OBJECTIVE

To determine the effect of two low-dose monophasic oral contraceptives containing either <em>2</em> mg chlormadinone acetate or <em>1</em>50 microg desogestrel on blood clotting and fibrinolysis.

METHODS

In vivo markers of intravascular coagulatory and fibrinolytic activity were measured in 45 volunteers randomly assigned to a 6-month treatment with one of the two study preparations.

RESULTS

During oral contraceptive use, the procoagulatory activity increased (increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>), the anticoagulatory capacity changed (increased protein C activity, decreased activated protein C sensitivity, decreased protein S activity and decreased antithrombin III activity) and the fibrinolytic system was activated (increased concentrations of plasmin-antiplasmin complexes and D-dimer as well as total fibrin degradation products). There were no relevant differences between the two medication groups.

CONCLUSIONS

Our results demonstrate that both oral contraceptive preparations have comparable effects on the hemostatic system. There was a shift towards a new equilibrium of hemostatic activities, both coagulatory and fibrinolytic, at a higher turnover rate. Changes did not exceed the range of normal variation and were comparable to the published effects of other low-dose oral contraceptives. There was no evidence ofa differential risk of deep vein thrombosis between the two preparations.

Microvascular thrombosis is a major cause of organ damage in Shiga toxin-mediated hemolytic uremic syndrome (Stx-HUS). In vitro and clinical studies implicate thrombin-mediated mechanisms in the pathogenesis of Stx microvascular thrombosis. In a greyhound model, administration of 0.03 microg/kg to 0.05 microg/kg Stx<em>1</em> or Stx<em>2</em> causes severe bloody diarrhea and HUS with microvascular thrombosis requiring humane euthanasia within 65 hours. Using a greyhound model of Stx-HUS we analyzed early hemostatic changes, and tested the hypothesis that thrombin blockade with lepirudin would prevent lethal Stx effects. Two Stx<em>1</em>-exposed greyhounds were analyzed for hemostatic changes prior to onset of clinical manifestations. Serial hemostasis studies after Stx<em>1</em> challenge revealed trends of increased aPTT, fibrinogen levels, and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, and appearance of abnormally large von Willebrand factor multimers. Three greyhounds were anticoagulated with lepirudin to maintain activated partial thromboplastin times (aPTT)>><em>2</em>.5-fold normal, followed by administration of Stx<em>2</em> and observation of clinical responses. Among the 3 lepirudin-treated, Stx<em>2</em>-challenged greyhounds, one developed severe illness requiring euthanasia. Remarkably, <em>2</em> of the 3 greyhounds developed only hypersalivation and restlessness that resolved (P <.03 compared to <em>1</em>4 historical controls). These two greyhounds were clinically, hematologically and biochemically normal 74 hours after Stx administration, well beyond the time of euthanasia of any previous greyhound. This study suggests that greyhounds exposed to Stx develop procoagulant changes similar to humans, and that thrombin may be a critical factor in the pathogenesis and treatment of Stx-HUS.

OBJECTIVE

Systemic thromboembolism is a major complication in patients of mitral stenosis (MS) with atrial fibrillation (AF) due to induction of hypercoagulable state. The aim was to assess the relationship, if any, between control of ventricular rate and systemic coagulation factors.

METHODS

70 patients of moderate to severe MS in AF were studied. 35 patients with average heart rate>> <em>1</em>00 beats/min over a <em>2</em>4 hour period assessed by Holter monitoring were considered as having a uncontrolled ventricular rate (Group A) and those with average heart rate ≤ <em>1</em>00 beats/min as controlled ventricular rate (Group B). 30 healthy volunteers acted as controls.

RESULTS

Plasma concentration of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (PF<em>1</em>+<em>2</em>) 6600 pmol/ml [interquartile range (IQR) 5400.0-9500], thrombin antithrombin III <em>2</em><em>2</em>.0 ng/ml [IQR <em>1</em>8.6-<em>2</em>8.0], and plasminogen activator inhibitor 46.8 ng/ml [IQR 44.0-54.0] were elevated in Group A as compared to Group B (5400 pmol/ml [IQR 3600-7700] p = 0.009, <em>1</em>6.0 ng/ml [IQR <em>1</em><em>1</em>.0-<em>1</em>8.5] p<0.00<em>1</em>, and <em>2</em>5.8 ng/ml [IQR <em>2</em>0.9-34.4] p < 0.00<em>1</em>), respectively. A significant correlation was found between heart rate and all three coagulation markers. Multivariate multiple regression analysis showed only heart rate to be an independent predictor of systemic coagulation activation and risk of thrombus formation.

CONCLUSIONS

Control of ventricular rate in subjects of MS with AF produces significant reduction in the activation of the coagulation system and may decrease risk of thrombosis.

BACKGROUND

Endothelial damage and hemostatic imbalance play an important role in the evolution of the Systemic Inflammatory Response Syndrome (SIRS) into the Multiple Organ Dysfunction Syndrome (MODS). In Acute Renal Failure associated with SIRS, different types of Continuous Renal Replacement Therapies (CRRT) may give non-renal benefits by modifying the levels of some factors related to those disturbances.

