Aprotinin reduces blood loss after cardiopulmonary bypass, but may sensitize recipients and is expensive. Tranexamic acid, a synthetic antifibrinolytic, has less disadvantages, but opinions differ regarding its efficacy. We studied three groups of patients undergoing cardiopulmonary bypass for coronary disease: recipients of aprotinin (total dose 4.<em>2</em> x <em>1</em>0(6) kallikrein inhibiting units, n = <em>1</em>4), recipients of tranexamic acid (total dose <em>2</em>0 mg/kg body weight, n = <em>1</em>5), and nonmedicated controls (n = <em>1</em>4) during <em>2</em>4 hours after cardiopulmonary bypass. Compared with controls, aprotinin reduced blood loss, the number of patients requiring transfusions, and the mean number of transfused red cell units (all with p < 0.05), whereas the recipients of tranexamic acid did not differ either from aprotinin recipients or from controls. Aprotinin and tranexamic acid both mitigated the early postoperative reduction of adenosine diphosphate-induced platelet aggregation seen in the controls (p < 0.05). Postoperative increases of plasma concentrations of the <em>prothrombin</em> activation <em>fragment</em> F<em>1</em> + <em>2</em> and the thrombin-antithrombin III complex showed an activation of intravascular coagulation, without any intergroup differences. The balance between concentrations of tissue plasminogen activator and the type <em>1</em> plasminogen activator inhibitor disclosed an activation of fibrinolysis, without differences between the groups. The concentrations of D-dimer, a breakdown product of cross-linked fibrin, remained at baseline in the recipients of aprotinin and tranexamic acid but tripled in the controls (p < 0.05). By contrast, the plasma antiplasmin activity was equally depressed in the tranexamic acid and the control groups but decreased less in the recipients of aprotinin (p < 0.05). This discrepancy may reflect the different modes of action of the two agents, which may make aprotinin more efficacious than tranexamic acid in the "nonfibrinolytic" act of protecting platelet function against attack by plasmin during cardiopulmonary bypass.

OBJECTIVE

Diurnal variations in levels of factor VII (FVII), FVIII, proteins C and S, antithrombin, plasminogen activator inhibitor-<em>1</em>, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, and D-dimers in healthy humans point to the existence of circadian rhythms of coagulation factors. We sought for temporal fluctuations of tissue factor pathway inhibitor (TFPI) activity in human and mouse plasma.

RESULTS

TFPI activity showed significant daily variations with highest levels in the morning in healthy men (+<em>1</em><em>1</em>%) and in mice at the light-to-dark transition (+63%), the beginning of the physically active period. Variations in FVII activity paralleled those in TFPI. In mice, the feeding schedule had a strong impact on these rhythms. Although restricted feeding and fasting shifted the peak of TFPI, the FVII peak disappeared. Investigation of temporal fluctuations in constant darkness indicated the existence of daily rhythms for TFPI and of true circadian rhythms for FVII.

CONCLUSIONS

For the first time, we report, both in humans and mice, temporal variations in TFPI activity. The coherent variations in FVII and TFPI activity could interplay to maintain the coagulation equilibrium. The chronobiological patterns should be considered to analyze activity levels of these factors. Moreover, the mouse model could be exploited to investigate modifiers of coagulation rhythms potentially associated to morning peaks of cardiovascular events.

BACKGROUND

Resistance to activated protein C (aPC) is usually linked to factor V Leiden, but may occur in other disorders associated with hypercoagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation.

METHODS

Patients (n = 39) had established diagnosis of advanced cancer; controls (n = <em>2</em>0) were healthy persons. aPC resistance was measured as the ratio of activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a polymerase-chain-reaction based technique. Other assays were performed by standard laboratory methods. Data were analyzed using t tests and the Pearson correlation.

RESULTS

aPC-SR was below <em>2</em> SD for 5 of the cancer patients (<em>1</em>3%), but none of the controls; only <em>1</em> of the 5 had the factor V Leiden mutation. aPC-SR was inversely correlated (p < 0.0<em>1</em>) with factor VIII and fibrinogen in patients and with <em>prothrombin</em> activation <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>) in controls. Patient factor VIII, von Willebrand factor, (vWF), fibrinogen, F<em>1</em>.<em>2</em> and D dimer were all significantly increased (p < 0.0<em>1</em>; antithrombin III, protein C and proteins were similar to controls. Factor VIII correlated with vWF (p < 0.00<em>1</em>) and F<em>1</em>.<em>2</em> with d-dimer (p < 0.00<em>1</em>). Other associations (p < 0.05) were observed between factor V and protein C, fibrinogen and protein C, factor V and antithrombin III and protein C and antithrombin III. Four cancer patients had a history of thromboembolism; their aPC-SR was similar to that of patients without thrombosis. Of the several coagulation measures examined, only vWF was higher in the patients with thrombosis (p = 0.0<em>1</em>).

CONCLUSIONS

Cancer patients have evidence of intravascular coagulation and increases in procoagulants and may have aPC resistance. The aPC resistance is not due to factor V Leiden, but is rather associated with elevated levels of factor VIII and fibrinogen, and in itself does not predict thrombosis.

In a prospective study, coagulation test results were compared in <em>1</em>37 patients with colorectal cancer (CRC) and 39 subjects with benign colorectal diseases. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complex (TAT), and soluble fibrin (SF) were measured in plasma before and after surgery. CRC patients presented with significantly higher values of F<em>1</em>+<em>2</em> and TAT than controls. Patients with localised CRC had elevated values of F<em>1</em>+<em>2</em> and TAT, whereas patients with advanced CRC also had elevated SF values. TAT and SF levels correlated with tumour spread, and normal values virtually excluded advanced cancer. Postoperative deep venous thrombosis (DVT) was diagnosed by phlebography in <em>2</em>0% of the CRC patients. Preoperative values of the markers did not predict postoperative DVT, but postoperative values did.

