Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD<em>1</em><em>1</em>b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD<em>1</em><em>1</em>b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, <em>2</em> hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. <em>2</em>000;96:4<em>2</em>6<em>1</em>-4<em>2</em>66)

OBJECTIVE

To quantify changes in variables of inflammation, coagulation, and fibrinolysis in blunt trauma patients with lower extremity fractures who underwent different types of surgical procedures.

METHODS

Prospective, cohort study.

METHODS

Level I university trauma center.

METHODS

We allocated 83 blunt trauma patients in stable condition and <em>2</em><em>2</em> patients eligible for elective hip replacement to four treatment groups.

METHODS

In 34 multiply traumatized patients with femoral fracture (group PTFF) and in <em>2</em>8 patients with an isolated femoral fracture (group IFF), primary unreamed intramedullary nailing for stabilization of the femoral shaft fracture was performed. In <em>2</em><em>2</em> patients, an elective uncemented total hip arthroplasty (group THA) was inserted for osteoarthritis, and in <em>2</em><em>1</em> control patients, an isolated ankle fracture (group AF) was acutely stabilized.

RESULTS

From serially sampled central venous blood, the perioperative concentrations of interleukin (IL)-6, of tumor necrosis factor-alpha, of <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, and of D-dimer cross-linked fibrin degradation products were evaluated. Intramedullary instrumentation for an isolated femur fracture caused a significant perioperative increase in the concentrations of IL-6 (preoperative IL-6, 5<em>2</em> +/- <em>1</em><em>2</em> pg/mL; IL-6 30 mins postinsertion, 78 +/- <em>1</em>4 pg/mL; p = .0<em>2</em>). This increase was comparable with group THA (preoperative IL-6, 46 +/- <em>1</em>6 pg/mL; IL-6 30 mins postinsertion, 67 +/- <em>1</em><em>1</em> pg/mL; p = .03). A positive correlation occurred between both groups (r = .83, p < .0004). Multiple trauma patients demonstrated significantly (p = .000<em>2</em>) higher IL-6 concentrations than all other groups throughout the study period and showed a significant increase after femoral nailing (preoperative IL-6, 570 +/- <em>2</em><em>1</em> pg/mL; IL-6 30 mins postinsertion, 690 +/- <em>2</em>4 pg/mL; p = .003), whereas no perioperative change was seen in group AF. The highest IL-6 increases were associated with a longer ventilation time (group PTFF) and a longer period of positive fluid balances (groups PTFF, IFF, THA). The coagulatory variables demonstrated similar perioperative increases in groups IFF and THA, but not in groups PTFF and AF. The IL-6 concentrations and the <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> concentrations correlated between groups THA and IFF at 30 mins and at <em>1</em> hr after surgery (r<em>2</em> = .64, p < .0<em>2</em>). In all patients the clinical variables were stable perioperatively.

CONCLUSIONS

Major surgery of the lower extremity causes changes to the inflammatory, fibrinolytic, and coagulatory cascades in patients with stable cardiopulmonary function. The inflammatory response induced by femoral nailing is biochemically comparable to that induced by uncemented total hip arthroplasty. In multiple trauma patients, increases, which occurred in addition to those induced by the initial trauma, were measured. Definitive primary femoral stabilization by intramedullary nailing imposes an additional burden to the patient with blunt trauma. A careful preoperative investigation is required to evaluate whether primary definitive stabilization can be performed safely.

Cancer patients are at increased risk of deep vein thrombosis and pulmonary embolism. The incidence among different groups of cancer patients varies considerably depending on clinical factors, the most important being tumor entity and stage. Biomarkers have been specifically investigated for their capacity of predicting venous thromboembolism (VTE) during the course of disease. Parameters of blood count analysis (elevated leukocyte and platelet count and decreased hemoglobin) have turned out to be useful in risk prediction. Associations between elevated levels and future VTE have been found for d-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, and soluble P-selectin and also for clotting factor VIII and the thrombin generation potential. The results for tissue factor-bearing microparticles are heterogeneous: an association with occurrence of VTE in pancreatic cancer might be present, whereas in other cancer entities, such as glioblastoma, colorectal, or gastric carcinoma, this could not be confirmed. Risk assessment models were developed that include clinical and laboratory markers. In the high-risk categories, patient groups with up to a>><em>2</em>0% VTE rate within 6 months can be identified. A further improvement in risk stratification would allow better identification of patients for primary VTE prevention using indirect or novel direct anticoagulants.

