Larry D. Brace

Development of aspirin resistance in persons with previous ischemic stroke.

The ex vivo effect of aspirin (ASA) on platelet aggregation, the platelet component of thrombosis, was studied at repeated intervals in a cohort of patients taking aspirin for recurrent ischemic stroke prevention to define the maintenance of efficacy over time. Methods We administered increasing doses of aspirin (from 325 to 1300 mg/d) to patients with previous ischemic stroke and determined the extent of inhibition of platelet aggregation after 2 weeks and thereafter at approximately 6-month intervals. Results Over 33 months, 306 patients had platelet aggregation studies performed to define their initial response to ASA therapy. Of these, 228 had complete and 78 had partial inhibition of platelet aggregation at initial testing. To date, 119 of those who had complete inhibition and 52 who had partial inhibition have undergone repeat testing at least once. At repeat testing 39 of the 119 (32.7%) with complete inhibition at initial testing had lost part of the antiplatelet effect of ASA and converted from complete to partial inhibition without change in ASA dosage. Of the 52 with partial inhibition at initial testing, 35 achieved complete inhibition either by ASA dosage escalation (in 325 mg/d increments) or fluctuation of response at the same dosage, but 8 of those 35 (22.8%) had reverted to partial inhibition when tested again. Overall, 8.2% of patients ultimately exhibited ASA resistance to 1300 mg/d-8 of 52 (15.4%) with partial inhibition and 6 of 119 (5.0%) with complete inhibition at initial testing. Conclusions The antiplatelet (and presumably the antithrombotic) effect of a fixed dose of ASA is not constant over time in all individuals. The mechanisms by which increased dosage requirement or ASA resistance develops and the clinical significance of this development are currently undefined.

Aspirin response and failure in cerebral infarction

Background and Purpose The purpose of this study was to assess the biological effect of aspirin as measured by the inhibition of platelet aggregation in patients taking aspirin for stroke prevention and in patients with acute stroke. Methods We administered increasing doses of aspirin (325,650,975, and 1,300 mg daily) to 113 patients for stroke prevention and measured the inhibition of platelet aggregation in these patients and in 33 patients with acute stroke taking aspirin before stroke onset. Results Eighty-five patients on ≤325 and six on ≥650 mg aspirin daily had complete inhibition of platelet aggregation. Increase of the dose by 325 mg in nine of the 22 patients with partial inhibition of platelet aggregation produced complete inhibition in five patients at 650 mg and in one at 975 mg. At 1,300 mg, three patients still had only partial inhibition of platelet aggregation (aspirin resistance). Of the 33 inpatients with acute stroke, 24 had platelet aggregation studies done before further administration of aspirin. Of these, 19 had complete inhibition of platelet aggregation and three had partial inhibition, with production of complete inhibition of platelet aggregation at dose escalation; one patient was aspirin-resistant and the other noncompliant. Conclusions How the inhibition of platelet aggregation relates to stroke prevention remains unclear. The ability of aspirin and the dose required to inhibit platelet aggregation may depend upon the individual.

EVI1 induces myelodysplastic syndrome in mice

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolemic humans

Tocotrienols inhibit cholesterol synthesis by post-transcriptionally suppressing β-hydroxy-β-methylglutaryl-coenzyme A reductase activity. A double blind, 12-week study was performed to investigate the effect of a novel tocotrienol-rich fraction (TRF25; obtained by molecular distillation from specially processed rice bran oil) on cardiovascular disease risk factors of hypercholesterolemic human subjects (serum total cholesterol >5.69 mmol/L). After acclimation to an alcohol-free regimen (baseline) participants were assigned to the National Cholesterol Education Program (NCEP) Step-1 diet (saturated fat <19%, total fat <30% of total calories and cholesterol <7.76 mmol/L). The participants were evaluated after 4 weeks of exposure to the NCEP Step-1 diet; one group of 21 participants was continued on the NCEP Step-1 diet for 4 weeks receiving an additional 1.2 gm corn oil (placebo group) and a second group of 20 received 200 mg TRF25 dissolved in 1.0 gm corn oil (TRF25 group). Serum total cholesterol and LDL-cholesterol levels of all the participants, stable during the baseline phase of the study, decreased 5% and 8%, respectively, during the 4-week NCEP Step-1 diet. Placebo continuing on the NCEP Step-1 diet for an additional 4 weeks experienced additional but modest decreases in serum total cholesterol (2%) and LDL-cholesterol (3%), yielding significant (P < 0.05) decreases when compared with the baseline values. These responses confirm the cholesterol-lowering action of a low fat, low cholesterol diet. Participants receiving TRF25 had 12% and 16% reductions (P < 0.05) in total cholesterol and LDL-cholesterol levels during the 4-week experimental phase; during the two phases (NCEP Step-1 diet plus treatment) the serum total cholesterol and LDL-cholesterol levels of these participants were decreased (P < 0.05) by 17% and 24%, respectively. TRF25-mediated decreases in Apo B, Lp(a), platelet factor 4 and thromboxane B2 (15%, 17%, 14%, and 31%, respectively) were significant (P < 0.05). There was no change in the levels of HDL-cholesterol and apolipoprotein A-I by this treatment. The treatments also resulted in remarkable increases in the levels of LDL-bound antioxidants, especially tocotrienols, which have substantially greater antioxidant activity than vitamin E.