OBJECTIVE

Visfatin and apelin are novel adipocytokines that have recently generated much interest. The aim of the study was to assess visfatin and apelin in correlation with markers of endothelial cell injury and inflammation in <em>2</em><em>2</em> patients with chronic kidney disease-CKD and <em>2</em><em>2</em> age- and sex-matched healthy volunteers.

METHODS

We assessed visfatin, apelin, markers of coagulation: TAT (thrombin-antithrombin complexes), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>; fibrinolysis: tPA (tissue plasminogen activator), PAI-<em>1</em> (plasminogen activator inhibitor), PAP (plasmin-antiplasmin complexes); endothelial function/injury: vWF (von Willebrand factor), thrombomodulin, ICAM (intracellular adhesion molecule), VCAM (vascular cell adhesion molecule), CD<em>1</em>46, CD40L, CD44, E-selectin, inflammation: hsCRP.

RESULTS

Triglycerides, hsCRP, creatinine, vWF, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, TAT, thrombomodulin, ICAM, VCAM, CD<em>1</em>46, CD44, CD40L, PAI-<em>1</em>, PAP, visfatin and E-selectin were elevated in chronic kidney disease patients when compared with the control group. Visfatin correlated significantly in patients with chronic kidney disease, in univariate analysis, with CD40L (r=-0.<em>2</em>7, p<0.05), apelin (r=0.<em>2</em>7, p<0.05), ICAM (r=0.<em>2</em>6, p<0.05), VCAM (r=0.3<em>1</em>, p<0.05) and tended to correlate with CD<em>1</em>46 (r=0.<em>2</em><em>1</em>, p=0.<em>1</em>0). Apelin correlated significantly with E-selectin (r=0.3<em>1</em>, p<0.05) and VCAM (r=0.3<em>1</em>, p<0.05). In the healthy volunteers visfatin correlated significantly with ICAM (r=-0.37, p<0.05) and serum creatinine (0.38, p<0.05).

CONCLUSIONS

Elevated visfatin in CKD patients may be due to renal failure and/or inflammation. Adipocytokines related to adhesion molecules might support the importance of inflammation/endothelial cell injury in the pathogenesis of atherosclerosis and its consequences in CKD.

The study was aimed to investigate the effect of two different statins on the levels of haemostatic variables reflecting procoagulant and fibrinolytic activity in patients with coronary heart disease (CHD), with the hypothesis that statins might beneficially modify these levels. Fifty-eight patients were randomized to treatment with atorvastatin (n=<em>2</em>8) or simvastatin (n=30) for <em>1</em> year. The starting dose in both groups was <em>2</em>0 mg/day. Fasting blood samples were collected before and after <em>1</em><em>2</em>-month treatment for determinations of fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), plasma D-dimer, soluble tissue factor, tissue plasminogen activator (tPA) antigen, tPA activity, plasminogen activator inhibitor type-<em>1</em> activity (PAI-<em>1</em> activity) and serum D-dimer as a global test of fibrinolytic activity. In the total population, improved fibrinolytic activity was observed after <em>1</em> year with increased levels of serum D-dimer (P=.00<em>1</em>) and tPA activity (P=.0<em>2</em>4) and a reduction in tPA antigen (P=.048). No statistically significant changes were observed in any of the measured coagulation variables. Separately examined, an improved fibrinolytic profile was seen in the atorvastatin group with a significant increase in serum D-dimer (P=.005), a borderline increase in tPA activity (P=.083) and a borderline reduction in tPA antigen (P=.069). Within the simvastatin group, a reduction in <em>prothrombin</em> F<em>1</em>+<em>2</em> was observed (P=.038). The differences in changes between the groups were statistically significant only for global fibrinolysis (serum D-dimer, P=.046). In conclusion, an improved fibrinolytic profile was observed after statin treatment, most pronounced with atorvastatin. The results indicate that the drugs promote a profibrinolytic profile, and may in part explain the benefit of statin treatment rendered in the prevention of CHD.

Upon incubation of human <em>prothrombin</em> with factor Xa bound to human umbilical vein endothelial cells (HUVEC) (0.5-0.6 fmol factor Xa/<em>1</em>0(5) cells), three bonds at Arg<em>2</em>73-Thr<em>2</em>74, Arg<em>2</em>86-Thr<em>2</em>87, and Arg3<em>2</em><em>2</em>-Ile3<em>2</em>3 were cleaved, yielding and releasing <em>fragment</em> <em>1</em>-<em>2</em> and a degraded form of alpha-thrombin, but not meizothrombin, into the fluid phase. The apparent Km for <em>prothrombin</em> and the Vmax were 0.<em>2</em>5 +/- 0.07 microM and <em>2</em><em>1</em>0 +/- 40 fmol thrombin/min/<em>1</em>0(5) cells, respectively. For the maximally bound factor Xa, the calculated catalytic efficiency (kcat = 6-7 s-<em>1</em>) was similar to those reported for the <em>prothrombin</em>ase complex formed on the phospholipid vesicles and natural membrane surfaces. The <em>prothrombin</em> derivatives lacking the <em>1</em>0 gamma-carboxyglutamic acid (Gla) residues-containing region were not activated by the cell-bound factor Xa. The activation rate of <em>prothrombins</em> with Gla residues variously modified to gamma-methyleneglutamic acids was reduced in accordance with the number of modified residues. For the inhibition of <em>prothrombin</em> activation, intact <em>fragment</em> <em>1</em> was needed; the Gla-domain alone did not affect the reaction. Binding of monoclonal antibodies to the region of <em>1</em>-48 or the kringle <em>1</em> region of <em>prothrombin</em> also interfered with the <em>prothrombin</em> activation. <em>Prothrombin</em> activation on the surface of HUVEC appeared to proceed via formation of a cellular <em>prothrombin</em>ase complex composed of phospholipids of HUVEC membrane, endogenous factor Va, factor Xa, and <em>prothrombin</em>. The Gla-domain and kringle <em>1</em> regions are indispensable for the molecule to serve as an effective substrate for the cell-bound factor Xa.

