it is not currently being developed for general clinical use.     

The assessment of platelet function in vivo by measurement of beta-thromboglobulin, platelet factor 4 and platelet survival

The measurement of platelet lifespan and the plasma concentration of beta-thromboglobulin and platelet factor 4 are potentially useful techniques for assessing platelet function in vivo. Recent clinical and laboratory studies are reviewed.     

The prothrombotic potential of platelet factor 4

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by heparin administration. It is caused by the formation of pathogenic antibodies to complexes of platelet factor-4 (PF4) and heparin on platelet surfaces that cause platelet activation, aggregation and thrombosis. There has been intense research on this intriguing, drug-related thrombocytopenia explaining several characteristic aspects of this condition. However, prothrombotic potential of the key player, PF4 has not been investigated in many studies although it has been shown to be critical in monocyte chemotaxis, monocyte–platelet interaction, and megakaryocyte suppression, all of which can contribute to the pathophysiology of HIT. This article explains the important role of PF4 released during platelet activation with the administration of heparin in the pathogenesis of thrombocytopenia and thrombosis in HIT.     

Effect of heparin on in vivo platelet factor 4 (PF4) release and platelet aggregation after aspirin administration

We studied the release in vivo of platelet specific proteins platelet factor 4 (PF4) and β-thromboglobulin (βTG), and confirmed that only PF4 is released, after heparin intravenous administration. A good correlation was found between platelet count and PF4 and this seems at variance with the idea that the source of released PF4 is vascular endothelium rather than platelets; however, such a possibility cannot be excluded. Although platelet function was blocked by aspirin injection heparin was still able to induce the release of PF4 and exerted a potentiating effect on platelet aggregation induced by ADP and collagen.     

The mechanism of thrombin-induced platelet factor 4 secretion

We have measured thrombin-induced secretion of platelet factor 4 antigen (PF4) and simultaneously followed its intracellular translocation by immunofluorescence. In permeable resting platelets, speckled intracellular immunofluorescent staining for PF4 was observed. Addition of thrombin to washed platelets at 22 degrees C resulted in secretion of PF4 and formation of large (approximately 0.5 micrometer) immunofluorescent masses. These masses moved to the cell periphery during secretion and were virtually absent at the conclusion of secretion. Ultrastructural examination of thrombin-treated platelets revealed vacuoles corresponding in size, shape, and time of occurrence to the large immunofluorescent masses of PF4. These vacuoles contained PF4 by immunoferritin staining of frozen thin sections; they therefore appear to represent the ultrastructural counterpart of the large PF4 masses. When intact cells were stained for PF4 after thrombin addition, only 5.6% of the large masses stained. Thus, during secretion, PF4 antigen is consolidated into large closed pools that appear as vacuoles in the electron microscope.     

Human platelet surface binding of endogenous secreted factor VIII-von Willebrand factor and platelet factor 4

Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.     

Plasma levels of beta-thromboglobulin and platelet factor 4 in relation to the venous platelet concentration

The plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) were determined in patients with various hematologic malignancies, and the results were related to simultaneously determined venous platelet counts. All studied patients were in a steady state. The plasma beta-TG concentrations were determined on 69 occasions and the values ranged from 0 to 82 ng/ml. In 33 instances, the venous platelet count was <25 x 10 (9/1) and in two thirds of these samples beta-TG was undedectable. The highest values for plasma beta-TG were found in patients with the highest venous platelet counts. A highly significant correlation (r=0.77, p <0.001) between the values for plasma beta-TG and venous platelet count was present. The plasma concentrations for PF-4 ranged from 0 to 50 ng/ml. Similarly, there was a highly significant relationship (r=0.78, p<0.001) between the values for PF-4 and venous platelet concentration. We conclude, if the plasma levels of beta-TG and PF-4 are used as markers of platelet activation in vivo, it is necessary to simultaneously consider the platelet concentration in the collected blood.     

