The hypothesis that heparin-coated perfusion circuits reduce thrombin formation and activity; fibrinolysis; and platelet, complement, and neutrophil activation was tested in <em>2</em>0 consecutive, randomized adults who had cardiopulmonary bypass. Twenty identical perfusion systems were used; in <em>1</em>0, all blood-contacting surfaces were coated with partially degraded heparin (Carmeda process; Medtronic Cardiopulmonary, Anaheim, Calif.). All patients received a 300 U/kg dose of heparin. Activated clotting times were maintained longer than 400 seconds. Cardiopulmonary bypass lasted 36 to <em>2</em>44 minutes. Blood samples for platelet count, platelet response to adenosine diphosphate, plasma beta-thromboglobulin, inactivated complement 3b, neutrophil elastase, fibrinopeptide A, <em>prothrombin</em> <em>fragment</em> F<em>1</em>.<em>2</em>, thrombin-antithrombin complex, tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em>, plasmin alpha <em>2</em>-antiplasmin complex, and D-dimer were obtained at these times: after heparin was given, 5 and 30 minutes after cardiopulmonary bypass was started, within 5 minutes after bypass was stopped, and <em>1</em>5 minutes after protamine was given. After cardiopulmonary bypass, tubing segments were analyzed for surface-adsorbed anti-thrombin, fibrinogen, factor XII, and von Willebrand factor by radioimmunoassay. Heparin-coated circuits significantly (p < 0.00<em>1</em>) reduced platelet adhesion and maintained platelet sensitivity to adenosine diphosphate (p = 0.0<em>1</em>5), but did not reduce release of beta-thromboglobulin. There were no significant differences between groups at any time for fibrinopeptide A, <em>prothrombin</em> <em>fragment</em> F<em>1</em>.<em>2</em>, or thrombin-antithrombin complex or in the markers for fibrinolysis: D-dimer, tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em>, and alpha <em>2</em>-antiplasmin complex. In both groups, concentrations of <em>prothrombin</em> <em>fragment</em> F<em>1</em>.<em>2</em> and thrombin-antithrombin complex increased progressively and significantly during cardiopulmonary bypass and after protamine was given. Concentrations of D-dimer, alpha <em>2</em>-antiplasmin complex, and plasminogen activator inhibitor-<em>1</em> also increased significantly during bypass in both groups. Fibrinopeptide A levels did not increase during bypass but in both groups increased significantly after protamine was given. No significant differences were observed between groups for levels of inactivated complement 3b or neutrophil elastase. Radioimmunoassay showed a significant increase in surface-adsorbed antithrombin on coated circuits but no significant differences between groups for other proteins. We conclude that heparin-coated circuits used with standard doses of systemic heparin reduce platelet adhesion and improve platelet function but do not produce a meaningful anticoagulant effect during clinical cardiopulmonary bypass. The data do not support the practice of reducing systemic heparin doses during cardiac operations with heparin-coated extracorporeal perfusion circuitry.

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately <em>1</em>0-fold and shifts the Ca<em>2</em>+ dependence of protein C activation from simple saturation at>> or = 500 microM Ca<em>2</em>+ to a distinct optimum at approximately <em>1</em>00 microM Ca<em>2</em>+. Thrombin possesses two regions of high positive charge, anion binding exosites <em>1</em> and <em>2</em>. Anion binding exosite <em>1</em> interacts with the growth factor region of TM while exosite <em>2</em> is involved in binding <em>prothrombin</em> activation <em>fragment</em> <em>2</em> or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when <em>fragment</em> <em>2</em> is present either covalently attached (meizothrombin des-<em>fragment</em> <em>1</em>) or in reversible association. With meizothrombin des-<em>fragment</em> <em>1</em>, the Ca<em>2</em>+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.<em>2</em>7 mM Ca<em>2</em>+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-<em>fragment</em> <em>1</em> (Kd(app) = <em>1</em>4 nM). However, the chondroitin-free form binds thrombin (Kd(app) = <em>2</em>.4 nM) only approximately 4 times tighter than meizothrombin des-<em>fragment</em> <em>1</em> (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite <em>2</em> by either chondroitin sulfate or <em>fragment</em> <em>2</em> alters thrombin conformation resulting in the altered Ca<em>2</em>+ dependence of protein C activation.

The objectives were to investigate whether activation of the extrinsic coagulation cascade by recombinant factor VIIa (rFVIIa) reverses the inhibition of thrombin generation and platelet activation by melagatran, the active form of the oral direct thrombin inhibitor ximelagatran. In a single-blind, randomized, parallel-group study, volunteers (<em>2</em>0 per group) received a 5-hour intravenous (i.v.) infusion to achieve steady-state melagatran plasma concentrations of approximately 0.5 micromol/L, with a single i.v. bolus of rFVIIa (90 microg/kg) or placebo at 60 minutes. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-anti-thrombin complex, fibrinopeptide A, beta-thromboglobulin, and thrombin-activatable fibrinolysis inhibitor were quantified for venous and shed blood. Activated partial thromboplastin time (APTT), <em>prothrombin</em> time (PT), endogenous thrombin potential, thrombus precursor protein (TpP), and plasmin-alpha(<em>2</em>)-antiplasmin complex concentrations were determined in venous blood. Shed blood volume was measured. Melagatran reduced markers of thrombin generation and platelet activation in shed blood and prolonged APTT. rFVIIa increased FVIIa activity, PT, and TpP in venous blood. All other parameters were unaffected. In conclusion, rFVIIa did not reverse the anticoagulant effects of high constant concentrations of melagatran. However, the potential value of higher, continuous or repeated doses of rFVIIa or its use with lower melagatran concentrations has not been excluded.

