The Aptamer ARC1779 Potently and Specifically Inhibits the Excessive von Willebrand Factor (vWF) Activity and vWF-Mediated Ex Vivo Platelet Function of Patients with Thrombotic Thrombocytopenic Purpura (TTP).

Abstract Background: ARC1779 is an aptamer which blocks the binding of the vWF A1 domain to platelet GPIb receptors. In TTP there is an excess of ultra-large multimers of vWF which are especially avid for binding GPIb and give rise to disseminated platelet thrombi which are fibrin-poor and vWF-rich in composition. ARC1779 is being evaluated for use as front-line therapy of acute TTP in conjunction with plasma exchange. ARC1779 has already been demonstrated in healthy volunteers to inhibit vWF activity and vWF-dependent platelet function. ARC1779 has no anticoagulant effect and does not inhibit other pathways of platelet activation. ARC1779 is expected to normalize platelet dysfunction and prevent the thrombotic end-organ complications of TTP based upon the mechanism of action defined for ARC1779 and the mechanism of thrombosis defined for TTP. Methods: We first assessed vWF activity (vWF:RiCO) and platelet function in blood samples taken from TTP patients and age-matched, healthy controls. We then studied the ex vivo dose response curves for ARC1779 on vWF activity (free A1 domain sites) and on platelet function assessed by the Platelet Function Analyzer (PFA-100®), cone and plate analyzer (IMPACT®), and agonist-induced impedence platelet aggregometry (Multiplate®) of TTP patients (N=10, 2 in acute phase and 8 in remission) and healthy age-matched controls (N=23). Results: vWF:RiCO activity (p=0.002) and vWF-dependent platelet plug formation (p=0.001) were increased in TTP patients relative to healthy controls, but agonist-induced platelet aggregation (ADP, arachidonic acid, collagen, TRAP) was not. ARC1779 fully blocked platelet plug formation as measured by PFA-100® with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation, and ∼ 3–4 mcg/mL with hirudin anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked shear-dependent platelet adhesion measured by the IMPACT® analyzer with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked vWF activity (free A1 domain sites) with an IC90 of ∼ 6 mcg/mL in TTP patients and ∼ 2 mcg/mL in young controls (p<0.001 between groups). ARC1779 did not inhibit platelet aggregation by ADP, collagen or arachidonic acid at concentrations (10mcg/mL) that fully inhibited vWF dependent platelet function. Conclusions: ARC1779 potently and specifically inhibits vWF activity and vWF dependent platelet function in the setting of TTP where vWF activity is increased. ARC1779 represents a novel therapeutic principle (vWF antagonism) and a novel therapeutic class (aptamers) with potential for the treatment of TTP. vWF Activity and Platelet Function in TTP Patients and Age-Matched Controls Population Healthy Controls TTP Patients values shown as mean +/− standard deviation Sample Size N=23 N=10 vWF:RiCO (%) 94.9 +/− 60.4 153.3 +/− 55.9 PFA-100® Closure Time in Citrate (sec) 90.9 +/− 16.0 66.8 +/− 12.7 PFA-100® Closure Time in Hirudin (sec) 84.0 +/− 12.9 64.6 +/− 11.9 ARC1779 IC100 PFA in Citrate (ARC1779 mcg/mL) 0.9 +/− 0.4 1.4 +/− 0.6 ARC1779 IC100 PFA in Hirudin (ARC1779 mcg/mL) 3.2 +/− 1.5 4.4 +/− 2.7 IC100 IMPACT in Citrate (ARC1779 mcg/mL) 0.8 +/− 1.2 0.8 +/− 0.8 IC90 vWF free A1 domain sites (ARC1779 mcg/mL) 1.8 +/− 0.8 6.2 +/− 2.7

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Abstract 2214: The Aptamer ARC1779 Is A Potent And Specific Inhibitor Of von Willebrand Factor (vWF) Mediated Ex Vivo Platelet Function In ST-elevation (STEMI) and non-ST elevation (NSTEMI) Acute Myocardial Infarction (AMI)

