Racial differences in endotoxin-induced tissue factor-triggered coagulation

Summary.  Background: Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (= DARC, Duffy antigen receptor of chemokines) and coagulation. Objectives: Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation. Patients/methods: Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg⁻¹ LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). Results: LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin–antithrombin (TAT) complexes (10.4 pg mL⁻¹ vs. 23.0 pg mL⁻¹, P  < 0.0001) and less prothrombin fragments (F₁₊₂) (337 pmol mL⁻¹ vs. 819 pmol mL⁻¹, P  < 0.0001). Consistently, Africans also had decreased fibrin formation ( d -dimer: 0.3 pg mL⁻¹ vs. 0.5 pg mL⁻¹, P  = 0.02). Conclusion: Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.     

High plasma levels of the soluble receptor for advanced glycation endproducts in patients with symptomatic carotid atherosclerosis

Background  Advanced glycation endproducts (AGEs), particularly carboxymethyl(lysine)-adducts (CML), exert part of their cellular effects by binding to a receptor, named receptor for AGEs (RAGE). The soluble form of this receptor (sRAGE) has been shown to have an athero-protective role. We hypothesized the existence of a relationship between the AGE–RAGE axis and the occurrence of symptoms related to carotid atherosclerosis in nondiabetic conditions.
Materials and methods  We evaluated plasma levels of CML and sRAGE (by ELISA), and tissue levels (tAGEs and tRAGE, semiquantitatively, by immunohistochemistry) in endarterectomy carotid plaque tissue in 29 nondiabetic patients. At the time of surgery, 10 patients were asymptomatic and 19 were symptomatic.
Results  Plasma levels of sRAGE were higher in symptomatic patients than in asymptomatic patients [median (interquartile range): 676 (394–858) pg mL⁻¹ vs. 347 (284–479) pg mL⁻¹, P  = 0·009]. In symptomatic patients, plasma levels of sRAGE correlated positively with CML ( r  = 0·60, P  < 0·01), C-reactive protein (CRP) ( r  = 0·618, P  < 0·01) and fibrinogen ( r  = 0·522, P <0·005), while in asymptomatic patients, no correlation was observed. Although tissue and plasma levels of AGEs and RAGE did not correlate between each other, tAGEs and tRAGE were also positively correlated only in symptomatic patients (χ² = 8·93, P  = 0·003).
Conclusions  Plasma levels of sRAGE are higher in symptomatic than asymptomatic carotid atherosclerosis. Higher levels of sRAGE in symptomatic patients may be markers of a higher degree of vascular inflammation in such patients.     

Stimulation of arginine vasopressin secretion by a small increase in blood ionized calcium in normal men

Evidence has been provided for an increase in baseline serum corticotrophin (ACTH) levels in response to a rise in circulating ionized calcium (Ca_i) levels within the physiological range. In order to establish whether small Ca_i increments are also able to modify the basal secretion of arginine vasopressin (AVP), we infused calcium gluconate through an intravenous infusion pump in eight healthy male subjects (25–31 years old). Serum Ca_i, ACTH and AVP concentrations were measured every 10 min over an infusion period lasting 90 min. A significant progressive rise in serum Ca_i (baseline: 42 ± 0.9 mg dL⁻¹; 90 min: 47.2 ± 0.9 mg dL⁻¹, P  < 0.001), ACTH (baseline: 30.7 ± 1.3 pg mL⁻¹; mean peak at 80 min: 37.4 ± 2.4 pg mL⁻¹, P  < 0.01) and AVP levels (baseline: 2.1 ± 0.6 pg mL⁻¹; mean peak at 80 min: 3.2 ± 0.5 pg mL⁻¹, P  < 0.01) was observed during calcium infusion. Furthermore, a significant positive correlation ( r  = 0.71; P  < 0.001) was observed between ACTH and AVP responses to calcium infusion at 60, 70, 80 and 90 min. These data demonstrate that AVP secretion is stimulated by a slight rapid increase in serum Ca_i levels even though absolute serum Ca_i levels remain within the normal range. In addition, the positive correlation between Ca_i-induced ACTH and AVP increments suggests that AVP plays a releasing role on ACTH secretion during calcium infusion.     