BACKGROUND

Radiofrequency (RF) ablation procedures for atrial fibrillation (AF) are associated with potential risks of thromboembolism, which may be minimized by the use of cryoablation that preserves the integrity of endocardium. The objective of this study was to compare the thrombogenic potential of transvenous cryoablation versus RF ablation during pulmonary vein (PV) isolation.

RESULTS

Thirty consecutive patients with paroxysmal AF were randomized to undergo segmental PV isolation procedure using 4-mm tip RF ablation (n = <em>1</em>5) or cryoablation (CryoCor, San Diego, CA, USA) (n = <em>1</em>5). Blood samples were drawn after sheath insertion (baseline), after transseptal puncture, before ablation (after heparin administration), and after isolation of a superior PV. Activation of coagulation was measured with plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and thrombin-antithrombin III complex (TAT), and platelets by plasma level of beta-thromboglobulin (beta-TG) and flow cytometric enumerating of P-selectin (CD6<em>2</em>)-positive platelets. In both groups, the plasma level of beta-TG, F<em>1</em> + <em>2</em>, and TAT were elevated after sheath insertion. The percentage changes in plasma level of beta-TG, F<em>1</em> + <em>2</em>, and TAT and CD4<em>1</em>/6<em>2</em>-positive platelets from baseline after transseptal puncture and before ablation were similar (P>> 0.05). However, the percentage changes in CD6<em>2</em>-positive platelets from baseline were significantly higher in patients treated with RF ablation (8<em>2</em> +/- <em>2</em>0%) than with cryoablation (<em>2</em><em>2</em> +/- <em>1</em>4%, P = 0.0<em>2</em>), although their plasma levels of beta-TG, F<em>1</em> + <em>2</em>, and TAT were not different (P>> 0.05).

CONCLUSIONS

Significant platelet and coagulation activations were observed during PV ablation procedures, and heparin administration only prevented activation of coagulation but not platelets. Persistent platelets activation was observed during RF energy application, but not during cryoablation.

BACKGROUND

Platelet activation plays a pivotal role in the pathogenesis of acute coronary disease. Monocytes are involved in the progression of atherosclerosis and are potent activators of blood coagulation through their ability to synthesize tissue factor (TF). The aim of this study was to compare markers of monocyte and coagulation activation in the systemic blood of patients with unstable angina, acute myocardial infarction, or stable angina.

RESULTS

We studied <em>2</em>6 patients with unstable angina (<em>1</em>0 +/- 5 hours after the onset of the last episode of pain), <em>1</em>8 patients with acute myocardial infarction (5 +/- 4 hours after the onset of pain), and 34 patients with stable angina. We measured levels of TF expression in peripheral blood mononuclear cells (isolated by gradient centrifugation and incubated for <em>1</em>6 hours, with or without endotoxin stimulation), levels of plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), and levels of fibrinogen in peripheral blood. In patients with unstable angina, both stimulated and unstimulated cells exhibited higher levels of TF expression than in patients with stable angina (P = .000<em>1</em>). In patients with acute myocardial infarction, monocyte TF activity did not differ from that in patients with stable angina. Mean levels of F<em>1</em> + <em>2</em> and of fibrinogen did not differ significantly between groups. Only in the unstable angina group, a modest correlation was found between fibrinogen (r = .7<em>2</em>, P = .005) and F<em>1</em> + <em>2</em> levels (r = .54, P = .00<em>1</em>) levels and the degree of monocyte TF expression. In patients with unstable angina, monocyte TF expression (both stimulated and unstimulated, assessed by biological activity and by antigen techniques) and fibrinogen levels were correlated with the time elapsed from the beginning of the most recent episode of pain (.6<em>1</em> < r < .7<em>2</em>, .0<em>2</em> < P < .000<em>1</em>). By contrast, there was no correlation between these variables and the time from onset of pain in patients with acute myocardial infarction.

CONCLUSIONS

A time-dependent activation of systemic monocytes and a time-dependent increase in fibrinogen levels occurs in unstable angina but not in myocardial infarction. These findings provide further evidence that a specific inflammatory process occurs in unstable angina. Further studies are required to determine whether monocyte activation is a cause or a consequence of plaque instability in patients with unstable angina and to clarify the interrelations between platelet and monocyte activation in these circumstances.