Platelet aggregation in patients with atrial fibrillation taking aspirin or warfarin.

Background and Purpose Although warfarin and perhaps aspirin may be effective in preventing thromboembolism in patients with nonvalvular atrial fibrillation, some patients develop cerebral infarction despite these therapies. The purpose of this study was to determine inhibition of platelet aggregation in patients on aspirin and platelet reactivity in those on warfarin in the Stroke Prevention in Atrial Fibrillation study. Methods Twenty-four patients in the Stroke Prevention in Atrial Fibrillation study at the University of Illinois at Chicago, 17 on enteric-coated aspirin 325 mg/d and 7 on warfarin to produce an International Normalized Ratio of 2.0 to 4.5, had platelet aggregation studies performed during a 10-month period and interpreted by an investigator blinded to therapy. Epinephrine, adenosine diphosphate, collagen, and arachidonic acid were used as aggregating agents. Compliance was determined by pill count for those patients on aspirin. Results Seven patients taking aspirin had partial and 10 had complete inhibition of platelet aggregation. Three of seven patients on warfarin had hyperaggregable platelets. Compliance was 80% or greater for those patients taking aspirin. One patient on warfarin had partial inhibition of platelet aggregation. Conclusions Some patients in the Stroke Prevention in Atrial Fibrillation trial on aspirin 325 mg/d did not achieve complete inhibition of platelet aggregation. Others had hyperaggregable platelets. These findings suggest platelet-dependent mechanisms for aspirin and warfarin failure to prevent stroke in these patients.

Sex Difference in the Antiplatelet Effect of Aspirin in Patients with Stroke

Background: There is substantial interpatient variability in response to aspirin after an ischemic stroke or transient ischemic attack (TIA), as assessed by ex vivo effects of aspirin on platelet aggregation. The factors contributing to this variability are not well defined. Objective: To determine whether demographic, social, or clinical characteristics are associated with ex vivo response to aspirin in patients with a history of stroke or TIA. Methods: Eighty-one patients who were taking aspirin for secondary stroke prevention and underwent ex vivo platelet aggregation studies were identified. The medical records of eligible patients were reviewed by clinicians who specialize in the management of stroke patients. Characteristics were compared between 45 patients who had a complete response to aspirin and 36 patients who exhibited an incomplete (partial) response to aspirin based on the results of platelet aggregation testing. Results: The median (range) aspirin dose was similar in complete (325; 81–1950 mg/day) and partial (325; 81–1300 mg/day) responders. There was no association between aspirin response and age, race, body mass index, medical history, smoking status, or use of statin or hormone replacement therapy. However, sex was significantly associated with response to aspirin, with more women in the partial versus complete responder group (75% vs 49%; p = 0.02). Conclusions: Our data suggest that aspirin may be less effective at inhibiting platelet aggregation in women compared with men who have a history of ischemic stroke or TIA.

Factitious lithium toxicity secondary to lithium heparin-containing blood tubes.

