Wolfgang Speiser

HIGH PLASMA LEVELS OF FACTOR VIII AND THE RISK OF RECURRENT VENOUS THROMBOEMBOLISM

BACKGROUND A high plasma level of factor VIII is a risk factor for venous thromboembolism. We evaluated the risk of a recurrence of thrombosis after an initial episode of spontaneous venous thromboembolism among patients with high plasma levels of factor VIII. METHODS We studied 360 patients for an average follow-up period of 30 months after a first episode of venous thromboembolism and discontinuation of oral anticoagulants. Patients who had recurrent or secondary venous thromboembolism, a congenital deficiency of an anticoagulant, the lupus anticoagulant, hyperhomocysteinemia, cancer, or a requirement for long-term treatment with antithrombotic drugs or who were pregnant were excluded. The end point was objectively documented, symptomatic recurrent venous thromboembolism. RESULTS Recurrent venous thromboembolism developed in 38 of the 360 patients (10.6 percent). Patients with recurrence had higher mean (+/-SD) plasma levels of factor VIII than those without recurrence (182+/-66 vs. 157+/-54 IU per deciliter, P=0.009). The relative risk of recurrent venous thrombosis was 1.08 (95 percent confidence interval, 1.04 to 1.12; P<0.001) for each increase of 10 IU per deciliter in the plasma level of factor VIII. Among patients with a factor VIII level above the 90th percentile of the values in the study population, the likelihood of recurrence at two years was 37 percent, as compared with a 5 percent likelihood among patients with lower levels (P<0.001). Among patients with plasma factor VIII levels above the 90th percentile, as compared with those with lower levels, the overall relative risk of recurrence was 6.7 (95 percent confidence interval, 3.0 to 14.8) after adjustment for age, sex, the presence or absence of factor V Leiden or the G20210A prothrombin mutation, and the duration of oral anticoagulation. CONCLUSIONS Patients with a high plasma level of factor VIII have an increased risk of recurrent venous thromboembolism.

Leptin Induces Endothelin-1 in Endothelial Cells In Vitro

Leptin, a protein encoded by the obese gene, is produced by adipocytes and released into the bloodstream. In obese humans, serum leptin levels are increased and correlate with the individual’s body mass index and blood pressure. Elevated serum concentrations of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, were also observed in obese subjects. The pathomechanisms underlying this ET-1 increase in obesity are poorly understood. In the present study, we investigated the influence of the ob gene product leptin on the expression of ET-1 in human umbilical vein endothelial cells (HUVECs). Binding studies using 125I-radiolabeled leptin revealed high- and low-affinity leptin binding sites on HUVECs (Kd1=13.1±3.1 nmol/L and Kd2=1390±198 nmol/L, respectively), mediating a time- and dose-dependent increase of ET-1 mRNA expression and protein secretion after incubation of HUVECs with leptin. This leptin-induced ET-1 expression was inhibited by preincubation of HUVECs with 0.75 &mgr;mol/L antisense phosphorothioate oligonucleotides directed against the leptin receptor Ob-Rb. Furthermore, after incubation with leptin, increased nuclear staining of c-fos and c-jun, the major components of the transcription factor AP-1, and increased AP-1 DNA binding were observed. Transient transfection studies with ET-1 promoter constructs showed that leptin-induced promoter activity was abolished in the absence of AP-1 binding sites or by cotransfection with a plasmid overexpressing a mutated jun, which is able to bind c-fos but not DNA. Thus, leptin upregulates ET-1 production in HUVECs via a mechanism potentially involving jun binding members of the bZIP family.

Clinical Studies and Thrombin Generation in Patients Homozygous or Heterozygous for the G20210A Mutation in the Prothrombin Gene

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.

Heparin Blunts Endotoxin-Induced Coagulation Activation

BACKGROUND Lipopolysaccharide (LPS) is a major trigger of sepsis-induced disseminated intravascular coagulation (DIC) via the tissue factor (TF)/factor VIIa-dependent pathway of coagulation. Experimental endotoxemia has been used repeatedly to explore this complex pathophysiology, but little is known about the effects of clinically used anticoagulants in this setting. Therefore, we compared with placebo the effects of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on LPS-induced coagulation. METHODS AND RESULTS In a randomized, double-blind, placebo-controlled trial, 30 healthy male volunteers received LPS 2 ng/kg IV followed by a bolus-primed continuous infusion of UFH, LMWH, or placebo. In the placebo group, activation of coagulation caused marked increases in plasma levels of prothrombin fragment F(1+2) (P<0.01) and polymerized soluble fibrin, termed thrombus precursor protein (TpP; P<0.01); TF-positive monocytes doubled in response to LPS, whereas levels of activated factor VII slightly decreased and levels of TF pathway inhibitor remained unchanged. UFH and LMWH markedly decreased activation of coagulation caused by LPS, as F(1+2) and TpP levels only slightly increased; TF expression on monocytes was also markedly reduced by UFH. TF pathway inhibitor values increased after either heparin infusion (P<0.01). Concomitantly, factor VIIa levels dropped by >50% at 50 minutes after initiation of either heparin infusion (P<0.01). CONCLUSIONS This experimental model proved the anticoagulatory potency of UFH and LMWH in the initial phase of experimental LPS-induced coagulation. Successful inhibition of thrombin generation also translates into blunted activation of coagulation factors upstream and downstream of thrombin.

