Loading solution prevents activation damage of human platelets before lyophilization

The current study aims to optimize the compositions of platelet activation-inhibitors for a loading solution of lyophilizing protectants and to establish a series of perfect pretreatment methods for platelet lyophilization. The optimal combination of six kinds of inhibitors and loading solutions of lyophilizing protectants, including prostaglandin E1 (PGE1), adenosine, l-arginine, phyticacid, bivalirudin, and cilostazol, was analyzed using the orthogonal experimental design. The values of the expression rates of p-selectin (CD62p) and platelet membrane glyeoprotein (PAC-1), as well as of platelet and mean platelet volume (MPV), were selected as indices of platelet activation. The values of CD62p and Pac-1 induced by thrombin were determined as indices of platelet reactivity. The maximal aggregation and slide platelet aggregation test (SPAT) induced by the inducer were calculated as indices of the aggregation function of platelets. Level I of the loading condition factor had no adverse action on MPV, CD62p, PAC-1, SPAT, and the maximum platelet aggregation rate. Level II of factors PGE1, l-arginine, phycicacid sodium, and Bivalirudin could inhibit the activation of platelets and enable them to retain their function. The results show that the optimal solution compounding was the third group. The loading solution, which includes plasma, 1 μM prostaglandin E1, 5 mM l-arginine, 0.5 mM phyticacid, and 0.5 μM bivalirudin, could prevent the activation damage of platelets before lyophilization.     

Mechanism of collagen activation in human platelets

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.     

Conditions of activation of guanylate cyclase from human platelets

Preincubation (50 min, 0 degree C) with nitroprusside increases 12-fold the activity of human platelet guanylate cyclase. The stimulating effect of nitroprusside is enhanced two-fold by dithiothreitol (2 mM) and by 60% by hemoglobin (20 micrograms/ml). Storage of guanylate cyclase preparations (105000 g supernatant) for 2-3 days at 4 degrees C causes a progressive increase of the enzyme activity and diminishes the stimulating effect of nitroprusside. After storage of guanylate cyclase preparations for 3 days, hemoglobin (20 micrograms/ml) augments the stimulating effect of nitroprusside by 130%. It is concluded that the degree of activation of guanylate cyclase by nitroprusside reflects the functional state of the enzyme.     

Activated human platelets induce factor XIIa-mediated contact activation

Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood.Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time.We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.     

Mathematical model of PAR1-mediated activation of human platelets

Thrombin, one of the major proteases in the coagulation cascade, activates protease activated receptors 1 and 4 (PAR 1 and PAR4) to generate a network of intracellular signals that lead to stable platelet aggregation. Abnormal platelet activation could lead to either thrombosis or bleeding disorders, thus a predictive model of platelet activation would be an invaluable tool for the study of platelet function. In this work, we developed a computational model of PAR1-stimulated human platelet activation fully based on experimental observations. The model is represented by a system of ordinary differential equations (ODEs) describing the kinetics of the interacting components. The model is able to reproduce experimental dose responses and time-courses of cytosolic calcium (Ca(2+)), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), GTP-bound Ras-proximate-1 (Rap1GTP), secretion of dense-granules, and activation of integrin α2bβ3 (GPIIbIIIa). Because of the inherent complexity of such a model, we also provide a simple way to identify and divide the system into interlinked functional modules to reduce the number of unknown parameters. Both the full and the reduced kinetic models are shown to predict platelet behavior in response to PAR1 activation.     

Epinephrine potentiates activation of human platelets by low density lipoproteins

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

Protein phosphorylation and activation of human platelets by sodium fluoride

The ability of sodium fluoride (NaF) and thrombin to stimulate aggregation and protein phosphorylation in intact human platelets was measured and compared. When platelets were stimulated by NaF, phosphorylation of the 20 KDa protein was transient and after 5-10 min returned to the same level as that of unstimulated cells. On the other hand, 47 KDa protein was slowly phosphorylated without obvious dephosphorylation. The slow activation of the 47 KDa protein phosphorylation correlated well with the time required for the aggregation and secretion. Phosphoamino acid analysis showed that the phosphorylated amino acids of the 47 KDa protein from platelets activated by NaF and thrombin were slightly different. These results suggest that different stimuli may lead to the same protein phosphorylation by different biochemical mechanisms of action.     

FGF-10 prevents mechanical stretch-induced alveolar epithelial cell DNA damage via MAPK activation

Cyclic stretch of alveolar epithelial cells (AEC) can alter normal lung barrier function. Fibroblast growth factor-10 (FGF-10), an alveolar type II cell mitogen that is critical for lung development, may have a role in promoting AEC repair. We studied whether cyclic stretch induces AEC DNA damage and whether FGF-10 would be protective. Cyclic stretch (30 min of 30% strain amplitude and 30 cycles/min) caused AEC DNA strand break formation, as assessed by alkaline unwinding technique and DNA nucleosomal fragmentation. Pretreatment of AEC with FGF-10 (10 ng/ml) blocked stretch-induced DNA strand break formation and DNA fragmentation. FGF-10 activated AEC mitogen-activated protein kinase (MAPK), and MAPK inhibitors prevented FGF-10-induced AEC MAPK activation and abolished the protective effects of FGF-10 against stretch-induced DNA damage. In addition, a Grb2-SOS inhibitor (SH(3)b-p peptide), a RAS inhibitor (farnesyl transferase inhibitor 277), and a RAF-1 inhibitor (forskolin) each prevented FGF-10-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in AEC. Moreover, N17-A549 cells that express a RAS dominant/negative protein prevented the FGF-10-induced ERK1/2 phosphorylation and RAS activation in AEC. We conclude that cyclic stretch causes AEC DNA damage and that FGF-10 attenuates these effects by mechanisms involving MAPK activation via the Grb2-SOS/Ras/RAF-1/ERK1/2 pathway.     

Redistribution of the fibrinogen receptor of human platelets after surface activation

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surface receptor sites suggesting a link between the surface receptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction.     

Monitoring of the activation of receptor-operated calcium channels in human platelets

Activation of receptor-operated calcium channels has been monitored by measurements of the quenching of the fluorescence of intracellularly trapped fura-2 by Mn entering from the extracellular medium. Release of calcium from intracellular stores was followed simultaneously by measurements of the ratio of the fluorescence excited at 340 and 380 nm. Thrombin, ADP, platelet-activating-factor (PAF) and collagen, all produced both release of calcium from the intracellular stores and uptake of Mn from the extracellular medium. The uptake of Mn, but not the increase of (Ca2+)i, was blocked by nickel. These results suggest the existence of plasma membrane calcium channels which can be activated by the different agonists tested here. The activation of calcium channels was very fast and transient with ADP and PAF, fast and maintained with thrombin, and delayed with collagen.     

Membrane dynamic alterations associated with activation of human platelets by thrombin

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.     

Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.