Cytokine profiles in bronchoalveolar lavage fluid and blood in HIV-seropositive patients with Pneumocystis carinii pneumonia

Concentrations and ex vivo production of interleukin 1β (IL-1), tumour necrosis α (TNF), interleukin 6 (IL-6), interleukin-1 receptor antagonist (IL-1RA) and TNF soluble receptors (sTNF-receptors, P55 and P75) were measured in bronchoalveolar lavage (BAL) fluid and blood in 23 HIV-seropositive (HIV+) patients with Pneumocystis carinii pneumonia (PCP) and compared with values found in healthy HIV-seronegative (HIV−) controls and asymptomatic HIV+ subjects. Concentrations of the proinflammatory cytokine IL-1β were increased in BAL fluid of HIV+ patients with PCP (184 ± 47 pg mL⁻¹) compared with undetectable levels in healthy control subjects ( P  = 0.0001). In plasma of these patients higher concentrations of the anti-inflammatory cytokine IL-1RA were found during acute PCP than after recovery (2.1 ± 0.7 vs. 0.5 ± 0.2 ng mL⁻¹, P  = 0.01). No correlations could be found between cytokine concentrations and clinical severity of the infection. Corticosteroid treatment did not influence cytokine concentrations in BAL or blood, nor did it suppress the production in alveolar cells. In whole-blood cultures, however, lipopolysaccharide (LPS)-stimulated production was significantly suppressed for IL-1 (1.3 vs. 5.5 ng mL⁻¹, P  = 0.009) and for IL-6 (0.6 vs. 2.5 ng mL⁻¹, P  = 0.01). The overall data show that in HIV+ patients with PCP (similar to what we had found previously in HIV− patients with PCP) proinflammatory cytokines are more prominently present in BAL, whereas anti-inflammatory reaction is predominant in the circulation.     

Responses of atrial natriuretic peptide and brain natriuretic peptide to exercise in patients with chronic heart failure and normal control subjects

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are known to be elevated in patients with chronic heart failure at rest. While it is known that during exercise the circulating level of ANP increases in patients with heart failure, the response of BNP to exercise in these patients relative to control subjects is unclear. Ten patients with stable chronic heart failure and 10 normal control subjects performed symptom-limited exercise with respired gas analysis. All patients had depressed left ventricular ejection fractions (LVEF). Patients had lower peak oxygen consumption P V ˙ O ₂) than the control group [median (range) 1.18 (0.98–1.76) vs. 1.94 (1.53–2.31) L min⁻¹; P  < 0.001]. Circulating plasma levels of ANP and BNP were higher at rest in patients than in control subjects [ANP 335 (140–700) vs. 90 (25–500) pg mL⁻¹; BNP 42 (25–50) vs. 20 (10–20) pg mL⁻¹], and at peak exercise [ANP 400 (200–1000) vs. 130 (10–590); BNP 46 (40–51) vs. 20 (10–30)]. The rise in ANP at peak exercise was significant in patients compared with the resting level, but not in control subjects. For BNP, there was a significant rise in patients but no change in control subjects. The circulating plasma levels of both peptides showed a strong negative correlation with LVEF (ANP, P  < 0.005; BNP, P  < 0.0001) and, to a less extent, with RVEF. It is possible that BNP may give a better indication of cardiac function.     

Defective interferon-gamma production by T-lymphocytes from patients with acute brucellosis

The authors investigated the production of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) by phytohaemagglutinin (PHA)-stimulated T-lymphocytes from 21 untreated patients with acute brucellosis. PHA-stimulated T-lymphocytes from acute brucellosis patients showed normal IL-2 production but defective IFN-γ production (brucellosis patients 531 ± 103 pg mL⁻¹ vs. healthy controls 1024 ± 212 pg mL⁻¹) after 72 h of culture. This altered pattern of IL-2 and IFN-γ production by T-lymphocytes was observed in seven brucellosis patients whose T-lymphocytes exhibited a normal proliferative response to PHA (61 612 ± 18 422 cpm) as well as in the 14 patients with a defective T-lymphocyte proliferative response to the PHA after 5 days of culture (19 479 ± 4409 cpm). After antibiotic therapy, production of the two lymphokines by the PHA-stimulated T-lymphocytes from acute brucellosis patients was similar to that of T-lymphocytes from healthy control subjects. The authors conclude that PHA-stimulated T-lymphocytes from untreated patients with acute brucellosis have defective INF-γ production but normal IL-2 production.     

