Polyamines and platelet aggregation

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polycythemia rubra vera. Spermidine and spermine inhibit $\text{in}$$\text{vitro}$ plate-let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents. The proposal is made that these organic cations may modulate $\text{in}$$\text{vivo}$ platelet agglutinability.     

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.     

Glycerol stress and platelet integrity

Platelet response to glycerol gradient was studied using a few in vitro parameters. These were platelet count, mean platelet size, platelet response to hypotonic stress (PHRS), and collagen-induced platelet aggregation. An equal volume of 1-10% (w/v) glycerol in plasma was added at once to the platelet concentrate resulting in 0.5-5% (w/v) final glycerol concentration. The concentrate was kept at 22 degrees C for 60 min. Platelets were then separated by one centrifugation and resuspended in glycerol-free plasma. A loss in platelet count was observed when the gradient of glycerol was more than 3%. This was associated with an increase in mean cell size and a reduction in aggregability. With 5% glycerol stress, a loss of 30% in cell count, an increase in 18% in cell size, and a 78% loss in aggregability was observed. Declining of PRHS was shown already with a 1% glycerol gradient and 69% of this function was suppressed by 5% glycerol stress. In other experiments, 5% glycerol was first added, them removed in 5 steps with a gradient of 1% each. When time interval between each step was less than 0.5 min, platelet loss and PRHS reduction were 17 and 47% respectively. These values were gradually improved to 4% and 11-20%, respectively, as increasing time interval up to 15 min. It was concluded that a gradient of 1% glycerol and a 15-min interval for each step minimizes the detrimental osmotic stress on platelets while glycerol is added or removed. Our findings may lead up to devising an improved protocol for platelet cryopreservation with glycerol.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

Regulation of platelet production: the normal response to perturbation and cyclical platelet disease

An age-structured model for the regulation of platelet production is developed, and compared with both normal and pathological platelet production. We consider the role of thrombopoietin (TPO) in this process, how TPO affects the transition between megakaryocytes of various ploidy classes, and their individual contributions to platelet production. After the estimation of the relevant parameters of the model from both in vivo and in vitro data, we use the model to numerically reproduce the normal human response to a bolus injection of TPO. We further show that our model reproduces the dynamic characteristics of autoimmune cyclical thromobocytopenia if the rate of platelet destruction in the circulation is elevated to more than twice the normal value.