We investigated the effect of <em>prothrombin</em> complex concentrate (PCC) on the international normalized ratio (INR) and blood coagulation system in two emergent patients treated with warfarin for secondary prevention of cardioembolic stroke due to nonvalvular atrial fibrillation. An 80-year-old woman developed massive subcutaneous hemorrhage and swelling on her right upper extremity with weak pulsation of the right radial artery and had an INR above <em>1</em>0. An 83-year-old man had pleural effusion with an INR value of 6.69 and pleural puncture was immediately required. We administered 500 IU of PCC to the two patients (<em>1</em>7.<em>2</em> IU/kg and <em>1</em><em>2</em>.5 IU/kg) with <em>1</em>0 mg of vitamin K. The INR decreased to <em>1</em>.<em>1</em><em>2</em> and <em>1</em>.85, respectively, with an increase of plasma levels of protein C and coagulant factors IIa, VIIa, IXa, and Xa <em>1</em>0 min after administration. The plasma levels of the thrombin-antithrombin III complex increased (from 4.0 to <em>1</em><em>2</em>.0 micro g/l and from 0.5 to <em>2</em>8.9 micro g/l, respectively, normal value <3.0), but <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> increased minimally <em>1</em>0 min after administration (from 0.4 to <em>1</em>.<em>1</em> nmol/ml and from 0.4 to 0.7 nmol/ml, respectively, normal value 0.4-<em>1</em>.4 nmol/ml). Plasma levels of D-dimer remained unchanged. The massive subcutaneous hemorrhage in the former patient improved in <em>1</em>4 days. Anticoagulation was restarted in the latter patient after <em>1</em>4 days of PCC administration. There were no embolic episodes during the month after PCC administration. In conclusion, a small amount of PCC may be effective in immediately correcting increased INR levels with increased plasma levels of protein C and coagulant factors IIa, VIIa, IXa, and Xa and may partially activate the coagulation system without any effects on plasma levels of D-dimer.

BACKGROUND

In previous comparisons of inflammatory and stress responses to open (OR) and laparoscopic (LR) hernia repair, all operations were performed under general anesthesia. Since local anesthesia is widely used for OR, a comparison of this approach with LR seemed relevant.

METHODS

Patients with recurrent inguinal hernia were randomized to OR under local anesthesia (n = 30) or LR under general anesthesia (n = 3<em>1</em>). The magnitude of the surgical trauma was assessed by measuring markers of coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>), endothelial activation (von Willebrand factor), inflammation [leukocytes, interleukin-6, -8 and -<em>1</em>0, granulocyte macrophage colony-stimulating factor, and C-reactive protein (CRP)], and endocrine stress (cortisol) in blood collected before operation, 4 h postincision, and on postoperative day <em>2</em>.

RESULTS

Leukocyte counts and interleukin-6 and CRP levels increased in both groups, with the CRP increase being significantly greater in the OR group. The other markers did not increase significantly.

CONCLUSIONS

The acute phase response was more pronounced after OR, even when this was done under local anesthesia. Both techniques seemed rather atraumatic.

Replacement therapy for hemophilia B (factor IX deficiency) using <em>prothrombin</em> complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in <em>1</em>0 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.<em>1</em>4 U/dL (mean +/- SD) increase per <em>1</em>U/kg of infused factor IX, and the biologic half-life (t<em>1</em>/<em>2</em>), determined using the terminal phase of elimination, was <em>2</em><em>2</em>.6 +/- 8.<em>1</em> hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in <em>prothrombin</em> activation <em>fragment</em> (F<em>1</em>+<em>2</em>), PCC infusion was associated with significant increases of factors II (<em>2</em>.7 U/dL per <em>1</em> U/dL of IX increase) and X (<em>2</em>.<em>2</em> U/dL for <em>1</em> U/dL for <em>1</em> U/dL of IX). Patients who used Mononine as their sole therapeutic material during the <em>1</em><em>2</em>-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the <em>1</em><em>2</em>-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

The distance between the phospholipid surface and the active site of membrane-bound meizothrombin, a derivative of <em>prothrombin</em>, was determined directly using fluorescence energy transfer. The active site of <em>prothrombin</em> was exposed after a single cleavage by Echis carinatus protease in the presence of [5-(dimethylamino)-<em>1</em>-naphthalenesulfonyl]glutamylglycylarginyl+ ++ (DEGR) chloromethyl ketone to yield DEGR-meizothrombin and thereby minimize secondary proteolysis. When DEGR-meizothrombin was titrated with 80% phosphatidylcholine, <em>2</em>0% phosphatidylserine vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes in the active sites of the membrane-bound proteins and the acceptor dyes at the outer surface of the phospholipid bilayer. This energy transfer required both Ca<em>2</em>+ and phosphatidylserine. Assuming k<em>2</em> = <em>2</em>/3, the dependence of the efficiency of energy transfer upon the acceptor density showed that the distance of closest approach between the active site probe and the bilayer surface was 7<em>1</em> +/- <em>2</em> A. In the presence of factor Va, the distance was 67 +/- 3 A. These direct measurements show that the active site of meizothrombin is located far above the membrane surface. Also, association of factor Va with meizothrombin on the phospholipid surface appears to cause a slight movement of the meizothrombin protease domain toward the membrane surface. The environment of the dansyl dye covalently attached to the active site of meizothrombin was particularly sensitive to the presence of calcium: addition of Ca<em>2</em>+ ions to metal-free DEGR-meizothrombin reduced the dansyl fluorescence lifetime from <em>1</em><em>1</em>.7 to 9.0 ns and the dansyl emission intensity by <em>2</em>4%. Hence, the conformation of the active site changed when Ca<em>2</em>+ ions bound to meizothrombin. Since the intensity change was half-maximal at 0.<em>2</em> mM and was also elicited by the binding of Mg<em>2</em>+ ions, this spectral change correlates with the calcium-dependent conformational change previously observed in <em>fragment</em> <em>1</em>. We conclude, therefore, that the binding of Ca<em>2</em>+ ions to meizothrombin and, by extension, perhaps to <em>prothrombin</em>, elicits a conformational change that extends beyond the <em>fragment</em> <em>1</em> domains into the distant (cf. above) active site or protease domain. The association of factor Va with membrane-bound DEGR-meizothrombin increased both the dansyl emission intensity (by 7%) and polarization. This intensity change and the factor-Va dependent change in energy transfer indicate that the cofactor of the <em>prothrombin</em>ase complex functions to modulate the conformation and orientation of both the substrate and the enzyme of the complex.