Effects of hemicolectomy on bile acid metabolism in relation to colon carcinogenesis in man

Bile acids are probably important in colon carcinogenesis. Regional differences in bile acid metabolism within the colon were studied to illuminate the preferential distal occurrence of colon cancer in Western countries. Faeces (24 h) were collected for bile acid measurement from 25 patients with hemicolectomy (nine left and 16 right) and 17 adenoma patients with an intact colon (control subjects). Duodenal bile and cytolytic and alkaline phosphatase activity of faecal water were also studied. The median percentage of deoxycholic acid (DCA) was lower in the hemicolectomy groups [left 48% (range 38–57%), right 45% (2–62%) vs. control subjects 59% (38–70%), P  < 0.05]. In duodenal bile, the proportion of DCA in left [4% (1–25%)] was lower than in the patients with right hemicolectomy [19% (0–69%)] and control subjects [24% (7–50%)], P  < 0.05. Faecal concentration of protonated DCA was higher in those with right hemicolectomy (0.101 μmol g⁻¹) than in those with left hemicolectomy (0.048 μmol g⁻¹), which coincided with a higher cytolytic [right 49% (3–93%), left 2% (1–37%)] and alkaline phosphatase activity [right 6.7 U mL⁻¹ (1.2–40.1 U mL⁻¹), left (2.0 U mL⁻¹ (1–25.7 U mL⁻¹), both P  < 0.02]. These findings suggest differences in bile acid metabolism between the proximal and distal colon that may contribute to the disparity in cancer risk.     

Soluble P-selectin levels, P-selectin polymorphisms and cardiovascular disease

Summary.  P-selectin is a member of the selectin family of cell adhesion molecules which are important in the transient attachment of leukocytes to endothelial cells and platelets. A number of polymorphisms in the gene encoding P-selectin have been identified. Objectives were to investigate the relationship of soluble P (sP)-selectin with P-selectin gene polymorphisms and coronary artery disease (CAD). Two hundred and forty-nine patients, with extent of CAD characterized by ≥50% stenosis in one or more coronary arteries, and 252 healthy controls were studied. Soluble P-selectin was significantly higher in the patients than controls after adjustment for age, sex and smoking [patients 49.8 (47.5–52.1) ng mL⁻¹; controls 46.7 (44.5–49.1) ng mL⁻¹, P  = 0.03). There was no association of sP-selectin with myocardial infarction (MI) or presence of ≥50% stenosis. The −1817 T/C, −1969 G/A and −2123 C/G (but not the Thr715Pro) polymorphisms were in strong linkage disequilibrium. The Thr715Pro polymorphism was significantly associated with sP-selectin even after adjustment for covariates [TT 48.9 (46.9–50.0) ng mL⁻¹; TP + PP 40.7 (38.1–43.6) ng mL⁻¹, P  < 0.0001]. A significant interaction of Thr715Pro and smoking status was identified in the determination of sP-selectin levels. There was no significant association of genotype at any of the polymorphism in relation to MI or stenosis. The Thr715Pro polymorphisms is associated with plasma sP-selectin. This association is modulated by smoking, although the underlying mechanism remains unclear.     

Long-term use of smokeless tobacco and physical performance in middle-aged men

To determine the influence of prolonged nicotine exposure on maximal physical working capacity, a study of clinical measures of physical fitness and cardiovascular response to exercise was performed in 144 healthy men, 35–60 years old, subdivided into smokeless tobacco users, smokers and non-users of tobacco. Regular users of smokeless tobacco, with exposures of more than 20 years, showed similar maximal oxygen uptake (mean 3.48 L min⁻¹, SD 0.49, n  = 48) to non-users (mean 3.51 L min⁻¹, SD 0.51, n  = 65). In smokeless tobacco users, higher blood pressure and heart rate values were observed at rest and at submaximal work, after exposure to tobacco shortly before the exercise test, but not at maximal work. However, significantly lower maximal oxygen uptake was found for smokers (mean 2.88 L min⁻¹, SD 0.49, n  = 31) compared with non-users ( P  < 0.001). Plasma concentration of cotinine, the main metabolite of nicotine, was significantly higher in smokeless tobacco users (mean 347 ng mL⁻¹, SD 175, n  = 48) than in smokers (mean 253 ng mL⁻¹, SD 153, n  = 31, P  < 0.001). The findings indicate that long-term use of smokeless tobacco does not significantly influence exercise capacity in healthy, physically well-trained subjects.     

