Exposure Of Human Platelets Research Articles

UVB Light Exposed Human Platelets Mediate Acute Lung Injury In a Two-Event Mouse Model of Transfusion

AbstractAbstract 3354Transfusion related acute lung injury (TRALI) has occurred in patients whose underlying condition led to an inflamed endothelium, and who were transfused with a transfusion product that contained either HLA or HNA antibodies or biologic modifiers such as lipids or antigens from stored cells. Similar two-event reactions can be modeled in animals pretreated with lipopolysaccharide (LPS) and infused with similar types of antibodies or media from stored transfusion products. The first event induces activation of the endothelium and sequestration of neutrophils in the lungs while the second event activates neutrophils to cause local tissue damage. In some animal models of acute lung damage platelet depletion reduces the lung damage while in other models infusion of activated platelets potentiates it. Ultraviolet B (UVB) light has been used on platelet transfusion products to prevent alloimmunization or with chemical sensitizers to reduce pathogens. Such processing may damage platelets and potentiate their storage lesion. UVB exposed human platelets (HPs) were evaluated in a two-event animal model of acute lung injury (ALI) where the sensitizing event was LPS and the second event was infusion of HPs or UVB HPs (2.4 J/cm2). Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). UVB exposure of HPs increased their p-selectin expression (control 17.8±0.3% vs. UVB 35.9±3.2%) and reduced their JC-1 dye ratio indicating mitochondrial damage (8.9±0.7 control vs 1.9±1.2 UVB). Internal organ distribution of intravenous (IV) infused HPs was followed with whole animal imaging, confocal microscopy and with pathophysiological changes in bronchoalveolar lavage fluid (BALF). In LPS-treated mice, UVB HPs labeled with fluorescent dye had more accumulation in lungs compared to untreated HPs (29±12% vs 15±5% respectively; % of total fluorescence recovered), while the accumulation in lungs of healthy animals was equivalent for both UVB treated and untreated HPs. In separate experiments, LPS pretreated mice were infused with UVB exposed HPs or control HPs, and lungs were examined by histology and with confocal microscopy for fluorescent staining for CD41 and CD62 expression. Histology sections revealed extensive changes in lungs, such as thickening of the alveolar septa and obliteration of lung architecture in LPS animals infused with UVB HPs as compared to healthy animals infused with control HPs or with HPs treated with UVB. Confocal microscopy with specific antibodies identified HP accumulation in lungs of LPS treated animals infused with UVB exposed HPs. However, HP accumulation in lungs did not occur with control HPs or in healthy mice. Accumulation of UVB HPs in lungs of LPS pretreated mice was associated with increased (3-4 fold compared to control HPs) protein concentration and leukocyte accumulation in BALF. Increased alveolar permeability to protein and leukocyte accumulation in alveoli is associated with acute lung damage. To examine whether lung damage occurred due to presence of UVB HPs in the lungs or due to biologic mediators released into plasma from UVB exposed HPs we separated plasma from HPs post UVB treatment and infused the UVB plasma alone. Plasma isolated from UVB HPs did not alter BALF protein levels or leukocyte counts even though the UVB HPs did. In conclusion, UVB HPs can accumulate in lungs of LPS primed animals and are associated with lung damage as indicated by histological changes and increased protein and WBCs in BALF fluid. The ALI is associated with direct platelet accumulation in the lungs but not with biologic modifiers released into plasma. Our animal model of ALI suggests that HPs exposed to high doses of UVB could mediate similar effects in patients predisposed to TRALI with sepsis or other causes of endothelial cell inflammation. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Exposure of Human Platelets to Plasmin Results in the Expression of Irreversibly Active Fibrinogen Receptors