Plasma levels of beta-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo

Measurement of plasma levels of two secreted platelet proteins (beta- thromboglobulin and platelet factor 4) has been suggested as a means for detecting increased platelet activation in vivo. A crucial question in the measurement is the distinction between in vivo and in vitro secretion of the proteins. One approach to this distinction is the measurement of both proteins in each sample. These proteins are present in platelets in similar amounts and are released in similar quantities, but the plasma levels of beta-thromboglobulin exceed the plasma levels of platelet factor 4. This difference in plasma level is presumably due to more rapid removal of platelet factor 4 from the plasma level, and there is suggestive evidence that the rapid removal of released platelet factor 4 is due to its binding to endothelial cells. It appears that when there is increased release of beta-thromboglobulin and platelet factor 4 in vivo, there is an increase in the ratio of plasma beta-thromboglobulin to plasma platelet factor 4 compared to that found in normal individuals, whereas when in vitro release is responsible for elevated levels, the ratio decreases. Thus measurements of both proteins in each blood sample will allow distinction between in vivo release and artefactual in vitro release.     

Human platelet factor 4: Preparation from outdated platelet concentrates and application in cerebral vascular disease

Platelet factor 4 (PF4), the platelet antiheparin protein, was isolated from both the supernatant and the cells of recently outdated platelet concentrates. Following purification by affinity chromatography, a competitive binding radioimmunoassay was developed to detect this protein in human plasma. The normal range was determined to be 9.4 ± 4.7 ng/ml (mean ± SD for 52 healthy adults). In order to determine whether individuals with transient ischemic attack (TIA) or stroke had measurable increments of PF4 in their plasma, radioimmunoassay studies were performed on 11 patients with well-documented TIA, 10 patients with well-documented stroke and on 16 age-matched controls hospitalized on a neurology service with disorders unrelated to arterial thrombosis. The 16 hospitalized controls had PF4 levels of 10.3 ± 9.1 ng/ml, a value not significantly different from the 52 normals (P > 0.50). Patients with TIA had PF4 levels of 24.6 ± 12.1 ng/ml, a value significantly higher than both the 52 normals (P < 0.001) and the 16 hospitalized control patients (P < 0.005). Patients with stroke had PF4 levels of 35.4 ± 29.2 ng/ml, a value significantly higher than both the 52 normals (P < 0.001) and the 16 hospitalized control patients (P < 0.005). Outdated platelet concentrates facilitate the development of a reproducible radioimmunoassay for PF4. The elevation of this platelet-derived protein in the plasma of patients with stroke and TIA provides evidence for recent or ongoing platelet activation in the cerebral vascular disease population.     

Amino acid sequence of human platelet factor 4.

Human platelet factor 4, a protein that binds heparin, has been purified to apparent homogeneity and the complete amino acid sequence of the protein has been determined. The 70-residue polypeptide chain contains no methionine, tryptophan, or phenylalanine, and contains only a single tyrosyl residue. The sequence analysis demonstrates a highly negatively charged amino-terminal region. The carboxyl-terminal region of the polypeptide is unusual in that it contains a repetitive clustering of positively charged and hydrophobic pairs of amino acids; preliminary evidence suggests that this domain may play a role in the binding of heparin.     

Interactions of platelet factor 4 with the vessel wall

Platelet factor 4 (PF4) is a platelet-specific protein that is stored in platelet alpha granules and released following platelet activation. PF4 was the first chemokine that was isolated, but unlike other chemokines, it may not have a clear role in inflammation. Gathering evidence suggests that unlike other chemokines that bind to specific receptors, PF4's biology depends on its unusually high affinity for heparan sulfates and other negatively charged molecules at concentrations attained in the immediate vicinity of activated platelets. There has been one report that PF4 binds to CXCR3B, a chemokine receptor isoform that may be present in some vascular beds, but the biological relevance of this single observation is not clear. We propose that the main biological role of PF4 and the basis for its presence in the alpha granules of all known mammalian platelets is to neutralize surface heparan sulfate side-chains of glycosaminoglycans and to optimize thrombus development at sites of vascular injury. In addition, the binding of PF4 to surface glycosaminoglycans may also underlie its angiostatic and proatherogenic properties. Additionally, PF4 binds to several other proteins that are central to thrombosis, angiogenesis, and atherogenesis. These interactions may also contribute to its biological and pathobiological effects. Certainly, future studies using in vivo models to test biological relevance of each of these proposed mechanisms by which PF4 interacts with the vasculature are needed, as are studies to define the importance of PF4 binding to CXCR3B.     

Antigenic and antiheparin properties of human platelet factor 4 (PF4)

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.