METHODS

Forty patients with SIRS-associated ARF were randomised to receive either continuous venovenous hemofiltration (CVVH) or continuous venovenous hemodiafiltration (CVVHDF) for the first <em>2</em>4 h. Afterwards the CRRT method was reversed. The group treated with CVVH moved to CVVHDF and that treated with CVVHDF to CVVH for the next <em>2</em>4 h. Plasma levels of: von Willebrand Factor (vWF), thrombomodulin, plasminogen activity inhibitor type <em>1</em> (PAI-<em>1</em>: antigen and activity), tissue type plasminogen activator (t-PA: antigen), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complexes (TAT) were measured previously to CRRT (base-line), and after <em>2</em>4 and 48 hours of therapy. Multivariate ANOVA was the statistical method used.

RESULTS

In the MANOVA study a significant decrease in PAI-<em>1</em> activity during the treatment procedure was observed (horizontality p <0.05). PAI-<em>1</em> antigen showed a tendency to decrease although without statististical significance. There were no significantly different changes in the other factors analysed during either CRRT (parallelism p >0.05). At the base-line point, all the factors were higher than normal values in healthy adults.

CONCLUSIONS

The present study suggests that CRRT, in patients with SIRS, may promote a decrease in PAI-<em>1</em> and consequently, a better outcome. There were no differences between the CVVH and the CVVHDF methods regarding the factors analysed. The present data confirms that there is an important endothelial and hemostatic dysfunction in SIRS from the early phases.

OBJECTIVE

To explore the hypothesis that a relationship exists between free radical activity and abnormalities in hemostasis in NIDDM.

METHODS

The use of the total radical-trapping antioxidant parameter (TRAP) has very recently been proposed to explore the antioxidant property of a plasma and their mutual cooperation. In the present study, TRAP, vitamin E, vitamin C, vitamin A, uric acid, protein-bound SH (thiol) groups, fibrinogen, <em>prothrombin</em> <em>fragments</em> F<em>1</em> + <em>2</em>, and D-dimer have been evaluated in 46 NIDDM patients and 47 healthy matched control subjects.

RESULTS

In NIDDM patients, TRAP, vitamin A, SH groups, and uric acid were significantly reduced, whereas the level of vitamin E was significantly increased. Vitamin C was similar in the two groups. Fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and D-dimer were increased in diabetic patients. TRAP, but no single other antioxidant, had a strong inverse association with fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and D-dimer.

CONCLUSIONS

These findings are consistent with the hypothesis that oxidative stress may condition coagulation activation in diabetics. However, the data suggest that it is the total antioxidant capacity rather than any single plasma antioxidant that is the most relevant parameter.

Evidence suggests that isolated intracranial hypertension (iIH) is often associated with cerebral venous thrombosis (CVT). In eight patients referred to our Institution for iIH who were later shown to harbor CVT we have performed a comprehensive coagulation work-up, including genetic tests for inherited predisposition to thrombophilia, to clarify the etiology of sinus venous thrombosis. All subjects were women. All but one were overweight. There were high plasma concentrations of D dimer, thrombin-anti-thrombin complexes or <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, further supporting the neuroimaging diagnosis of CVT. Importantly, seven of eight cases had a raised level of plasminogen activator inhibitor <em>1</em>, a well known inhibitor of fibrinolysis related to obesity. Tissue plasminogen activator levels were elevated accordingly. Factor V gene mutation was present in one subject, and the <em>2</em>0,<em>2</em><em>1</em>0 <em>prothrombin</em> gene mutation was found in another individual. Three patients had elevated plasmatic levels of homocysteine. In conclusion, the present study provides solid evidence that impaired fibrinolysis probably related to overweight, acting in concert with other prothrombotic abnormalities, is involved in the pathogenesis of CVT presenting as iIH.

When <em>1</em><em>2</em>5I-labeled canine <em>prothrombin</em> was given to normal adult dogs intravenously, it was calculated that <em>2</em>40% of the plasma <em>prothrombin</em> crossed the capillary barrier per day, 4<em>1</em>0% of the interstitial <em>prothrombin</em> returned to the blood stream daily, and 79% of the plasmatic <em>prothrombin</em> was catabolized per day. These data are in close agreement with those observed for bovine <em>prothrombin</em> in calves by Takeda (<em>1</em>970). When derived from normal dog <em>prothrombin</em>, prethrombin-<em>1</em> is a mixture of <em>2</em> polypeptides, one larger than the other, and both present in about equal amounts. The longer peptide, "prethrombin-<em>1</em>-long," was catabolized twice as fast as <em>prothrombin</em>, and the shorter, "prethrombin-<em>1</em>-short," 4 times faster. <em>Prothrombin</em> <em>fragment</em>-<em>1</em> was catabolized by the normal dog still more rapidly. The catabolism of <em>prothrombin</em> was not accelerated in 3 dogs receiving continuous infusions of a thromboplastic emulsion of dog brain. Nor was the level of <em>prothrombin</em> in their plasma remarkably altered.

BACKGROUND

Thromboembolism is considered a crucial event in the pathogenesis of retinal occlusion, resulting in a severe damage of central or peripheral visual function.