Although plasmin can trigger strong platelet responses such as shape change and exocytosis of internal granules, limited platelet aggregation is induced by this proteinase, owing to its capacity to rapidly proteolyse secreted adhesive proteins. In this context, we have investigated the state of activation of the fibrinogen receptor, the integrin alpha IIb beta 3, on platelets exposed to plasmin. Following incubation with plasmin at 37 degrees C, washing, and resuspension, platelets exhibit a moderate, low-velocity aggregation when stirred in the presence of fibrinogen. Optimum aggregability is observed when platelets have been exposed to plasmin activity of approximately 0.5 CU/ml for 20 min, and aggregation is insensitive to the presence of antagonists such as prostaglandin (PG) E1 and apyrase. Plasmin-induced platelet aggregability is associated with the expression of active fibrinogen receptors on the cell surface, which, using a 125I-fibrinogen binding assay, can be quantified to approximately 2,300 molecules per platelet. Exposure of active alpha IIb beta 3 receptors appears to depend partially, but not totally on a metabolic activation and granule exocytosis at the time of incubation with plasmin. In contrast with alpha-thrombin, plasmin-induced activation of alpha IIb beta 3 is sustained and cannot be reversed by exposure of platelets to PGE1. Immunoblotting analysis of the receptor subunits shows no extensive proteolytic modification of alpha IIb beta 3 by plasmin, and only reveals a limited proteolysis of the aminoterminal domain of the alpha IIb subunit. In addition to their capacity to aggregate in the presence of fibrinogen alone, plasmin-treated platelets also show a potentiated aggregability in response to low doses of ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of methaqualone on blood platelet function.

To study the mechanism whereby toxic doses of methaqualone cause a bleeding tendency in humans, the effects of methaqualone, diphenhydramine, and the combination of methaqualone plus diphenhydramine on blood platelet function were investigated. Exposure of human platelets in platelet-rich plasma in vitro to final concentrations of methaqualone ranging from 1.1 to 4.5 X 10(-4)) M resulted in nearly complete inhibition of the secondary phase and significant inhibition of the primary phase of adenosine diphosphate (ADP)--induced aggregation. Both the slope and height of collagen-induced aggregation responses were reduced significantly in vitro by the drug. When methaqualone final concentrations of 1.1, 2.3, and 4.5 X 10(-4) M were studied in the presence of diphenhydramine (1.1, 2.3, and 4.5 X 10(-5) M, respectively), the degree of inhibition of ADP-induced aggregation was only slightly greater (not significant) than that observed with methaqualone. The platelets of rabbits injected intravenously with methaqualone, 10 mg/kg, demonstrated a significantly decreased ability to aggregate with ADP and collagen 30 and 60 min after administration of the drug. These results suggest that a drug-induced defect of blood platelet function may play a role in the bleeding associated with methaqualone toxicity.

Influence of lysolecithin on platelet aggregation initiated by 5-hydroxytryptamine.

PREVIOUS studies from this laboratory1,2 have shown that irreversible platelet aggregation initiated in vitro by adenosine diphosphate (ADP), adrenaline or collagen was inhibited by lysolecithin, whilst reversible ADP-induced aggregation was unaffected. This indicated that lysolecithin blocked the platelet release reaction which precedes secondary or irreversible platelet aggregation. Born3 has reported that exposure of human platelets to 5-hydroxytryptamine (5-HT) does not result in a release reaction and that platelet aggregation initiated by 5-HT is small, rapidly reversed and never enters the second phase which can be induced in human platelets by ADP or adrenaline. However, our results failed to confirm this and 5-HT was found on occasions to initiate biphasic aggregation resembling that induced by ADP. Table 1 shows the frequency of biphasic platelet aggregation, initiated by 5-HT, ADP and adrenaline, recorded during platelet studies of a group of normal male subjects and a group of male patients suffering from chronic peripheral arterial disease, none of whom was known to be taking drugs affecting platelet behaviour. The frequency of biphasic responses within both groups did not differ significantly and the response for individuals was usually the same when repeated at a later date. From the individual data summarized in Table 1 it was found that biphasic 5-HT induced aggregation was always accompanied by biphasic responses in parallel samples treated with ADP and adrenaline. Biphasic platelet aggregation curves initiated by 5-HT were all similar to that shown in Fig. 1 (control), in which secondary aggregation was preceded by partial disaggregation.