J. Thom

Homocysteine-Lowering Treatment With Folic Acid, Cobalamin, and Pyridoxine Does Not Reduce Blood Markers of Inflammation, Endothelial Dysfunction, or Hypercoagulability in Patients With Previous Transient Ischemic Attack or Stroke: A Randomized Substudy of the VITATOPS Trial

Background and Purpose— Epidemiological and laboratory studies suggest that increasing concentrations of plasma homocysteine (total homocysteine [tHcy]) accelerate cardiovascular disease by promoting vascular inflammation, endothelial dysfunction, and hypercoagulability. Methods— We conducted a randomized controlled trial in 285 patients with recent transient ischemic attack or stroke to examine the effect of lowering tHcy with folic acid 2 mg, vitamin B12 0.5 mg, and vitamin B6 25 mg compared with placebo on laboratory markers of vascular inflammation, endothelial dysfunction, and hypercoagulability. Results— At 6 months after randomization, there was no significant difference in blood concentrations of markers of vascular inflammation (high-sensitivity C-reactive protein [P=0.32]; soluble CD40L [P=0.33]; IL-6 [P=0.77]), endothelial dysfunction (vascular cell adhesion molecule-1 [P=0.27]; intercellular adhesion molecule-1 [P=0.08]; von Willebrand factor [P=0.92]), and hypercoagulability (P-selectin [P=0.33]; prothrombin fragment 1 and 2 [P=0.81]; D-dimer [P=0.88]) among patients assigned vitamin therapy compared with placebo despite a 3.7-&mgr;mol/L (95% CI, 2.7 to 4.7) reduction in total homocysteine (tHcy). Conclusions— Lowering tHcy by 3.7 &mgr;mol/L with folic acid-based multivitamin therapy does not significantly reduce blood concentrations of the biomarkers of inflammation, endothelial dysfunction, or hypercoagulability measured in our study. The possible explanations for our findings are: (1) these biomarkers are not sensitive to the effects of lowering tHcy (eg, multiple risk factor interventions may be required); (2) elevated tHcy causes cardiovascular disease by mechanisms other than the biomarkers measured; or (3) elevated tHcy is a noncausal marker of increased vascular risk.

Rapid detection of hereditary and acquired platelet storage pool deficiency by flow cytometry

Platelet aggregation is commonly used to investigate patients with possible dense granule storage pool deficiency (delta SPD), but recent studies have shown that this investigation is not specific or sensitive for this disorder. We describe a simple one-step technique to detect mepacrine loaded platelets by flow cytometry and found a good correlation (r = 0.83) between this method and the enumeration of platelet dense granules by conventional fluorescent microscopy. Seven patients with congenital delta SPD had significantly (P < 0.001) reduced mepacrine labelling detected by flow cytometry (mean 15%; range 5-23%) compared to normal controls (mean 48%; normal range 32-64%). Six patients with other hereditary platelet disorders had normal mepacrine labelling (mean 49%; range 34-66%) and were clearly distinguished from patients with delta SPD despite similar platelet aggregation patterns. Acquired delta SPD is frequently associated with the platelet function defect described in the myeloproliferative and myelodysplastic disorders (MPD/MDS) and we compared platelet aggregation and mepacrine labelling in 15 of these patients. The results confirm that delta SPD occurs commonly in MPD/MDS with 7/15 patients having reduced mepacrine staining but, like the findings in hereditary delta SPD, 3/7 patients with normal platelet aggregation had delta SPD. Similarly abnormal platelet aggregation was not diagnostic of delta SPD as 4/8 of these patients had normal mepacrine levels. These results may contribute to the known lack of correlation between the limited assessment of platelet function and bleeding events in MPD/MDS. We found mepacrine labelling of platelets detected by flow cytometry to be a useful, simple and inexpensive method to detect hereditary and acquired delta SPD which will improve the definition of the platelet defect in these disorders in the clinical laboratory.