Low-density lipoprotein-induced formation of nitric oxide by cultured vascular smooth muscle cells of the rat

There is evidence that low-density lipoprotein (LDL) plays a crucial role in atherogenesis. On the cellular level, LDL has been shown to activate a number of mechanisms involved in atherogenesis and vasoconstriction. Local immoderate vasoconstriction is physiologically antagonized by nitric oxide, which is released from the endothelium. To find out whether LDL also influences the synthesis of nitric oxide in vascular smooth muscle cells, both the conversion of arginine to citrulline and the production of nitrite were determined as a measure of nitric oxide formation. After incubation of rat vascular smooth muscle cells with native LDL (25 μg mL⁻¹) for 24 h, the production of both l -[¹⁴C]-citrulline [39 600 (3600) cpm mg⁻¹ cell protein] and nitric oxide [2.95 (0.56) μmol L⁻¹] were about twice and 1.5-fold the amount of the corresonding values in untreated cells (mean ± SD, P  < 0.05, n  = 4). Oxidized LDL was less effective than the native form. The presence of the arginine analogue N ^G-methyl- l -arginine reduced citrulline production dose-dependently but augmented DNA synthesis, both induced by LDL. In addition, the lipoprotein caused a 1.6-fold increase in cyclic GMP production following a 24-h incubation [control = 10.9 (3.8) pmol mg⁻¹ cell protein, P  = 0.016]. The results suggest that native LDL might partly impair its atherogenic potential on the vasculature by stimulating the production by smooth muscle cells of both nitric oxide and cyclic GMP.     

Plasminogen activator inhibitor-1 predicts coronary in-stent restenosis of drug-eluting stents

Background : We tested the hypothesis that plasma levels of plasminogen activator inhibitor-1 (PAI-1) are influenced by percutaneous coronary intervention (PCI) with the implantation of drug eluting stents (DES) and are able to predict the occurrence of in-stent restenosis (ISR). Methods and results : PAI-1 active antigen plasma levels were determined in 75 patients before and 24 h after PCI with DES implantation. Patients with ISR after six to eight months (16%) showed significantly lower PAI-1 plasma levels before PCI (ISR, 11.7 ± 8.1 ng mL⁻¹; non-ISR, 22.8 ± 18.8 ng mL⁻¹; P  < 0.05). PAI-1 levels in the lowest tertile were associated with a 9.5-fold increased risk of ISR, independent of clinical risk factors, angiographic or procedural characteristics, compared to the highest tertile ( P  <   0.05). The induced change of PAI-1 active antigen 24 h after PCI was significantly higher in patients with ISR (ISR, +5.6 ± 8.0 ng mL⁻¹; non-ISR, −3.2 ± 12.1 ng mL⁻¹; P  <   0.05) with positive correlation to late lumen loss ( r  =   0.30; P  <   0.05). Conclusions : ISR after DES implantation is significantly related to plasma levels of PAI-1 active antigen before and after PCI. If confirmed by larger multicenter studies, the determination of PAI-1 plasma levels might be clinically helpful in the identification of patients at high risk of developing of ISR, even after DES implantation.     

Tumour-specific induction of immune complexes: DCP-IgM in hepatocellular carcinoma

Background   In the sera of liver, colorectal and prostate cancer patients, several biomarkers may be detected as IgM immune complexes. To determine whether the presence of immune complexes was correlated to an increase of IgMs, we measured the IgM content in the sera of patients with hepatocellular carcinoma (HCC) and cirrhosis, and evaluated the occurrence of des-gamma-carboxy prothrombin (DCP) as immune complexes ( DCP-IgM ) compared to the levels of DCP and alpha-fetoprotein (AFP).
Patients and methods   Serum samples from 31 patients with cirrhosis, 33 untreated HCC patients diagnosed by ultrasound, computed tomography and/or magnetic resonance and confirmed by histopathology, when indicated, and 30 healthy controls were analysed. Concentrations of IgM and DCP-IgM were determined by ELISAs.
Results   Circulating IgM in patients with HCC (median level = 1·79 mg mL⁻¹) and cirrhosis (1·09 mg mL⁻¹) were not significantly different ( P  = 0·1376) while DCP-IgM were significantly higher in HCC patients (median level = 2171·2 AU mL⁻¹) than in those with cirrhosis (1152 AU mL⁻¹, P  = 0·0047). No correlation was found between DCP-IgM and IgM in HCC ( r  = 0·227) and cirrhosis patients ( r  = 0·475). DPC-IgM was positive in 55% (18/33) of HCC patients and in 26% (8/31) of cirrhosis patients compared to 39% and 26% for DCP and 48% and 13% for AFP. DCP-IgM, DCP and AFP tests had 100% specificity in healthy controls.
Conclusions   DCP-IgM in HCC patients was not associated with an increase in IgM concentration. DCP-IgM was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes for liver cancer.     