Complement activation has been reported after major trauma. However, little is known about the clinical relevance and the mechanisms of complement activation early after trauma. Therefore, the aim of this study was to measure complement activation, to identify the roles of injury severity and hypoperfusion, to determine the predominant activated pathway, and to identify the clinical significance of early complement activation in trauma patients. A total of <em>2</em>08 adult trauma patients were enrolled in this prospective single-center cohort study of major trauma patients. Blood samples were obtained within 30 min after injury before any significant fluid resuscitation. Complement (C5b-9) was activated early after trauma, correlated with injury severity and tissue hypoperfusion, and was associated with increased mortality rate and with the development of organ failure such as acute lung injury and acute renal failure. The alternative pathway seems to be the predominant activated complement pathway early after trauma. However, the classical and/or the lectin pathway initiated complement activation because of the correlation between plasma levels of C4d and C3a/C5b-9. Finally, in patients with low C3a levels, C5b-9 levels correlated with plasma levels of <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, a marker of thrombin generation, suggesting additional C3-independent complement activation by thrombin after severe trauma. In summary, complement activation via its amplification by the alternative pathway is observed early after trauma and correlates with injury severity, tissue hypoperfusion, and worse clinical outcomes. Besides complement activation by the classical and/or lectin pathways, there is an independent association between thrombin generation and complement activation.

OBJECTIVE

The pathogenesis of cerebral small vessel disease (SVD) is poorly understood, but endothelial activation and dysfunction may play a causal role. Cross-sectional studies have found increased circulating markers of endothelial activation, but this study design cannot exclude causality from secondary elevations. Confluent white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI) appear to represent asymptomatic cerebral SVD. In a prospective study, we determined whether circulating markers of endothelial activation predicted progression of WMH.

METHODS

In the community-based Austrian Stroke Prevention Study, MRI was performed at baseline in <em>2</em>96 subjects and repeated at 3 and 6 years. The following were measured on baseline plasma samples: intercellular adhesion molecule (ICAM), thrombomodulin, tissue factor plasma inhibitor, <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, and D-dimers.

RESULTS

ICAM was associated with age- and gender-adjusted WMH lesion progression at both 3 and 6 years, respectively; (odds ratio [OR], <em>1</em>.007; 95% confidence interval [CI], <em>1</em>.00<em>2</em> to <em>1</em>.0<em>1</em><em>2</em>; P=0.004; and OR, <em>1</em>.004; 95% CI, <em>1</em>.000 to <em>1</em>.009 per ng/mL; P=0.057). After multivariate analysis controlling for other cardiovascular risk factors and C-reactive protein, 3-year OR was <em>1</em>.0<em>1</em>0 (95% CI, <em>1</em>.004 to <em>1</em>.0<em>1</em>7; P=0.00<em>1</em>) and 6-year OR was <em>1</em>.008 (<em>1</em>.00<em>2</em> to <em>1</em>.0<em>1</em>4 per ng/mL; P=0.006). Baseline log lesion volume was a strong independent predictor of progression but associations remained after controlling for this (3-year OR, <em>1</em>.0<em>1</em><em>1</em>; 95% CI, <em>1</em>.00<em>2</em> to <em>1</em>.0<em>2</em>0; P=0.0<em>1</em>3; and 6-year OR, <em>1</em>.009; 95% CI, <em>1</em>.000 to <em>1</em>.0<em>1</em>7; P=0.039 per ng/mL). There was no association between WMH progression and other markers.

CONCLUSIONS

ICAM levels are related to progression of WMH on MRI. The prospective study design increases the likelihood that this association is causal and supports a role of endothelial cell activation in disease pathogenesis. In contrast, we found no evidence for coagulation activation being important.

OBJECTIVE

To examine the hypothesis that the higher rates of coronary heart disease (CHD) in Indians (South Asians) compared with Malays and Chinese is at least partly explained by central obesity, insulin resistance, and syndrome X (including possible components).

METHODS

Cross sectional study of the general population.

METHODS

Singapore.

METHODS

Random sample of 96<em>1</em> men and women (Indians, Malays, and Chinese) aged 30 to 69 years.

RESULTS

Fasting serum insulin concentration was correlated directly and strongly with body mass index (BMI), waist-hip ratio (WHR), and abdominal diameter. The fasting insulin concentration was correlated inversely with HDL cholesterol and directly with the fasting triglyceride concentration, blood pressures, plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>), and tissue plasminogen activator (tPA), but it was not correlated with LDL cholesterol, apolipoproteins B and A<em>1</em>, lipoprotein(a), (Lp(a)), fibrinogen, factor VIIc, or <em>prothrombin</em> <em>fragment</em> (F)<em>1</em> + <em>2</em>. This indicates that the former but not the latter are part of syndrome X. While Malays had the highest BMI, Indians had a higher WHR (men 0.93 and women 0.84) than Malays (men 0.9<em>1</em> and women 0.8<em>2</em>) and Chinese (men 0.9<em>1</em> and women 0.8<em>2</em>). In addition, Indians had higher fasting insulin values and more glucose intolerance than Malays and Chinese. Indians had lower HDL cholesterol, and higher PAI-<em>1</em>, tPA, and Lp(a), but not higher LDL cholesterol, fasting triglyceride, blood pressures, fibrinogen, factor VIIc, or <em>prothrombin</em> F<em>1</em> + <em>2</em>.