Visfatin, a ubiquitous adipokine, was first described in <em>2</em>005. It was found to be selectively up-regulated in the adipose tissue and to have insulin-mimetic effects. It has been reported that visfatin is associated with endothelial damage in chronic kidney disease. We investigated plasma visfatin levels (using commercially available kits) in <em>1</em>00 clinically stable kidney allograft recipients. We assessed visfatin markers of coagulation: thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>; fibrinolysis: tissue plasminogen activator, plasminogen activator inhibitor, plasmin-antiplasmin complexes; endothelial function/injury: von Willebrand factor, thrombomodulin, intracellular adhesion molecule, vascular cell adhesion molecule (VCAM); inflammation: hsCRP and interleukin-6. Visfatin was significantly higher in kidney allograft recipients than in healthy volunteers. Visfatin did not differ significantly between diabetic and nondiabetics, hypertensives and normotensives, patients with and without coronary artery disease, and between male and female subjects. Type of immunosuppressive regimen (mycophenolate mofetil vs azathioprine) did not affect visfatin levels. On univariate analysis, visfatin correlated positively with <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, VCAM, creatinine, high-sensitivity C-reactive protein, and negatively with albumin. In multivariate analysis, only VCAM was associated with visfatin in kidney allograft recipients. Visfatin, which is related to markers of inflammation, may represent a novel link between inflammation and adipocytokines among long-term kidney transplant recipients.

BACKGROUND

We evaluated the newly introduced Bioline heparin coating and tested the hypothesis that surface heparinization limited to the oxygenator and the arterial filter will ameliorate systemic inflammation and preserve platelets during cardiopulmonary bypass (CPB).

METHODS

In a prospective double-blind study, <em>1</em>59 patients underwent coronary revascularization using closed-system CPB with systemic heparinization, mild hypothermia (33 degrees C), a hollow-fiber oxygenator, and an arterial filter. The patients were randomly divided in three groups. In group A (controls, n = 5<em>1</em>), surface heparinization was not used. In group B (n = 5<em>2</em>), the extracorporeal circuits were totally surface-heparinized with Bioline coating. In group C (n = 56), surface heparinization was limited to oxygenator and arterial filter.

RESULTS

No significant difference was noted in patient characteristics and operative data between groups. Operative (30-day) mortality was zero. Platelet counts dropped by <em>1</em><em>2</em>.3% of pre-CPB value among controls at <em>1</em>5 minutes of CPB, but were preserved in groups B and C throughout perfusion (p = 0.0<em>1</em><em>2</em>7). Platelet factor 4, plasmin-antiplasmin levels, and tumor necrosis factor-alpha increased more in controls during CPB than in groups B or C (p = 0.0443, p = 0.0<em>2</em>38 and p = 0.0<em>1</em>54 respectively). Beta-thromboglobulin, fibrinopeptide-A, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, factor XIIa levels, bleeding times, blood loss, and transfusion requirements were similar between groups. Intensive care unit stay was shorter in groups B and C than in controls (p = 0.037).

CONCLUSIONS

Surface heparinization with Bioline coating preserves platelets, ameliorates the inflammatory response and is associated with a reduced fibrinolytic activity during CPB. Surface heparinization limited to the oxygenator and the arterial filter had similar results as totally surface-heparinized circuits.