Protein Z Gene Polymorphisms, Protein Z Concentrations, and Ischemic Stroke

Background and Purpose— We aimed to determine whether A-13G or G79A polymorphisms of the protein Z gene that have been reported to be an important determinant of blood concentrations of protein Z are associated with risk of ischemic stroke in a broad range of stroke patients and controls. Methods— We conducted a case control study of 151 hospital cases of first-ever ischemic stroke and 164 randomly selected community controls. Protein Z genotype was determined for the A-13G promoter polymorphism and the G79A intron F polymorphism, and plasma protein Z concentrations were measured during the first 7 days and at 3 to 6 months after the acute stroke event. Results— Geometric mean concentrations of protein Z measured within 7 days of acute stroke were significantly higher in cases compared with controls (1.51 &mgr;g/mL versus 1.13 &mgr;g/mL; P<0·0001). Protein Z concentrations were highest among subjects with the A-13G AA genotype, intermediate among those with the AG genotype, and lowest among those with the GG genotype (1.39 &mgr;g/mL versus 1.05 &mgr;g/mL versus 0.76 &mgr;g/mL; P<0.0001); and highest among those with the G79A GG genotype, intermediate among those with the GA genotype, and lowest among those with the AA genotype (1.47 &mgr;g/mL versus 1.13 &mgr;g/mL versus 0.66 &mgr;g/mL; P<0.0001). The prevalence of A-13G and G79A genotypes was not significantly different between cases of ischemic stroke and controls. However, compared with the G79A GG genotype (reference), the odds of ischemic stroke was progressively lower for the heterozygote GA (odds ratio [OR], 0.83; 95% CI, 0.52 to 1.33) and the homozygote AA genotype (OR, 0.63; 95% CI, 0.20 to 1.98). A pooled analysis showed that compared with the G79A GG genotype (reference), the odds of ischemic stroke was progressively lower for the heterozygote GA (OR, 0.78; 95% CI, 0.57 to 1.07) and the homozygote AA genotype (OR, 0.31; 95% CI, 0.14 to 0.69). Conclusion— The consistency of the association between protein Z genotypes, blood concentrations of protein Z, and ischemic stroke, determined using 2 different methods that have different sources of bias strengthens the evidence that increased blood concentrations of protein Z concentrations are associated causally with an increased risk of ischemic stroke.

Enhanced antiplatelet effect of clopidogrel in patients whose platelets are least inhibited by aspirin: a randomized crossover trial.

Summary.  Objective: We aimed to determine whether adding clopidogrel to aspirin in patients at high risk of future cardiovascular events would suppress laboratory measures of the antiplatelet effects of aspirin; and have greater platelet inhibitory effects in patients with the least inhibition of platelets by aspirin. Methods: We performed a randomized, double‐blind, placebo‐controlled, crossover trial, comparing clopidogrel 75 mg day−1 versus placebo, in 36 aspirin‐treated patients with symptomatic objectively confirmed peripheral arterial disease. Results: The addition of clopidogrel to aspirin did not suppress platelet aggregation induced by arachidonic acid, urinary 11 dehydro thromboxane B2 concentrations, or soluble markers of platelet activation markers (P‐selectin, CD40‐ligand) and inflammation (high sensitivity serum C‐reactive protein, interleukin‐6). Clopidogrel significantly inhibited platelet aggregation induced by ADP (reduction 26.2%; 95% CI: 21.3–31.1%, P < 0.0001) and collagen (reduction 6.2%; 95% CI: 3.2–9.3%, P = 0.0003). The greatest inhibition of collagen‐induced platelet aggregation by clopidogrel was seen in patients with the least inhibition of arachidonic acid induced aggregation by aspirin [lower tertile of arachidonic acid‐induced platelet aggregation: 2.8% (95% CI: −0.8 to 6.3%) reduction in mean collagen‐induced aggregation by clopidogrel; middle tertile: 4.0% (95% CI: 0.4–7.6%); upper tertile 12.6% (95% CI: 4.5–20.8%); P‐value for interaction 0.01]. Conclusions: The greatest platelet inhibitory effect of clopidogrel occurs in patients with the least inhibition of arachidonic acid‐induced platelet aggregation by aspirin. This raises the possibility that the clinical benefits of adding clopidogrel to aspirin may be greatest in patients whose platelets are least inhibited by aspirin. Confirmation in clinical outcome studies may allow these patients to be targeted with antiplatelet drugs that inhibit the ADP receptor, thereby overcoming the problem of laboratory aspirin resistance.

