Cytoplasmic free calcium mobilization in platelets, expression of P-selectin, phosphatidylserine, and microparticle formation, measured by whole blood flow cytometry, in hypertensive patients. Effect of doxazosin GITS

The effects of doxazosin on expression of CD62 (P-selectin) and phosphatidylserine on platelet membrane and platelet calcium flux were studied in 50 uncomplicated essential hypertensive patients (World Health Organization stages 1-2) and 80 normotensive control subjects, matched for age, sex, and cardiovascular risk factors. Hypertensive patients showed greater in vivo platelet activation at baseline than control patients (percentage of CD62-positive platelets, 4.1+/-2.2% versus 2.4+/-1.5%, p<0.001; percentage of phosphatidylserine-positive platelets, 0.8+/-0.5% versus 0.5+/-0.3%, p<0.001). Increased platelet activation was associated with significant changes in the mobilization of free intraplatelet calcium, evaluated by a whole blood flow cytometric kinetic method. With this method, an arbitrary Ca(2+) mobilization index was defined as the ratio of cytoplasmic free calcium before activation with thrombin to the slope of the calcium removal rate following the action of the agonist. This index was significantly higher in untreated hypertensive patients than in normotensive controls (0.12+/-0.06 versus 0.05+/-0.08, p<0.001). Treatment of hypertensive patients with doxazosin gastrointestinal therapeutic system (4 mg/day as a single dose) for 2 months normalized both platelet activation and Ca(2+) mobilization. Changes in the expression of CD62 and phosphatidylserine in the platelet membrane after treatment with doxazosin gastrointestinal therapeutic system may be related to normalization of the kinetics of cytoplasmic free Ca(2+). Normalization of platelet activation may represent an additional beneficial effect to the known antihypertensive action of doxazosin gastrointestinal therapeutic system.     

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.     

Effects of glycoprotein IIb/IIIa antagonists on platelet activation: development of a transfer method to mimic peak to trough receptor occupancy

Several oral glycoprotein (GP) IIb/IIIa antagonists, Sibrafiban, Orbofiban and Lotrafiban, have been studied in large phase III trials; each has failed to provide efficacy and has been associated with increased mortality. Roxifiban has pharmacokinetic and pharmacodynamic properties believed to be more favorable than the earlier oral agents. Here, we revisit the controversial hypothesis of platelet activation liabilities of GP IIb/IIIa antagonists. The effects of site occupancy by four fibans (Roxifiban, Sibrafiban, Orbofiban and Lotrafiban) on platelet activation was assessed using P-selectin expression, fibrinogen binding and microaggregate formation. All four fibans inhibited ADP and TRAP-stimulated fibrinogen binding and microaggregate formation in a concentration-dependent manner, whereas P-selectin expression was relatively unaltered. To more vigorously test for activation liabilities, the effects of transition from peak to trough receptor occupancy upon platelet stimulation was analyzed. The high affinity of Roxifiban for resting platelets precluded reduction of site occupancy by dialysis or gel filtration. A method was developed that takes advantage of the rapid equilibrium of Roxifiban between platelets and soluble GPIIb/IIIa. The platelet occupancy is controlled by the ratio of platelet GPIIb/IIIa to soluble GPIIb/IIIa. This method allows in vitro investigation of peak/trough transitions on platelet activation. A decrease in site occupancy from peak to trough of Roxifiban or Sibrafiban did not result in increased activation of platelets. The loss of platelet-bound antagonist upon incubation with purified soluble GPIIb/IIIa returned fibrinogen binding/microaggregate formation to no drug levels. In conclusion, these studies do not provide evidence for an activation liability of GPIIb/IIIa antagonists in vitro.     

Arginyl-glycyl-aspartyl (RGD) epitope of human apolipoprotein (a) inhibits platelet aggregation by antagonizing the IIb subunit of the fibrinogen (GPIIb/IIIa) receptor

