Platelet aging in vivo is associated with activation of apoptotic pathways: studies in a model of suppressed thrombopoiesis in dogs

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial deltapsi (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5+/-1.7 vs 4.7+/-1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial deltapsi alteration. The results show that platelet aging in vivo is associated with a decrease in mitochondrial deltapsi and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.     

Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood--different effects of collagen,TRAP and calcium ionophore A23187

We have studied the effects of different platelet agonists on phosphatidylserine (PS) exposure and clotting times in blood without anticoagulants. Similar reductions in clotting time were obtained for collagen, TRAP-6 or calcium ionophore A23187 (50 micro mol/L), in spite of huge differences in PS expression [6.7 +/- 2.4%, 2.3 +/- 0.5% and 99.9 +/- 0.1%, respectively (mean +/- SD, n = 5)]. Furthermore, the clotting times were much longer for samples with A23187 exposing the same amounts of PS as samples with collagen or TRAP-6. Annexin V reversed the clotting time reduction, but could not prevent coagulation. Addition of phospholipid vesicles containing 20% PS neither affected the clotting times nor induced clotting in recalcified, platelet-free plasma. We conclude that platelet PS exposure is necessary, but not sufficient, for the coagulation amplification observed when platelets are stimulated via physiological receptors in a whole blood environment.     

Membrane fluidity is related to the extent of glycation of proteins, but not to alterations in the cholesterol to phospholipid molar ratio in isolated platelet membranes from diabetic and control subjects.

Platelets from diabetic subjects are hypersensitive to aggregating agents in vitro. Membrane fluidity modulates cell function and we previously reported reduced membrane fluidity associated with hypersensitivity to thrombin in intact platelets from diabetic subjects. Reduced membrane fluidity and hypersensitivity to agonists has also been reported in platelets from non-diabetic subjects whose platelets have an increased cholesterol/phospholipid molar ratio. Glycation of platelet membrane proteins is enhanced in diabetic subjects, and could contribute to the decreased membrane fluidity in these platelets. We examined the relation among fluidity, cholesterol/phospholipid molar ratio, and glycation of proteins in isolated platelet membranes from diabetic and control subjects. Seven poorly controlled diabetic subjects were compared with 7 age- and sex-matched control subjects. The mean steady-state fluorescence polarization value in 1,6-diphenyl-1,3,5-hexatriene-labeled isolated platelet membranes from diabetic subjects (0.184 +/- 0.004) was significantly greater than from control subjects (0.171 +/- 0.004, p less than 0.01); thus, fluidity in platelet membranes from diabetic subjects is decreased. Reduced fluidity in platelet membranes from diabetic subjects could not be attributed to changes in the cholesterol/phospholipid molar ratio. Total or very low density (VLDL), low density (LDL), or high density (HDL3) lipoprotein cholesterol concentration in plasma was not significantly different between groups, but the ratio of VLDL+LDL to HDL2 + HDL3 cholesterol was significantly greater in diabetic subjects (4.79 +/- 0.73) than in control subjects (2.54 +/- 0.30, p less than 0.02). Proteins were glycated significantly more extensively in platelet membranes from diabetic subjects (25.5 +/- 0.9 nmol glucose/mg protein) than those from control subjects (21.0 +/- 0.6 nmol glucose/mg protein, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of platelet-derived factor Va to thrombin generation on immobilized collagen- and fibrinogen-adherent platelets

Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e.g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 +/- 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.     

PS-liposome and ox-LDL bind to different sites of the immunodominant domain (#155-183) of CD36: a study with GS95, a new anti-CD36 monoclonal antibody

CD36, a multifunctional adhesive receptor on a variety of cells such as monocytes and platelets, has been implicated in clearance of modified LDL and in the removal of apoptotic or senescent cells. We recently developed a new anti-CD36 monoclonal antibody, GS95. We determined the binding site of phosphatidylserine (PS)-liposome on CD36 by flow cytometric analysis of competitive bindings between phospholipid-liposomes or synthetic CD36 peptides and FITC-labeled anti-CD36 antibodies (GS95, OKM5, and FA6-152). The epitope of GS95 was mapped to the amino acid sequence #162-183 of CD36 that was partially overlapped with, but distinct from, #155-183, which has been reported as the epitopes of two commercially available antibodies, OKM5 and FA6-152. Oxidized-LDL dose-dependently inhibited bindings of both GS95 and OKM5 antibodies to platelet CD36, while PS-liposome inhibited the binding of GS95 but not OKM5 or FA6-152. These results indicate that the binding site of PS-liposome on platelet CD36 is not identical to that of oxidized-LDL and may be located in the amino acid sequence #162-183.     

