Biological action of nitric oxide donor compounds on platelets from patients with sickle cell disease.

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP-induced platelet aggregation, CD62P expression, PAC-1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC-1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.

Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization.

Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction; however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADP addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa- mediated ligand binding can be separated mechanistically from GPIIb-IIIa-mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.

Restoration of in vitro responses in platelets stored in plasma.

Conventional platelet storage in a blood bank is up to 5 days at room temperature in plasma. We investigated the optimal medium for assessing the quality of stored platelets by comparing in vitro test responses after resuspension in autologous plasma prepared from platelet-rich plasma after 5 days of storage at room temperature, autologous plasma stored cell-free for 5 days at room temperature, or autologous plasma stored cell-free for 5 days at -20 degrees C. Five-day-old platelets were prepared from aliquots of the same unit and resuspended in I of the 3 plasma preparations. The platelet-plasma mixtures were monitored for changes in pH, mean platelet volume, hypotonic shock response, P-selection expression, and aggregation. There were statistically significant differences between platelets resuspended in original plasma and platelets resuspended in either plasma stored cell-free at room temperature or frozen, with regard to hypotonic shock response, agonist-induced aggregation, and P-selectin expression. Plasma stored with platelets for 5 days yielded inferior platelet function test responses when compared with plasma stored cell-free at room temperature or frozen. Therefore, for direct comparison of platelet responses following novel storage methods, the resuspending plasma should be stored under the same conditions as the control platelet unit.

Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets.

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).

Hemoglobin A0 and alpha-crosslinked hemoglobin (alpha-DBBF) potentiate agonist-induced platelet aggregation through the platelet thromboxane receptor.

Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.

Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets.

Peroxynitrite (ONOO-) induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. We investigated the effect of peroxynitrite on protein tyrosine nitration and phosphorylation in resting or thrombin-activated platelets. Peroxynitrite (150 microM) rapidly induced tyrosine nitration of 187, 164, 113, 89, and 61 kDa proteins in gel-filtered platelets which persisted up to 4.5 h. Repeated exposure of platelets to peroxynitrite produced increasing levels of nitration. Peroxynitrite also rapidly increased tyrosine phosphorylation of 120, 117, 95, 80-85, and 70 kDa platelet proteins, but this decreased by 5 min. The same pattern of tyrosine phosphorylation, but with higher intensity, was induced by thrombin in control platelets. Pretreatment of platelets with peroxynitrite decreased thrombin-induced tyrosine phosphorylation at 0.05 and 1 U/ml thrombin but not at 2 U/ml thrombin. Platelet activation responses such as P-selectin expression, serotonin secretion, and aggregation were also decreased by peroxynitrite treatment at low thrombin concentrations. Peroxynitrite exposure and tyrosine nitration decreased platelet sensitivity to thrombin but did not absolutely prevent tyrosine phosphorylation and other platelet responses.

Endogenous ADP prevents PGE1-induced tyrosine dephosphorylation of focal adhesion kinase in thrombin-activated platelets.

Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.

Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor.

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex-activating antibody induces fibrinogen- and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD-containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand-receptor interactions that directly mediate platelet aggregation.

Immunological comparisons of integrin alpha IIb beta 3 (GPIIb-IIIa) expressed on platelets and human erythroleukemia cells: evidence for cell specific differences.

Platelet glycoprotein IIb-IIIa (GPIIb-IIIa, alpha IIb beta 3) is expressed on the cell surface of the human erythroleukemia (HEL) cell line. Previous studies have demonstrated differences in GPIIb-IIIa ligand binding properties of HEL cells when compared to platelets. Although the mRNA sequences for GPIIb and GPIIIa are identical in platelets and HEL cells, cell specific differences in the conformation states of the GPIIb-IIIa complex may exist and may explain in part the contrasting functional properties. Two monoclonal antibodies (mAbs), an anti-GPIIb mAb C3 and an anti-GPIIIa mAb D3, were used to determine whether differences in GPIIb-IIIa conformational states could be measured. Initial studies in a purified system showed that the mAbs' binding to isolated GPIIb-IIIa conformers was increased to the active GPIIb-IIIa and to dissociated receptor subunits when compared to the inactive form. Furthermore, soluble active GPIIb-IIIa was a much better inhibitor of D3 binding to the immobilized receptor compared to soluble inactive GPIIb-IIIa. Extending these studies with intact cells, we detected at least two classes of binding sites for each mAb on each cell type. Differences in Bmax and in the relative affinities of the mAbs were identified and may represent subpopulations of GPIIb-IIIa conformations. Total HEL cell and platelet GPIIb-IIIa was determined in our binding assays using a radiolabeled GPIIb-IIIa complex specific mAb, 10E5. HEL cells express approximately five times more GPIIb-IIIa on a per cell basis. The percent of total GPIIb-IIIa that represented each class of mAb binding sites was determined. In summary, the relative differences in GPIIb-IIIa conformation found on platelets and HEL cells may be related to cell-specific ligand binding properties and activation states of the receptor.