Quantitative detection of circulating endothelial cells in vasculitis: comparison of flow cytometry and immunomagnetic bead extraction

Summary.  Background:  Circulating endothelial cells (CECs) are biomarkers for endothelial cell (EC) injury and are quantified using immunomagnetic bead extraction (IBE), or flow cytometry (FC). Reports suggest that there is good agreement between these methods for CEC quantification. Objectives:  We examined levels of agreement between these techniques in children with systemic vasculitis. Methods:  We added HUVEC or human pulmonary artery EC to whole blood to optimize FC gating strategies for EC. EC-optimized FC was then compared with IBE for CEC enumeration in 25 children with vasculitis and 20 healthy controls. Results:  Using Bland–Altman analysis, agreement between IBE and EC-optimized FC was poor in children with vasculitis ( n  = 25) and healthy controls ( n  = 20): IBE consistently detected higher values than the EC-optimized FC method: the mean difference between the two techniques was 60 CECs mL⁻¹, 95% CI ±374 CECs mL⁻¹ (paired analyses of 45 individuals). Agreement was poorest for vasculitis patients: mean difference (IBE – EC-optimized FC) 120 CECs mL⁻¹, 95% CI ±460 CECs mL⁻¹ ( P  = 0.018). We identified three reasons for this discrepancy: (i) sub-optimal FC gating parameters previously used for detecting CECs; (ii) inherent lack of sensitivity of FC compared with IBE for CEC rare event detection; and (iii) use of lysis buffers required for FC causing CEC lysis. Conclusions:  There was poor agreement between EC-optimized FC and IBE for the quantification of CECs from children with active vasculitis and controls. We emphasize that in this clinical setting the two techniques are not directly comparable when comparing results obtained using these different methodologies.     

An enzyme immunoassay for polymorphonuclear leucocyte-mediated fibrinogenolysis

Upon stimulation, polymorphonuclear leucocytes (PMNs) release potent serine proteases, i.e. elastase, cathepsin G and proteinase 3, which contribute to the degradation of tissue and plasma components. Here, we describe the development of a plasma test to assess PMN-mediated fibrinogenolysis as a biochemical marker for actual PMN-derived proteolysis in vivo , useful for monitoring therapeutic efficacy, i.e. of elastase inhibitors. We generated a monoclonal antibody (MAb), designated 1-1/B3, with a high affinity for elastase-degraded fibrinogen (EDF). The epitope for 1-1/B3 becomes exposed in a time-dependent manner during digestion of fibrinogen with purified PMN-derived serine proteases and with isolated PMNs in vitro . However, 1-1/B3 does not react with plasma fibrinogen or with fibrin(ogen) degradation products generated by plasmin or by other active proteases that may occur locally, i.e. metalloproteases and lysosomal cathepsins. On the basis of MAb 1-1/B3, we developed a plasma test for the assessment of PMN-mediated fibrin(ogen) degradation products (PMN-FDP). In a panel of control plasmas, we observed concentrations of PMN-FDP of 8.2 ± 0.9 ng mL⁻¹ ( n  = 18). These values were increased twofold in patients with α₁-proteinase inhibitor deficiency (18.6 ± 3.3 ng mL⁻¹; n  = 12;  P  < 0.0001) and even more in patients with sepsis (365.7 ± 97.7 ng mL⁻¹; n  = 16;  P  < 0.0001). Furthermore, synovial tissue extracts from patients with rheumatoid arthritis contained increased levels of PMN-FDP, compared with synovial tissue extracts ( P  < 0.005) from patients with osteoarthritis.     