An unknown epitope of apolipoprotein (a) antagonizes fibrinogen binding to agonist-stimulated platelet's fibrinogen (GPIIb/IIIa) receptor yielding lipoprotein (a) mediated decreased platelet aggregation. The purpose of this study was to test the hypothesis that human apolipoprotein (a)'s single arginyl-glycyl-aspartyl (RGD) epitope, unique to apolipoprotein (a) in lipoprotein (a) binds to the RGD binding motif on the IIb subunit of the GPIIb/IIIa receptor thus reducing platelet-bound fibrinogen and consequently decreasing agonist-stimulated platelet aggregation. Platelets (N=30 subjects) were prepared from fresh plasma, washed three times in Tyrode's buffer and stimulated using 10 microM ADP or 2 microg/ml collagen. Lipoprotein (a) was isolated from plasma using lectin affinity chromatography followed by ultracentrifugation. The peptide RGDS inhibited (125)I-labelled lipoprotein (a) binding to autologous platelets with IC-50's of 25.1+/-2.2 (mean+/-SEM) and 15.4+/-1.3 microM for collagen- and ADP-stimulation respectively. Further, RGDS reduced platelet binding of (125)I-labelled fibrinogen IC-50's of 35.5+/-3.2 (mean+/-SEM) and 20.7+/-2.2 microM for collagen- and ADP-stimulation respectively. The monoclonal antibody PAC-1, uniquely directed at the RGD binding motif on the IIb subunit on collagen- and ADP-stimulated platelets, inhibited binding of (125)I-labelled lipoprotein (a) with IC-50's of 6.4+/-0.7 and 2.5+/-2.2 microg/10(8) platelets for collagen- and ADP-stimulation respectively. Additionally, PAC-1 reduced platelet bound of (125)I-labelled fibrinogen with IC-50's of 9.0+/-1.4 and 4.1+/-2.2 microg/10(8) platelets for collagen- and ADP-stimulation respectively. In a dose-related fashion, a polyclonal antibody, specific for the RGD epitope on apolipoprotein (a), restored platelet aggregation to control levels, inhibited (125)I-labelled lipoprotein (a) binding, and increased (125)I-labelled fibrinogen by displacing lipoprotein (a) from the GPIIb/IIIa receptor. Thus a never before demonstrated aspect of the mechanism of lipoprotein (a)'s suggested novel role as an endogenous regulator of fibrinogen binding to collagen- and ADP-stimulated platelets has been shown. In conclusion, lipoprotein (a), via apolipoprotein (a)'s RGD epitope, binds to the RGD binding motif on the IIb protein of the GPIIb/IIIa receptor consequently reducing platelet-bound fibrinogen which results in decreased platelet aggregation.     

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 GPIIb/IIIa receptor occupancy studies using a novel fluoresceinated cyclic Arg-Gly-Asp peptide

DMP 728 is a potent and specific platelet GPIIb/IIIa antagonist. Like all GPIIb/IIIa antagonists, DMP 728 has a steep dose-response relationship in inhibiting platelet aggregation. In this study the relationships between receptor occupancy, platelet aggregation and bleeding time was determined in anesthetized dogs after intravenous infusion of DMP 728 (0.01 and 0.1 mg/kg/2h). Receptor occupancy was determined by flow cytometry using XL086, a novel fluorescent cyclic RGD peptide that binds to GPIIb/IIIa with high specificity and affinity (k_d ~55 nM). Mean number of GPIIb/IIIa as determined by flow cytometric assay was ~53,800 and 79,000 on unactivated and ADP-activated platelets respectively. After DMP 728 intravenous infusion, there was a dose and time-dependent increase in receptor occupancy, inhibition of platelet aggregation and bleeding time. The two methods of receptor occupancy determination correlate with each other with an r² = 0.78. The present data suggest that blockade of only 40–60% (~40,000 receptors) of the total platelet GPIIb/IIIa was required to achieve >90% inhibition of platelet aggregation and >15 min bleeding time. Our results showed the potential clinical utility of this approach in the study of GPIIb/IIIa dose-response relationship.     

Effects of fish oil supplementation on platelet survival and ex vivo platelet function in hypercholesterolemic patients

