Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time.Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay.ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca²⁺ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation.ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.     

Computational study on effect of stenosis on primary thrombus formation

The purpose of this study was to evaluate the effects of stenosis geometry on primary thrombogenesis with respect to the dynamics of the blood flow. A two-dimensional computer simulation was carried out to simulate the formation of a primary thrombus under blood flow in two geometrically different blood vessels: one straight and the other stenosed. In the simulation, blood was modeled by particles that have characteristics of plasma and of platelets. Plasma and platelet flow was analyzed using the Moving Particle Semi-implicit (MPS) method, while the motion of adhered and aggregated platelets was expressed by mechanical spring forces. With these models, platelet motion in the flowing blood and platelet aggregation and adhesion were successfully coupled with viscous blood flow. The results of the simulation demonstrated that the presence of a stenosis induced changes in blood flow and thereby altered the formation, growth, and destruction of a thrombus. In particular, whereas in the absence of stenosis, the thrombus evenly covered the injured site, in the presence of a stenosis, thrombus formation was skewed to the downstream side. The number of platelets that adhered to the injured site increased earlier as the stenosis became more severe. These results suggest that dynamic changes in blood flow due to the presence of a stenosis affect primary thrombogenesis.     

Growth kinetics of platelet thrombi

A model for the kinetics of a platelet thrombus growth is presented, which takes into account the principal hydrodynamic and cellular adhesion features of thrombus development. These consist of the rate at which platelets encounter the growing thrombus, their residence time near the surface of the thrombus, the rate of escape over the potential barrier between the free platelets and the surface of the thrombus, and the influence of ADP or related agents on the height of this potential barrier. The latter is explained in terms of the changes in the shape and surface potential of platelets, which are induced by exposure to ADP or related agents.In qualitative agreement with available experimental data, the model predicts an approximately exponential growth of the thrombus volume, a maximum in the growth rate with respect to the blood flow rate, and a plateau value which is reached by the thrombus volume with time. It is shown that at low flow rates the rate of provision of platelets by the blood stream is the determining factor, while at high flow rates the kinetics of adhesion to the surface of the thrombus are predominant. Under all circumstances, when the blood conduit becomes more than about half occluded, the resulting decrease in the blood flow rate decreases, in turn, the growth rate of the thrombus.     

Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.     

Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth

Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time.     

Sustained hypothermia accelerates microvascular thrombus formation in mice

Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thrombosis. We studied the effect of clinically encountered systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric chloride-induced microvascular thrombus formation was analyzed in cremaster muscle preparations from hypothermic mice. Additionally, flow cytometry and Western blot analysis was used to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombosis, experiments were repeated after hypothermia and rewarming to 37 degrees C. Control animals revealed complete occlusion of arterioles and venules after 742 +/- 150 and 824 +/- 172 s, respectively. Systemic hypothermia of 34 degrees C accelerated thrombus formation in arterioles and venules (279 +/- 120 and 376 +/- 121 s; P < 0.05 vs. 37 degrees C). This was further pronounced after cooling to 31 degrees C (163 +/- 57 and 281 +/- 71 s; P < 0.05 vs. 37 degrees C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as shown by 1.8- and 3.0-fold increases in mean fluorescence after PAC-1 binding to glycoprotein (GP)IIb-IIIa and 1.6- and 2.9-fold increases of fibrinogen binding on incubation at 34 degrees C and 31 degrees C. Additionally, tyrosine-specific protein phosphorylation in platelets was increased at hypothermic temperatures. In rewarmed animals, kinetics of thrombus formation were comparable to those in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37 degrees C. Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function

Various in vitro and in vivo techniques exist for study of the microcirculation. Whereas in vivo systems impress with their physiological fidelity, in vitro systems excel in the amount of reduction that can be achieved. Here we introduce the autoperfused ex vivo flow chamber designed to study murine leukocytes and platelets under well-defined hemodynamic conditions. In our model, the murine heart continuously drives the blood flow through the chamber, providing a wide range of physiological shear rates. We used a balance of force approach to quantify the prevailing forces at the chamber walls. Numerical simulations show the flow characteristics in the chamber based on a shear-thinning fluid model. We demonstrate specific rolling of wild-type leukocytes on immobilized P-selectin, abolished by a blocking MAb. When uncoated, the surfaces having a constant shear rate supported individual platelet rolling, whereas on areas showing a rapid drop in shear platelets interacted in previously unreported grapelike conglomerates, suggesting an influence of shear rate on the type of platelet interaction. In summary, the ex vivo chamber amounts to an external vessel connecting the arterial and venous systems of a live mouse. This method combines the strengths of existing in vivo and in vitro systems in the study of leukocyte and platelet function. autoperfused flow chamber; intravital microscopy; inflammation; thrombus formation     

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.     