Advanced chronic heart failure (CHF) is associated with abnormal haemostasis and inflammation, but it is not known how these abnormalities are related, whether they are modified by oral anticoagulants (OAT), or if they persist after successful heart transplantation. We studied <em>2</em>5 patients with CHF (New York Heart Association class IV, <em>1</em>0 of whom underwent heart transplantation) and <em>2</em>5 age- and sex-matched healthy controls by measuring their plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin (TAT) complexes, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), D-dimer, factor VII (FVII), fibrinogen, von Willebrand factor (VWF), tumour necrosis factor (TNF), soluble TNF receptor II (sTNFRII), interleukin 6 (IL-6), soluble intercellular adhesion molecule-<em>1</em> (sICAM-<em>1</em>), soluble vascular cell adhesion molecule-<em>1</em> (sVCAM-<em>1</em>), endothelial-selectin (E-selectin) and thrombomodulin. CHF patients had higher plasma levels of TAT, D-dimer, t-PA, fibrinogen, VWF, TNF, IL-6, sTNFRII, sVCAM-<em>1</em> (P = 0.000<em>1</em>), sICAM-<em>1</em> (P = 0.003) and thrombomodulin (P = 0.007) than controls. There were significant correlations (r = 0.4<em>1</em>4-0.595) between coagulation, fibrinolysis, endothelial dysfunction and inflammation parameters, which were lower in those patients treated with OATs. Heart transplantation led to reductions in fibrinogen (P = 0.00<em>1</em>), VWF (P = 0.05), D-dimer (P = 0.05) and IL-6 levels (P = 0.05), but all the parameters remained significantly higher (P = 0.0<em>1</em>-0.000<em>1</em>) than in the controls. Advanced CHF is associated with coagulation activation, endothelial dysfunction and increased proinflammatory cytokine levels. Most of these abnormalities parallel each other, tend to normalize in patients treated with OATs and, although reduced, persist in patients undergoing successful heart transplantation, despite the absence of clinical signs of CHF.

BACKGROUND

Thrombotic microangiopathy is the fundamental lesion in diarrhea-associated hemolytic uremic syndrome. The extent of the lesion in the renal parenchyma determines the severity and outcome of the disorder, bilateral renal cortical necrosis being the worst end of the spectrum. In the early years, intravascular coagulation was considered the most important pathogenic mechanism. Yet, individual coagulation factors were normal in the vast majority of patients and therapy with anticoagulants did not alter the course. Recent studies indicate that impaired fibrinolysis might be of importance.

METHODS

We studied seven variables of the coagulation pathway (PT, aPTT, fibrinogen, FVIII:c, von Willebrand factor, thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>) and seven parameters of the fibrinolytic system (plasminogen, alpha<em>2</em>-antiplasmin, C<em>1</em>-esterase inhibitor, tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor type <em>1</em>, D-dimer) in <em>2</em>4 pediatric patients with diarrhea-associated hemolytic uremic syndrome and in <em>1</em>5 children with acute renal failure not due to hemolytic uremic syndrome. Samples were collected at diagnosis and every second day thereafter for a period of ten days. Additional samples were collected from patients who underwent dialysis, that is, before and after each session from those subjected to hemodialysis and every day from those subjected to peritoneal dialysis. The obtained data were compared with data from a control group consisting of healthy children.

RESULTS

Our data show four important features. (<em>1</em>) A significant increase in both thrombin-antithrombin complexes (P < 0.005) and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (P < 0.00<em>1</em>) is observed in hemolytic uremic patients as compared to patients with acute renal failure of other causes. This finding is clearly indicative for an activation of the coagulation pathway. (<em>2</em>) Patients with the hemolytic uremic syndrome have significantly higher D-dimer levels, a sensitive marker of fibrin-specific fibrinolysis, as compared to patients with acute renal failure of other causes (P < 0.005). (3) Levels of plasminogen activator inhibitor-<em>1</em> (active antigen as well as plasminogen activator inhibitor-<em>1</em> activity) are not different in both patient groups. In contrast, plasma levels of tissue-type plasminogen activator and urokinase-type plasminogen activator are significantly higher in the hemolytic uremic patients than in those with acute renal failure of other causes (P < 0.0<em>1</em> and P < 0.05 respectively). (4) Hemodialysis leads to an increase in tissue-type plasminogen activator antigen and a decrease of plasminogen activator inhibitor-<em>1</em> activity levels.

CONCLUSIONS

Our data demonstrate that in children with diarrhea-associated hemolytic uremic syndrome, limited intravascular coagulation occurs, without evidence of impaired fibrinolysis.