Little is known about the effects of dietary supplementation on platelet survival with low doses of n-3 and n-6 fatty acids in patients with hypercholesterolemia. The effects of a 6-week intervention with fish oil capsules (daily intake: 216 mg eicosapentaenoic acid, 140 mg docosahexaenoic acid, 390 mg gamma-linolenic acid, and 3480 mg linoleic acid) on in vivo platelet survival (111 In-oxine labeled platelets) and on ex vivo markers of platelet activation were investigated in a placebo-controlled, double-blind study with 26 hypercholesterolemic patients. In vivo platelet survival increased in the fish oil group (T) from a mean of 159+/-14 hours to a mean of 164+/-12 hours (p=0.025), whereas it remained unchanged in the placebo (P) group (T vs. P; p=0.055). Ex vivo, thromboxane B2 decreased from a mean of 225+/-16 to 212+/-21 ng/mL (p=0.003) in T but did not change in P (T vs. P: p=0.002). Malondialdehyde formation was lowered significantly by fish oil supplementation from a mean of 5.49+/-1.3 to 5.12+/-1.05 nM/10(9) platelets, p=0.005, as compared with P (T vs. P; p=0.018). The trendwise decrease in 11-DH-thromboxane B2 plasma levels was not significant nor was the increase in platelet sensitivity to prostaglandin I2 by fish oil. Baseline platelet survival in patients with hyperlipoproteinemia type IIa was not different from those with hyperlipoproteinemia IIb and response to treatment in terms of platelet activation markers was not either. The changes in platelet activation parameters in T were associated with significant reductions in cholesterol (-2.9%), low density lipoprotein cholesterol (-3.5%), and triglycerides (-12.4%). Both ex vivo and in vivo platelet activation parameters exhibited signs of decreased activation by a 6-week diet supplemented with n-3 and n-6 fatty acids, which might be beneficial in reducing atherothrombotic risk, in patients with hyperlipoproteinemia type IIa and IIb.     

The anti-platelet effect of niceritrol in patients with arteriosclerosis and the relationship of the lipid-lowering effect to the anti-platelet effect

A hypolipidemic agent, pentaerythritol tetranicotinate (niceritrol) yields nicotinic acid upon hydrolysis in vivo, but niceritrol is hardly soluble in distilled water, so that the effects of nicotinic acid on platelet aggregation in vitro were studied. Nicotinic acid inhibited in vitro platelet aggregation induced by ADP, collagen and adrenaline. Twenty patients (61.4 +/- 2.4 years (mean +/- S.E.)) with ischemic heart disease, cerebral infarction, transient cerebral ischemic attack and hypercholesterolemia were given niceritrol orally at 750 mg per day for 8 weeks. Significant decreases in ADP-, collagen- and adrenaline-induced platelet aggregation were observed at 4 and 8 weeks after niceritrol treatment. There was a significant correlation between the rates of changes in platelet aggregation and those in plasma total cholesterol or plasma LDL-cholesterol before treatment and 8 weeks following treatment. The results indicate that niceritrol has inhibitory effects on platelet aggregation not only caused by its direct action on platelet but mediated by its secondary action due to decrease in blood lipids.     

Glycoprotein IIb/IIIa Receptor Antagonists Inhibit the Development of Platelet Procoagulant Activity

We examined the effects of glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists c7E3 Fab and DMP728 on the development of platelet prothrombinase (PT) activity. c7E3 Fab dose-dependently inhibited the rate of thrombin-stimulated thrombin generation over a 1-minute reaction time. The IC50 was 11 nM with an IC90 of 1000 nM. DMP728 inhibited PT activity maximally by 60% at 100 nM. A similar profile was observed for the inhibition of platelet tenase activity. Inhibition was platelet specific up to approximately 200 nM c7E3 Fab. Above 200 nM, inhibition was platelet independent, as shown by the inhibition of activity assembled on PS/PC vesicles. c7E3 Fab and DMP728 did not inhibit calcium ionophore-induced activity. DMP728 potency diminished with reaction time (over 6 minutes) whereas c7E3 Fab potency did not. Inhibition by 2 μM DMP728 was not further increased by 20 nM c7E3 Fab. Heparin inhibition of platelet PT activity was additive to that of c7E3 Fab. Studies with added von Willebrand factor (vWf) indicate that in the context of thrombin activation vWf activates platelets through mechanisms independent of GPIIb/IIIa to promote PT activity. Thrombin activation induced binding of FITC-AnnexinV to a subpopulation of platelets which was reduced by approximately 50% by pretreatment with either c7E3 Fab or DMP728. Together, these data indicate that c7E3 Fab and DMP728 inhibit the development of GPIIb/IIIa-mediated platelet PT activity at events during platelet activation. The inhibitory activities are not additive, suggesting these agents compete for the same site or inhibit via the same mechanism. Inhibition accompanies a reduction in the number of phosphatidylserine binding sites, implying that GPIIb/IIIa receptor antagonists reduce platelet membrane scrambling induced by thrombin. The additivity of inhibition with heparin by c7E3 Fab suggests a combination of these agents might have a greater bleeding liability than the use of either agent alone.     

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.     

