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.     

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.     

Specific detection of human coagulation factor IX in cynomolgus macaques

Summary.  After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg⁻¹; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 ± 37.6 ng mL⁻¹ at 1 h after the injection and gradually decreased to 1.79 ± 1.1 ng mL⁻¹ by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.     

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.     

Neointima formation and thrombosis after vascular injury in transgenic mice overexpressing plasminogen activator inhibitor-1 (PAI-1)

Summary.  The controversial role of plasminogen activator inhibitor-1 (PAI-1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI-1 (PAI-1 Tg) and in wild-type (WT) controls. Despite the high circulating PAI-1 levels in the PAI-1 Tg mice (52 ± 9.8 ng mL⁻¹ vs. 0.76 ± 0.17 ng mL⁻¹ in WT mice), no significant fibrin deposition was observed in non-injured femoral arteries of 8- to 12-week-old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situ fibrinolytic activity in arterial sections of the PAI-1 Tg mice. The neointimal and medial areas were similar in WT and PAI-1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 ± 0.25 and 1.04 ± 0.17 at the center of the injury). Nuclear cell counts in cross-sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI-1 Tg mice (224 ± 63, 233 ± 20), and the distribution pattern of α-actin-positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI-1 does not affect neointima formation.     

Sustained prothrombotic profile after hip replacement surgery: the influence of prolonged prophylaxis with dalteparin

Summary.  In a randomized trial on the effect of dalteparin for 5 weeks after HRS we evaluated hemostatic variables in plasma sampled before and 1, 6 and 35 days postoperatively. In 218 patients we found that prothrombin fragment 1 + 2 (F1 + 2), thrombin–antithrombin complexes (TAT), d -dimer and fibrinogen were significantly higher on day 35 as compared with baseline values in the placebo group ( P  < 0.001 for all). The same pattern was found in the dalteparin group, but with significantly lower values for F1 + 2, TAT and d -dimer. In patients in the placebo group with venographically proven deep vein thrombosis (DVT) on day 35 (33%), significantly higher values were found for F1 + 2, TAT and d -dimer than in patients without DVT. Patients in the highest quartile of d -dimer (>2850 ng mL⁻¹) had an odds ratio for the presence of DVT of 24.0 when compared with patients in the lowest quartile (<1625 ng mL⁻¹). It is concluded that a substantial hypercoagulability is sustained until day 35 after HRS, significantly reduced with prolonged administration of dalteparin.     

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.     

Vasoactive substances in early dumping syndrome: effects of dumping provocation with and without octreotide

In patients after gastric surgery, early dumping symptoms can be provoked by oral glucose challenge. Octreotide effectively prevents the occurrence of dumping symptoms. We have studied plasma renin activity (PRA), aldosterone and atrial natriuretic peptide (ANP) concentrations in nine patients with early dumping, 10 surgical control subjects and nine healthy control subjects after an oral glucose challenge preceded by either placebo or 25 μg of octreotide subcutaneously (s.c.). In the dumping group, basal PRA was signifi-cantly ( P  < 0.01) higher (3.9 ± 0.6 μg L⁻¹ h⁻¹) than in either surgical or healthy control subjects (1.1 ± 0.3 μg L⁻¹ h⁻¹ and 1.1 ± 0.2 μg L⁻¹ h⁻¹ respectively) and showed a significant rise after glucose ingestion to 5.4 ± 0.9 μg L⁻¹ h⁻¹ that did not occur in control subjects. Aldosterone concentration showed a concomitant rise. In dumping patients, plasma ANP decreased after glucose ingestion from 31 ± 6 ng L⁻¹ to 21 ± 5 ng L⁻¹ ( P  < 0.05). This decrease did not occur in control subjects. Early dumping is associated with an activation of the renin–aldosterone axis and a decrease in plasma ANP, reflecting a hypovolaemic state. Octreotide prevents the occurrence of these changes.     

Maternal plasma levels of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by pre-eclampsia

We have measured the level of vascular endothelial growth factor (VEGF) in maternal plasma during normotensive pregnancy and in pregnancies complicated by pre-eclampsia. VEGF was measured using a competitive enzyme immunoassay. Plasma VEGF was significantly elevated ( P <0.0001) in the pre-eclamptic group (median value 32.7 ng mL⁻¹, range 10.3–64.0), compared with the normotensive group (median value 11.7 ng mL⁻¹, range 6.3–24.3). VEGF is a potent regulator of endothelial cell function. The increased level found in women with pre-eclampsia indicates that VEGF may be involved in the maternal endothelial cell dysfunction associated with this condition. An increase in VEGF, a potent regulator of microvascular permeability, may also contribute to the extravasation of plasma proteins and the subsequent development of proteinuria, both characteristic features of pre-eclampsia.     