Circulation ◽

10.1161/circ.116.suppl_16.ii_481-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Bernd Jilma ◽

Florian B Mayr ◽

Alexander O Spiel ◽

Patricia G Merlino ◽

Harold N Marsh ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Adhesion ◽

Ex Vivo ◽

Specific Inhibitor ◽

Citrate Anticoagulation ◽

St Elevation ◽

A1 Domain ◽

The Impact ◽

Novel Therapeutic

Background: ARC1779 is an aptamer which blocks the A1 domain binding of the vWF A1 domain to platelet GPIb receptors that is now in development for the treatment of AMI. vWF is increased in the elderly and in the setting of AMI, as reflected in higher vWF levels in circulation and in increased shear-dependent platelet function as measured by the platelet function analyzer (PFA-100) and cone and plate analyzer (IMPACT). Conventional therapy of AMI partially reduces platelet activation and aggregation, but does not address excessive vWF activity or platelet adhesion. Methods: We studied the ex vivo dose response curves for ARC1779 on PFA-100 and IMPACT platelet function tests, agonist-induced platelet aggregation, and vWF activity (free A1 domain sites) of patients with AMI on standard treatment including aspirin and clopidogrel (n=40), young (n=20) and elderly controls (n=20). Results: ARC1779 fully blocked collagen ADP induced platelet plug formation as measured by PFA-100 with an IC100 of ~ 1–2 mcg/mL with citrate anticoagulation, and 3–5 mcg/mL with hirudin anticoagulation. ARC1779 fully blocked shear-dependent platelet adhesion measured by the IMPACT analyzer with an IC100 of ~ 1 mcg/mL with citrate anticoagulation. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation by ADP, collagen or arachidonic acid at concentrations (10mcg/mL) that fully inhibited vWF dependent platelet function. ARC1779 fully blocked vWF activity ex vivo with an IC90 of ~ 1 mcg/mL in young controls and 6 – 8 mcg/mL in STEMI and NSTEMI patients. Conclusions: ARC1779 potently and specifically inhibits vWF activity and vWF dependent platelet function, even in the setting of AMI where vWF activity is increased. ARC1779 represents a novel therapeutic principle (vWF antagonism) and a novel therapeutic class (aptamers). Potent and specific inhibition of VWF makes ARC1779 a promising development candidate for patients with AMI. Results

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Increased Response to Arachidonic Acid and U-46619 and Resistance to Inhibitory Prostaglandins in Patients with Chronic Myeloproliferative Disorders

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642568 ◽

1988 ◽

Vol 59 (01) ◽

pp. 073-076 ◽

Cited By ~ 9

Author(s):

Sergio Cortelazzo ◽

Monica Galli ◽

Donatella Castagna ◽

Piera Viero ◽

Giovanni de Gaetano ◽

...

Keyword(s):

Bone Marrow ◽

Arachidonic Acid ◽

Platelet Aggregation ◽

Myeloproliferative Disorders ◽

Bone Marrow Stem Cell ◽

Healthy Controls ◽

Aggregation Response ◽

Thrombotic Complications ◽

Cyclic Endoperoxide ◽

Marrow Stem Cell

SummaryIn patients with myeloproliferative disorders (MPD) a group of related diseases of the bone marrow stem cell and recurrent haemorrhagic and/or thrombotic complications, the production of aggregating prostaglandins (PGs) may be normal or slightly reduced, while PGI2 production is normal. However, MPD platelet sensitivity to antiaggregatory PGs is still unknown.We studied the potency of PGD2, PGI2 and PGEi as inhibitors of platelet aggregation induced by threshold aggregating concentrations of arachidonic acid and U-46619-analogue of the cyclic endoperoxide PGH2 in 20 patients with MPD in comparison with healthy controls, with the aim of evaluating the sensitivity of MPD platelets to antiaggregatory PGs. In these patients platelet prostanoid metabolism was normal. However, the functional response of platelets to aggregating and antiaggregating prostanoids was shifted towards potentially increased platelet aggregation response. These findings could have a clinical relevance in view of the haemostatic and thrombotic complications so frequent in MPD.