CONCLUSIONS

Indians are more prone than Malays or Chinese to central obesity with insulin resistance and glucose intolerance and there are no apparent environmental reasons for this in Singapore. As a consequence, Indians develop some but not all of the features of syndrome X. They also have higher Lp(a) values. All this puts Indians at increased risk of atherosclerosis and thrombosis and must be at least part of the explanation for their higher rates of CHD.

BACKGROUND

The hemolytic-uremic syndrome is a thrombotic complication of Escherichia coli O<em>1</em>57:H7 infection. It is not known whether the coagulation abnormalities precede, and potentially cause, this disorder.

METHODS

In 53 children infected with E. coli O<em>1</em>57:H7, we measured a panel of markers indicating activation of the clotting cascade and renal function within four days after the onset of illness. These markers were measured again in as many as possible of the <em>1</em>6 children in whom the hemolytic-uremic syndrome developed.

RESULTS

The children in whom the hemolytic-uremic syndrome subsequently developed had significantly higher median plasma concentrations of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, tissue plasminogen activator (t-PA) antigen, t-PA-plasminogen-activator inhibitor type <em>1</em> (PAI-<em>1</em>) complex, and D-dimer than children with uncomplicated infection. These abnormalities preceded the development of azotemia and thrombocytopenia. When the hemolytic-uremic syndrome developed, the urinary concentrations of beta<em>2</em>-microglobulin and N-acetyl-beta-glucosaminidase rose significantly (P=0.03 for both increases); the plasma concentrations of t-PA antigen, t-PA-PAI-<em>1</em> complex, D-dimer, and plasmin-antiplasmin complex also increased significantly. The concentration of t-PA antigen correlated with that of the t-PA-PAI-<em>1</em> complex in a linear regression model (squared correlation coefficient, 0.80; P<0.00<em>1</em>).

CONCLUSIONS

In the hemolytic-uremic syndrome, thrombin generation (probably due to accelerated thrombogenesis) and inhibition of fibrinolysis precede renal injury and may be the cause of such injury.

BACKGROUND

Platelet-derived microparticles (PMPs) are generally considered a marker of platelet activation in cardiovascular disease. We studied the extent to which PMP subpopulations parallel platelet activation in vitro and in vivo.

METHODS

Using flow cytometry, we analyzed PMP subpopulations from resting and activated platelets in vitro (n = 6) as well as from plasma samples of patients with stable angina, peripheral arterial disease, or myocardial infarction [non-ST-elevation (NSTEMI) and ST-elevation (STEMI)] and from older, age- and sex-matched and young healthy individuals [n = <em>1</em>0 for all groups except NSTEMI (n = <em>1</em><em>1</em>)]. Coagulation markers <em>prothrombin</em> <em>fragment</em> F(<em>1</em> + <em>2</em>) and thrombin-antithrombin complexes were determined by ELISA. The PMP-associated fraction of soluble (s)P-selectin was estimated by ELISA.

RESULTS

In vitro, stimulation of platelets with thrombin receptor-activating peptide (<em>1</em>5 micromol/L) or the calcium ionophore A<em>2</em>3<em>1</em>87 (<em>2</em>.5 micromol/L) increased fractions of both platelets and PMPs exposing P-selectin or CD63 (P <0.00<em>1</em> for all). Whereas the number of PMPs released by A<em>2</em>3<em>1</em>87-stimulated platelets increased significantly (P <0.00<em>1</em>), the number of PMPs released from thrombin receptor-activating peptide-stimulated platelets remained constant (P >0.05). Ex vivo, numbers of circulating PMPs were comparable in all groups. Compared with young persons, P-selectin-exposing PMPs were increased in older persons (P = 0.0<em>2</em>) and were further increased in patients with NSTEMI (P = 0.007) and STEMI (P = 0.045). CD63-exposing PMPs were increased in patients with peripheral arterial disease (P = 0.04<em>1</em>), NSTEMI (P = 0.00<em>1</em>), and STEMI (P = 0.049). Subpopulations exposing P-selectin or CD63 correlated with each other (r = 0.58<em>1</em>; P <0.00<em>1</em>), but neither correlated with the plasma concentrations of F(<em>1</em> + <em>2</em>) or thrombin-antithrombin complexes. The PMP-associated fraction of sP-selectin constituted only <em>2</em>.<em>2</em> (4.7)% [mean (SD)] of total sP-selectin.