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor

Summary.  Background:  Oral contraceptives (OC) containing different types of progestogens induce different sensitivities to activated protein C (APC) measured with the thrombin generation-based APC-resistance test. These differences in APC resistance may be the biological explanation for the differences in thrombotic risk of the various pills. The mechanistic basis of APC resistance observed in OC users is unknown. Our objective was to study the effect of OC on the two main determinants of the APC-resistance test, free protein S and free tissue factor pathway inhibitor (TFPI). Patients/methods:  We measured free protein S and free TFPI in 156 users of various types of OC. Results:  Users of desogestrel-containing OC, known to double the risk of thrombosis compared with levonorgestrel-containing OC, had lower free protein S (24 vs. 33 U dL⁻¹) and TFPI free antigen (2.9 vs. 3.6 ng mL⁻¹) levels than users of OC containing levonorgestrel. Women using cyproterone acetate-containing OC, known to confer a high thrombotic risk, had the lowest free protein S (19 U dL⁻¹) and free TPFI antigen (2.5 ng mL⁻¹) levels. Users of OC containing drospirenone had lower free protein S (23 U dL⁻¹) and TFPI antigen levels (3.2 ng mL⁻¹) than users of levonorgestrel-containing OC. Low free protein S and low free TFPI antigen levels were associated with an increased resistance to APC, an established risk factor for thrombosis. Conclusions:  This study observed that the differences in APC resistance induced by OC containing different progestogens can at least in part be explained by different effects of OC on free protein S and TFPI.     

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.     

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.     

Prolonged procoagulant activity on overstretch-injured coronary arteries in pigs

Summary.  This study was designed to assess the time course and nature of the vascular procoagulant response after 1.5-fold balloon overstretch injury of the coronary arteries in pigs. Arteries were excised for chromogenic assay of bound factor (F)Xa and thrombin at 24 h, 3 days, 1 week, or 2 weeks after injury. FXa at the site of injury remained elevated for 1 week (4.9 ± 5.9 µg cm⁻², n  = 10), compared with non-injured control arteries (0.4 ± 0.2 µg cm⁻², n  = 18, P  = 0.00025), while thrombin was increased only at 24 h. Tissue factor protein was abundant in non-injured coronaries (10 ± 6 ng µg⁻¹ total protein, n  = 9) and levels were unchanged by injury (13 ± 11 ng µg⁻¹, n  = 6) or 24-h administration of tissue factor pathway inhibitor (16 ± 6 ng µg⁻¹, n  = 6). Persistent tissue factor-mediated procoagulant activity may explain the need for prolonged anticoagulation to attenuate neointimal formation after balloon-induced coronary injury.     

The association between birth weight and plasma fibrinogen is abolished after the elimination of genetic influences

Summary.  Low birth weight is associated with an increased risk of atherothrombosis, which may be related in part to the association between low birth weight and high plasma fibrinogen. The association between birth weight and fibrinogen may be explained by intrauterine, socio-economic or genetic factors. We examined birth weight and fibrinogen in 52 dizygotic and 56 adolescent monozygotic (genetically identical) twin pairs. The dizygotic but not the monozygotic twins with the lowest birth weight from each pair had a fibrinogen level that was higher compared with their co-twins with the highest birth weight [dizygotic twins: 2.62 ± 0.46 g L⁻¹ vs. 2.50 ± 0.41 g L⁻¹ ( P  = 0.04); monozygotic twins: 2.42 ± 0.45 g L⁻¹ vs. 2.49 ± 0.39 g L⁻¹ ( P  = 0.2)]. These findings suggest that the association between birth weight and plasma fibrinogen is abolished after the elimination of genetic influences and therefore that this association has genetic causes. Improvement of intrauterine nutrition may not lower fibrinogen levels in later life.     