Unstable coronary artery disease (UCAD) is associated with an increased risk of further coronary events. In the FRISC study, the risk was decreased during treatment with a high, twice-daily, dose of dalteparin, a low-molecular-weight heparin. However, lowering the dose resulted in raised risk of recurrences. To investigate the underlying pathophysiology, the thrombin generation and activity in patients with UCAD randomized to a 6-week placebo-controlled treatment with dalteparin were evaluated. Plasma <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) (n = 34<em>2</em>), thrombin-antithrombin complex (TAT) (n = <em>1</em>86) and soluble fibrin (SF) (n = <em>2</em>98) were analyzed before and during treatment with dalteparin/placebo administered subcutaneously, <em>1</em><em>2</em>0 IU/kg bw twice daily for 5-8 days and 7.500 IU once daily the following 35-40 days. High-dose treatment with dalteparin resulted in significantly reduced levels of all coagulation markers, demonstrating diminished thrombin generation and activity. When reducing the dalteparin dose, plasma TAT and SF remained low, indicating minimal fibrin formation. However, F<em>1</em>+<em>2</em> increased during this period. though the level at day 45 was still lower than in the placebo group. In the placebo group elevated thrombin generation and activity persisted during the entire period. In conclusion, high-dose treatment with dalteparin twice daily resulted in significantly reduced thrombin generation and activity. However, after changing to a lower, once-daily dose, the treatment was not sufficient in preventing a return to a procoagulable state. These changes of the coagulation activity might explain the changes in event rate observed during dalteparin treatment.

The bleeding diathesis in patients with acute promyelocytic leukemia (APL) is generally attributed to disseminated intravascular coagulation (DIC), initiated by the release of procoagulant activity from leukemic cells. Primary fibrinogenolysis, mediated by the release of leukocyte proteases, may also contribute to this disorder. We analyzed coagulation parameters in <em>1</em>5 non-septic APL patients. Before treatment, there was evidence of thrombin activation with DIC: increased levels of circulating thrombin-antithrombin III complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and D-Dimer complexes. This DIC syndrome was probably limited, since no <em>prothrombin</em> time decrease, no significant factor V consumption, and normal levels of coagulation inhibitors (antithrombin III and protein C) were observed in APL patients when compared to normal controls. In this context, marked hypofibrinogenemia suggested primary fibrinogenolysis as the predominant etiology. Despite normal or high tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-<em>1</em>) antigen levels, the plasma PAI-<em>1</em> activity and the formation of tPA/PAI-<em>1</em> complexes were lower in APL patients than in normal controls, suggesting a proteolytic degradation of PAI-<em>1</em>, not able to complex tPA. Two other fibrinolytic inhibitor molecules (alpha-<em>2</em> plasmin inhibitor antigen and histidine-rich glycoprotein antigen) were also significantly reduced, as well as the two subunits of fibrin stability factor XIII, although only subunit A is known to be susceptible to thrombin action. Evidence of degraded forms of von Willebrand factor in the plasma suggested an extended proteolytic activity. Four patients treated with all-trans-retinoic acid (ATRA) as a single differentiating agent were studied serially. A dissociation between these two syndromes--DIC and fibrinogenolysis/proteolysis--was observed. The rapid correction of the lysis markers contrasted with a more prolonged persistence of the procoagulant activity. We observed persistently high elastase/alpha <em>1</em>-proteinase inhibitor complex levels during ATRA therapy, despite progressive correction of all lysis markers. Thus, the release of elastase from promyelocytic leukemic cells is probably not the only determinant of the fibrinogenolytic/proteolytic syndrome. In summary, the present findings provide new arguments for the association of DIC and proteolysis syndromes in APL-associated coagulation disorders. Further prospective studies are needed in order to confirm the persistence of thrombin activation in course of ATRA therapy.

Tissue factor (TF) plays a pivotal role in thrombus formation. Statins and angiotensin converting enzyme inhibitors attenuate expression of TF by distinct mechanism. Therefore, we hypothesized that combined therapy with simvastatin and ramipril may have additive beneficial anti-atherogenic effects to lower TF activity when compared with either drug alone. This was a randomized, double-blind, placebo-controlled cross-over trial with three treatment arms (each <em>2</em> months) and two washout periods (each <em>2</em> months). Fifty patients with type <em>2</em> diabetes were given simvastatin <em>2</em>0 mg and placebo, simvastatin <em>2</em>0 mg and ramipril <em>1</em>0 mg, or ramipril <em>1</em>0 mg and placebo daily during each treatment period. Simvastatin and ramipril monotherapy tended to reduce TF activity (0.53 to 0.46 nM, P=0.056; 0.54 to 0.50 nM, P=0.<em>1</em>67, respectively) while combined therapy had a significant effect (0.64 to 0.43 nM, P<0.00<em>1</em>). All three therapies significantly reduced <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) levels from their respective baselines (P=0.037, P<0.00<em>1</em>, and P=0.057, respectively). Combined therapy significantly reduced TF activity and F<em>1</em>+<em>2</em> levels to a greater extent than either simvastatin or ramipril alone (P=0.0<em>2</em>9 and P=0.040 by ANOVA, respectively). Percent changes in TF activity and percent changes in F<em>1</em>+<em>2</em> levels were significantly correlated. All three therapies reduced CD40 ligand levels from their respective baselines (P=0.098, P<0.00<em>1</em>, and P=0.00<em>2</em>, respectively) with no significant differences among these three therapies (P=0.<em>2</em>04 by ANOVA). Ramipril combined with simvastatin significantly reduces plasma TF activity and F<em>1</em>+<em>2</em> levels to a greater extent than monotherapy with either drug in patients with type <em>2</em> diabetes.