Endoscopic biliary drainage (EBD) for unresectable hepatocellular carcinoma (HCC) associated with obstructive jaundice remains controversial because of the short survival of these patients. To evaluate the effectiveness of this procedure, we retrospectively studied <em>1</em>8 patients who had unresectable HCC with obstructive jaundice and underwent EBD with polyethylene stents, over a <em>1</em>0-year period. Nine patients with tumor thrombus involving the first branches of the portal vein or portal trunk (Vp3) formed group A and the other 9 (Vp0-Vp<em>2</em>) formed group B. The serum albumin level and serum total bililubin level differed significantly between the two groups (P < 0.05 and P < 0.005. Student's t-test), but <em>prothrombin</em> time did not. The obstructive jaundice was mainly caused by direct tumor invasion in 6 patients from group A and 3 from group B, by blood clots and/or tumor <em>fragments</em> in <em>2</em> patients from group A and 3 from group B, by the tumor protruding into the common hepatic duct in <em>2</em> patients from group B. and by tumor compression of the common bile duct in <em>1</em> patient from each group. Drainage was successful in 4 patients (44%) from group A and in all 9 patients (<em>1</em>00%) from group B. Among the 5 patients with unsuccessful drainage in group A, 4 had obstruction of both the left and right hepatic ducts and 3 had multiple tumors in both lobes. The mean survival time (mean +/- SD) after EBD was 47 +/- 44 days in group A and <em>1</em>8<em>1</em> +/- 70 days in group B. In group A. the average survival time was only 85 days in the 4 patients with successful drainage. However, an improvement in the quality of life after EBD was observed in one-third of the Vp3 patients and in all of the Vp0-Vp<em>2</em> patients. In summary, satisfactory palliation is possible with successful EBD, but this is difficult in most patients with Vp3 portal thrombus, direct tumor invasion involving both hepatic ducts, and multiple tumors in both lobes. It is important to determine the site, extent, and nature of the obstruction, as well as liver function and the presence of portal thrombus, before performing EBD.

In a recent randomized, double-blind, placebo-controlled trial of women with a history of venous thromboembolism (VTE), we found that hormone replacement therapy (HRT) was associated with an early excess risk of recurrent thrombosis. The aims of the present study were to characterize the effects of HRT on coagulation in these women to elucidate the mechanism(s) by which HRT increases the risk of thrombosis. The study comprised <em>1</em>40 women who were randomized to receive continuous treatment for <em>2</em>4 months with once daily <em>2</em> mg <em>1</em>7-beta-estradiol plus <em>1</em> mg norethisterone acetate (n = 7<em>1</em>) or placebo (n = 69). HRT caused significant increases in <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex, and D-Dimer after 3 months, but these changes were less pronounced on prolonged treatment. The increases in markers of activated coagulation was higher in those women who subsequently developed recurrent thrombosis, but was similar in carriers and non-carriers of the factor V Leiden mutation. HRT had no effects on fibrinogen and factor VIII. Activated factor VII, but not factor VII antigen, decreased significantly on HRT as compared with placebo. The coagulation inhibitors antithrombin, protein C, and TFPI, but not protein S, all showed significant sustained decreases in the HRT group as compared with placebo. Antithrombin and protein C decreased by 8-<em>1</em><em>2</em>% on HRT, whereas TFPI activity decreased by <em>1</em><em>2</em>-<em>1</em>7% and TFPI free antigen by <em>2</em>9-30%. In multivariate analysis, only TFPI activity was a significant predictor for the increased activation of coagulation. We conclude that HRT was associated with early activation of coagulation, which corroborates the finding of an early risk of recurrent VTE. This activation may in part be explained by reduction in circulating anticoagulants.

OBJECTIVE

To investigate the expression of monocyte tissue factor (MTF) and adhesion molecules in patients with chronic renal failure (CRF) and to look for any correlation with thrombin generation and Lp(a) lipoprotein.

METHODS

A study of MTF expression and adhesion molecules, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (PTf<em>1</em>+<em>2</em>), an index of thrombin generation, and lipoproteins in patients with CRF and in normal control subjects.

BACKGROUND

Patients with end stage renal failure have an increased risk of coronary artery disease despite advances in therapy. Stimulated monocytes are potent activators of blood coagulation through the generation of MTF, which was recently implicated in the aetiology of acute coronary ischaemic syndromes.

METHODS

MTF expression and adhesion molecules were measured in whole blood using immunofluorescence of monocytes labelled with anti-tissue factor antibody and CD<em>1</em><em>1</em>b and c by flow cytometry. PTf<em>1</em>+<em>2</em> and Lp(a) lipoprotein in plasma were measured by enzyme linked immunosorbent assay (ELISA).

METHODS

70 patients with CRF without documented coronary artery disease (30 patients with CRF undialysed, <em>2</em>0 patients undergoing chronic ambulatory peritoneal dialysis (CAPD), and <em>2</em>0 undergoing haemodialysis (HD)), together with <em>2</em>0 normal controls, were studied.

RESULTS

The (mean (SD)) increased MTF of CRF (48.0 (<em>2</em>9) v 33.3 (7.<em>2</em>) mesf unit/<em>1</em>00 monocytes in controls, p = 0.04) was more pronounced in patients undergoing dialysis (HD 73.<em>1</em> (3<em>2</em>.8) (p < 0.003) and CAPD 6<em>2</em>.8 (<em>2</em>8.9) mesf unit/<em>1</em>00 monocytes, p < 0.04). MTF activity showed a positive correlation with both PTf<em>1</em>+<em>2</em> and serum creatinine (p < 0.003) but not with Lp(a) lipoprotein. Lp(a) lipoprotein was significantly increased in both dialysis groups compared with controls (p < 0.005) and non-dialysis CRF groups (p < 0.0<em>2</em>). Monocyte adhesion molecule (CD<em>1</em><em>1</em>b) was significantly higher in all three CRF groups than in the controls (p = 0.006).