Compromised ITAM‐based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background: Receptors on platelets that contain immunoreceptor tyrosine‐based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives: We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen‐related peptide, or to FcγRII‐activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti‐GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa‐blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full‐length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10‐kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL−1 soluble GPVI by ELISA (normal plasma, ∼15 ng mL−1) and IgG purified from patient plasma caused FcγRIIa‐mediated, EDTA‐sensitive cleavage of both GPVI and FcγRIIa on normal platelets. Conclusions: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

Measurement of soluble P-selectin and soluble CD40 ligand in serum and plasma.

Harvey VS, Bach R, Davis GL, DeWolf W, Carvalho AC. Procoagulant activity associated with plasma membrane vesicles shed by cultured tumor cells. Cancer Res 1983; 43: 4434–42. 5 Giesen PL, Nemerson Y. Tissue factor on the loose. Semin Thromb Hemost 2000; 26: 379–84. 6 Bogdanov VY, Balasubramanian V, Hathcock J, Vele O, Lieb M, Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat Med 2003; 9: 458–62. 7 Nishibe T, Parry G, Ishida A, Aziz S, Murray J, Patel Y, Rahman S, Strand K, Saito K, Saito Y, Hammond WP, Savidge GF, Mackman N, Wijelath ES. Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation. Blood 2001; 97: 692–9. 8 Higgins DL,Mann KG. The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles. J Biol Chem 1983; 258: 6503–8. 9 Ginestra A, Monea S, Seghezzi G, Dolo V, Nagase H, Mignatti P, Vittorelli ML. Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells. J Biol Chem 1997; 272: 17216–22. 10 Dolo V, Adobati E, Canevari S, Picone MA, Vittorelli ML. Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens. Clin Exp Metastasis 1995; 13: 277–86. 11 Kim CW, Lee HM, Lee TH, Kang C, Kleinman HK, Gho YS. Extracellular membrane vesicles from tumor cells promote angiogenesis via sphingomyelin. Cancer Res 2002; 62: 6312–7.

Restored platelet function after romiplostim treatment in a patient with immune thrombocytopenic purpura.

Whilst the precipitating aetiology of immune thrombocytopenic purpura (ITP) remains unclear, the predominant cause of the thrombocytopenia is the presence of circulating antiplatelet autoantibodies that coat platelets, leading to platelet destruction and clearance, primarily in the spleen. Traditional clinical management of patients with ITP, including treatment with corticosteroids, intravenous immunoglobulins, splenectomy, rituximab, and cyclophosphamide, aims to curb platelet destruction (Nurden et al, 2009a). Newer reagents approved for trial to treat ITP patients, such as thrombopoietin mimetics, romiplostim (Kuter et al, 2008) and eltrombopag, act primarily in the bone marrow to stimulate thrombopoiesis. Whilst neither plasma thrombopoietin levels nor platelet production kinetics are markedly altered in ITP patients, ITP anti-platelet autoantibodies interfere with megakaryocyte proliferation and platelet production in vitro (Chang et al, 2003). Here we describe a significant improvement to the nature and function of platelet immunoreceptor tyrosinebased activation motif (ITAM) receptors in an ITP patient with an autoantibody to platelet glycoprotein (GP)VI receiving romiplostim.

Sustained Homocysteine-Lowering Effect over Time of Folic Acid-Based Multivitamin Therapy in Stroke Patients despite Increasing Folate Status in the Population