CONCLUSIONS

PMP subpopulations reflect platelet activation status better than the total number of PMPs. Increased concentrations of circulating PMP subpopulations are found in aging, and further increases are encountered in peripheral arterial disease and myocardial infarction.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S<em>1</em>), after defervescence (S<em>2</em>), and <em>1</em> month after onset of disease (S4). Patients were classified as DF (n = <em>2</em><em>1</em>) and DHF grades <em>1</em>, <em>2</em>, and 3; (DHF<em>1</em>, n = 8; DHF<em>2</em>, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of <em>prothrombin</em> <em>fragment</em> F<em>1</em>.<em>2</em> in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

BACKGROUND

Type <em>2</em> diabetes mellitus (T<em>2</em>DM) is a hypercoagulable state. Tissue factor (TF) is the principal initiator of blood coagulation.

OBJECTIVE

Our objective was to examine the effects of hyperglycemia and hyperinsulinemia on the TF pathway of blood coagulation in T<em>2</em>DM.

METHODS

Three study protocols were used: 1) acute correction of hyperglycemia (with iv insulin) followed by <em>2</em>4 h of euglycemia, <em>2</em>) <em>2</em>4 h of selective hyperinsulinemia, and 3) <em>2</em>4 h of combined hyperinsulinemia and hyperglycemia.

METHODS

The study took place at a clinical research center.

METHODS

Participants included 18 T<em>2</em>DM patients and <em>2</em><em>2</em> nondiabetic controls.

RESULTS

Basal TF-procoagulant activity (TF-PCA), monocyte TF mRNA, plasma coagulation factor VII (FVIIc), and thrombin-anti-thrombin complexes were higher in T<em>2</em>DM than in nondiabetic controls, indicating a chronic procoagulant state. Acutely normalizing hyperglycemia over <em>2</em>-4 h resulted in a small ( approximately 7%) but significant decline in TF-PCA with no further decline over <em>2</em>4 h. Raising insulin levels alone raised TF-PCA by 30%, whereas raising insulin and glucose levels together increased TF-PCA (by 80%), thrombin-anti-thrombin complexes, and prothrombin fragment 1.<em>2</em>. Plasma FVIIa and FVIIc declined with increases in TF-PCA.

CONCLUSIONS

We conclude that the combination of hyperglycemia and hyperinsulinemia, common in poorly controlled patients with T<em>2</em>DM, contributes to a procoagulant state that may predispose these patients to acute cardiovascular events.

OBJECTIVE

Thrombosis, usually venous, occurs in <em>1</em>0% to <em>2</em>5% of patients with Behçet's disease, but its pathogenesis is poorly understood. We evaluated parameters of hemostasis and their relation with thrombosis in a series of patients with Behçet's disease.

METHODS

We studied 38 patients with Behçet's disease (<em>1</em>3 with venous thrombosis), 38 patients with venous thrombosis without thrombophilia, and <em>1</em>00 control subjects. Levels or presence of protein C, protein S, antithrombin, methylenetetrahydrofolate reductase C677T, factor V Leiden, <em>prothrombin</em> gene G<em>2</em>0<em>2</em><em>1</em>0A, antiphospholipid antibodies, plasminogen, tissue-type plasminogen activator (tPA), type-<em>1</em> tPA inhibitor (PAI-<em>1</em>), PAI-<em>1</em> 4G/5G polymorphism, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, plasmin/alpha(<em>2</em>)-antiplasmin complexes, thrombomodulin, and activated factors VII and XII were determined.

RESULTS

There were no deficiencies in protein C, protein S, antithrombin, or factor V Leiden in the patients with Behçet's disease, nor was there evidence of most other thrombotic abnormalities. Compared with control subjects, however, the Behçet's disease group had elevated mean (+/- SD) levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (<em>2</em>09<em>1</em> +/- <em>1</em>3<em>2</em>3 pmol/L vs. 804 +/- 398 pmol/L, P <0.00<em>1</em>), plasmin/alpha<em>2</em>-antiplasmin complexes (4<em>1</em>0 +/- <em>2</em><em>2</em>0 microg/L vs. <em>2</em><em>1</em>4 +/- 9<em>2</em> microg/L, P <0.00<em>1</em>), and thrombomodulin (37 +/- <em>2</em>4 ng/mL vs. <em>2</em>7 +/- <em>1</em>0 ng/mL, P <0.00<em>1</em>). These levels did not differ between patients with or without thrombosis.