CONCLUSIONS

This study has demonstrated a hypercoagulable state in patients with CRF. This was especially pronounced in the dialysis patients. These findings provide a possible explanation for the increased cardiovascular and cerebrovascular morbidity and mortality in these patients.

OBJECTIVE

To determine the impact of improved glycemic control on the development and progression of retinopathy after the institution of insulin therapy in patients with type <em>2</em> diabetes and to assess the relation to IGF-<em>1</em> and hemostatic variables.

METHODS

In a prospective observational study, 45 type <em>2</em> diabetic patients were examined at baseline and <em>1</em>, 3, 6, <em>1</em><em>2</em>, and <em>2</em>4 months after change to insulin therapy. Retinopathy was graded on fundus photographs using the Wisconsin scale; HbA<em>1</em>c, IGF-<em>1</em>, and hemostatic variables were measured.

RESULTS

During the observation period of <em>2</em> years, <em>2</em>3 patients progressed in the retinopathy scale; 8 progressed>> or = 3 levels. After <em>2</em> years of insulin treatment, HbA<em>1</em>c and IGF-<em>1</em> were significantly lower than at baseline, whereas the hemostatic variables had not changed significantly. Progression of retinopathy>> or = 3 levels was related to the degree of HbA<em>1</em>c reduction, the duration of diabetes, a higher <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels (F<em>1</em> + <em>2</em>), but not to other hemostatic variables or IGF-<em>1</em>. The relative risk for progression>> or = 3 levels was <em>2</em>.6 when HbA<em>1</em>c had been reduced>> or = 3 percent units (95% CI <em>1</em>.<em>1</em>-6.<em>1</em>).

CONCLUSIONS

The magnitude of improvement of HbA<em>1</em>c by the institution of insulin treatment over a <em>2</em>-year period may be associated with progression of retinopathy in patients with type <em>2</em> diabetes.

Hypercoagulable states can be detected by measuring activation peptides, enzyme-inhibitor complexes, and fibrin/fibrinogen degradation products, which are markers of hemostatic activation. A series of these prethrombotic markers has been evaluated in the elderly, pregnancy, diabetes and acute myocardial infarction patients (n=30 in each group) as well as in hematologic malignancies (n=4<em>2</em>). The parameters assayed were: <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA), plasmin-alpha<em>2</em> antiplasmin complexes (PAP) and D-Dimer. Results were compared with those obtained in a group of 30 healthy subjects. We found a significant increase of F<em>1</em>+<em>2</em>, TAT and FPA in elderly (p<0.05), acute myocardial infarction (AMI) (p<0.0<em>1</em>), hematologic malignancies (p<0.0<em>1</em>), and pregnancy (p<0.000<em>1</em>), indicating a marked clotting activation. Diabetic patients under strict metabolic control only presented a moderate increase of TAT (p<0.05), suggesting a slight activation. We also observed a highly significant elevation of PAP and D-Dimer in elderly (p<0.00<em>1</em>), AMI (p<0.000<em>1</em>), and malignancy (p<0.000<em>1</em>), indicating an activation of the fibrinolytic system. The combination of selected fibrinolytic and coagulation measurements is useful for the detection of a hypercoagulable state in conditions characterized by a risk of thrombosis.

OBJECTIVE

Mechanisms underlying the morning increase in platelet aggregation produced by arising and assuming the upright posture were studied by examining <em>1</em>) the expression on the platelet surface of activation-dependent markers; <em>2</em>) platelet aggregation in whole blood; and 3) hematologic factors likely to influence aggregation.

BACKGROUND

The morning increase in thrombotic cardiovascular events has been attributed, in part, to the morning surge in platelet aggregability, but its mechanisms are poorly understood.

METHODS

Expression of seven platelet surface antigens (including P-selectin, activated GPIIb,IIIa and GPIb-IX), whole-blood platelet aggregation, platelet count and hematocrit were measured before and after arising in <em>1</em>7 normal volunteers. The fibrinolytic variables, tissue-type plasminogen activator, plasminogen activator inhibitor <em>1</em> and catecholamine levels were also measured.

RESULTS

On arising and standing, platelet aggregation increased by 7<em>1</em>% (p < 0.0<em>1</em>) and <em>2</em>7% (p < 0.03) in response to collagen and adenosine diphosphate, respectively. However, there was no change in any of the activation-dependent platelet surface markers. Whole-blood platelet count and hematocrit increased by <em>1</em>5% and 7% (both p < 0.000<em>1</em>), respectively. Norepinephrine and epinephrine levels increased by <em>1</em>89% (p < 0.000<em>1</em>) and <em>1</em>30% (p < 0.0<em>1</em>), respectively. Tissue-type plasminogen activator antigen increased (3<em>1</em>%, p < 0.0<em>1</em>), but there was no significant increase in plasminogen activator inhibitor <em>1</em>, suggesting an overall increase in fibrinolysis on standing. Prothrombin fragment <em>1</em>.<em>2</em> increased by <em>2</em>8% (p < 0.0<em>2</em>), indicating a small increase in thrombin generation. The increases in hematocrit and platelet count that occurred on standing were carefully mimicked in vitro and resulted in a <em>1</em><em>1</em>5% (p < 0.05) increase in platelet aggregation in response to adenosine diphosphate.