Morning Hypercoagulability and Hypofibrinolysis Diurnal Variations in Circulating Activated Factor VII, Prothrombin Fragment F1+2, and Plasmin–Plasmin Inhibitor Complex

BACKGROUND Diurnal fluctuations of blood coagulation and fibrinolysis activity are thought to play a role in the observed circadian variation in the frequency of onset of acute cardiovascular events. In the present study, the diurnal variations in blood coagulation and fibrinolysis activity were investigated in 10 young, healthy control subjects by use of specific molecular activation markers. METHODS AND RESULTS The plasma levels of activated factor FVII (FVIIa), the active portion of the main coagulation activator, decreased during the day (8 AM: 2.03 ng/mL, CI 1.16 to 2.88 ng/mL; 8 PM: 1.16 ng/mL, CI 0.81 to 1.5 ng/mL; P = .005), whereas FVII antigen did not change significantly. In parallel with the diurnal variations of FVIIa, we found a decrease of prothrombin fragment F1+2 (8 AM: 0.97 nmol/L, CI 0.79 to 1.15 nmol/L; 8 PM: 0.78 nmol/L, CI 0.64 to 0.93 nmol/L; P = .005), a molecular marker of intravasal thrombin generation. Evidence for a possible functional relevance of circulating FVIIa was found because this parameter was significantly correlated with prothrombin fragment F1+2 in 72 fasting healthy individuals (r = .29, P = .011). Plasminogen activator inhibitor-1 levels decreased (8 AM: 9.9 ng/mL, CI 7.7 to 12.1 ng/mL; 8 PM: 5.4 ng/mL, CI 3.8 to 6.9 ng/mL; P < .005), whereas plasmin-plasmin inhibitor complex levels, representing the degree of intravascular plasmin generation, concomitantly increased (8 AM: 235 micrograms/L, CI 198 to 272 micrograms/L; 8 PM: 449 micrograms/L, CI 391 to 507 micrograms/L; P = .008). CONCLUSIONS Our data suggest that the diurnal changes in the plasma levels of activators and inhibitors of coagulation and fibrinolysis lead to corresponding changes in the activity state of these systems, leading to morning hypercoagulability and hypofibrinolysis.

Effect of hydroxyethyl starch on the activity of blood coagulation and fibrinolysis in healthy volunteers : comparison with albumin

ObjectiveThe aim of this study was to investigate whether hydroxyethyl starch of medium molecular weight (200 daltons), compared with albumin, has specific effects on blood coagulation and fibrinolysis. DesignA prospective, randomized, double-blind, crossover trial. SettingLaboratory at a university hospital. PatientsTen healthy male volunteers, 23 to 39 yrs old, with no history of hypersensitivity, who had normal physical examinations, and were free of a bleeding disorder. All patients did not ingest any medications for ≥2 wks before or during the study period.Intervention: Each volunteer received either 500 mL of hydroxyethyl starch (6% wt/vol, average molecular weight 200 kilodaltons, molar substitution 0.5 [ratio hydroxyethyl groups/glucose units] in 0.9% sodium chloride; average molecular weight of 200 kilodaltons) or a control of 500 mL of human albumin (5% albumin solution). After a washout period of 4 wks, subjects crossed over to the alternate treatment. Measurements and Main ResultsBlood samples were taken immediately before infusion and 20, 45, 75, 105, 165, 285, 405, and 1485 mins after the infusion started. Hematocrit, the blood coagulation parameters fibrinogen, activated partial thromboplastin time, factor VIII:C, thrombin-antithrombin III complexes, and the fibrinolytic parameters fibrin-split product D-Dimer, tissue type plasminogen activator, urokinase plasminogen activator, plasminogen activator inhibitor, and plasmin-antiplasmin complexes were measured. Except for factor VIII:C levels, which were significantly lower in the hydroxyethyl starch group, no other significant differences in the plasma levels of the parameters mentioned were detected between hydroxyethyl starch and albumin infusions. In one volunteer, who had a low initial factor VIII:C level of 51%, a decrease to 28% at 165 mins during hydroxyethyl starch infusion was found (corresponding albumin value at 165 mins was 41%). Conclusionsa) Medium molecular weight hydroxyethyl starch has a specific lowering effect on factor VIII:C concentrations; this phenomenon may be hazardous to patients who need full hemostatic competence and who receive medium molecular weight hydroxyethyl starch (e.g., as a plasma expander), b) Medium molecular weight hydroxyethyl starch does not specifically influence the activity of the fibrinolytic system. (Crit Care Med 1994; 22:606–612)