METHODS

We evaluated hemostatic system parameters in the plasma of <em>1</em>4 patients (<em>1</em><em>1</em> males and 3 females aged 59-73 years) affected by acute retinal ischemia (central retinal arterial occlusion or arterial branch occlusion). The diagnosis of retinal arterial occlusion was established according to clinical symptoms, ophthalmoscopic findings and fluorescein angiography. In addition to routine coagulation tests, antithrombin III, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin III complex (TAT), and D-dimer were measured in the plasma both at the moment of diagnosis (before therapy initiation) and 3-6 months later (at least <em>1</em> months after antithrombotic therapy discontinuation).

RESULTS

We found a marked increase in the plasma levels of F<em>1</em>+<em>2</em>, TAT, and D-dimer during the acute event, compared with healthy control values. F<em>1</em>+<em>2</em> and TAT significantly decreased during follow-up, whereas D-dimer was unchanged.

CONCLUSIONS

Our data indicate accelerated conversion of <em>prothrombin</em> to thrombin (high F<em>1</em>+<em>2</em>) and an increase in circulating thrombin (high TAT) during retinal arterial occlusion, providing evidence that increased thrombin generation may play a role in acute retinal ischemia.

BACKGROUND

exercise in IC leads to ischaemia-reperfusion injury of leg muscles and a systemic inflammatory response, but the effect of on coagulation is unknown.

OBJECTIVE

to compare the effect of exercise on thrombin formation and fibrin turnover in patients with IC (n = <em>1</em>0), and age and sex matched smokers ([S] n = 5) and non-smokers ([NS] n = 5) without peripheral vascular disease.

METHODS

blood was taken from subjects 60 and 30 min before, and <em>1</em>, 5, <em>2</em>0, 40, 60 and <em>1</em><em>2</em>0 min after, treadmill exercise. Markers of thrombin generation (thrombin-antithrombin complexes [TAT] and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> [PF<em>1</em> + <em>2</em>]) and fibrin turnover (D-dimer and fibrin degradation products [FbDP]) were assayed at each time point.

RESULTS

following exercise, thrombin generation was significantly greater in the claudicant group compared to the control groups (Area Under Curve [AUC] post exercise IC vs S vs NS; TAT 3960 vs <em>1</em>6<em>2</em>3 vs <em>1</em>476 vs = 0.007 Kruskal-Wallis [KW]; PF<em>1</em> + <em>2</em> <em>1</em>63 vs <em>1</em>07 vs <em>1</em><em>2</em>3 p = 0.0<em>2</em>4 KW). Pre and post-exercise, fibrin turnover in claudicants was similar to smoking controls, but higher than non-smoking controls. (AUC post exercise IC vs NS; D-dimer 6340 vs <em>2</em>754 p = 0.055 Mann-Whitney U[MW]; FbDP 45<em>1</em><em>1</em>3 vs <em>2</em><em>1</em>5<em>1</em><em>1</em> p = 0.009 MW).

CONCLUSIONS

when compared to non-claudicants, exercise in IC is associated with excessive production of thrombin. Despite this, claudicants have a similar level of fibrin turnover suggesting a possible defect in fibrinolysis. This prothrombotic state may contribute to the excess thrombotic morbidity and mortality suffered by claudicants.

An augmented systemic production of thromboxane (TX) A<em>2</em>, as assessed by urinary excretion of the thromboxane metabolites, has been described in severe liver cirrhosis. However, the significance of this finding remains unclear since in liver cirrhosis a number of phenomena i.e. altered hepatic TXA<em>2</em> metabolism, increased intrasplenic platelet destruction, may affect TXA<em>2</em> entry into systemic circulation as well as its metabolism. In order to further clarify this, we measured both major enzymatic metabolites of TXB<em>2</em> in the urine of 44 patients affected by liver cirrhosis, subdivided in three classes on the basis of Child-Pugh criteria. Urinary <em>1</em><em>1</em>-dehydro-TXB<em>2</em> and <em>2</em>,3-dinor-TXB<em>2</em> were assayed with previously validated RIA techniques. The urinary excretion rate of <em>1</em><em>1</em>-dehydro-TXB<em>2</em> was significantly (p = 0.000<em>1</em>) increased in the cirrhotic patients (673.5 pg/mg cr, median) in comparison with the controls (<em>2</em>75 pg/mg cr, median) but no significant difference could be demonstrated among the excretion rates of the three patient subgroups. The excretion rate of <em>2</em>,3 dinor-TXB<em>2</em> was also significantly (p = 0.000<em>1</em>) increased in the patients (8<em>2</em>4 pg/mg cr, median) in comparison with controls (<em>1</em>75 pg/mg cr, median), with a significant (p < 0.05) increase from class A (38<em>1</em> pg/mg cr) to class C (<em>1</em>337 pg/mg cr). The sum of the two enzymatic metabolites was significantly (p = 0.000<em>1</em> ) increased in the cirrhotic patients in comparison to controls, with a progressive increase from class A (<em>1</em>003 pg/mg cr, median) to class C (<em>2</em><em>2</em>40 pg/mg cr, median). The urinary excretion of <em>2</em>,3 dinor-TXB<em>2</em> was significantly (p = 0.008) related to plasma <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>). This study provides further evidence of increased thromboxane biosynthesis in liver cirrhosis. Moreover, we demonstrate intraliver shift of thromboxane metabolic disposition, due to progressive liver decompensation, because only the fraction undergoing beta-oxidation to <em>2</em>,3-dinor-TXB<em>2</em> was progressively increased with the degree of liver failure. We, also, find a significant correlation between urinary excretion of <em>2</em>,3-dinor-TXB<em>2</em> and plasma F<em>1</em>+<em>2</em>, suggesting that clotting activation could partly account for in vivo platelet activation.