Background and Aims: It is uncertain what impact increasing voluntary folate fortification may be having on the statistical power of randomized trials testing the homocysteine hypothesis of atherothrombosis. The objective of this study was to determine whether there has been a change in folate status between 1998 and 2002 in stroke patients randomized into the VITAmins TO Prevent Stroke (VITATOPS) Study at a single center in Perth, Australia, and what impact this may have had on the magnitude of the homocysteine-lowering effect achieved over time with folic acid-based multivitamin therapy. Methods: We conducted a randomized, double-blind, placebo-controlled study involving 285 patients with stroke or transient ischemic attack who were recruited between 1998 and 2002 and randomized to long-term folic acid 2.0 mg/day, pyridoxine 25 mg/day and cobalamin 0.5 mg/day (active VITATOPS medication) or placebo. Fasting plasma total homocysteine, red cell folate, serum cobalamin and serum pyridoxine levels were measured at baseline and 6 months, and the change in blood levels over 4 time quartiles and differences in levels between the two randomized treatments were examined. Results: Between 1998 and 2002, there was a significant rise in baseline mean red cell folate levels over 4 time quartiles among the entire stroke cohort (723.3, 780.1, 922.6 and 1,023.7 nmol/l in the first, second, third and fourth quartiles, respectively; p < 0.0001), but this was not associated with a spontaneous reduction in mean baseline total homocysteine levels during the same time period (12.7, 14.3, 12.1 and 12.8 µmol/l in the first, second, third and fourth quartiles, respectively; p = 0.55). The homocysteine-lowering effect of the active VITATOPS trial medication at 6 months after randomization also did not change significantly between 1998 and 2002 (difference between randomized groups: –4.1, –4.1, –3.1 and –3.6 µmol/l in the first, second, third and fourth quartiles, respectively; p = 0.56). Conclusions: The homocysteine-lowering effect of the active VITATOPS trial medication has not attenuated significantly in the past 5 years despite increasing voluntary fortification of foods with folic acid as reflected by a progressive rise in baseline folate status. These data suggest that in the continuing absence of a program of mandatory folate fortification of food in populations served by centers participating in the VITATOPS trial, the study will remain adequately powered to test the homocysteine-lowering hypothesis for which it was designed.

A novel ABCA1 nonsense mutation, R1270X, in Tangier disease associated with an unrecognised bleeding tendency

The ATP binding cassette transporter A1 (ABCA1) is involved in the regulation of lipid trafficking and export of cholesterol from cells to high density lipoprotein (HDL). ABCA1 gene defects cause Tangier disease, an autosomal recessive disorder characterised by the absence of HDL-cholesterol in plasma, abnormal deposition of cholesteryl esters in the reticuloendothelial system, defective platelet dense and lysosomal granule release, and disordered cellular cholesterol efflux. We describe the case of a 62-year-old man with Tangier disease who presented with severe anaemia secondary to a spontaneous splenic haematoma. He underwent elective splenectomy without haemorrhage and his thrombocytopaenia resolved with a platelet count rising from 97 to 560 x 10(9)/L. Macroscopically, the resected spleen was enlarged with evidence of splenic haematoma. Histologic analysis of sections of spleen revealed lipid histiocytosis consistent with the diagnosis of Tangier disease. DNA sequence analysis revealed the subject to be a homozygote for a novel ABCA1 mutation c.4121C>T, which changes arginine 1270 to a stop codon (R1270X). In conclusion, we describe a case of Tangier disease in association with an unrecognised bleeding tendency, in a man homozygous for a novel ABCA1 gene mutation, R1270X.

Single or duplicate analysis for automated prothrombin time and activated partial thromboplastin time

Summary The introduction of automated coagulation analysers raises the possibility of performing routine coagulation tests as single rather than duplicate analyses. This study compares the duplicate results of the prothrombin time (PT) and activated partial thromboplastin time (aPTT) to that of a single result obtained by the MLA Electra 1000c using two different methods. The first method looks specifically at the difference between the mean duplicate result and the single result to determine if they alter patient management. This method found that two of 4152 PTs (0.048%) and two of 3047 aPTTs (0.065%) were clinically significant. The second method statistically assessed the agreement between the traditional mean duplicate result and the single test. This supported the introduction of single testing as the calculated 95% confidence intervals demonstrate that each result is interchangeable for purposes of clinical interpretation. These results supported the introduction of single testing for PT and aPTT estimation in our laboratory. The introduction of single testing was accompanied by changes to quality control and machine maintenance to ensure that the functions previously covered by duplicate testing were maintained. A strict protocol for repeat testing has also been adopted.