Relationship between platelet phospholipase activity and plasma in ischemic heart disease

Platelet phospholipase plays an important role in the metabolic responses of platelets to exogenous stimuli. The platelet phospholipase activity (PLA) was therefore studied in 38 patients with ischemic heart disease (IHD) and in 26 age-matched normal subjects who served as controls. The mean platelet PLA in the IHD group was 12.72 +/- 1.03 nmol/mg protein/30 sec which was significantly (p less than 0.005) higher than that of the normal controls (8.72 +/- 0.76). When they were classified into acute stage, such as unstable angina or acute myocardial infarction (AMI), and chronic stage, such as stable angina or old myocardial infarction (OMI), there was no significant difference between them. On the other hand, about two-fold activation of platelet PLA was observed in acute stage IHD, and 20-30% inhibition of it was demonstrated in chronic stage IHD following the addition of autologous plasma to washed platelet suspensions, suggesting that certain plasma factor(s) are responsible for such phenomena. In an attempt to identify these plasma factor(s), various substances such as serum albumin, high density lipoprotein, prostaglandin E1 (PGE1) and E2 (PGE2), and platelet activating factor were assessed by in vitro experiments. Only PGE1 and PGE2 revealed a significant effect on the platelet PLA. The relationship between plasma and platelet activity in terms of platelet PLA deserves attention since it varies according to the type and stage of IHD.     

Platelets release their lysosomal content in vivo in humans upon activation

Platelets contain, besides alpha- and delta-granules, lysosomes which store glycohydrolases able to degrade glycoproteins, glycolipids and glycosaminoglycans. While several studies have shown that alpha- and delta-granule secretion takes place "in vivo" in humans upon platelet activation, no data are available on the "in vivo" release of lysosomes. We have studied the release of platelet lysosomal contents "in vivo" in healthy volunteers at a localized site of platelet activation by measuring markers of lysosomal secretion in the blood oozing from a skin wound inflicted for the measurement of the bleeding-time. The levels of beta-N-acetylhexosaminidase (Hex) were 13.1 +/- 0.85 mU/ml in bleeding-time blood and 10.2 +/- 0.66 mU/ml in plasma (p <0.001). Hex in serum was 16.4 +/- 0.72 mU/ml. The levels of beta-galactosidase were also higher in bleeding-time blood than in plasma (0.85 +/- 0.07 mU/ml vs 0.4 +/- 0.05 mU/ml, p <0.001). In bleeding-time blood collected at one minute intervals, Hex rose progressively consistent with ongoing platelet activation and flow-cytometry showed a progressive increase of the expression of LIMP and LAMP-2, two lysosomal associated proteins. In conclusion, our data demonstrate that platelet lysosomal glycohydrolases are released "in vivo" in humans upon platelet activation.     

Effect of doxazosin gastrointestinal therapeutic system on platelet degranulation and platelet-leukocyte microaggregate formation induced by physiologic shear stress in hypertension

INTRODUCTION: In this prospective, ex vivo, single-blind study, the effect of doxazosin on platelet function was studied in patients with hypertension. MATERIALS AND METHODS: Platelet activation by shear stress was measured in whole blood samples of 22 hypertensive patients and 22 normotensive controls, using flow cytometry. Sheared samples were evaluated for CD62 expression, microaggregate formation, and Ca2+ mobilization. Results were collected at baseline and after 1 and 2 months of single-dose (4 mg/d) extended-release doxazosin gastrointestinal therapeutic system therapy. RESULTS: Doxazosin normalized blood pressure in hypertensive patients after 1 and 2 months of treatment. Hypertensive patients had a higher baseline percentage (mean+/-SD) of degranulated platelets (CD62+) than the normotensive control group (4.14+/-1.05 vs. 2.47+/-0.68, P<0.01). After 2 months of doxazosin gastrointestinal therapeutic system treatment, the percentage of CD62+ in the experimental group significantly decreased (P<0.05). At baseline, the number of platelet-leukocyte aggregates in vivo was greater in hypertensive patients (P<0.01); doxazosin did not normalize this measurement. Following shearing, platelet expression of CD62 increased significantly in the hypertensive group (P<0.001 vs. control). Shear stress-induced platelet activation and microaggregate formation were also greater in hypertensive patients. Intraplatelet-free calcium concentration was higher in hypertensive patients at baseline than in the normotensive group (P<0.001). At 2 months, doxazosin significantly reduced thrombin-stimulated Ca2+ mobilization in hypertensive patients (P<0.01 vs. baseline). CONCLUSIONS: Platelets from hypertensive patients are more readily activated by shear stress and demonstrate significant alterations in cytoplasmic-free calcium mobilization. Doxazosin treatment reduced blood pressure and normalized alterations in platelet function.     