Obstructive sleep apnoea is a risk factor for pulmonary embolism. Elevated D-dimer levels and other biomarkers are associated with recurrent pulmonary embolism. The objectives were to compare the frequency of elevated D-dimer levels (>500 ng·mL(-<em>1</em>)) and further coagulation biomarkers after oral anticoagulation withdrawal in pulmonary embolism patients, with and without obstructive sleep apnoea, including two control groups without pulmonary embolism.We performed home respiratory polygraphy. We also measured basic biochemical profile and haemogram, and coagulation biomarkers (D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex, plasminogen activator inhibitor <em>1</em>, and soluble P-selectin).64 (74.4%) of the pulmonary embolism cases and 4<em>1</em> (46.<em>1</em><em>1</em>%) of the controls without pulmonary embolism had obstructive sleep apnoea. Plasmatic D-dimer was higher in PE patients with OSA than in those without obstructive sleep apnoea. D-dimer levels were significantly correlated with apnoea-hypopnoea index, and nocturnal hypoxia. There were more patients with high D-dimer after stopping anticoagulants in those with pulmonary embolism and obstructive sleep apnoea compared with PE without obstructive sleep apnoea (35.4% versus <em>1</em>9.0%, p=0.003). Apnoea-hypopnoea index was independently associated with high D-dimer.Pulmonary embolism patients with obstructive sleep apnoea had higher rates of elevated D-dimer levels after anticoagulation discontinuation for pulmonary embolism than in patients without obstructive sleep apnoea and, therefore, higher procoagulant state that might increase the risk of pulmonary embolism recurrence.

BACKGROUND

Scientific and technical advances made in transfusion medicine sustain the need for more comprehensive understanding of the impact of collection procedures on the quality of plasma for fractionation and for transfusion. This prospective work evaluated protein composition and markers of activation in plasma donations collected with three different automatic collection procedures (performed on Haemonetics machines), including a new procedure using a high-separation core-molded bowl.

METHODS

A total of 90 collection procedures have been performed from a population of 37 donors, under comprehensively standardized conditions. Plasma aliquots were taken from the plasma units within 30 minutes of the end of the collection procedures and immediately frozen at -70 degrees C. Content in an extended range of proteins and of markers of activation of the coagulation and fibrinolytic systems has been measured using standard in vitro testing methods.

RESULTS

Plasma donations had normal mean total protein, IgG, IgM, and fibrinogen content. The mean levels in coagulation FV, FVII, FVIII, and FXI and in antithrombin were above the standard international requirements. There was no sign of activation of the hemostasis system, as assessed by activated FVII, thrombin antithrombin complex, <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, and D-dimers. Activated complement component C3 and C5 were low.

CONCLUSIONS

Data indicates the good and consistent protein composition of plasma obtained by those automatic apheresis procedures. In particular, the new high-separation core procedure yields a high-quality plasma meeting requirements for transfusion and fractionation.

OBJECTIVE

Thrombo-embolic events are one of the most feared complications related to atrial fibrillation (AF) ablation. Since radiofrequency (RF) energy is thought to be associated with a higher risk of thrombus formation than cryoenergy, the purpose of this study was to assess if the degree of activation of coagulation and inflammatory markers differed between ablation procedures performed with a cryoballoon catheter vs. a RF energy-based pulmonary vein ablation catheter (PVAC), respectively.

RESULTS

Thirty patients referred for AF ablation were randomized to pulmonary vein isolation with either the cryoballoon or the PVAC. Biomarkers were studied for endothelial damage (von Willebrand factor antigen), platelet activation (soluble P-selectin), and coagulation activity [<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and D-dimer] at five different time points during the procedure. Troponin I (Trop I) and C-reactive protein were analysed to reflect myocardial destruction and inflammatory activity. Markers of endothelial damage and platelet activation increased after ablation in both the cryo and the RF group. Similarly, the D-dimer levels increased significantly (P = 0.00<em>1</em>) in both groups, whereas the F<em>1</em> + <em>2</em> levels increased after the transseptal puncture only (P = 0.00<em>1</em>). The overall activation of the coagulation system was, however, comparable between the groups. The cryoballoon was associated with higher Trop I compared with the PVAC (P < 0.00<em>1</em>), but the ratios between biomarkers and Trop I were higher with the PVAC than with the cryoballoon.