CONCLUSIONS

These data demonstrate that the morning increase in platelet aggregation is not accompanied by expression of activation-dependent platelet surface receptors and suggest that the increase in whole-blood aggregation may be primarily due to the increases in catecholamine levels, platelet count and hemoconcentration.

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (<em>1</em>993) Biochemistry 3<em>2</em>, <em>1</em>0794-<em>1</em>080<em>2</em>], is a <em>2</em>7 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite <em>1</em> reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite <em>1</em> is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or <em>prothrombin</em> <em>fragment</em> <em>2</em> for binding to thrombin indicates that bothrojaracin not only binds exosite <em>1</em> but also binds exosite <em>2</em> or in close proximity. Bothrojaracin binds to the thrombin precursor, <em>prothrombin</em>. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite <em>2</em>. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.

BACKGROUND

Excess risk of cardiovascular disease occurs in effectively treated individuals with human immunodeficiency virus (HIV) infection. Although elevated plasma D-dimer levels are associated with increased morbidity and mortality, the impact of HIV infection on coagulation in vivo has not been well studied.

METHODS

We measured D-dimers, antithrombin, endogenous thrombin potential (ETP; a functional measure of thrombin generation in vitro), thrombin/antithrombin complexes (TAT; a measure of thrombin generation in vivo), tissue factor, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>), and normalized APC sensitivity ratio (nAPCsr) in <em>1</em>99 HIV-positive men who were receiving antiretroviral therapy and had an undetectable HIV RNA level, in 79 HIV-positive untreated men, and in 39 uninfected controls.

RESULTS

Median antithrombin levels were higher while the ETP was lower among HIV-infected adults (treated and untreated), compared with controls. There were few differences between coagulation markers in the <em>2</em> HIV groups. Compared with controls, the nAPCsr was lower in treated men and the TAT level was lower in untreated individuals. We observed little difference among measured levels of D-dimer, tissue factor, or F<em>1</em>+<em>2</em> between HIV-infected individuals and controls. Antiretroviral therapy exposure was associated with a lower antithrombin level, a lower nAPCsr, and a lower ETP, while history of opportunistic infection was associated with a higher nAPCsr.

CONCLUSIONS

HIV infection is associated with decreased thrombin generation, as measured by the ETP, and an increased antithrombin level. These data suggest that HIV infection may not be associated with increased propensity toward clotting, as has been suggested on the basis of isolated measures of D-dimer levels.

BACKGROUND

The present study was conducted to investigate the relation between the accumulation of the risk factors of thromboembolism and the levels of hemostatic markers in patients with nonvalvular atrial fibrillation (NVAF).

METHODS

Five hundred ninety-one NVAF patients and <em>1</em><em>2</em>9 control subjects were categorized into low, moderate or high risk of thromboembolism, according to CHADS(<em>2</em>) index. One point each was given to patients with advanced age >> or =75 years), hypertension, congestive heart failure, and diabetes mellitus, and <em>2</em> points, to those with prior ischemic stroke or transient ischemic attack. Patients with CHADS(<em>2</em>) score of 0, <em>1</em> or <em>2</em>, and>> or =3 were classified as low, moderate and high risk, respectively. Levels of hemostatic markers (platelet factor 4, beta-thromboglobulin, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> and D-dimer) were determined.

RESULTS

Of 59<em>1</em> patients with NVAF, 30<em>2</em> were treated with warfarin (mean international normalized ratio <em>1</em>.88). D-dimer levels increased as the risk level increased irrespective of warfarin use. Particularly, NVAF patients without receiving warfarin (n=<em>2</em>89) had significantly higher D-dimer levels than control patients (e.g., for high risk patients, <em>1</em>75+/-<em>1</em>44 vs 75+/-87 ng/ml, p<0.00<em>1</em>), while NVAF patients receiving warfarin had intermediate levels (<em>1</em>36+/-<em>1</em>56 ng/ml). F<em>1</em>+<em>2</em> levels increased as the risk level increased, and were significantly suppressed by warfarin. Levels of markers of platelet activation (platelet factor 4 and beta-thromboglobulin) were increased in NVAF patients but not affected by the risk level.

CONCLUSIONS

Coagulation and fibrinolytic activity is increased along with the accumulation of the risk factors of thromboembolism in NVAF patients.

OBJECTIVE

To determine whether obesity increases platelet reactivity and thrombin activity in patients with type <em>2</em> diabetes plus stable coronary artery disease.

METHODS

We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation <em>2</em> Diabetes (BARI <em>2</em>D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.<em>2</em> (PT1.<em>2</em>, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.<em>2</em>, and 1 micromol/l adenosine diphosphate (ADP).

RESULTS

Concentrations of FPA, PT1.<em>2</em>, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 microm ADP as assessed by surface expression of P-selectin (r = 0.<em>2</em>9, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.<em>2</em>. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS Among patients enrolled in this substudy of BARI <em>2</em>D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type <em>2</em> diabetes.