Estimation of platelet size by measurement of intracellular water space using an oil technique

The intracellular water space of human platelets has been measured after equilibration with tritiated water and then separating these cells by centrifugation through phthalate oil of density 1.042. The mean intracellular water space of platelets in citrated plasma was 0.52 +/- 0.09 microliter/10(8) cells for 19 normal subjects. The gravimetric water content of platelets was 784 +/- 4 mg water/g cells. From these values the mean platelet volume was calculated to be 6.2 fl which agrees closely with values based on Coulter size distribution and thrombocytocrit. Gel filtration alters platelets such that a mean 19% of the platelets could not be centrifuged through phthalate oils of density 1.031 or 1.042. The measurement of tritiated water space of platelets centrifuged from their own plasma through oil provides a simple and reliable estimate of the mean platelet size.     

βγ-CAT, a non-lens betagamma-crystallin and trefoil factor complex, induces calcium-dependent platelet apoptosis

In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets. βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, caused several in vivo toxic effects on mammals. Through determined haematological parameters of rabbits, it has been found that βγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect of βγ-CAT on platelets, washed platelets were incubated with various concentrations of βγ-CAT for 30 minutes. We found that βγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarisation of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However, βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation. In addition, we observed that βγ-CAT-induced PS exposure and ΔΨm depolarisation in platelets are Ca2+-dependent. Taken together, βγ-CAT can induce Ca2+-dependent platelet apoptosis but does not cause platelet activation.     

Factor Xa-driven thrombin generation in plasma: dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins

Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP). The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS. Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa. With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 microM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to four-fold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10-20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.     

Structural aspects of in vivo aging rabbit blood platelets

The structural and density changes that occur in aging blood platelets may help to evaluate the age distribution of platelets in a blood sample. Separation of normal rabbit platelets on human serum albumin discontinuous density gradient (HSAG), provided four bands of cells, usually in the 18,19,22 and 23% solutions of HSA. Large and small platelets were found in each of the four bands. No significant differences were found in the mean size of the cells taken from the light fractions (18% and 19%) when compared to cells taken from the heavier fractions (22% and 24%). However cells of the light fractions usually contained less Dense Bodies (DB) than cells from the heavier fractions. Newly formed platelets obtained from rabbits recovering from induced thrombocytopenia after injection of goat antirabbit platelet serum or neuraminidase, are generally large and found in the solution of 21% HSA (S.G. 1.0606). These platelets contain glycogen particles and practically no DB. After several days of maturation and regeneration, smaller platelets containing higher number of DB, are found in the circulation. On the 10th day, after the beginning of regeneration, platelets are distributed above and below the solution of 21% HSA, similarly to the normal platelet population. The $\text{in vivo}$ aging of platelets, seems to be associated with accumulation of DB within these cells.     

Caspase inhibition decreases both platelet phosphatidylserine exposure and aggregation: caspase inhibition of platelets

Apoptosis of nucleated cells is regulated by caspases, a group of cysteine proteases, and is characterized by phosphatidylserine expression on the outer leaflet of the plasma membrane. Reports indicate that platelets contain caspases. However, the role of caspases in platelet function is not well understood. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane. In addition, platelets aggregate when activated. The aims of this study were to determine if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk): (1) decreased PS expression and (2) decreased platelet aggregation following activation. Flow cytometry was used to determine PS expression and a platelet aggregometer was used to assess aggregation. We found that platelets treated with zVAD-fmk significantly decreased both A23187-induced PS exposure (total fluorescence index, TFI: A23187=791.42±174; zVAD+A23187=92.97±57, p≤0.05) and ADP-induced PS exposure (TFI: ADP=669.24±145, zVAD+ADP=174.6±151, p≤0.05). Further, treatment with zVAD-fmk significantly decreased ADP-induced platelet aggregation (%: UNTREATED=80±1.5, zVAD TREATED=69±3.0, p≤0.05). These results indicate that caspases play a role in platelet activation, suggesting a unique physiologic role for these proteases.     

Anti-beta2 glycoprotein I antibodies cause inflammation and recruit dendritic cells in platelet clearance

Scavenger phagocytes are mostly responsible for the in vivo clearance of activated or senescent platelets. In contrast to other particulate substrates, the phagocytosis of platelets does not incite proinflammatory responses in vivo. This study assessed the contribution of macrophages and dendritic cells (DCs) to the clearance of activated platelets. Furthermore, we verified whether antibodies against the beta2 Glycoprotein I (beta2GPI), which bind to activated platelets, influence the phenomenon. DCs did not per se intemalise activated platelets. In contrast, macrophages efficiently phagocytosed platelets. In agreement with the uneventful nature of the clearance of platelets in vivo, phagocytosing macrophages did not release IL-1beta, TNF-alpha, or IL-10, beta2GPI bound to activated platelets and was required for their recognition by anti-beta2GPI antibodies. DCs internalised platelets opsonised by anti-beta2GPI antibodies. The phagocytosis of opsonised platelets determined the release of TNF-alpha and IL-1beta by DCs and macrophages. Phagocytosing macrophages, but not DCs, secreted the antiinflammatory cytokine IL-10. We conclude that anti-beta2GPI antibodies cause inflammation during platelet clearance and shuttle platelet antigens to antigen presenting DCs.     