Platelet activation induced by combined effects of anticardiolipin and lupus anticoagulant IgG antibodies in patients with systemic lupus erythematosus--possible association with thrombotic and thrombocytopenic complications

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p<0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.     

Effect of Ca2+ chelation on the platelet inhibitory ability of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban

OBJECTIVE: Enhanced GPIIb/IIIa binding and inhibition of platelet aggregation of eptifibatide by the reduction of ionized plasma calcium concentrations have been reported. The present study compared the importance of Ca2+ chelation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban. METHODS AND RESULTS: Turbidimetric platelet aggregation dose response curves of the various GPIIb/IIIa antagonists were performed using platelet rich plasma (PRP) anticoagulated with either trisodium citrate, or the non-chelating anticoagulant, PPACK. The concentrations of antagonist that resulted in 50% inhibition of TRAP-induced (10 microM) platelet aggregation (IC50) were measured in the presence of either citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 values for citrate and PPACK were 88.2 +/- 12.2 nM and 126.1 +/- 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 +/- 13.3 nM and 142.6 +/- 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260.2 +/- 62.5 nM and 810.3 +/- 182.5 nM for eptifibatide (3.1-fold enhancement; p = 0.001). A similar shift in effective inhibitor concentrations for abciximab was observed with ADP (10 microM). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the presence of trisodium citrate and PPACK were equivalent. CONCLUSIONS: These data confirm previous observations that Ca2+ chelation afforded by citrate decreases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of decrease was less for abciximab and tirofiban, compared to eptifibatide.     

Expression of fibrinogen receptors in platelets of migraine patients--correlation with platelet GPIIb content and plasma cholesterol

Binding of fibrinogen to platelets washed from blood of migraine patients (n = 30) and control donors (n = 24) was compared. In addition, contents of platelet glycoprotein IIb and platelet fibrinogen were determined in both groups by radioimmunoassay. The receptor capacity for fibrinogen in platelets activated by ADP was significantly higher (p less than 0.01) in migraine patients (52,505 +/- 4,925) than in controls (33,881 +/- 3,965). The mean contents of GPIIb (3.51 +/- 0.34 micrograms/10(8) platelets) and fibrinogen (37.26 +/- 4.05 micrograms/10(8) platelets) in migraine platelets were also markedly increased (p less than 0.01 and p less than 0.001, respectively) when compared to controls (2.21 +/- 0.18 micrograms of GPIIb and 18.75 +/- 2.29 micrograms of fibrinogen per 10(8) platelets, respectively). There was a high correlation between the number of fibrinogen receptors exposed by ADP and the total amount of platelet GPIIb both in migraine patients (R = 0.69, p less than 0.01) and controls (R = 0.62 p less than 0.01), as well as plasma cholesterol in the case of migraine patients (R = 0.82, p less than 0.001).     

Kinetics and in vivo distribution of in-111-labelled platelets and platelet function in familial hypercholesterolaemia

The kinetics, in vivo distribution and sites of sequestration of autologous In-111-labelled platelets and other platelet function parameters were studied in ten patients with type IIa or IIb familial hypercholesterolaemia and thrombotic complications of atherosclerosis. The in vitro platelet aggregation response to ADP (P = 0.50) and collagen (P = 0.46); binding of fibrinogen to platelets (P = 0.61); and plasma beta-thromboglobulin levels (P = 0.42) of the patients and normal reference subjects did not differ significantly. The in vivo distribution of In-111-labelled platelets at equilibrium was within normal limits, and at the end of platelet life-span the sequestration pattern of labelled platelets in the reticuloendothelial system was also normal (spleen P = 0.31; liver P = 0.54). There was minimal evidence of in vivo platelet activation: only mean platelet lifespan (MPLS), 195 +/- 57 hours (difference between mean MPLS of patients and controls was 25 hours, with a 95% confidence interval from 23 to 31 hours; P = 0.02); mean platelet platelet turnover, 2298 +/- 824 platelets/microliter/hour (P = 0.005); plasma platelet factor 4 (P = 0.02); and the mean circulating platelet aggregate ratio, 0.8 +/- 0.1 (P = 0.02); differed significantly from normal. These results suggest that abnormalities of platelet function and kinetics observed in type II hyperlipoproteinaemia cannot be ascribed wholly to the hyperlipidaemia, but may be induced by the associated atherosclerosis.     