This is the first double-blind, controlled, randomized study comparing the effect of different estrogen components in oral contraceptives (OCs) on hemostasis variables. Four groups of <em>2</em>5 women each were treated for six cycles with monophasic combinations containing <em>2</em><em>1</em> tablets with either 30 microg ethinylestradiol (EE) + <em>2</em> mg dienogest (DNG) (30EE/DNG), <em>2</em>0 microg EE + <em>2</em> mg DNG (<em>2</em>0EE/DNG), <em>1</em>0 microg EE + <em>2</em> mg estradiol valerate (EV) + <em>2</em> mg DNG (EE/EV/DNG) or <em>2</em>0 microg EE + <em>1</em>00 microg levonorgestrel (LNG) (EE/LNG). Blood samples were taken on Days <em>2</em><em>1</em>-<em>2</em>6 of the control cycle and on Days <em>1</em>8-<em>2</em><em>1</em> of the first, third and sixth treatment cycle. Treatment with all four OCs caused an increase in levels of fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer, plasminogen, plasmin-antiplasmin complex and an increase in protein C activity, a decrease in antithrombin activity, tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI), and a slight decrease in the sensitivity to activated protein C, but no significant change in that of the thrombin-antithrombin complex. In users of the DNG-containing OCs, the reduction in total and free protein S, and in t-PA and PAI was dependent on the EE dose, while factor VII activity was elevated, but not significantly different from EE/LNG. The results are in agreement with those of previous studies. The effects of EE/EV/DNG on total and free protein S and on t-PA and PAI were lower than those of <em>2</em>0EE/DNG, suggesting that the impact of <em>2</em> mg EV on several hemostasis variables is less than that of <em>1</em>0 microg EE. The results show an antagonistic effect of LNG on the EE-induced rise of factor VII activity and <em>fragment</em> <em>1</em>+<em>2</em> and on the EE-dependent reduction of total and free protein S.

<i>Background:</i> Coronavirus Disease <em>2</em>0<em>1</em>9 (COVID-<em>1</em>9)-associated coagulopathy is characterized by a prothrombotic state not yet comprehensively studied. We investigated the coagulation pattern of patients with COVID-<em>1</em>9 acute respiratory distress syndrome (ARDS), comparing patients who survived to those who did not. <i>Methods:</i> In this prospective cohort study on <em>2</em>0 COVID-<em>1</em>9 ARDS patients, the following biomarkers were measured: thrombin generation (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (PF <em>1</em> + <em>2</em>)), fibrinolysis activation (tissue plasminogen activator (tPA)) and inhibition (plasminogen activator inhibitor <em>2</em> (PAI-<em>2</em>)), fibrin synthesis (fibrinopeptide A) and fibrinolysis magnitude (plasmin-antiplasmin complex (PAP) and D-dimers). Measurements were done upon intensive care unit (ICU) admission and after <em>1</em>0-<em>1</em>4 days. <i>Results:</i> There was increased thrombin generation; modest or null release of t-PA; and increased levels of PAI-<em>2</em>, fibrinopeptide A, PAP and D-dimers. At baseline, nonsurvivors had a significantly (<i>p</i> = 0.0<em>1</em>4) higher PAI-<em>2</em>/PAP ratio than survivors (<em>1</em>09, interquartile range (IQR) <em>1</em>8.<em>1</em>-<em>2</em><em>1</em>6, vs. 8.7, IQR <em>2</em>.9-<em>1</em><em>2</em>.6). At follow-up, thrombin generation was significantly (<i>p</i> = 0.0<em>2</em>5) reduced in survivors (PF <em>1</em> + <em>2</em> from 396 pg/mL, IQR <em>1</em>85-585 to <em>2</em>37 pg/mL, IQR <em>1</em><em>2</em>0-393), whereas it increased in nonsurvivors. Fibrinolysis inhibition at follow-up remained stable in survivors and increased in nonsurvivors, leading to a significant (<i>p</i> = 0.0<em>2</em>6) difference in PAI-<em>2</em> levels (<em>1</em>6<em>1</em> pg/mL, IQR 50-334, vs. <em>1</em>088 pg/mL, IQR <em>1</em>77-<em>1</em>565). <i>Conclusion:</i> Severe patterns of COVID-<em>1</em>9 ARDS are characterized by a thrombin burst and the consequent coagulation activation. Mechanisms of fibrinolysis regulation appear unbalanced toward fibrinolysis inhibition. This pattern ameliorates in survivors, whereas it worsens in nonsurvivors.

<strong class="sub-title"> Keywords: </strong> COVID-<em>1</em>9; fibrinolysis; heparin; pulmonary thromboembolism; sepsis; thrombin generation.

BACKGROUND

After tissue injury caused by trauma or surgery, alterations of hemostasis are observed and there is a risk for postoperative thromboembolic complications. Laparoscopic surgery, by causing limited tissue injury, appears to be associated with a lower risk for thromboembolism than open surgery. We conducted a prospective randomized study in order to detect potentially existing differences in activation of coagulation and fibrinolytic pathways between open and laparoscopic surgery.

METHODS

Forty patients suffering from chronic cholelithiasis were randomly assigned to undergo open (group A n = <em>2</em>0) or laparoscopic cholecystectomy (group B n = <em>2</em>0) by the same surgical and anesthesiology team. Demographic data were comparable. Blood samples were taken (a) preoperatively, (b) at the end of the procedure, (c) <em>2</em>4 h postoperatively and (d) 7<em>2</em> h postoperatively. The following parameters were measured and compared within each group and between groups: platelets (PLT), soluble fibrin monomer complexes (SFMC), fibrin degradation products (FDP), D-dimers (D-D), fibrinogen (FIB), activated partial thromboplastin time (APTT), <em>prothrombin</em> time (PT). Thrombin-antithrombin III complexes (TAT) were measured at <em>2</em>4 and 7<em>2</em> h postoperatively. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) was measured at <em>2</em>4 and 7<em>2</em> h postoperatively in <em>1</em><em>1</em> patients of group A and <em>1</em>3 patients of group B, respectively.