Assessment of platelet activation in several different anticoagulants by the Advia 120 Hematology System, fluorescence flow cytometry, and electron microscopy

In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.     

Platelet PAR1 receptor density--correlation to platelet activation response and changes in exposure after platelet activation

INTRODUCTION: A polymorphism (-14 A/T) affecting PAR1 expression on the platelet surface has recently been identified. A two-fold variation in receptor density, which correlated with the platelet response to PAR1-activating peptide (PAR1-AP), has been reported. MATERIALS AND METHODS: We used flow cytometry to measure the correlation between the number of PAR1 receptors and platelet activation. We also measured the changes in receptor exposure after platelet activation with PAR1-AP, ADP, PAR4-AP or a collagen-related peptide (CRP). RESULTS: In our study, the PAR1 receptor number varied almost four-fold, from 547 to 2063 copies/platelet (mean+/-S.D. 1276+/-320, n=70). The number of PAR1 receptors on resting platelets correlated to platelet fibrinogen binding and P-selectin expression following platelet activation with PAR1-AP (r(2)=0.30, p<0.01 and r(2)=0.15, p<0.05, respectively, n=36). The correlation was not improved by exclusion of the ADP-component from the PAR1-AP-induced response. We found a trend, but no statistically significant differences in PAR1 receptor number and platelet reactivity between A/A individuals and T/A or T/T individuals. Ex vivo activation with PAR1-AP decreased PAR1 surface exposure to 71+/-19% of the exposure on resting platelets (mean+/-S.D., p<0.01, n=19), while activation by ADP, PAR4-AP or CRP significantly increased the exposure, to 151+/-27%, 120+/-21% and 138+/-25%, respectively (n=11, 11 and 10). CONCLUSIONS: This study shows a large variation in PAR1 receptor number in healthy individuals, a variation correlated to the platelet activation response. We found a significant reduction in PAR1 surface exposure after adding PAR1-AP, while activation with ADP, PAR4-AP or CRP increased the exposure.     

CD32-dependent platelet activation by a drug-dependent antibody to glycoprotein IIb/IIIa antagonists

Thrombocytopenia is observed with a frequency of up to 2% in patients treated with glycoprotein (GP) IIb/IIIa antagonists. We recently provided evidence that thrombocytopenia is caused by antibody binding to drug-induced conformational changes in GP IIb/IIIa. Here, we report that a murine monoclonal antibody binds to GP IIb/IIIa in an antagonist-dependent manner and activates platelets. Platelet stimulation is associated with a disruption of the phospholipid asymmetry, resulting in the assembly of catalytic active intrinsic Xase and prothrombinase complexes. Further mechanistic studies revealed that this response is (I) mediated in cis, (II) not associated with the formation of prothrombotic microparticles, and (III) requires intact platelet signaling and (IV) is blocked by increases in cAMP. The prothrombotic response is not observed using F(ab')2 fragments and is blocked by incubation of platelets with neutralizing antibodies to the platelet FcgammaRIIa receptor (CD 32).Taken together, these observations suggest that GPIIb/IIIa antagonist-dependent antibody binding to the platelet fibrinogen receptor has the propensity to lead to CD32-mediated platelet activation and accelerated platelet clearance, leading to thrombocytopenia.     

Indium-111-labelled human platelets: a method for use in severe thrombocytopenia

We describe and evaluate a simple method for labelling autologous human platelets with Indium-111-oxine in patients with severe thrombocytopenia. Twenty patients with immune thrombocytopenia and platelet counts ranging from 5 to 119 X 10(9)/1 were investigated. Platelets were isolated from blood by differential centrifugation, residual platelets were repeatedly washed from the red cell layer and buffy coat and labelled with In 111 in saline. A mean of 55% +/- 21 of platelets were harvested from the blood, labelled with 49% +/- 24 efficiency and 15.8 X 10(8) labelled platelets reinjected to the patients. Contamination of the platelets with red cells and plasma was low. The labelled platelets were viable as assessed by in vitro aggregation, recovery in the circulation and mean survival time. This method permits quantitative platelet imaging with autologous labelled platelets in patients with severe thrombocytopenia.     