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The d-Enantiomer Form of Indobufen Totally Accounts for the Anti-Cyclooxygenase and Antiplatelet Activity Ex Vivo and for the Increase in Bleeding Time by Indobufen in Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648422 ◽

1992 ◽

Vol 67 (02) ◽

pp. 258-263 ◽

Cited By ~ 12

Author(s):

Raffaele De Caterina ◽

Rosa Sicari ◽

An Yan ◽

Walter Bernini ◽

Daniela Giannessi ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Plasma Levels ◽

Bleeding Time ◽

Ex Vivo ◽

Random Sequence ◽

Antiplatelet Agent ◽

Antiplatelet Drug ◽

Double Blind

SummaryIndobufen is an antiplatelet drug able to inhibit thromboxane production and cyclooxygenase-dependent platelet aggregation by a reversible inhibition of cyclooxygenase. Indobufen exists in two enantiomeric forms, of which only d-indobufen is active in vitro in inhibiting cyclooxygenase. In order to verify that also inhibition of platelet function is totally accounted for by d-indobufen, ten patients with proven coronary artery disease (8 male, 2 female, age, mean ± S.D., 58.7 ± 7.5 years) were given, in random sequence, both 100 mg d-indobufen and 200 mg dl-indobufen as single administrations in a double-blind crossover design study with a washout period between treatments of 72 h. In all patients thromboxane (TX) B2 generation after spontaneous clotting (at 0, 1, 2, 4, 6, 8, 12, 24 h), drug plasma levels (at the same times), platelet aggregation in response to ADP, adrenaline, arachidonic acid, collagen, PAF, and bleeding time (at 0, 2, 12 h) were evaluated after each treatment. Both treatments determined peak inhibition of TXB2 production at 2 h from administration, with no statistical difference between the two treatments (97 ±3% for both treatments). At 12 h inhibition was 87 ± 6% for d-indobufen and 88 ± 6% for dl-indobufen (p = NS). Inhibition of TXB2 production correlated significantly with plasma levels of the drugs. Maximum inhibitory effect on aggregation was seen in response to collagen 1.5 pg/ml (63 ± 44% for d-indobufen and 81 ± 22% for dl-indobufen) and arachidonic acid 0.5-2 mM (78 ± 34% for d-indobufen and 88 ± 24% for dl-indobufen) at 2 h after each administration. An effect of both treatments on platelet aggregation after 12 h was present only for adrenaline 2 μM (55 ± 41% for d-indobufen and 37 ± 54% for dl-indobufen), collagen 1.5 pg/ml (69 ± 30% for d-indobufen and 51 ± 61% for dl-indobufen), arachidonic acid 0.5-2 mM (56 ± 48% for d-indobufen and 35 ± 49% for dl-indobufen). The extent of inhibition of TX production and the extent of residual platelet aggregation were never significantly different between treatments. Bleeding time prolongation was similar in the two treatment groups without showing a pronounced and long lasting effect (from 7.0 ± 2.0 min to 10.0 ± 3.0 min at 2 h and 8.0 ± 2.0 min at 12 h for d-indobufen; from 6.0 ±1.0 min to 8.5 ± 2.0 min at 2 h and 8.0 ± 1.0 min at 12 h for dl-indobufen). These results demonstrate that the biological activity of dl-indobufen as an antiplatelet agent in vivo is totally accounted for by d-indobufen.