RESULTS

Demographics were comparable between groups. Immediately postoperatively, TAT and F<em>1</em> + <em>2</em> were significantly higher in group A as compared to group B (p < 0.05). They also increased significantly postoperatively as compared to preoperative levels within each group (p < 0.05). D-dimers were significantly higher in group A as compared to group B (p < 0.0<em>1</em>) immediately postoperatively. D-dimers also increased significantly postoperatively in group B as compared to preoperative levels (p < 0.00<em>1</em>). FIB decreased slightly in both groups at <em>2</em>4 h postoperatively but there was a significant increase in group A as compared to group B (p < 0.0<em>1</em>). SFMC were detected twice in group A and only once group B. FDP levels over 5 mug/ml were detected more often in group A than in group B (p < 0.05). No patient from either group suffered thromboembolism or abnormal bleeding as a postoperative complication.

CONCLUSIONS

Open surgery as compared to laparoscopic procedures leads to activation of the clotting system of a higher degree. Although of a lower degree, hypercoagulability is still observed in patients undergoing laparoscopic surgery and, therefore, routine thromboembolic prophylaxis should be considered.

BACKGROUND

Danazol is a synthetic androgen derivative frequently used as prophylaxis in patients with hereditary angioedema (HAE) due to complement-<em>1</em> esterase inhibitor deficiency. However, danazol has been reported to decrease high-density lipoprotein cholesterol (HDL-C) levels and to adversely affect coagulation parameters, which are considered to be proatherothrombotic.

OBJECTIVE

The short- and long-term effects of danazol were evaluated on proatherogenic intermediate end points in healthy volunteers and patients with HAE.

METHODS

Short-term effects were evaluated in healthy men randomly assigned to 200 mg/d of danazol or placebo for 4 weeks in a crossover trial with no washout period. Long-term effects of danazol on lipoproteins, coagulation, and carotid intima-media thickness (CIMT) were evaluated in a cross-sectional study in which patients with HAE treated with danazol, a mean dose of <em>1</em>70 mg/d for>>or=2 years, were compared with healthy controls matched for age, sex, and body mass index (BMI). Drug tolerability was assessed by questionnaires and adherence was measured by pill count when drug bottles were returned after every study visit.

RESULTS

Patients in the short-term study were <em>1</em>5 men with a mean (SD) age of 32.6 (6.9) years and BMI of 24.3 (4.<em>1</em>) kg/m(2). In the long-term study, patients with HAE were <em>1</em>0 women and 7 men with a mean (SD) age of 4<em>1</em>.<em>1</em> (<em>1</em>2.9) years and BMI of 25.4 (2.6) kg/m(2); the <em>1</em>7 matched controls had a mean (SD) age of 39.8 (<em>1</em><em>1</em>.8) years and BMI of 25.4 (2.6) kg/m(2). Short-term danazol treatment was associated with a decrease from baseline in apolipoprotein A-I of 2<em>1</em>% and in HDL-C of 23%. Flow-mediated dilation and coagulation parameters were unaffected after 4 weeks. Longterm danazol treatment did not adversely affect HDL-C concentration (<em>1</em>.<em>1</em> [0.5] vs baseline, <em>1</em>.2 [0.5] pmol/L), HDL-related transfer proteins such as paraoxonase-<em>1</em> activity (92 [62] vs 80 [40] U/mM), cholesteryl-ester transfer protein mass (<em>1</em>.5 [0.4] vs 2.2 [0.6] microg/mL), lecithin cholesterol acyltransferase activity (2<em>1</em>.2 [4.5] vs 32.<em>1</em> [7.2] nmol CE . mL(-<em>1</em>) . h(-<em>1</em>)), plasma phospholipid transfer protein activity (<em>1</em>5.4 [<em>1</em>.5] vs <em>1</em>4.9 [<em>1</em>.2] AU), and apolipoproteins between patients with HAE and controls. The mean (SD) CIMT was similar between patients with HAE and controls (0.62 [0.09] vs 0.59 [0.08] mm; P = NS). However, HAE patients using danazol had increased coagulation activation when compared with controls (prothrombin fragments, 286 [<em>1</em><em>1</em>9] vs <em>1</em>64 [57] pmol/L, P = 0.002; thrombinantithrombin complex, 3.9 [<em>1</em>.4] vs 2.6 [<em>1</em>.<em>1</em>] microg/L, P = 0.0<em>1</em>).

CONCLUSIONS

Short-term danazol treatment in healthy volunteers was associated with a reduction in HDL-C levels without a significant effect on endothelial function or coagulation parameters. In contrast, patients with HAE treated for >2 years with danazol had increased activation of coagulation, but there were no significant differences in HDL-C or CIMT compared with matched healthy controls.

Total internal reflection fluorescence microscopy has been used to compare the membrane binding characteristics of fluorescein-labeled bovine <em>prothrombin</em> and fluorescein-labeled bovine <em>prothrombin</em> <em>fragment</em> <em>1</em>. The Ca(<em>2</em>+)-dependent association of these proteins with quartz-supported planar membranes composed of mixtures of phosphatidylserine (<em>2</em>-<em>1</em>0 mol%) and phosphatidylcholine was examined. Equilibrium binding measurements showed that the apparent equilibrium dissociation constants increased with decreasing molar fractions of phosphatidylserine and that the dissociation constants were somewhat lower for intact <em>prothrombin</em>. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissociation rates were approximately equivalent for <em>prothrombin</em> and <em>fragment</em> <em>1</em> and did not change with the protein solution concentration or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equilibrium and kinetic constants are reported and compared for <em>prothrombin</em> and <em>fragment</em> <em>1</em> on planar membranes.