IntroductionLithium concentrations analyzed from blood collected in inappropriate tubes may lead to misdiagnosis and lead to unnecessary hospitalization and intervention. We sought to assess the magnitude of falsely elevated lithium levels in green top lithium heparin-containing blood tubes.MethodsSerum lithium concentrations from two types of commonly used standard green top tubes were evaluated against a control tube. Blood samples obtained from 5 healthy volunteers who have never ingested pharmaceuticals containing lithium were aliquoted into a control serum separator tube (SST), a light green heparin-containing tube, and a dark green heparin tube at the following volumes: full draw, 2cc, and 1cc. Serum lithium levels in lithium heparin blood tubes were compared to standard blood tubes.ResultsAll levels are reported as mmol/L. Mean serum lithium level for the control was 0.16 [range: 0.1–0.2]. Levels for the light-green tubes at full-draw, 2cc, and 1cc were 1.05 [1.0–1.1], 1.99 [1.68–2.3], and 3.31 [2.8–4.2] respectively. Levels for the dark-green tubes at full-draw, 2cc, and 1cc were 1.07 [1.0–1.14], 2.35 [2.1–2.55], and 4.04 [3.8–4.4] respectively.ConclusionsFalsely elevated lithium levels may occur when using green-top lithium containing blood tubes and may contribute as much as 4 mmol/L to the level in tubes not completely filled.

Platelet aggregation and recruitment with aspirin-clopidogrel therapy.

Background: Aspirin-clopidogrel combination therapy inhibits platelet aggregation. The effect on platelet recruitment is unknown. Methods: Thirty chronic ischemic stroke patients taking aspirin alone followed by aspirin-clopidogrel combined therapy had platelet reactivity tests performed over 3 months: ex vivo platelet aggregation, platelet recruitment and urinary 11-dehydro-thromboxane B2 (11-dhTxB2)excretion. Statistical analysis of variance compared platelet aggregation and recruitment between aspirin alone and aspirin-clopidogrel, and longitudinal regression analysis estimated platelet recruitment over time. Nonlinear mapping defined variable connections in each patient. Results: Statistically significant differences were found between aspirin alone and aspirin-clopidogrel for (1) adenosine-diphosphate- and collagen-induced platelet aggregation and maximum inhibition of platelet recruitment and (2) increasing inhibition of platelet recruitment over time. Urinary 11-dhTxB2 excretion did not predict platelet aggregation response. Nonlinear mapping showed patient-unique variable interconnections. Conclusions: Platelet inhibition with aspirin-clopidogrel may increase over time, and future studies should focus on this finding in the context of vascular complications.

Influence of cyclooxygenase-1 genotype on ex vivo aspirin response in patients at risk for stroke.

Background: We sought to determine whether cyclooxygenase-1 (PTGS1) genotype is associated with the ability of aspirin to inhibit platelet aggregation in patients at risk for stroke. Methods: Blood and urine samples were collected from 60 subjects, including 28 African Americans, who were taking aspirin for primary or secondary stroke prevention. Samples were analyzed for the PTGS1 A–707G, PTGS1 P17L, and glycoprotein IIIa (ITGB3)P1A1/A2 genotypes, ex-vivo platelet aggregation, serum cholesterol, plasma salicylate levels, and urinary 11-dehydrothromboxane B2 (11-dhTxB2) concentrations. The association between PTGS1 A–707G and P17L genotypes and aspirin response, as assessed by ex vivo studies and 11-dhTxB2 concentrations, was evaluated by statistical testing and nonlinear mapping. Results: Salicylate concentrations, ITGB3 genotype distribution and 11-dhTxB2 concentrations were similar among PTGS1 genotype groups. More subjects with the PTGS1 17PP versus PL genotype had incomplete ex-vivo inhibition of platelet aggregation by aspirin (57 vs. 20%; p = 0.04). Fifty-nine percent of subjects homozygous for both the PTGS –707A and 17P alleles, but none with both the PTGS1 –707G and 17L alleles had incomplete inhibition with aspirin; p = 0.04. Similarly, nonlinear mapping showed a direct relationship between the PTGS1 17P allele and decreased aspirin response. When analyzed separately by ethnicity, the association with the P17L genotype and aspirin response persisted in African Americans, but not Caucasians. Conclusions: Our data suggest that the PTGS1 P17L genotype contributes to response to aspirin as assessed by ex-vivo platelet aggregation. Our data further suggest that the association between PTGS1 genotype and aspirin response might vary by ethnicity.