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Assessment of Antithrombotic Properties of Sodium Ibuprofen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657223 ◽

1982 ◽

Vol 48 (01) ◽

pp. 087-090 ◽

Cited By ~ 2

Author(s):

Carlos O Esquivel ◽

David Bergqvist ◽

Claes-Göran Björck ◽

Stan N Carson ◽

Bodil Nilsson

Keyword(s):

Drug Administration ◽

Platelet Function ◽

Ex Vivo ◽

Inhibitory Effect ◽

Formation Time ◽

Platelet Activity ◽

Laser Injury ◽

Sodium Ibuprofen ◽

Plug Formation

SummaryThe effect of sodium ibuprofen on platelet activity in vivo and the lysability of ex vivo thrombi was investigated. The formation of a hemostatic platelet plug in the rabbit mesentery and platelet embolism as a response to a laser-induced injury in the ear chamber of rabbits were used as models for determining platelet activity. Ibuprofen at a dose of 25 mg/kg i.v. was found to increase the primary (PHT) and the total hemostatic plug formation time (THT). The same dose decreased the number of cumulative emboli over a 10 min period after a laser injury to arterioles. A dose of 10 mg/kg i.v. did not affect the formation of the hemostatic platelet plug. In dogs, doses of 10, 25 und 50 mg/kg did not enhance the release of 125I-FDP from the thrombi after incubation in plasmin, but the largest dose which is approximately five times the recommended dose in humans, did significantly decrease the thrombus weight 90 and 180 min after the drug administration. In conclusion, sodium ibuprofen was shown to have an inhibitory effect on platelet function in vivo and in large doses was also found to diminish the thrombus weight.

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Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00622.2002 ◽

2003 ◽

Vol 285 (2) ◽

pp. H637-H642 ◽

Cited By ~ 26

Author(s):

Maurice Beghetti ◽

Catherine Sparling ◽

Peter N. Cox ◽

Derek Stephens ◽

Ian Adatia

Keyword(s):

Nitric Oxide ◽

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Human Platelet ◽

Healthy Human ◽

Human Volunteers ◽

Independent Mechanism ◽

Inhaled No ◽

Maximal Extent

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine ( P < 0.005), 56% with collagen ( P < 0.005), and 20% with arachidonic acid ( P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen ( P < 0.005), 60% with epinephrine ( P < 0.05), 33% with arachidonic acid ( P < 0.05), and 14% with ADP ( P > 0.05). Plasma cGMP levels increased from 2.58 ± 0.43 to 9.99 ± 5.57 pmol/ml ( P < 0.005), intraplatelet cGMP levels were unchanged (means ± SD: 1.96 ± 0.58 vs. 2.71 ± 1.67 pmol/109platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.

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Modulation of platelet function by reactive oxygen metabolites

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1994.267.1.h308 ◽

1994 ◽

Vol 267 (1) ◽

pp. H308-H318 ◽

Cited By ~ 2

Author(s):

