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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cisplatin-induced renal effects and thromboxane A2 receptor blockade

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A₂ receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 ± 25 mL min⁻¹ ( P  = 0.049 vs. baseline) and effective renal plasma flow by 94 ± 150 mL min⁻¹ ( P  = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 ± 18 mL min⁻¹ ( P  = 0.050 vs. baseline) and in effective renal plasma flow by 26 ± 63 mL min⁻¹ ( P  = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.     

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 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.     

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.     

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.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Predictive value of plasma plasminogen activator inhibitor-1 for coronary restenosis: dependence on stent implantation and antithrombotic medication.

BACKGROUND: The plasmin activation system is involved in the development of restenosis after percutaneous coronary interventions (PCI). Conflicting data exist concerning the role of plasminogen activator inhibitor-1 (PAI-1) and its predictive value for restenosis. OBJECTIVES: To evaluate the fibrinolytic response to injury after PCI with or without stent implantation on different antithrombotic medications and its relation to late restenosis. PATIENTS AND METHODS: Eighty consecutive patients with successful PCI without (balloon only; n = 37) or with stent implantation (stent; n = 43) on different antithrombotic regimes (balloon only, aspirin; stent, aspirin/coumadin/dipyridamole vs. aspirin/ticlopidine). Blood samples were taken at baseline and up to 7 days after PCI and PAI-1 active antigen and tissue plasminogen activator (t-PA) antigen were determined. Restenosis was angiographically determined after 6 months. RESULTS: PCI increased both t-PA and PAI-1 levels (P < 0.001), with a significant prolonged and pronounced increase in stent vs. balloon-only patients (P < 0.05). Restenosis (stent 26%; balloon 38%) was significantly correlated to an attenuated PAI-1 increase after 24 h in the ticlopidine group (P = 0.007; restenosis, relative Delta PAI-1 + 50 +/- 28%; non-restenosis, + 139 +/- 50%), but not in the coumadin group. In the balloon-only group late restenosis (ISR) was associated with a trend for an augmented PAI-1 increase after 24 h. CONCLUSIONS: Coronary stent implantation significantly increases t-PA and PAI-1 plasma levels up to 1 week compared with balloon angioplasty alone. ISR in ticlopidine-treated patients was associated with an attenuated early PAI-1 active antigen increase. A less than 50% increase 24 h after stent implantation under ticlopidine treatment may identify patients at risk for the development of ISR.     

Low-density lipoprotein metabolism and its association to plasma lipoprotein(a) in the nephrotic syndrome

Patients with nephrotic syndrome have multiple abnormalities of lipoprotein metabolism, but the cause and exact nature of these abnormalities have not been established. In the present study we have determined the kinetics of plasma low-density lipoprotein (LDL) apoB in seven nephrotic patients demonstrating an elevated LDL apoB production rate (25.7 ± 6.4 vs. 13.1 ± 0.3 mg kg^–1 day^–1; P  < 0.001) but a normal LDL apoB fractional catabolic rate (FCR) (0.31 ± 0.04 vs. 0.33 ± 0.008 pools day^–1; NS) compared with 41 healthy control subjects. However, two out of the seven patients had a markedly low LDL apoB-FCR. Serum albumin was inversely correlated with the LDL apoB production rate ( R  = –0.82; P  < 0.05). Plasma lipoprotien (a) [Lp(a)] levels were significantly ( P  < 0.001) increased in the nephrotic patients compared with control subjects. Significant correlations were observed between log Lp(a) and LDL apoB production rate ( R  = 0.90; P  < 0.01), VLDL-cholesterol ( R  = 0.95; P  < 0.001) and VLDL-triglycerides ( R  = 0.80; P  < 0.05) respectively. In summary, the present study suggests that nephrotic hyperlipidaemia may be caused by at least two independent mechanisms. The elevated LDL apoB production rate is highly correlated with the prevailing levels of serum albumin, whereas some nephrotic patients seem to have a decreased LDL apoB clearance, suggesting impaired LDL receptor-mediated clearance. The present results also suggest that the elevated plasma Lp(a) levels in nephrosis are related to an increased hepatic synthesis rather than a decreased catabolism of lipoproteins.