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.     

Absence of platelet activating factor (PAF) mediated platelet aggregation: A new platelet defect

The failure of normal human platelets to aggregate in response to platelet activating factor (PAF) has not been previously observed. We report here the first case of a patient whose platelets did not aggregate to PAF on multiple occasions.     

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 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.     

Analysis of platelet von Willebrand factor antigen

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand's disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.     

Evidence that platelet accelerator (platelet factor 1) is adsorbed plasma proaccelerin

1. Human platelets possess an accelerator activity equal to about six per centof the proaccelerin activity of normal citrated plasma. This accelerator activityis only slightly reduced by 10 washings.

2. Thrombin increases platelet accelerator activity as much as ten fold. Thismeans that platelet accelerator activity behaves like proaccelerin (plasma Acglobulin) rather than accelerin (serum Ac-globulin).

3. Platelets from a patient with parahemophilia (congenital proaccelerindeficiency) possess only a trace of accelerator activity. They acquire a normalamount of accelerator activity after contact with normal platelet-poor plasma.

4. Trypsin destroys 90 per cent or more of the platelet accelerator activityof normal platelets without altering their appearance. Trypsinized plateletsregain normal accelerator activity upon incubation with normal platelet-poorplasma.

5. These findings strongly suggest that the platelet accelerator (plateletfactor 1) is adsorbed plasma proaccelerin.

Submitted on April 15, 1955 Accepted on July 5, 1955     

Human platelet membrane receptor for bovine von Willebrand factor (platelet aggregating factor): An integral membrane glycoprotein

The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property of a soluble glycoprotein, glycocalicin, that is loosely attached to the platelet surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose-response curve obtained with Brij 99-solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or Lens culinaris lectin. By using wheat germ agglutinin-Sepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4 degrees C in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents.     

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

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.     

N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor

Recent evidence suggests that endothelium-derived relaxing factor exhibits properties of nitric oxide. Like nitric oxide, it inhibits platelet function and mediates its effects by elevating intracellular cyclic GMP. In this study we have investigated the role of reduced thiol in the mechanism of action of endothelium-derived relaxing factor on platelets. Bovine aortic endothelial cells were grown on microcarrier beads and pretreated with aspirin before use. Endothelial cells stimulated with bradykinin or exposed to stirred medium expressed a dose-dependent inhibition of platelet aggregation that was potentiated by the reduced thiol, N-acetylcysteine. Endothelial cell-mediated platelet inhibition was attenuated by methylene blue. Inhibition of platelet aggregation by endothelial cells was associated with a rise in platelet intracellular cyclic GMP, an effect that was enhanced by N-acetylcysteine. These data show that 1) the reduced thiol N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor and 2) this effect is associated with increasing intracellular platelet cyclic GMP levels.     

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.