G. Ambrosio ◽

P. Golino ◽

I. Pascucci ◽

M. Rosolowsky ◽

W. B. Campbell ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Guanylate Cyclase ◽

Reactive Oxygen Metabolites ◽

Cyclic Monophosphate ◽

Cgmp Formation ◽

Oxygen Metabolites ◽

Exogenous H2o2

Reactive oxygen metabolites have been reported to affect platelet aggregation. However, this phenomenon is still poorly understood. In the present study we investigated the effects of superoxide radical and hydrogen peroxide (H2O2) on platelet function in vitro and correlated those effects to possible changes of platelet concentrations of cyclic nucleotides and thromboxane, since these systems play a key role in the response of platelets to activating stimuli. Human platelets were exposed to xanthine-xanthine oxidase (X-XO), a system that generates both superoxide radicals and H2O2. Sixty seconds of incubation with X-XO impaired aggregation in response to ADP (by 48%), collagen (by 71%), or the thromboxane mimetic U-46619 (by 50%). This effect was reversible and occurred in the absence of cell damage. Impairment of aggregation in platelets exposed to X-XO was due to H2O2 formation, since it was prevented by catalase but not by superoxide dismutase. Similarly, incubation with the pure H2O2 generator glucose-glucose oxidase also markedly inhibited ADP-induced platelet aggregation in a dose-dependent fashion. Impaired aggregation by H2O2 was accompanied by a > 10-fold increase in platelet concentrations of guanosine 3',5'-cyclic monophosphate (cGMP), whereas adenosine 3',5'-cyclic monophosphate levels remained unchanged. The inhibitory role of increased cGMP formation was confirmed by the finding that H2O2-induced impairment of platelet aggregation was largely abolished when guanylate cyclase activation was prevented by incubating platelets with the guanylate cyclase inhibitor, LY-83583. Different effects were observed when arachidonic acid was used to stimulate platelets. Exposure to a source of H2O2 did not affect aggregation to arachidonate. Furthermore, in the absence of exogenous H2O2, incubation with catalase, which had no effects on platelet response to ADP, collagen, or U-46619, virtually abolished platelet aggregation and markedly reduced thromboxane B2 production (to 44% of control) when arachidonic acid was used as a stimulus. In conclusion, our data demonstrate that H2O2 may exert complex effects on platelet function in vitro. Low levels of endogenous H2O2 seem to be required to promote thromboxane synthesis and aggregation in response to arachidonic acid. In contrast, exposure to larger (but not toxic) concentrations of exogenous H2O2 may inhibit aggregation to several agonists via stimulation of guanylate cyclase and increased cGMP formation.

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Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

Thrombosis and Haemostasis ◽

10.1160/th13-11-0919 ◽

2014 ◽

Vol 112 (08) ◽

pp. 412-418 ◽

Cited By ~ 15

Author(s):

Nima Vaezzadeh ◽

Ran Ni ◽

Paul Y. Kim ◽

Jeffrey I. Weitz ◽

Peter L. Gross

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Saphenous Vein ◽

Ex Vivo ◽

Arterial Injury ◽

Inhibitory Antibody ◽

Study Objective ◽

Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

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Abstract 4023: Aspirin “Resistance”: Role of Pre-existent Platelet Reactivity and Correlation Between Tests

Circulation ◽

10.1161/circ.118.suppl_18.s_821 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Andrew L Frelinger ◽

Youfu Li ◽

Matthew D Linden ◽

Inge Tarnow ◽

Marc R Barnard ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Normal Subjects ◽

Aspirin Resistance ◽

The Other ◽

List Type ◽

Platelet Function Test ◽

Function Test

Background: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a platelet function test) has been widely reported, but the underlying mechanism is unclear. We examined the role of pre-existent platelet hyperreactivity in aspirin “resistance”. We also determined the correlation between aspirin resistance defined by serum thromboxane (TX) B 2 (the most specific test of aspirin’s effect) and other assays of platelet function. Methods: Platelet function measured before and after aspirin 81 mg daily for 7 days was analyzed by Spearman’s rank correlation. Normal subjects (n=165) were studied because virtually all clinically relevant patients are already taking aspirin. An additional advantage of the use of normal subjects is that the platelet response to stimuli is not influenced (with resultant increased scatter of the data) by an underlying disease, e.g. coronary artery disease, which causes platelet hyperreactivity. Results: The proportion of the post-aspirin platelet function predicted by the pre-aspirin platelet function was 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB 2 , 39.3 ± 6.8% for urinary 11-dehydro TXB 2 , 4.4 ± 7.7% for arachidonic acid-induced platelet aggregation, 40.4 ± 7.1% for ADP-induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay®, and 45.0 ± 10.9% for the TEG® PlateletMapping ™ System with arachidonic acid. Spearman rank order correlations were highly significant for comparisons between assays when both pre-aspirin and post-aspirin results were included in the analysis. However, residual serum TXB 2 levels post-aspirin treatment were not significantly associated with post-treatment results of any of the other assays. Platelet count correlated with pre-aspirin serum TXB 2 and VerifyNow Aspirin Assay, but not with any post-aspirin platelet function test. Conclusions: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. Individuals identified as aspirin “resistant” defined by serum TXB 2 are not the same individuals identified by the other tests.