CONCLUSIONS

Thrombophilic factors do not seem to explain most thromboses in Behçet's disease. There is increased thrombin generation, fibrinolysis, and thrombomodulin in Behçet's disease, but these abnormalities are not related to thrombosis.

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(<em>2</em>+) ions in the Gla, one in the EGF<em>1</em>, and one in the protease domain. However, blood contains both Ca(<em>2</em>+) and Mg(<em>2</em>+), and the Ca(<em>2</em>+) sites in FVIIa that could be specifically occupied by Mg(<em>2</em>+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(<em>2</em>+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(<em>2</em>+), Mg(<em>2</em>+), Na(+), and Zn(<em>2</em>+). The crystal diffracted to <em>1</em>.8A resolution, and the final structure has an R-factor of <em>1</em>9.8%. In this structure, the Gla domain has four Ca(<em>2</em>+) and three bound Mg(<em>2</em>+). The EGF<em>1</em> domain contains one Ca(<em>2</em>+) site, and the protease domain contains one Ca(<em>2</em>+), one Na(+), and two Zn(<em>2</em>+) sites. (45)Ca(<em>2</em>+) binding in the presence/absence of Mg(<em>2</em>+) to FVIIa, Gla-domainless FVIIa, and <em>prothrombin</em> <em>fragment</em> <em>1</em> supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(<em>1</em>93) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at <em>1</em>.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the <em>1</em>9<em>2</em>-<em>1</em>93 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Postmenopausal hormone replacement therapy is associated with a reduction in the incidence of coronary heart disease. However, inconclusive results have been reported with respect to the risk of stroke, and recent studies consistently showed an increased risk of venous thromboembolism in postmenopausal women using oral estrogen. There are surprisingly few interventional studies to assess the true effects of estrogen-progestin regimens on blood coagulation and fibrinolysis, and the impact of the route of estrogen administration on hemostasis has not been well documented. Therefore, we investigated the effects of oral and transdermal estradiol/progesterone replacement therapy on hemostatic variables. Forty-five healthy postmenopausal women, aged 45 to 64 years, were assigned randomly to one of the three following groups: cyclic oral or transdermal estradiol, both combined with progesterone, or no hormonal treatment. Hemostatic variables were assayed at baseline and after a 6-month period. Pairwise differences in the mean change between the three groups were compared using nonparametric tests. Oral but not transdermal estradiol regimen significantly increased the mean value of <em>prothrombin</em> activation peptide (F<em>1</em> + <em>2</em>) and decreased mean antithrombin activity compared with no treatment. Differences in <em>fragment</em> F<em>1</em> + <em>2</em> levels between active treatments were significant. The oral estrogen group was associated with a significant decrease in both mean tissue-type plasminogen (t-PA) concentration and plasminogen activator inhibitor (PAI-<em>1</em>) activity and a significant rise in global fibrinolytic capacity (GFC) compared with the two other groups. A transdermal estrogen regimen had no significant effect on PAI-<em>1</em>, t-PA, and GFC levels. There were no significant changes in mean values of fibrinogen, factor VII, von Willebrand factor, protein C, fibrin D-dimer, and plasminogen between and within the three groups. We conclude that oral estrogen/progesterone replacement therapy may result in coagulation activation and increased fibrinolytic potential, whereas opposed transdermal estrogen appears without any substantial effects on hemostasis. Whereas these results may account for an increased risk of venous thromboembolism in users of oral postmenopausal estrogen, they emphasize the potential importance of the route of estrogen administration in prescribing hormone replacement therapy to postmenopausal women, especially to those at high risk of thrombotic disease.

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, <em>1</em>0 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, <em>1</em>, <em>2</em>, 3, 4, 6, 8, and <em>2</em>4 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.0<em>2</em>. A progressive and significant (P <.000<em>1</em>) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.<em>1</em>, +<em>1</em> hour: <em>1</em>.3 +/- 0.9, +<em>2</em> hours: 4.<em>1</em> +/- 0.9), peaking at +3 hours (<em>1</em>0 +/- <em>1</em>.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F<em>1</em> + <em>2</em> were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F<em>1</em> + <em>2</em> levels returned to baseline after <em>2</em>4 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a <em>1</em><em>2</em>5-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. <em>2</em>000;96:554-559)

Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F<em>1</em>+<em>2</em> and the protein C activation peptide, which measure the cleavage of the <em>prothrombin</em> molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and <em>fragment</em> B beta <em>1</em>-4<em>2</em>, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F<em>1</em>+<em>2</em> and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta <em>1</em>-4<em>2</em> in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that <em>prothrombin</em> activation as measured by the F<em>1</em>+<em>2</em> assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.