Antiplatelet and antithrombotic activity of RWJ-53308, a novel orally active glycoprotein IIb/IIIa antagonist.

RWJ-53308 is a novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist that inhibits fibrinogen binding to GPIIb/IIIa with an IC(50) of 0.4+/-0.3 nM. RWJ-53308 inhibits thrombin-induced platelet aggregation in human gel-filtered platelets (IC(50)=60+/-12 nM) and platelet aggregation in human platelet-rich plasma (PRP) in response to collagen, arachidonic acid, ADP, and SFLLRN-NH(2) (IC(50)=60+/-10, 150+/-30, 70+/-4, and 160+/-80 nM, respectively). The potency of RWJ-53308 in dog and guinea pig PRP is similar to human PRP. RWJ-53308 inhibits ex vivo collagen- and ADP-induced platelet aggregation in conscious dogs for up to 4 h following 0.3 mg/kg iv, and through 4 and 6 h following 1 and 3 mg/kg po. Oral bioavailability is 16+/-7%. RWJ-53308 reduces thrombus weight in a canine arteriovenous (AV) shunt model following intravenous (0.01-0.1 mg/kg) and oral (3 mg/kg) administration. In a guinea pig carotid artery pinch-injury model, RWJ-53308 completely suppresses thrombus-induced cyclic flow reductions (CFR) at 0.7 mg/kg iv. RWJ-53308 also blocks thrombus formation in photoactivation- and ferric chloride-induced models of thrombosis in guinea pigs at 0.3 and 1 mg/kg iv, respectively. In summary, RWJ-53308 is a potent orally active GPIIb/IIIa antagonist that may be useful for both acute and chronic treatment of arterial thrombotic disorders.     

Plasma 11-dehydrothromboxane B2: a reliable indicator of platelet hyperfunction in patients with ischemic stroke

The plasma level of 11-dehydrothromboxane B2 (11-dehydroTXB2) is free from artifactual increase during blood sampling, and it can be reliable indicator of TXA2 production in vivo. We have estimated plasma 11-dehydroTXB2 in patients with ischemic stroke. Subjects studied were 29 patients with cerebral thrombosis (62 +/- 9 years old) and 41 healthy controls (61 +/- 7 years old). Plasma 11-dehydroTXB2 and TXB2 were determined by radioimmunoassay. Plasma 11-dehydroTXB2 levels in patients and controls were 5.4 +/- 2.5 and 1.8 +/- 0.9 pg ml, respectively, and the difference was significant (p less than 0.001). Plasma TXB2 also was higher in patients than in controls: 401 +/- 61 vs 311 +/- 51 pg/ml (p less than 0.05). However, the 11-dehydroTXB2 was found to be a more effective parameter to distinguish between stroke patients and controls. Estimation of plasma 11-dehydroTXB2 levels is a reliable method to detect platelet hyperfunction in stroke patients.     

Impaired Platelet Binding of Fibrinogen Due to a Lower Number of GPIIB/IIIA Receptors in Polycythemia Vera

We have previously described a stimulus-specific defect in platelet aggregation in polycythaemia vera (PV) after stimulation with surface receptor dependent agonists such as platelet activating factor (PAF). In contrast, responses to phorbol myristate acetate (PMA) were normal. We now report that after PAF stimulation, using flow cytometry, the amount of fibrinogen bound to its receptor was significantly lower in PV platelets with a median MFI of 6.0 (range 4.1–17.3) compared to controls, 12.8 (range 8–21.3; n=11; p<0.01). We found no evidence of preactivation of PV platelets. Quantitative analysis of GPIIIa gave a significantly lower number of GPIIIa on resting PV platelets, 14300 subunits of GPIIIa (range 8500–15500) vs. 19800 for controls (range 13400–26800; n=12; p<0.01). Both patients and controls increased their number of receptors on the cell surface after stimulation with PAF and PMA, but the significant difference in the number of receptors per cell remained. Indirect evaluation of PAF receptor function showed that activation of CD 62 did not differ in PV and controls after PAF stimulation. Additionally, although the basal level of serotonin in platelet-rich plasma was significantly lower in PV, there was a threefold increase of the basal level after stimulation with PAF for both PV and control platelets, also indicating a normal interaction of PAF with its receptor. Although our results indicate both an impaired PAF induced aggregation in PV and a lower number of GPIIb/IIIa complexes on single platelets, whether these phenomena are related remains uncertain.