A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro

Barbourin is a 73 amino acid venom protein that inhibits platelet aggregation. Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahistidine (H6) tags. BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP. BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin alphaIIbbeta3. The terminal catabolic half-life of BLAH6 approached that of RSAH6 [3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 +/- SD)], but was substantially increased relative to that of BARH6 [0.15 +/- 0.03 days (n = 3 +/- SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.     

Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome

Recent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin+ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity.     

Differences between sodium-dependent and desipramine-defined [3H]imipramine binding in intact human platelets

Measurements of sodium-dependent [3H]imipramine binding to intact human platelets from 20 human volunteers were made and compared to desipramine-defined binding, a method commonly employed in population studies of platelet [3H]imipramine sites. The density (Bmax) of sodium-dependent [3H]imipramine sides in platelets was significantly lower (449 +/- 36 sites/platelet) and the affinity (Kd) significantly higher (1.15 +/- 0.12 nM) than those obtained when excess desipramine was used to define specific binding (Bmax 654 +/- 33 sites/platelet, p less than 0.001; Kd 1.52 +/- 0.11 nM, p less than 0.001). There was no significant correlation between the density (Bmax) of sodium-dependent and desipramine-defined binding in individual subjects, suggesting that a different proportion of sites are labeled under the two assay conditions. No age-dependent variation was found in either Kd or Bmax values of sodium-dependent or desipramine-defined [3H]imipramine binding. The results suggest determination of sodium-dependent [3H]imipramine binding to intact platelets may be a useful measure for the estimation of [3H]imipramine recognition sites relevant to the serotonin uptake in studies of patients with affective disorders.     

Flow cytometric analysis of platelet activation in hypertensive patients. Effect of doxazosin

The percentage of spontaneously activated platelets and the platelet response to several agonists were studied in 26 hypertensive patients. The percentage of platelets expressing glycoprotein (GP) IIb/IIIa in its active conformation (GPIIb/IIIa*), P-selectin and phosphatidylserine (PS) was measured by flow cytometry at baseline and 1 and 2 months after treatment with doxazosin (4 mg/day). The response to ADP and Ca2+ ionophore was also evaluated. The results were compared with those of a control group of 71 normotensive volunteers. Spontaneous platelet activation was higher in patients than in controls (P-selectin-positive results in 4.4+/-2.0% patients vs. 2.7+/-1.7 controls, p<0.05; phosphatidylserine-positive results in 0.7+/-0.4% vs. 0.5+/-0.3%, respectively, p<0.05), and higher in response to ionophore action (phosphatidylserine-positive results 51.8+/-11.1% vs. 43.4+/-11.7%, p<0.01). Platelet activation in patients decreased after 2 months of doxazosin administration compared to baseline (P-selectin-positive results 2.7+/-1.4% vs. 4.4+/-2.0%, p<0.05; phosphatidylserine-positive results 0.3+/-0.2% vs. 0.7+/-0.4%, p<0.05). No significant differences were noted in GPIIb/IIIa*. The clinical significance of normalization of platelet activity by doxazosin remains to be established.     

Platelets in nonresponders to epinephrine stimulation showed reduced response to ADP.

It has been reported that platelets from some healthy donors did not respond to epinephrine (Epi). To identify the cause for the lack of response, we examined the alpha(2) adrenoceptor in the platelets and their signal transduction pathways. No differences in the genomic (-2076 to 1526 bp) and coding region of alpha(2A) adrenoceptor complementary DNA (cDNA) were found between the responders (R) and nonresponders (NR). No expression of alpha(2B) or alpha(2C) adrenoceptor was detected in platelets. When UK14,304 was used to induce platelet aggregation, similar effect to Epi was observed between R and NR, and any involvement of the alpha(1) and beta adrenoceptor was ruled out. Radioligand binding assay showed similar number of alpha(2) binding sites between the two groups (139+/-25/platelet vs. 145+/-37/platelets). However, platelets from NR showed a weaker response to adenosine diphosphate (ADP, 52.3+/-17.8% vs. 80.5+/-8.7% from R, P<.01). In the presence of P2Y(1) antagonist adenosine 3',5'-diphosphosulfate (A3P5PS), ADP failed to induce platelet aggregation in NR (7.8+/-4.7% vs. 64.7+/-11.2% in R, P<.01). Addition of SQ22,536 to inhibit adenylyl cyclase did not convert NR to R. These observations demonstrate that there is an impaired platelet responsiveness to ADP as well as to Epi in NR, due to a difference in downstream of the signal transduction pathway but independent of adenylyl cyclase inhibition.