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.

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.

Plasma and platelet von Willebrand factor defects in uremia.

PURPOSE: Several mechanisms have been proposed to explain the prolonged bleeding times and clinical bleeding in chronic renal failure. Recent evidence has implicated an abnormality in the structure or function of the von Willebrand factor or in its interaction with uremic platelets. We investigated this factor in 11 patients with chronic renal failure. PATIENTS AND METHODS: Blood samples for cell counts, chemistries, and coagulation studies were obtained from 11 patients with chronic renal failure and prolonged bleeding times. Concentrations of von Willebrand factor antigen and ristocetin cofactor activity were determined in plasma and platelets. Multimeric analysis of von Willebrand factor in plasma and platelets was conducted. In eight cases, the platelets of uremic patients were purified, and the thrombin- and ristocetin-induced binding of normal von Willebrand factor to these platelets was examined. RESULTS: The mean plasma von Willebrand factor antigen and activity (ristocetin cofactor assay) were elevated 2.77 mu/ml and 1.88 mu/ml, respectively (normal, 1.01 mu/ml and 1.07 mu/ml, respectively). The ratio of activity to antigen in uremic plasma was 0.67 (normal, 1.05). The mean platelet von Willebrand factor antigen and activity in the uremic patients was decreased (0.26 and 0.50 mu/10(9) platelets, respectively) compared with normal patients (0.46 and 0.93 mu/10(9) platelets, respectively). The oligomeric structure of the uremic plasma von Willebrand factor lacked the largest multimers. Collection of the blood for analysis in several protease inhibitors and/or EDTA did not change the multimeric structure. The von Willebrand factor multimeric structure of platelets from uremic patients was normal. The ristocetin-induced platelet aggregation of the uremic platelet-rich plasma was decreased compared with normal plasma samples. Thrombin and ristocetin-induced binding of normal von Willebrand factor to uremic patients' platelets was indistinguishable from the binding to normal platelets. CONCLUSION: These data suggest that the uremic platelet-binding sites for von Willebrand factor are intact and that the defect in ristocetin-induced platelet aggregation is most likely plasmatic in nature. At least one plasmatic defect was the observed reduction or absence of the largest plasma von Willebrand factor multimer in uremic patients. The platelet von Willebrand content was significantly decreased. These defects may play a role in the prolonged bleeding time and the clinical bleeding observed in patients with uremia.

Platelet density-dependent partition of platelet-von Willebrand factor between alpha granule and non-alpha granule pools.

In this study, we demonstrate that platelets contain a small but significant amount of platelet-von Willebrand factor (vWf) not associated with alpha-granules. When platelets free of plasma proteins are exposed to micromolar concentrations of digitonin, plasma membrane permeabilization occurs without disruption of platelet granules. Employing this technique, we have found that upon exposure of a total platelet population to 8 microM digitonin, 5% of total platelet-vWf is released into the supernatant; this occurs without release of beta-TG from alpha-granules. When platelets of discrete buoyant density profiles are tested, this extragranular platelet-vWf increased with decreasing platelet density. These findings suggest that a redistribution of platelet-vWf from alpha-granule to non-granule sites occurs coincident with a decrease in platelet buoyant density.

Platelet von Willebrand factor: comparison with plasma von Willebrand factor.

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that the multimeric organization was different, larger multimers were present, and the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.

Factor VIII/von Willebrand factor binding to von Willebrand's disease platelets.

A form of von Willebrand's disease has been described with enhanced ristocetin-induced platelet aggregation and anodal migration of the factor VIII/von Willebrand factor protein (type IIb). We studied two families with this form of von Willebrand's disease and macrothrombocytopenia. We have found that these platelets bind more of the normal and intermediate-sized multimers of the factor VIII/von Willebrand factor than normal platelets. Analysis of the binding data show an increased affinity of these vWd platelets for the factor VIII/von Willebrand factor. These findings are consistent with an increased number of platelet receptors, which, either by their native topography or migration on the platelet surface, bind factor VIII/von Willebrand factor protein with greater affinity than normal platelets, platelets of other vWd patients, and large platelets of other etiologies.

Carbon monoxide production associated with ineffective erythropoiesis.

The rate of endogenous carbon monoxide production ( Vco), determined by the closed rebreathing system technique, was elevated above the normal range in four of five patients studied with ineffective erythropoiesis (four patients with primary refractory anemia, one with thalassemia). The mean molar ratio of Vco to Vheme (rate of circulating heme catabolism, determined from (51)Cr red cell survival curves) was 3.0 +/- 0.6 (SE), indicating that most of the CO originated from sources other than circulating erythrocyte hemoglobin, in contrast to previous findings in patients with hemolytic anemia, where Vco paralleled Vheme closely.After administration of glycine-2-(14)C to these patients, endogenous CO was isolated by washout of body CO stores at high pO(2) or by reacting peripheral venous blood samples with ferricyanide. The CO was then oxidized to CO(2) by palladium chloride and trapped for counting in a liquid scintillation spectrometer. "Early labeled" peaks of (14)CO were demonstrated which paralleled "early labeled" peaks of stercobilin and preceded maximal labeling of circulating heme. Production of "early labeled" (14)CO in patients with ineffective erythropoiesis was greatly increased, up to 14 times that found in a normal subject. The increased Vco and "early (14)CO" production shown by these patients are presumably related mainly to heme catabolism in the marrow. The possibility exists that hepatic heme and porphyrin compounds may also contribute significantly to Vco, as suggested by the finding of a high Vco in an additional patient with porphyria cutanea tarda.