CONCLUSIONS

Even though the cryoballoon causes a higher degree of myocardial destruction than the PVAC, markers of coagulation, endothelial damage, and inflammation were comparable between the two techniques.

BACKGROUND

The improved biocompatibility of the cardiopulmonary bypass circuits made possible by the use of surface-immobilized heparin may allow for a reduction in the amount of heparin administered systemically. This study was performed to elucidate the effects of cardiopulmonary bypass using heparin-coated circuits and reduced heparinization on hemostatic variables and clinical outcome.

METHODS

Thirty patients scheduled to undergo myocardial revascularization were randomized to have either a heparin-coated or an uncoated cardiopulmonary bypass circuit. Anticoagulation was induced with heparin (<em>1</em>00 IU/kg in the coated group and 300 IU/kg in the uncoated group) and the activated clotting time was kept over <em>2</em>00 and 480 seconds in the coated and uncoated groups, respectively.

RESULTS

The postoperative overnight loss of hemoglobin through the drains was lower in the heparin-coated group (43.6 g; range, <em>1</em>8.5-69.0 g) than in the uncoated group (73.0 g; range, 3<em>2</em>.<em>2</em>-<em>1</em>37.7 g) (p = 0.00<em>1</em>5). Plasma concentrations of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and D-dimer were significantly more elevated after cardiopulmonary bypass in the coated group than they were in the uncoated group. Two patients in the coated group had a stroke postoperatively.

CONCLUSIONS

The reduction in systemic heparinization was associated with thrombin formation, which may predispose to intravascular and cardiopulmonary bypass circuit clotting. Therefore, generous systemic heparinization may still be prudent despite the improved biocompatibility offered by heparin-coated surface.

"Silent" lacunar stroke, often found in the elderly, has been proposed as a predisposing condition for clinically overt stroke. However, the risk factors related to this condition have not been studied thoroughly. We conducted brain magnetic resonance imaging and measured the levels of fibrinogen, molecular markers of coagulation activation [<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>)] and endothelial cell damage [von Willebrand factor (vWF) and thrombomodulin], and lipid profiles including lipoprotein (a) [Lp(a)] in <em>1</em>78 asymptomatic, high-risk, Japanese subjects aged 44 to 93 years. We also studied 3<em>2</em> symptomatic patients with lacunar stroke (symptomatic lacunar group). The prevalence of silent lacunar stroke increased with age up to 85 years but decreased with age in those 85 years old and older. Of the <em>1</em>60 elderly subjects (>> or = 60 years) 84 (53%) had>> or = <em>1</em> lacunar infarcts (silent lacunar group) and the remaining 76 were considered as the nonlacunar group. Fibrinogen and F<em>1</em> + <em>2</em> levels in the silent lacunar group were significantly higher than those in the nonlacunar group (P < .0<em>1</em>). Mean Lp(a) levels and the prevalence of subjects with an Lp(a) level>> 30 mg/dL were significantly higher in the symptomatic lacunar group than the nonlacunar group (P < .05), whereas these levels in the silent lacunar group were intermediate to those of the other two groups. When we further classified the silent lacunar group into three subgroups based on the number of lacunes (few lacunes, <em>1</em> or <em>2</em>; moderate number of lacunes, 3 or 4; and numerous lacunes,>> or = 5), levels of Lp(a), F<em>1</em> + <em>2</em>, vWF, and thrombomodulin were significantly higher and Lp(a) levels>> 30 mg/dL more common in the numerous-lacune than in the few-lacune subgroup. We conclude that silent lacunar stroke is often found in asymptomatic, high-risk, elderly Japanese patients and that silent multiple lacunar stroke is associated with hypercoagulability, endothelial cell damage, and high Lp(a) levels.

BACKGROUND

Exercise is frequently recommended for the treatment of patients with arterial hypertension. Previous studies have shown an enhanced coagulation state after exercise. Our study investigates the alterations observed after a single session of submaximal aerobic exercise concerning coagulation, fibrinolysis, platelet activation as well as endothelial function in patients with recently diagnosed essential hypertension.

METHODS

Twenty non-diabetic patients with recently diagnosed essential hypertension participated in a 45 min submaximal exercise test on a bicycle ergometer. Blood samples were drawn before and after exercise in order to determine parameters of coagulation activation (<em>Prothrombin</em> time [PT], activated Partial Thromboplastin time [aPTT], fibrinogen, D-Dimers, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> [PF<em>1</em>+<em>2</em>], thrombin-antithrombin III complex [TAT] and factors VII, VIII and XII), platelet activation (Platelet count, Platelet factor 4 [PF4] and beta-thromboglobulin [beta-TG]), fibrinolysis activation (Plasmin-a(<em>2</em>) antiplasmin complex, PAP) and endothelial function (soluble Thrombomodulin [sTM] and von Willebrand factor [vWf]). Soluble P-selectin served as a marker for endothelial and platelet activation.