Elevated cell-associated levels of interleukin 1β and interleukin 6 in inflamed mucosa of inflammatory bowel disease

The aim of this study was to investigate the involvement of the monocyte-derived cytokines interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor-α (TNF-α) in idiopathic inflammatory bowel disease. Endoscopic biopsies of normal and inflamed intestinal mucosa were obtained from patients with ulcerative colitis ( n  = 11) and with Crohn's disease ( n  = 10). Intestinal mucosal cells were isolated by collagenase digestion. Cell viability, morphology and CD14 expression were determined. To measure cell-associated cytokine levels, cells were lysed and analysed for IL-1β and TNF-α in specific radioimmunoassays and for IL-6 using a biological assay. Compared with mucosal cells from control patients without inflammatory bowel disease the inflamed intestine in ulcerative colitis and Crohn's disease displayed markedly enhanced levels of IL-1β (median 245 pg 10⁻⁶ cells, range 30–1275) and IL-6 (median 22 U 10⁻⁶ cells, range 1–298). Non-inflamed mucosa in patients with ulcerative colitis and Crohn's disease did not shown elevated levels of IL-1β (median 50 pg 10⁻⁶ cells, range 33–90) or IL-6 (mean below detection limit of assay, i.e. 1 U 10⁻⁶ cells). In contrast, no clear cut difference between inflamed and non-inflamed mucosa could be detected for TNF-α. High tissue levels of IL-6 were associated with a high endoscopic grade of local inflammation. These results suggest that the monocyte-derived cytokines IL-1β and IL-6 are mediators of inflammation in inflammatory bowel disease.     

Elevated tryptase levels selectively cluster in myeloid neoplasms: a novel diagnostic approach and screen marker in clinical haematology

Background  Recent data suggest that tryptase, a mast cell enzyme, is expressed in neoplastic cells in myeloid leukaemias. In several of these patients, increased serum tryptase levels are detectable.
Materials and methods  We have determined serum tryptase levels in 914 patients with haematological malignancies, including myeloproliferative disorders ( n  = 156), myelodysplastic syndromes (MDS, n  = 241), acute myeloid leukaemia (AML, n  = 317), systemic mastocytosis (SM, n  = 81), non-Hodgkin's lymphoma ( n  = 59) and acute lymphoblastic leukaemia ( n  = 26). Moreover, tryptase was measured in 136 patients with non-neoplastic haematological disorders, 102 with non-haematological disorders and 164 healthy subjects.
Results  In healthy subjects, the median serum tryptase was 5·2 ng mL⁻¹. Elevated serum tryptase levels were found to cluster in myeloid neoplasm, whereas almost all patients with lymphoid neoplasms exhibited normal tryptase. Among myeloid neoplasms, elevated tryptase levels (> 15 ng mL⁻¹) were recorded in > 90% of patients with SM, 38% with AML, 34% with CML and 25% with MDS. The highest tryptase levels, often > 1000 ng mL⁻¹, were found in advanced SM and core-binding-factor leukaemias. In most patients with non-neoplastic haematological disorders and non-haematological disorders analysed in our study, tryptase levels were normal, the exception being a few patients with end-stage kidney disease and helminth infections, in whom a slightly elevated tryptase was found.
Conclusions  In summary, tryptase is a new diagnostic marker of myeloid neoplasms and a useful test in clinical haematology.     

The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma

Summary.  Annexin A5 has been proposed to be important for shielding of negatively charged phospholipids from blood, thereby preventing the binding of clotting factors. It has been suggested that antiphospholipid antibodies can disrupt the binding of annexin A5 from negatively phospholipid-containing surfaces, resulting in uncontrolled coagulation. If this hypothesis is correct, than the plasma levels of annexin A5 will be increased in patients with antiphospholipid antibodies. Therefore, we have measured plasma levels of annexin A5 of 175 patients with systemic lupus erythematosus (SLE), of which 104 had antiphospholipid antibodies and 23 patients had primary antiphospholipid syndrome. The annexin A5 levels were compared with the annexin A5 plasma levels measured in 23 patients with diabetes mellitus type 2 and 35 healthy volunteers. We found a significant increase of annexin A5 plasma levels in patients with SLE (median 6.7 ng mL⁻¹) and primary antiphospholipid syndrome (median 7.1 ng mL⁻¹) as compared to patients with diabetes mellitus type 2 (median 3.3 ng mL⁻¹) and healthy volunteers (median 3.9 ng mL⁻¹). However, no correlation was found with the presence of antiphospholipid antibodies or with a history of thromboembolic complications. Based on these observations, we conclude that displacement of annexin A5 from cellular surfaces by antiphospholipid antibodies is not a common mechanism in patients with antiphospholipid antibodies.     