BACKGROUND

Leptin, a hormone secreted by the adipose tissue, might be a link between obesity and increased morbidity for cardiovascular disease. Leptin exerts proinflammatory, pro-angiogenic actions by activating a specific receptor (Ob-Rb) which is expressed in human endothelial cells. Thus, a link may exist between leptin expression and endothelial dysfunction.

OBJECTIVE

We sought to determine whether in obese women there is a correlation between leptin levels, endothelial perturbation and coagulative activation.

METHODS

Circulating levels of leptin, von Willebrand Factor (VWF), factor (F)VIIa, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>), were measured in 5<em>1</em> non-diabetic, obese women and in 5<em>1</em> normal-weight subjects, using immunoenzymatic assays.

RESULTS

Obese women had significantly higher levels of leptin, VWF, FVIIa, F<em>1</em>+<em>2</em> compared with healthy women. Simple correlation coefficients showed significant correlation between leptin and either VWF, FVIIa, or F<em>1</em>+<em>2</em> concentrations. A multiple linear regression analysis, performed to quantify further the relationship between leptin levels and the above-mentioned variables as well as the inflammatory marker C-reactive protein (CRP) and including age, body mass index (BMI), waist-hip ratio (WHR) and lipid parameters as potential confounders, revealed that only FVIIa and VWF were independently related to leptin levels. Reduction in adipose tissue after weight loss resulted in a decrease in both circulating leptin and endothelial and coagulative activation markers.

CONCLUSIONS

We suggest that leptin might have pro-atherogenic effects in vivo, with a mechanism involving endothelial cell activation.

BACKGROUND

The activation of plasma enzyme systems contributes to hereditary angioedema attacks. We aimed to study the activation markers of the fibrinolytic, coagulation, and contact systems in a larger number of paired samples obtained from the same C<em>1</em>-INH-HAE patients in symptom-free periods and during attacks.

METHODS

Eleven parameters (Factors XI, XII, and C<em>1</em>-inhibitor activity; the concentrations of the D-dimer, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, plasminogen, plasminogen activator inhibitor-<em>1</em> [PAI-<em>1</em>], thrombin-anti-thrombin III [TAT] complex, fibrinogen) were measured along with <em>prothrombin</em> time and activated partial thromboplastin time (aPTT), using commercial kits. We compared these markers in samples obtained from the same 39 patients during attack-free periods and during 6<em>2</em> edematous episodes. Forty healthy subjects of matching sex and age served as controls.

RESULTS

Compared with the healthy controls, significantly higher FXI and FXII activity (p = 0.0007, p = 0.005), as well as D-dimer (p < 0.000<em>1</em>), <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (p < 0.000<em>1</em>), and TAT (p = 0.0303) levels were ascertained in the patients during symptom-free periods. The evaluation of samples from symptom-free periods or obtained during attacks revealed the increase of FXII activity, as well as of the concentration of D-dimer, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, and TAT during edematous episodes. PAI-<em>1</em> level, <em>prothrombin</em> time, and aPTT decreased significantly during attacks, compared with symptom-free periods. D-dimer level was significantly higher during multiple- vs. single-site attacks.

CONCLUSIONS

Comparing a large number of paired samples from symptom-free periods or from edematous episodes allowed accurate appraisal of the changes occurring during attacks. Moreover, our study pointed out that individual episodes may be characterized by different marker patterns.

Cancer is often associated with abnormal activation of coagulation leading to a prothrombotic state. Some chemotherapeutic agents used for cancer may induce thrombosis but their biological alterations in the hemostatic system are not yet well understood. This study evaluated alterations of coagulative and fibrinolytic parameters following chemotherapy. In plasma samples of 38 patients (median age: 49 years) receiving CMF (schedule <em>1</em>-<em>2</em><em>1</em> or <em>1</em>-8) for Stage II breast cancer, we evaluated: PT, aPTT, antithrombin III (AT-III), protein C (PC), protein S (PS), thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragment</em> F <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>), fibrinogen (Fbg), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-<em>1</em>) and D-dimer (D-D). PT, aPTT, and Fbg were determined with routine methods; AT-III, PC, and PS were measured with coagulative tests; PC and PS were also evaluated with immunoenzymatic methods, t-PA, PAI-<em>1</em>, D-D, TAT, and F <em>1</em> + <em>2</em> were measured with immunoenzymatic methods. All tests were performed immediately before starting therapy and after each cycle. A PC antigen decrease appeared soon after beginning therapy and lasted throughout chemotherapy. The lowest values were present after the first treatment both in the CMF <em>1</em>-<em>2</em><em>1</em> group (mean +/- SD = 7<em>2</em>.5 +/- <em>1</em>0.8%) and in the CMF <em>1</em>-8 group (mean +/- SD = 77.<em>2</em> +/- 6.9%): PC activity was also decreased. PS antigen decreased after the first administration (mean +/- SD = 73.3 +/- <em>1</em>0% in CMF <em>1</em>-<em>2</em><em>1</em> group, and 7<em>2</em>.5 +/- 4.9% in CMF <em>1</em>-8 group): PS activity also decreased. PAI-<em>1</em> antigen levels increased (mean +/- SD = 43.<em>1</em> +/- <em>2</em>0.4 ng/ml in the CMF <em>1</em>-<em>2</em><em>1</em> group, and 37.5 +/- <em>1</em><em>2</em>.<em>2</em> ng/ml in CMF <em>1</em>-8 group) lasting up to the last cycle. CMF provokes a trend toward hypercoagulability; this effect should be considered when chemotherapy is employed in advanced cancer patients at high risk for thrombosis, or in patients with other risk factors.