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation

Amyloid precursor protein (APP) is the precursor of amyloid β (Aβ) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aβ peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aβ peptides Aβ_1–40, Aβ_1–42 and Aβ_25–35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aβ peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aβ peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aβ_1–42 and Aβ_25–35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aβ under static condition but not platelet aggregation, and is responsible for Aβ-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aβ peptides accumulate in cerebral vessel walls or atherosclerotic plaques.     

Model for the regulation of platelet volume and responsiveness by the trans-membrane Na+/K(+)-pump.

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.     

The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

Platelet adhesion to collagen-coated wells: analysis of this complex process and a comparison with the adhesion to matrigel-coated wells.

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.     

Original article submission: Platelet stress accumulation analysis to predict thrombogenicity of an artificial kidney

An implantable artificial kidney using a hemofilter constructed from an array of silicon membranes to provide ultrafiltration requires a suitable blood flow path to ensure stable operation in vivo. Two types of flow paths distributing blood to the array of membranes were evaluated: parallel and serpentine. Computational fluid dynamics (CFD) simulations were used to guide the development of the blood flow paths. Pressure data from animal tests were used to obtain pulsatile flow conditions imposed in the transient simulations. A key consideration for stable operation in vivo is limiting platelet stress accumulation to avoid platelet activation and thrombus formation. Platelet stress exposure was evaluated by CFD particle tracking methods through the devices to provide distributions of platelet stress accumulation. The distributions of stress accumulation over the duration of a platelet lifetime for each device revealed that stress accumulation for the serpentine flow path exceeded levels expected to cause platelet activation while the accumulated stress for the parallel flow path was below expected activation levels.     

A multiscale biomechanical model of platelets: Correlating with in-vitro results

Using dissipative particle dynamics (DPD) combined with coarse grained molecular dynamics (CGMD) approaches, we developed a multiscale deformable platelet model to accurately describe the molecular-scale intra-platelet constituents and biomechanical properties of platelets in blood flow. Our model includes the platelet bilayer membrane, cytoplasm and an elaborate elastic cytoskeleton. Correlating numerical simulations with published in-vitro experiments, we validated the biorheology of the cytoplasm, the elastic response of membrane to external stresses, and the stiffness of the cytoskeleton actin filaments, resulting in an accurate representation of the molecular-level biomechanical microstructures of platelets. This enabled us to study the mechanotransduction process of the hemodynamic stresses acting onto the platelet membrane and transmitted to these intracellular constituents. The platelets constituents continuously deform in response to the flow induced stresses. To the best of our knowledge, this is the first molecular-scale platelet model that can be used to accurately predict platelets activation mechanism leading to thrombus formation in prosthetic cardiovascular devices and in vascular disease processes. This model can be further employed to study the effects of novel therapeutic approaches of modulating platelet properties to enhance their shear resistance via mechanotransduction pathways.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemostatic effects of phospholipid vesicles carrying fibrinogen gamma chain dodecapeptide in vitro and in vivo

We studied prototypes of platelet substitutes that bear on their surface a dodecapeptide, HHLGGAKQAGDV (H12). The peptide is a fibrinogen gamma chain carboxy-terminal sequence (gamma400-411) and recognizes specifically the active form of glycoprotein (GP) IIb/IIIa on the surface of activated platelets. We conjugated H12 to the end of poly(ethylene glycol) chains on the surface of a phospholipid vesicle with an average diameter of 220 nm to prepare H12-PEG-vesicles. The half-life of the H12-PEG-vesicles was significantly prolonged by PEG modification, and the ability of H12 on the surface of the vesicle to recognize GPIIb/IIIa was maintained even though the surface was modified with PEG chains. The H12-PEG-veiscles enhanced the in vitro thrombus formation of platelets that were adhering to a collagen-immobilized plate, when thrombocytopenia-imitation blood was passed over the plate. Based on the flow cytometric analyses of PAC-1 binding and P-selectin expression, the H12-PEG-vesicles were shown not to cause platelet activation. Furthermore, the H12-PEG-vesicles dose-dependently shortened the tail bleeding time of thrombocytopenic rats. It was confirmed that the H12-PEG-vesicles had a hemostatic effect and may be a suitable candidate for an alternative to human platelet concentrates transfused into thrombocytopenic patients.