Sulfatides inhibit platelet adhesion to von Willebrand factor in flowing blood

Summary.  Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins such as von Willebrand factor (VWF), P-selectin, laminin and thrombospondin. Previous studies have localized the sulfatide-binding site of VWF to amino acid residues Gln626–Val646 in the A1 domain. The A1 domain also contains the binding site for platelet glycoprotein Ib (GP Ib), a site that has been reported to be distinct from the sulfatide-binding site. In this study, we analyzed the interaction of sulfatides with VWF and its effect on GP Ib-mediated platelet adhesion under flow conditions. Recombinant VWF A1 domain (rVWF-A1) bound specifically and saturably to sulfatides (half-maximal concentration of ∼12.5 µg mL⁻¹), binding that was blocked by dextran sulfate (IC₅₀≈100 µg mL⁻¹) but not by heparin at concentrations up to 100 U mL⁻¹. Furthermore, sulfatides (125 µg mL⁻¹) prevented the adhesion of platelets or glycocalicin-coupled polystyrene beads to a rVWF-A1-coated surface under high shear stress. In addition, plasma VWF prebound to a sulfatide-coated surface failed to support subsequent platelet adhesion. These results provide firm evidence that sulfatides bind the VWF A1 domain at a site overlapping the GP Ib-binding site.     

Genetic variants and haplotypes of lipoprotein associated phospholipase A2 and their influence on cardiovascular disease (The Ludwigshafen Risk and Cardiovascular Health Study)

Summary.  Background:   There is increasing evidence that lipoprotein-associated phospholipase A2 (LpPLA2) is associated with cardiovascular disease. However, it is still unclear whether LpPLA2 is simply a marker or has a causal role as either a pro- or anti-atherogenic factor. Methods:   We analyzed the association of five polymorphisms (−1357G>A, −403T>C, Arg92His, Ile198Thr, Ala379Val) and related haplotypes at the PLA2G7 locus with angiographic coronary artery disease (CAD), plasma LpPLA2 activity, and long-term survival in 3234 patients scheduled for coronary angiography. Results:   The promoter variant −403C and His⁹² were associated with a decrease and Val³⁷⁹ with an increase in plasma LpPLA2 activity. Both coding variants revealed a clear gene-dose effect. Interestingly, the rare Thr¹⁹⁸ allele, which was not associated with any change in plasma LpPLA2 activity, was more frequent in subjects without CAD ( P  = 0.009), with an adjusted odds ratio for CAD of 0.69 (95% CI: 0.49–0.96; P  = 0.029). None of the analyzed variants showed any robust association with all-cause or cardiovascular mortality. Conclusion:   Irrespective of the significant association between some variants with plasma LpPLA2 activity, it is still unclear whether these polymorphisms or haplotypes are associated with the risk and outcome of cardiovascular disease in Caucasians.     

CD31+/Annexin V+ microparticles in healthy offsprings of patients with coronary artery disease

Background  First-degree relatives of patients with premature coronary artery disease (CAD) develop endothelial dysfunction even in the case they are apparently healthy. In this study we wanted to clarify whether reduced blood levels of circulating endothelial progenitor cells (EPCs), an endogenous repair mechanism to replace dysfunctional endothelium, or elevated endothelial-derived microparticles (EMPs), an indicator and a mediator of increased endothelial cell damage/apoptosis, are an initial step in the pathogenesis of endothelial dysfunction in genetically predisposed subjects.
Materials and methods  Fifty-six healthy young men (aged 23 to 31 years) from a fire brigade were enrolled, of which 20 subjects had a positive family history (FH) for premature CAD. Subjects with or without a positive FH did not differ with respect to age, body mass index, risk factors and C-reactive protein. Endothelial function was assessed by hyperaemia-mediated relaxation of the brachial artery, blood levels of EPCs (VEGFR2⁺CD34⁺ cells) and number of EMPs (CD31^+(bright)/Annexin V⁺ particles) were analysed by flow cytometry.
Results  Hyperaemia-mediated relaxation of the brachial artery was similar in both groups, and the blood levels of EPCs were comparable. However, the number of EMPs were significantly increased in subjects with a positive FH compared to those with a negative FH (neg. FH: 55·31 ± 4·88 vs. pos. FH: 70·37 ± 6·32 particles µL⁻¹ platelet poor plasma; P  < 0·05). Number of EMPs correlate inversely with the FMD response.
Conclusions  These results suggest that increased plasma levels of EMPs may be an initial step in the development of endothelial dysfunction in genetically predisposed subjects.