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Sex-Associated Differences On The Effect Of Asa Or Flurbiprofen On Platelet Function Ex Vivo

10.1055/s-0038-1652096 ◽

1981 ◽

Author(s):

E E Nishizawa ◽

B A Molony ◽

M M Meinzinger ◽

D J Williams

Keyword(s):

Arachidonic Acid ◽

Platelet Function ◽

Bleeding Time ◽

Ex Vivo ◽

Normal Male ◽

Normal Response ◽

Beneficial Effects ◽

Acid Pathway ◽

Induced Aggregation ◽

Arachidonic Acid Pathway

It has been reported that although ASA may have some beneficial effects in males, no differences from placebo was detected in females in clinical trials on venous thrombosis, TIA and stroke. Since ASA inhibits platelet function and because the end-points measured in the above clinical studies may be related to platelet function, we measured bleeding time and platelet aggregation induced by collagen or arachidonic acid following oral administration of ASA (650 mg) or flurbiprofen (100 mg) in normal male and female volunteers (10 per group, total of 40 subjects) between the age of 50 and 70.No sex-associated differences in response with either drug were observed in bleeding time or platelet function. Thus, we were unable to confirm that the observation of beneficial effect of ASA in males was due to its effect on platelets. However, more interestingly there was a statistically shorter bleeding time in males (p = 0.026) before administration of drug. It was also found that despite the normal response to collagen, PRP prepared from pre-drug blood samples from 6 individuals (15%) did not aggregate when stimulated with arachidonic acid at concentrations as high as 1 mM. These results suggest that, at least in some individuals, collagen-induced aggregation may proceed by a pathway independent of the arachidonic acid pathway.

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Lack Of Inhibition Of Thromboxane Production Despite Inhibition Of Platelet Function By 1,3-Bis(2 Chloroethyl)-1-Nitrosourea (BCNU)

10.1055/s-0038-1652605 ◽

1981 ◽

Author(s):

R McKenna ◽

T Ahmad ◽

A Prancan ◽

D Simon ◽

H Frischer

Keyword(s):

Arachidonic Acid ◽

Oxygen Consumption ◽

Platelet Aggregation ◽

Acetylsalicylic Acid ◽

Platelet Function ◽

Human Platelet ◽

Thromboxane A2 ◽

Rabbit Aorta ◽

Thromboxane Production ◽

Platelet Thromboxane

We have previously shown that BCNU inhibits human platelet glutathione reductase (GSSG-R) prior to inhibiting platelet function; since thromboxane production is important in platelet function, we evaluated the effect of BCNU induced inhibition of GSSG-R on platelet thromboxane production.Control platelet GSSG-R activity was 0.091 ]jmoles NAD(P)H oxidized min-1lmg-1 protein at 37°C (±0.015 S.D.; n=9); inhibition was detectable at 10-7M% BCNU (70% of control) with a >90% inhibition at and above 10-5M BCNU. Platelet aggregation in response to 1.5×10-3M Arachidonic acid (AA), 10 μM epinephrine, 6 μg/ml equine collagen and 3 μM ADP were inhibited at 10-5M BCNU and abolished at 10-4 BCNU.BCNU (10-3M) did not affect the increase in oxygen consumption induced by AA. Using the rabbit aorta superfusion bioassay for thromboxane A2 (TXA2), threshold concentrations of AA in 10-5 and 10-4 BCNU platelets resulted in an increased measure of aortic tension 13.5 ± 9.4 mm S.D. (n=6) and 23.2 ± 9.5 mm respectively, compared with control values of 4.5 ± 2.4. Acetylsalicylic acid (5 × l0-4M) inhibited the contraction: 1.7 ± 1.1 (n=5). The conversion of 14C AA to thromboxane B2 (TXB2) and PGE2, as measured by radio TLC, was not decreased in BCNU treated platelets. There is a significant increase in TXB2 (p<0.05;n=4) and in the ratio of TXB2:PGE2 in platelets treated with 10-4M BCNU and 10-3M imidazole when compared to platelets treated with imidazole alone.In conclusion BCNU induced inhibition of platelet GSSG-R and platelet function occurs despite preservation of thromboxane production

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