RESULTS

All patients completed the exercise test. aPTT (P < 0.00<em>1</em>) and factor VII (P = 0.0<em>1</em>) significantly decreased while PT (P = 0.04), fibrinogen (P = 0.008), factor VIII (P < 0.00<em>1</em>), platelet count (P = 0.00<em>2</em>) and beta-TG levels (P = 0.0<em>1</em>) significantly increased as a result of exercise. Compared to baseline there was an <em>1</em><em>1</em>% increase in TAT (P = 0.04) and a <em>2</em>8% increase in PAP (P < 0.00<em>1</em>) at peak exercise. One hour post exercise, there was a 43% increase in PAP whereas TAT levels became similar to those at baseline. Additionally vWf (P = 0.0<em>1</em>) and sP-selectin (P = 0.0<em>2</em>) levels significantly increased throughout the exercise protocol.

CONCLUSIONS

Patients with recently diagnosed and never treated mild to moderate essential hypertension undergoing submaximal aerobic exercise present evidence of enhanced fibrinolysis compared with a mild increase of coagulation indices. However, whether there is a favourable effect of exercise on fibrinolysis over coagulation and/or endothelial involvement during exercise needs to be further investigated.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine <em>prothrombin</em> and <em>prothrombin</em> <em>fragment</em> <em>1</em> to acidic lipid membranes. <em>Prothrombin</em> and <em>prothrombin</em> <em>fragment</em> <em>1</em> were examined under four different conditions: in the presence of (a) Na<em>2</em>EDTA, (b) 5 mM CaCl<em>2</em>, and in the presence of CaCl<em>2</em> plus membranes containing <em>1</em>-palmitoyl-<em>2</em>-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) <em>1</em>,<em>2</em>-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(<em>2</em>+)-induced conformational change in bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> was properly detected by our procedures, although Ca(<em>2</em>+)-induced changes in whole <em>prothrombin</em> spectra were too small to be reliably interpreted. Binding of <em>prothrombin</em> in the presence of Ca<em>2</em>+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (<em>2</em>% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in <em>prothrombin</em> <em>fragment</em> <em>1</em> secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in <em>prothrombin</em> in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding <em>fragment</em> <em>1</em> region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.

BACKGROUND

Sickle cell trait donations can block leukodepletion (LD) filters or fail to LD, but the variables affecting blockage are unclear.

METHODS

To identify critical variables for further study, the relationship was investigated between filter blockage and donor characteristics, processing conditions, PLT and coagulation system activation, and microvesicle formation in donations with (n = 63) and without (n = 40) sickle trait. With eight filter types whole blood was LD either at ambient temperature on Day 0 or after overnight 4 degrees C hold. Markers of PLT activation (CD6<em>2</em>P and CD63 expression and soluble CD6<em>2</em>P) and coagulation activation (activated FXII and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> [F<em>1</em> + <em>2</em>]), RBC microvesicles, blood gases, and residual WBCs were measured.

RESULTS

All Day 0 filtrations blocked (n = 7). On Day <em>1</em>, no filter tested was <em>1</em>00 percent successful, with most achieving an approximate 50 percent success rate. Two filters blocked consistently and an additional filter did not block, but resulted in 50 percent of units with high residual WBC counts (30 x <em>1</em>0(6)-394 x <em>1</em>0(6)/unit). Day <em>1</em> filtration was not improved if performed at 4 degrees C. Donor RBC variables and prefiltration measures varied little between blocked and successful filtrations except pO<em>2</em>, where 9 of <em>1</em>7 blockages had a pO<em>2</em> of less than 5.0 kPa, compared with 0 of <em>1</em>3 completed filtrations. F<em>1</em> + <em>2</em> levels increased after filtration in sickle trait units, a consequence of slow flow rate.

CONCLUSIONS

Filter blockage in sickle trait donors cannot be predicted by donor characteristics or filter type and is not related to PLT or coagulation activation, but can be reduced by storing units at 4 degrees C before filtration.

We studied <em>1</em>7<em>2</em> Greek patients (7<em>2</em> men aged 44.0 +/- <em>1</em>6.7 years and <em>1</em>00 women aged 46.5 +/- <em>1</em>4.<em>1</em> years) with an unexplained thrombophilic tendency. One hundred and four apparently healthy persons (63 men aged 34.<em>2</em> +/- <em>1</em>0.0 years and 4<em>1</em> women aged 37.<em>1</em> +/- <em>1</em>3.3 years) were included as a control group. We performed the activated protein C resistance (APC-r) test using a clotting test (Chromogenix kit), detection of factor V Leiden using polymerase chain reaction (PCR)-restriction <em>fragment</em> length polymorphisms and measurement of thrombin-antithrombin complexes (TAT) and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) levels with an immunoenzymatic assay. The normal range for the APC-r test >> <em>2</em>.<em>1</em><em>2</em>) was determined from the controls. The factor V Leiden mutation was found in 3<em>1</em>.9% of all the patients tested, in <em>2</em>8.<em>1</em>% of the unrelated patients with documented thrombophilic tendency of unknown origin and in 4.8% of the healthy controls. The APC-r test had a sensitivity of 0.4<em>2</em> and a specificity of 0.9<em>1</em> for the detection of factor V Leiden. Furthermore, we found no significant difference in levels of TAT and F<em>1</em> + <em>2</em> between patients with and without the mutation and there was no correlation between aPC-r values and levels of TAT and F<em>1</em> + <em>2</em>.