The promoting effect of epinephrine on lipopolysaccharide-induced interleukin-8 production in whole blood may be mediated by thromboxane A2

Summary.  Epinephrine is known to enhance lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion in a platelet dependent manner. To determine whether thromboxane A₂ (TxA₂; a product from activated platelets) is involved in this process, blood samples drawn either before or 2 h after oral administration of 440 mg acetylsalicylic acid (ASA) were stimulated with LPS (5 ng mL⁻¹) and different concentrations of epinephrine were added (0.1–100.0 µmol L⁻¹). ASA ingestion significantly (global P  < 0.05) reduced the enhancing effect of epinephrine on LPS-induced IL-8 release by 15–28%. To further explore whether TxA₂ may be involved in this process, a TxA₂ agonist (U46619) was added to whole blood together with LPS instead of epinephrine. U46619 mimicked the epinephrine effect: 20 ng mL⁻¹ U46619 enhanced LPS-induced IL-8 release by 39% ( P  < 0.05). Furthermore, preincubation of whole blood with 75 µmol L⁻¹ or 150 µmol L⁻¹ SQ29548, a TxA₂ receptor antagonist, completely blocked epinephrine's promoting effect on LPS-induced IL-8 release. Since thrombin-activated platelets have been reported to be important in the production of IL-8 in monocytes through the activation of monocytes by exposed RANTES in a P-selectin-dependent reaction, we suggest that the epinephrine effect is mediated by enhanced TxA₂ production and subsequent rise in the exposure of RANTES and P-selectin on the platelets of whole blood.     

Plasma endothelin-1 levels during transient acute myocardial ischaemia in men: effects of coronary revascularization

The endothelium-derived peptide endothelin-1 (ET-1) was evaluated in 14 male patients [mean age 52.74 years (SEM 1.10)] affected by coronary artery disease during a bicycle electrocardiographic stress test and dipyridamole echocardiogram. Both tests were performed before and after coronary revascularization. Fourteen healthy male subjects served as controls [mean age 53.21 years (SEM 1.63)]. Baseline plasma endothelin-1 levels were higher ( P  < 0^.0001) in ischaemic patients [1^.81 pg mL⁻¹ (0^.15, n  = 14)] than in control subjects [0^.61 pg mL⁻¹ (0^.03, n  = 14)], but did not increase with exercise in both groups. Similar results were obtained with dipyridamole infusion. Endothelin-1 levels significantly decreased after coronary revascularization [before: mean 1^.81 pg mL⁻¹ (SEM 0^.15, n  = 14); after: mean 1^.16 pg mL⁻¹ (SEM 0^.11), P  < 0^.002], without changes in the peptide response to both tests. In conclusion, elevated plasma endothelin-1 concentrations were found in patients with stable angina compared with non-ischaemic subjects. No changes were observed during exercise or dipyridamole infusion in both groups. Coronary revascularization was followed by a significant decrease in plasma endothelin-1 levels.     

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.     

Inhibition of paracrine angiotensin-converting enzyme in vivo: effects on interstitial glucose and lactate concentrations in human skeletal muscle

Microdialysis was used to selectively assess the effect of the paracrine renin–angiotensin system (RAS) on interstitial glucose and lactate concentration profiles in skeletal muscle of healthy volunteers ( n  = 8) during basal and insulin-stimulated conditions. Paracrine RAS was selectively inhibited by local retrodialysis with enalaprilate. Under basal conditions, local administration of enalaprilate (2 μg mL⁻¹) increased interstitial dialysate glucose concentration from 0.71 ± 0.14 mmol L⁻¹ to 0.84 ± 0.14 mmol L⁻¹ and decreased the serum interstitial gradient (SIG_glu) compared with baseline ( P  < 0.02). Under clamp conditions, enalaprilate, even at the lowest concentration (0.02 μg mL⁻¹), increased interstitial dialysate glucose concentration from 0.77 ± 0.11 mmol L⁻¹ to 1.02 ± 0.09 mmol L⁻¹ and decreased SIG_glu compared with baseline ( P  < 0.01). Interstitial lactate concentrations slightly increased during basal as well as during clamp conditions ( P  < 0.05 vs. baseline). Selective inhibition of paracrine muscle angiotensin-converting enzyme (ACE) increases interstitial glucose and lactate concentrations and decreases SIG_glu in muscle by facilitating transcapillary glucose transport. This effect is more pronounced during hyperinsulinaemia and may be of clinical relevance in diabetic patients treated with therapeutic doses of enalapril.     

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model

Summary.  Objective: The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 n m , followed by injection of human B domain-deleted FVIII at 180 U kg⁻¹, producing peak plasma concentrations of ∼2 n m . At 2 h, blood loss following a 4-mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti-A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti-C2 inhibitors, (iii) 2–77 and B45, non-classical type II anti-C2 inhibitors, and (iv) 2–117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg⁻¹ overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL⁻¹ and 3 U mL⁻¹ FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose FVIII rather than bypassing agents may be warranted.     

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.