Activation of the contact and complement systems in C<em>1</em>-inhibitor deficiencies is thought to contribute to the pathogenesis of angioedema attacks by releasing kinins. Trigger stimuli of attacks may also activate coagulation. This is particularly important because experimental data suggest that thrombin, the main enzyme of the coagulation cascade, increases vascular permeability and can thus influence edema formation. We have studied <em>1</em>9 patients with hereditary angioedema (HAE) during remission, 5 HAE patients during acute attacks, and 6 patients with acquired angioedema (AAE) during remission and during seven attacks. Thirty normal subjects, matched for sex and age, served as controls. Generation of thrombin was measured by enzyme-linked immunosorbent assay (ELISA) as plasma levels of the <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>); the initiators of the tissue factor and contact coagulation pathways were investigated by measuring plasma levels of activated factor VII (FVIIa) coagulometrically and activated factor XII (FXIIa) by ELISA. Cleavage of high molecular weight kininogen (HK) was evaluated by immunoblotting analysis. F<em>1</em> + <em>2</em> was slightly increased during remission and further significantly increased during attacks in both HAE (P = .0<em>1</em><em>1</em>5) and AAE. FVIIa and FXIIa, normal during remission, increased strikingly during attacks in both HAE (P = .00<em>2</em><em>2</em> and P = .0044) and AAE. During remission, cleaved HK was normal in HAE and high in AAE; during attacks it increased in HAE (P = .0008) and remained elevated in AAE. Our data indicate that in C<em>1</em>-inhibitor deficient patients there is increased generation of thrombin during attacks, with signs of activation of both the contact and tissue factor coagulation pathways. In conclusion, C<em>1</em>-inhibitor deficiency, whether hereditary or acquired, has demonstrable activation of the coagulation and kinin-forming cascades during attacks and that thrombin should be considered a possible contributing factor in the pathogenesis of edema in HAE and AAE.

BACKGROUND

Hypercholesterolemia is associated with inflammation and the prothrombotic state. CD40-CD40 ligand (CD40L) interactions promote a prothrombotic response in nucleated cells. The aim of this study was to characterize the in vivo expression of soluble CD40L (sCD40L) in hypercholesterolemia, to correlate it with the extent of the prothrombotic state, and to investigate whether it may be modified by statins.

RESULTS

We studied 80 hypercholesterolemic patients and 80 matched healthy subjects. Hypercholesterolemic subjects had enhanced levels of sCD40L, factor VIIa (FVIIa), and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) compared with healthy subjects. sCD40L correlated with total cholesterol and LDL cholesterol. Moreover, sCD40L was positively associated with in vivo platelet activation, as reflected by plasma P-selectin and urinary <em>1</em><em>1</em>-dehydro-thromboxane B<em>2</em>, and with procoagulant state, as reflected by FVIIa and F<em>1</em>+<em>2</em>. Inhibition of cholesterol biosynthesis by pravastatin or cerivastatin was associated with comparable, significant reductions in sCD40L, FVIIa, and F<em>1</em>+<em>2</em>.

CONCLUSIONS

This study suggests that sCD40L may represent the molecular link between hypercholesterolemia and the prothrombotic state and demonstrates that statin therapy may significantly reduce sCD40L and the prothrombotic state.

There is strong evidence linking venous thromboembolic events and malignancy. Laboratory markers of coagulation activation such as thrombin-antithrombin complex or <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> support the premise that malignancy is a hypercoagulable state. Inflammatory cytokines (e.g. tumor necrosis factor and interferon-gamma), coagulation proteins (e.g. tissue factor and factor VIII), and procoagulant microparticles may be elevated in patients with malignancy. However, the molecular basis for cancer associated thrombosis remains unknown and the relative contribution of chemotherapeutics, tumor cells, endothelium, and circulating procoagulants in promoting thrombus formation continues to be investigated.

BACKGROUND

Several aspects of the pathogenesis of chronic urticaria (CU) remain contradictory. Autologous serum skin tests (ASSTs) and in vitro histamine release assays seem to look into distinct aspects of the disease, and the specificity of ASST has been questioned.

OBJECTIVE

We compared the autologous plasma skin test (APST) with ASST to detect autoreactivity in patients with CU. The clotting process was investigated as well by measuring in vivo thrombin generation.

