Release Of Alpha-granule Contents Research Articles

Constricted microfluidic devices to study the effects of transient high shear exposure on platelets.

Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and αIIbβ3 activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s-1 for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (βTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, βTG, and sP-selectin).

Platelets from platelet-rich-plasma versus buffy-coat-derived platelets: what is the difference?

Platelets from platelet-rich-plasma versus buffy-coat-derived platelets: what is the difference?

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

Storage of buffy coat–derived platelets in additive solutions: in vitro effects of storage at 4°C

The aims of this in vitro study were to compare the storage of platelets (PLTs) at 4 degrees C with those stored at 22 degrees C and to determine the in vitro effects of preincubation at 37 degrees C for 1 hour before the analysis on the basis of the maintenance of PLT metabolic and cellular integrity. PLT concentrates (PCs) were prepared from pooled buffy coats (BCs) for paired studies (total eight pools from 160 BCs). Each pool was divided into four PCs and stored under different conditions: at 20 to 24 degrees C on a flatbed agitator, at 20 to 24 degrees C on a flatbed agitator and with incubation of the samples at 37 degrees C for 1 hour before the analysis, at 4 degrees C, and at 4 degrees C and with incubation of the samples at 37 degrees C for 1 hour before the analysis. Storage of PLTs at 4 degrees C resulted in reductions in the rate of glycolysis and better retention of pH after Day 10 than in PCs stored at 22 degrees C (Day 14, 7.003 +/- 0.047 vs. 7.201 +/- 0.146). Hypotonic shock response and extent of shape change were higher at 22 degrees C than at 4 degrees C and in preincubated PCs stored at 22 degrees C than in reference PCs stored at the same temperature (Day 5, 45.6 +/- 2.7 vs. 36.5 +/- 3.9 and 24.1 +/- 2.0 vs. 15.5 +/- 1.8). The concentration of RANTES was higher in PCs stored at 22 degrees C than at 4 degrees C (Day 7, 179 +/- 25 vs. 79 +/- 32). PLTs stored at 4 degrees C without agitation maintain metabolic and cellular characteristics to a great extent during 21 days of storage. These studies confirm the view that PLTs lose their discoid shape and that this loss with storage at 4 degrees C is associated with reductions in metabolic rate and in their release of alpha-granule content.

Distinguishing features of basophil and neutrophil activation by major basic protein

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Storage of single‐donor platelet concentrates: metabolic and functional changes

During the last decade, the trend toward intensifying chemotherapeutic regimens in patients with hematologic malignancies rapidly increased the demand for single-donor platelet concentrates (PCs). The logistics of such supply, however, necessitated the storage of these blood components prior to transfusion. Today, most blood centers use di(2-ethylhexyl)phthalate-free blood bags, which are assumed to allow a storage period of up to 5 days. This report describes biochemical and functional changes of stored single-donor PCs, which may influence the expected quality of PCs. The acid-base status is characterized by an initial respiratory alkalosis compensated by a metabolic acidosis. Changes in extracellular electrolyte, lactate dehydrogenase, glucose, lactate, elastase, and complement levels, as well as in the release of alpha granule content and the initial activation of plasma coagulation, are demonstrated. These changes result in a functional impairment of stored PCs as reflected by thromboxane and serotonin release reaction and by aggregation and in vitro bleeding time studies. In contrast, in vivo recovery and survival rates have been reported to be unaffected. Whether the good recovery and survival rates are caused by a rejuvenescence of stored PCs in vivo or are due to injured circulating platelets has not yet been proven.

Shear stress activation of platelets with subsequent refractoriness

Platelet rich plasma was prepared with soybean trypsin inhibitor (SBTI) as anticoagulant, to preserve normal Ca++-concentration, and subsequently tested in an aggregometer. Shear stress alone, induced by stirring, resulted in platelet aggregation and disaggregation. Such platelets showed only minor aggregational response on the addition of ADP, indicating that the platelets had been partly desensitized towards ADP-stimulation. When the stirrer was started at the same time as the addition of 1.6 pM ADP, the aggregation velocity was related to the speed of the stirrer. Disaggregation always started 1.5 min after the stimulus. Platelet shape change was apparently not influenced by the stirring. Since platelets from SBTI-anticoagulated blood could be aggregated by stirring alone, we examined the effect of centrifugation of whole blood at 50xg for 3 and 10 min. Signs of transient aggregation and release of alpha-granule content were respectively observed.