OBJECTIVE

to determine whether Behçet's disease affects haemostatic function.

METHODS

University Hospital, Turkey.

METHODS

one hundred and twenty-seven consecutive patients with Behçet's disease, 34 of whom with a history of vascular involvement.

METHODS

<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> tissue plasminogen activator, protein S and C, antithrombin, fibrinogen, von Willebrand factor, thrombomodulin and <em>prothrombin</em> time (PT) were measured in patient plasma.

RESULTS

soluble thrombomodulin was significantly lower and von Willebrand factor (vWF) and tissue plasminogen activator (tPA) significantly higher in Behçet's patients. Patients with vascular involvement showed the highest levels of vWF and tPA. There was no activation of coagulation, not even in patients with an active disease at the time of sampling.

CONCLUSIONS

there were indirect signs of endothelial activity or damage, particularly in patients with vascular involvement. Coagulation was not activated.

Bothrojaracin (BJC) is a <em>2</em>7 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human <em>prothrombin</em> (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci <em>2</em>00<em>1</em>; <em>1</em>0: <em>1</em>897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable <em>1</em>:<em>1</em>, non-covalent complex with the zymogen whereas no interaction with <em>fragment</em> <em>1</em> or <em>2</em> domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human <em>prothrombin</em> activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, <em>2</em>5% phosphatidylserine), BJC also inhibited (35%) <em>prothrombin</em> activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by <em>prothrombin</em>ase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of <em>prothrombin</em> by platelet-assembled <em>prothrombin</em>ase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of <em>prothrombin</em> as substrate by factor Xa-factor Va complex and <em>prothrombin</em>ase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of <em>prothrombin</em>.

OBJECTIVE

Venous lesions, including obstruction and thromboembolism (VTE), are not uncommon after pacemaker implantation. The purpose of this prospective study was to assess the role of various patient and procedure-related risk factors in the development of these complications.

RESULTS

A prospective venography-based study of <em>1</em>50 consecutive pacemaker implantations with a 6-month follow-up was conducted. Current case-control study included all cases (n = 47) with a new venous lesion, and their matched controls. Several surgical and technical factors, i.e. lead burden, choice of venous access, operator experience and procedure duration, as well as patient-related classic risk factors of VTE were assessed. Plasma markers of coagulation and endothelial activation [<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), D-dimer (DD), von Willebrand factor (vWF), thrombomodulin (Tm)] were used to evaluate the extent of acute surgical trauma. All cases with venous lesions were also screened for thrombophilia. None of the procedure-related variables were predictive of VTE. Mean levels of vWF, F<em>1</em> + <em>2</em> and DD increased significantly (P < 0.00<em>1</em>) and equally in both cases and controls. No single clinical factor predicted venous lesions, but significant (P < 0.05) clustering of classic clinical VTE risk factors was seen among the cases. Thrombophilia was overrepresented in patients with symptomatic pulmonary embolism (<em>2</em>/5, 40%).

CONCLUSIONS

Pacemaker implantation induces a transient hypercoagulable state, but its degree does not predict subsequent venous thromboembolism, and neither did the grade of endothelial damage as reflected by plasma markers. The aetiology of these lesions seems to be multifactorial, and clustering of classic thrombotic risk factors plays a role in the pathogenesis.

Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human <em>prothrombin</em> (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC <em>fragment</em>, SC-(<em>1</em>-3<em>2</em>5), bound to human prethrombin <em>2</em> showed that the SC-(<em>1</em>-3<em>2</em>5) N terminus inserts into the Ile(<em>1</em>6) pocket of prethrombin <em>2</em>, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(<em>1</em>-3<em>2</em>5) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the approximately <em>2</em>-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(<em>1</em>-3<em>2</em>5).human (pro)thrombin complexes, SC-(<em>1</em>-3<em>2</em>5).bovine ProT shows a 3,500-fold lower k(cat)/K(m) compared with free bovine thrombin, because of a 47-fold increase in K(m) and a 67-fold decrease in k(cat). The SC-(<em>1</em>-3<em>2</em>5).bovine ProT complex is approximately 5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(<em>1</em>-3<em>2</em>5).(pro)thrombin complexes indicates that the species specificity of SC-(<em>1</em>-3<em>2</em>5) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(<em>1</em>-3<em>2</em>5).bovine ProT complex is incompletely formed. The current crystal structure of SC-(<em>1</em>-3<em>2</em>5).bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg(<em>1</em>44) and Arg(<em>1</em>45) would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.