Sandwich ELISA for t-PA antigen employing a monoclonal antibody

A sandwich enzyme-linked immunosorbent assay (ELISA) for determination of tissue-type plasminogen activator (t-PA) was developed. 96-well flat-bottom polystyrene plates were coated with polyclonal (goat) anti t-PA IgG (10 micrograms/ml). After addition of samples monoclonal (murine) anti t-PA IgG (1.5 micrograms/ml) was added. Finally, peroxidase labelled anti-mouse IgG (goat) was used to quantify the bound second antibody. The assay can be used for determination of t-PA antigen in purified systems, in cell culture supernatants, and in human plasma, provided that EDTA (0.005 M) is present in the sample. In 78 healthy volunteers, t-PA antigen levels at rest were 0.4 - 15.2 ng/ml (4.3 +/- 2.7, means +/- S.D.); A significant positive correlation between t-PA antigen and age could be demonstrated.

Effects of desmopressin on circulating P-selectin.

Von Willebrand factor (VWF) and P‐selectin share an identical intracellular storage compartment and transport to the cell surface. We induced degranulation of the Weibel‐Palade bodies and measured circulating (c)P‐selectin in plasma to test whether soluble P‐selectin is present in the Weibel‐Palade bodies, or if P‐selectin bound to the cell membrane of endothelial cells (EC) is rapidly proteolytically cleaved in vivo.  A dose of 0.3 μg/kg of desmopressin (DDAVP) or placebo was infused over 30 min to eight healthy men in a double‐blind cross‐over study. Plasma levels of cP‐selectin and VWF‐Ag were followed for 24 h. Despite a twofold increase in VWF‐Ag levels immediately after and 2 h after infusion of DDAVP, no significant change in plasma concentrations of cP‐selectin were observed.  In summary, degranulation of the Weibel‐Palade bodies is an unlikely source of cP‐selectin. Thus cP‐selectin in healthy subjects is conceivably attributable to other mechanisms, such as minor degrees of platelet activation or transcription induction of an alternatively spliced P‐selectin.

Thromboembolism and resistance to activated protein C in patients with inflammatory bowel disease.

OBJECTIVE: Thromboembolic events are serious complications in patients with inflammatory bowel disease (IBD). Resistance of factor V to degradation by activated protein C (APC) is a major cause for venous thrombosis and is found in approximately 30% of patients with thromboembolism. The aim of the present study was to assess the prevalence of APC resistance and clinical risk factors in patients with IBD. METHODS: One-hundred-two patients with IBD (64 women and 38 men; median age, 35 yr; range, 17–77 yr; 77 with Crohn’s disease, 25 with ulcerative colitis) and 102 gender- and age-matched healthy control subjects were investigated prospectively for the presence of APC resistance. None of the healthy controls but 16 patients with IBD had a history of thromboembolism. RESULTS: Patients with IBD and thromboembolism were young, with a median age of 37 yr (range, 17–61 yr). Five (31.3%) of them had APC resistance, which was more common than in patients with IBD without thromboembolism (7%) and in controls (5.9%) (p < 0.01). Three patients had two thromboembolic events, the other 13 each had one. Deep vein thrombosis of the leg and pulmonary emboli were the most common thromboembolic complications (84.2%). Active disease, fistula, or bowel stenosis were found in 10 (52.6%) of 19 thromboembolic events; in three (15.8%) cases thromboembolism happened postoperatively. CONCLUSIONS: APC resistance is not associated with IBD but, when present, increases the risk of thromboembolism. Patients with IBD and thromboembolism are mostly young and clinical risk factors can be found in one-half of cases.

Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen.

During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression. TF expression is mediated by nuclear factor kappaB and amplified by activated platelets. TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa). Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor kappaB-dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans. Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model. Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg). Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis. Neither aspirin nor acetaminophen inhibited LPS-induced coagulation. However, LPS increased the percentage of circulating TF(+) monocytes by 2-fold. This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion. Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected. In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation. However, our study showed, for the first time, that LPS increases TF(+) monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.