METHODS

A total of 96 adults with CU underwent ASST; 7<em>1</em> of them underwent APST with Na citrate-anticoagulated plasma. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> plasma levels were measured by a sandwich ELISA in Na citrate-anticoagulated plasmas from <em>2</em>8 patients and <em>2</em>7 controls.

RESULTS

Fifty-one of 96 (53%) patients scored positive on ASST, whereas 6<em>1</em> of 7<em>1</em> (86%) patients scored positive on APST (<em>2</em><em>1</em>/30 [70%] ASST-negative and 40/4<em>1</em> [98%] ASST-positive). Plasma prothrombin <em>fragment</em> <em>1</em>+<em>2</em> was higher in patients than controls (3.06 [SD 3.36] vs 0.80 [0.34]; P < .00<em>1</em>) and in ASST-positive/APST-positive than in ASST-negative/APST-positive patients (3.89 [SD 3.68] vs <em>1</em>.33 [<em>1</em>.64]; P = 0.058) and was directly related to urticaria severity (r = 0.37; P < .05).

CONCLUSIONS

Most patients with CU are positive on APST-Na citrate. CU is associated with the generation of thrombin, a serine protease able to activate mast cells and to cause relevant increase in permeability of endothelium. APST and ASST only partially depend on the presence of circulating antibodies to FcepsilonRI or to IgE.

CONCLUSIONS

These findings provide new insights into the pathogenesis of CU and suggest new therapeutic opportunities for treating this disease.

In the mid <em>1</em>800s Trousseau observed cancer-associated thrombosis, of which the underlying pathogenesis still remains unknown. We performed a prospective study on platelet-derived microparticles (PMP) and their procoagulant potential in breast cancer patients. Fifty-eight breast cancer patients and <em>1</em>3 women with benign breast tumors were included in the study. Microparticles (MP) were examined by electron microscopy and FACS analysis using labels for annexin V (total numbers), CD6<em>1</em> (PMP), CD6<em>2</em>P and CD63 (activated platelets), CD6<em>2</em>E (endothelial cells), CD45 (leukocytes) as well as CD<em>1</em>4<em>2</em> (tissue factor). <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin generation were measured as blood coagulation markers. Numbers of annexin V+-MP were highest in breast cancer patients with larger tumor size (T<em>2</em>; median = 5,637 x <em>1</em>0(6)/l; range = <em>2</em>,85<em>2</em>-8,6<em>1</em>3) and patients with distant metastases (M<em>1</em>; median = 6,<em>1</em>0<em>2</em> x <em>1</em>0(6)/l; range = 3,350-7,445), and differed significantly from patients with in-situ tumor (Tis; median = 3,<em>2</em><em>2</em>0 x <em>1</em>0(6)/l; range = <em>2</em>,<em>2</em>77-4,<em>1</em><em>2</em>4; p = 0.0<em>1</em>9), small tumor size (T<em>1</em>; median = 3,<em>2</em>8<em>1</em> x <em>1</em>0(6)/l; range = <em>2</em>,356-4,86<em>1</em>; p = 0.043) and women with benign breast tumor (median = 4,<em>1</em>08 x <em>1</em>0(6)/l; range = <em>2</em>,530-4,874; p = 0.040). A total of 8<em>2</em>.3% of MP were from platelets, <em>1</em>4.6 % from endothelial cells and 0.3% from leukocytes. Less than <em>1</em>0% of PMP showed degranulation markers. Larger tumor size (T<em>2</em>) and metastases correlated with high counts of PMP and with highest F<em>1</em>+<em>2</em> levels. Since <em>prothrombin</em> levels and thrombin generation did not parallel MP levels, we speculate that MP act in the microenvironment of tumor tissue and may thus not be an exclusive parameter reflecting in-vivo procoagulant activity.

Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to <em>1</em> year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, β<em>2</em>-microglobulin, and liver fatty acid binding protein-<em>1</em>) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-<em>1</em>), coagulation (<em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> and d-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-<em>1</em>) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT0<em>1</em><em>1</em>94973.

BACKGROUND

The novel anticoagulant fondaparinux proved to be effective and safe in the postoperative prevention of venous thrombosis. Current phase III trials with this synthetic selective factor Xa inhibitor focus on its use in the treatment of patients with venous and arterial thrombosis. As with any anticoagulant therapy, there is a risk of bleeding complications; hence, a strategy to reverse the effects of fondaparinux is desirable. The aim of this study was to investigate whether recombinant factor VIIa (rFVIIa) could neutralize the anticoagulant effects of subcutaneously administered fondaparinux.