Prolonged standing activates the coagulation cascade by the activation of endothelial cells, and probably the haemoconcentration effect contributes to this "orthostatic hypercoagulability". It was the objective of this study to assess whether rehydration (haemodilution) prevents or attenuates orthostatic induced thrombin formation. Twelve healthy young subjects were studied during two separate visits. Haematocrit (Hct), total plasma protein, coagulation profile tests, including endothelial activation related factors, and protein C global pathway were studied at rest supine, and while standing at <em>1</em>5 and 30 minutes (min). During the second visit the study was repeated after intravenous <em>1</em>.5 liter 0.9% saline. While in supine posture, intravenous rehydration resulted in Hct reduction of <em>1</em>4.<em>2</em> +/- <em>2</em>% (haemodilution), a decrease of <em>1</em><em>1</em>.5 +/- <em>1</em>.3% in total protein, as well as a significant dilutional effect on most of the coagulation parameters. Still standing for 30 min, with and without rehydration caused a comparable increase in tissue factor by 49.83 +/- <em>1</em>3.6%, and 35.34 +/- 8.55% (p>0.05), respectively and in von Willebrand factor (vWF) 9.5 +/- <em>2</em>.4% and <em>1</em>3.59 +/- <em>2</em>.<em>1</em>7% (p>0.05), respectively. At 30 min standing, after intravenous rehydration, factor V and VIII activities, and fibrinogen rose by <em>2</em><em>2</em> +/- <em>1</em>.9%, 3<em>1</em>.<em>2</em> +/- 6.<em>2</em>%, 9.<em>1</em>5 +/- <em>2</em>.64%, (p<0.00<em>2</em> for all), respectively. <em>Prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> elevated by 84.84 +/- <em>1</em>5.3% (p<0.00<em>1</em>). Comparable results were obtained with and without the rehydration. Additionally, protein C assay results decreased by <em>1</em>9.4 +/-<em>1</em>.7% and <em>1</em>7.5 +/- <em>2</em>.6%, with and without fluids (p<0.05 for both). In healthy subjects, intravenous prophylactic rehydration with normal saline resulted in a haemodilution of all the coagulation parameters, but did neither attenuate nor prevent the orthostatic hypercoagulability.

A condition of oxidative stress, due to perturbation of oxidant/antioxidant balance, has been suggested to play a role not only in the pathogenesis of human immunodeficiency virus (HIV) infection, but also in the promotion of a thrombophilic condition. Because various hemostatic dysfunctions usually considered as risk factors for thrombotic events were reported in HIV infection, this study was undertaken to investigate whether the oxidative phenomenon could promote a prothrombotic state in such condition. Erythrocyte glutathione peroxidase (GSH-Px), the major free-radical scavenger enzyme, and serum tumor necrosis factor-alpha (TNF-alpha) were evaluated in 33 consecutive HIV-infected out-patients and 35 matched HIV-negative healthy controls at a distance of any acute episode. Thrombin generation was explored by measuring the plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), whereas fibrin degradation products (D-dimer) and plasminogen activator inhibitor (PAI-<em>1</em>) activity were evaluated as indices of plasmin activity and fibrinolytic derangement. The anticoagulant pathway was investigated by measuring the plasma levels of antithrombin and protein C. Erythrocyte GSH-Px activity and serum TNF-alpha were significantly higher in HIV-infected patients when compared to controls. F<em>1</em> + <em>2</em>, D-dimer, and PAI-<em>1</em> activity were increased in HIV-infected patients by comparison with controls. Normal antithrombin, but decreased protein C, was instead detected in HIV-infected patients. In the latter patients, serum TNF-alpha negatively correlated with both erythrocyte GSH-Px activity and plasma D-dimer. On the other hand, a positive correlation was shown between F<em>1</em> + <em>2</em> and D-dimer and between D-dimer and GSH-Px activity. Furthermore, a trend toward increasing levels of GSH-Px with increasing PAI-<em>1</em> activity was reported. These findings suggest a relationship between erythrocyte oxidative stress and the hypercoagulable condition during HIV infection.