RESULTS

In a randomized, placebo-controlled design, <em>1</em>6 healthy male subjects received either a single subcutaneous dose of fondaparinux (<em>1</em>0 mg) and a single intravenous bolus of rFVIIa (90 microg/kg; n=8), fondaparinux and placebo (n=4), or placebo and rFVIIa (n=4). Fondaparinux (or placebo) was administered <em>2</em> hours before rFVIIa (or placebo). Injection of rFVIIa after fondaparinux normalized the prolonged activated partial thromboplastin and <em>prothrombin</em> times and reversed the decrease in <em>prothrombin</em> activation <em>fragments</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)), as observed with fondaparinux alone. Thrombin-generation time and endogenous thrombin potential, which were inhibited by fondaparinux, normalized up to 6 hours after rFVIIa injection.

CONCLUSIONS

rFVIIa is capable of normalizing coagulation times and thrombin generation during fondaparinux treatment. The duration of this effect ranged from <em>2</em> to 6 hours after rFVIIa injection. These results suggest that rFVIIa may be useful to reverse the anticoagulant effect of fondaparinux in case of serious bleeding complications or need for acute surgery during treatment with fondaparinux.

OBJECTIVE

An elevated plasma D-dimer level indicates the activation of coagulation and fibrinolysis. In the present study, we investigated the association of pre-treatment haemostatic parameters (D-dimer, fibrinogen and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) with clinicopathological parameters and outcome in patients with lung cancer.

METHODS

Plasma levels of D-dimer and other parameters were measured in 78 evaluable patients with lung cancer (60 non-small cell lung cancers, <em>1</em>8 small cell lung cancers). At diagnosis, 35 patients (44.9%) were locally advanced stage (IIIA/B) and 43 patients (55.<em>1</em>%) had metastatic disease (IV). Multivariate statistical analysis was carried out using Cox's proportional hazards model. The receiver operating characteristic curve was used to determine the cut-off values for D-dimer, fibrinogen and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>.

RESULTS

The median survival for all patients was <em>2</em>64 days (95% confidence interval <em>2</em>00-3<em>2</em>8 days). A significant association between the plasma levels of D-dimer and the response to chemotherapy was observed (P=0.03). With the univariate analysis, tumour stage, pre-treatment plasma levels of D-dimer, fibrinogen, platelet count, lactate dehydrogenase concentration and Karnofsky performance status were predictive for survival. With the multivariate analysis (P< or =0.<em>1</em>), the plasma level of D-dimer (P<0.00<em>1</em>), tumour stage (P=0.0<em>1</em>) and Karnofsky performance status (P=0.0<em>2</em>) were identified as independent predictive factors. The median survival times were 405 days (95% confidence interval <em>1</em>65-644 days) and <em>2</em>07 days (95% confidence interval <em>1</em>46-<em>2</em>67 days, P<0.00<em>1</em>), respectively, for patients with a low D-dimer level (< or =0.65 microg/ml) and a high D-dimer level (>0.65 microg/ml).

CONCLUSIONS

Elevated plasma levels of D-dimer in patients with lung cancer are associated with decreased survival and a poor response to treatment. Pre-treatment for the D-dimer level may be useful in the prediction of survival and the response to treatment.

BACKGROUND

Beyond lipid lowering, various antiinflammatory properties have been ascribed to statins. Moreover, in vitro studies have suggested the presence of anticoagulant effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, as lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) was suppressed. In this study, we examined the role of statins in experimental endotoxemia on inflammatory and procoagulant responses in vivo.

RESULTS

In this double-blind, placebo-controlled, parallel-group study, <em>2</em>0 healthy, male subjects were randomized to receive either simvastatin (80 mg/d) or placebo for 4 days before intravenous administration of LPS (<em>2</em>0 IU/kg IV). Plasma high-sensitive C-reactive protein (hsCRP), monocyte chemoattractant protein (MCP-<em>1</em>), sCD40L, sCD40, and <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>.<em>2</em>) were determined by ELISAs at baseline and at 4 and 8 hours after LPS administration. Monocyte TF expression and monocyte-platelet aggregates were measured by whole-blood flow cytometry over the same time course. The increases in hsCRP and MCP-<em>1</em>, both known inducers of TF, were significantly suppressed by statin treatment after LPS challenge. Statin premedication blunted the increase of monocyte TF expression in response to LPS. In parallel, endotoxin-induced formation of F<em>1</em>.<em>2</em> was significantly reduced by simvastatin after 4 and 8 hours. LPS infusion affected neither the formation and activation of monocyte-platelet aggregates nor plasma levels of sCD40 and sCD40L.

CONCLUSIONS

Simvastatin suppresses the inflammatory response to endotoxin and blunts monocyte TF expression but does not affect platelet activation.