Microalbuminuria and haemostasis derangements have been considered as independent risk factors for cardiovascular death in type <em>2</em> (non-insulin-dependent) diabetic patients. Few studies have assessed coagulation inhibitors in type <em>2</em> diabetic patients with normoalbuminuria and microalbuminuria. Therefore, 3<em>2</em> type <em>2</em> diabetic patients with normoalbuminuria (albumin excretion rate, AER < <em>2</em>0 mg/min, mean 7 +/- <em>1</em>) and <em>2</em>8 type <em>2</em> diabetic patients with microalbuminuria (AER <em>2</em>0-<em>2</em>00 mg/min, mean 84 +/- <em>1</em><em>1</em>) were studied. The patients were matched for age, sex, disease duration and treatment, body mass index (BMI), blood pressure and glycohaemoglobin. Protein C and S activity, antithrombin III, thrombomodulin and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>) were assessed together with fibrinogen, triglycerides, total and high density lipoprotein (HDL)-cholesterol concentrations. Fibrinogen, total and low density lipoprotein (LDL) concentrations were similar in the two groups, while a significant difference was observed for triglycerides (normoalbuminuric group: <em>1</em><em>2</em>8 +/- <em>1</em>0 mg/dl, microalbuminuric group: <em>1</em>84.<em>1</em> +/- <em>1</em>7 mg/dl; P < 0.007) and HDL-cholesterol (normoalbuminuric group: 45 +/- <em>2</em> mg/dl, microalbuminuric group: 39 +/- <em>2</em> mg/dl; P < 0.05). The coagulation parameters were as follows: normoalbuminuric group: protein C activity <em>1</em>09% +/- 5%, protein S 95.4% +/- 5%, thrombomodulin 49.3 +/- 3 ng/ml, antithrombin III 93.3% +/- 3%, F <em>1</em> + <em>2</em> <em>1</em>.05 +/- 0.04 nmol/l; microalbuminuric group: protein C activity <em>1</em>07% +/- 4%, protein S 98.4% +/- 4%, thrombomodulin 64.4 +/- 4 ng/ml, antithrombin III 93.3% +/- 3%, F <em>1</em> + <em>2</em> <em>1</em>.03 +/- 0.05 nmol/l. The difference was significant for thrombomodulin (P < 0.007). A significant direct correlation was observed in the microalbuminuric group between AER and thrombomodulin (r = 0.38, P < 0.05). In conclusion, our data do not support the hypothesis that a reduction in the activity of anticoagulant physiological inhibitors (protein C, protein S, antithrombin III) could contribute to explain the higher cardiovascular risk in type <em>2</em> diabetic patients with microalbuminuria. The elevation of plasma thrombomodulin concentration in type <em>2</em> diabetic patients could be the consequence of widespread vascular damage in diabetic patients with incipient nephropathy.

BACKGROUND

During commencement of oral anticoagulant therapy (OAT) a theoretical possibility of a transient hypercoagulable state emerges from the difference in plasma half-life between the vitamin K-dependent pro-coagulation factors II and X, and the vitamin K-dependent anticoagulant proteins C and S. In the present study, markers reflecting the activity in the haemostatic system (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>], D-dimer and soluble fibrin) was assessed during initiation of OAT compared to subcutaneously administered low-molecular weight heparin (LMWH) which does not cause any imbalance between the concentrations of the pro- and anticoagulation proteins.

METHODS

Thirty-three patients with atrial fibrillation were randomly treated either with OAT (warfarin <em>1</em>0, 7.5, and 5 mg for three consecutive days) or LMWH administered in a fixed dose of <em>2</em>00 anti-Xa IU/kg body weight in one subcutaneous injection daily. The biochemical markers were measured at baseline, and after <em>1</em><em>2</em>, 36 and 60 h of treatment.

CONCLUSIONS

After introducing antithrombotic therapy, none of the biochemical markers increased within the study period in the two treatment groups. The level of F<em>1</em>+<em>2</em> had declined significantly at 60 h in both groups. The level of soluble fibrin showed a significant decrease within the first 60 h in the OAT group, and no significant changes were seen in the LMWH group. No significant change in the level of D-dimer was seen during the first 60 h of treatment in either group. Taken together, no transient hypercoagulable state could be identified within the first 60 h of commencing OAT in patients with atrial fibrillation.

A multicenter study of a recently developed ELISA for the determination of <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> was performed in order to evaluate analytical and clinical aspects. Mean intra-assay and inter-assay reproducibility were found to be <em>1</em><em>1</em>.0 and <em>1</em><em>2</em>.6%, respectively. The measuring range covered by the calibration curve reaches from 0.04 to <em>1</em>0.0 nM/l F<em>1</em>+<em>2</em>. Testing <em>1</em>33 healthy subjects a reference range of 0.37 to <em>1</em>.<em>1</em><em>1</em> nM/l F<em>1</em>+<em>2</em> (<em>2</em>.5-97.5 percentile) with a median of 0.66 nM/l F<em>1</em>+<em>2</em> was calculated. Minor difficulties with blood sampling (venous occlusion for <em>2</em> min) did not affect F<em>1</em>+<em>2</em> plasma concentrations. Significantly increased F<em>1</em>+<em>2</em> levels were measured in patients with leukemia (p < 0.000<em>1</em>), severe liver disease (p < 0.005) and after myocardial infarction (p < 0.0<em>1</em>). Elevated F<em>1</em>+<em>2</em> concentration before the beginning of heparin therapy (<em>1</em>.<em>2</em>5 nM/l) decreased to 0.77 nM/l (p < 0.000<em>1</em>) after <em>1</em> day of therapy. For patients in the stable phase of oral anticoagulant therapy decreasing F<em>1</em>+<em>2</em> concentrations were measured with increasing INR. F<em>1</em>+<em>2</em> levels were already significantly reduced in patients with INR < <em>2</em>.0 (0.56 nM/l; p = 0.0005). Thus F<em>1</em>+<em>2</em> determination may be helpful in identifying activation processes as well as in monitoring anticoagulant therapy.