To investigate how cigarette smoking increases the risk of cardiovascular disease, risk factors were compared between <em>1</em>66 cigarette smokers and 3<em>1</em><em>2</em> non-smokers, in a random sample of males (Chinese, Malays and Asian Indians) aged 30-69 years from the general population of Singapore. There was adjusted for age and ethnic group. The prevalence of hypertension was lower in cigarette smokers (<em>1</em>5.<em>2</em>%) than non-smokers (<em>2</em><em>1</em>.9%), with the difference reduced by adjustment for body mass index (BMI). Smokers had: lower mean serum HDL-cholesterol (0.76 versus 0.8<em>1</em> mmol/l) and higher mean serum fasting triglyceride (<em>1</em>.9<em>2</em> versus <em>1</em>.7<em>1</em> mmol/l), which will increase atherosclerosis; higher mean plasma fibrinogen (<em>2</em>.75 versus <em>2</em>.67 g/l) and plasminogen activator inhibitor <em>1</em> [PAI-<em>1</em>] (<em>2</em>4.9 versus <em>2</em><em>2</em>.<em>2</em> ng/ml), which will increase thrombosis; and lower mean plasma vitamin C (4.4 versus 6.4 mg/l) and serum selenium (<em>1</em><em>1</em>8 versus <em>1</em><em>2</em>3 microg/l), which may increase atherosclerosis. Adjustment for BMI slightly increased the differences for HDL-cholesterol, fasting triglyceride, fibrinogen and PAI-<em>1</em>, indicating that less generalised obesity among smokers reduces their increased cardiovascular disease risk. Smoking was not found to be related to: diabetes mellitus; serum total cholesterol, LDL-cholesterol, apolipoproteins A<em>1</em> and B and lipoprotein(a); plasma factor VIIc and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>; and plasma vitamins A and E and serum ferritin. There was no evidence of increased insulin resistance in smokers, as measured by mean fasting serum insulin.

OBJECTIVE

Coagulation factor XIII is a plasma transglutaminase involved in crosslinking of fibrin, the last step of the coagulation system and a connective tissue factor contributing to the wound healing process. It circulates as a heterotetrameric molecule consisting of two identical proenzyme subunits (factor XIIIA) and two carrier protein subunits (factor XIIIS). The aim of this study was to determine the disease features associated with the diminution of factor XIII in Crohn's disease.

METHODS

Factor XIIIA and factor XIIIS levels were assessed in patients presenting with Crohn's disease, ulcerative colitis, infectious colitis, or diverticulitis, in patients with rheumatoid arthritis, and in control subjects. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> assay, as a marker of the generation of thrombin and measurement of C-terminal telopeptide of type I collagen as an estimate of degradation of collagen type I, were performed.

RESULTS

Factor XIIIA was significantly decreased in Crohn's disease, in ulcerative colitis, and in infectious colitis by comparison with subjects presenting with diverticulitis, normal, and rheumatoid subjects p = 0.000<em>1</em>). Factor XIIIS was unmodified in patients with Crohn's disease by comparison with controls but was reduced in those presenting with intestinal bleeding (p = 0.000<em>2</em>). In Crohn's disease, the lowest level of factor XIIIA was observed in patients with intestinal bleeding (p = 0.0003). Factor XIIIA was correlated with the Van Hees index (r = -0.566<em>1</em>; p = 0.000<em>1</em>) and with the C-terminal telopeptide of type I collagen (r = -0.4<em>1</em><em>1</em>0; p = 0.00<em>1</em><em>1</em>) but not with prothrombin <em>fragment</em> <em>1</em> + <em>2</em>. The multiple regression analysis showed that only Van Hees index and intestinal bleeding were independent variables for explaining the diminution of Factor XIIIA in Crohn's disease.

CONCLUSIONS

Factor XIIIA subunit is an indicator of Crohn's disease activity. Our study suggests that a low factor XIIIA level is related to the presence of intestinal lesions and might be linked to intestinal repair mechanisms; loss in intestinal lumen could be also involved, especially in patients with intestinal bleeding.

BACKGROUND

The incidence of ischemic heart disease (IHD) in Crete was lower than expected on the basis of blood lipid concentrations of participants in the Seven Countries Study. A favorable effect of a high intake of olive oil on thrombogenesis may have contributed to this finding.

OBJECTIVE

We compared the effects of virgin olive oil with those of rapeseed and sunflower oils on blood coagulation factor VII (FVII), a key factor in thrombogenesis.

METHODS

In a randomized and strictly controlled crossover study, <em>1</em>8 healthy young men consumed diets enriched with 5 g/MJ (<em>1</em>9% of total energy) olive oil, sunflower oil, or rapeseed oil for periods of 3 wk. On the final day of each period, participants consumed standardized high-fat meals (4<em>2</em>% of energy as fat). Fasting and nonfasting blood samples were collected after each period.

RESULTS

Mean (+/-SEM) nonfasting peak concentrations of activated FVII (FVIIa) were <em>1</em><em>1</em>.3 +/- 5.<em>1</em> U/L lower after olive oil than after sunflower oil, an <em>1</em>8% reduction (P < 0.05). Olive oil also tended to cause lower FVIIa peak concentrations than did rapeseed oil (mean difference: 8.6 U/L, a <em>1</em>5% reduction; P = 0.09). There were no significant differences between diets with respect to nonfasting factor VII coagulant activity (FVII:c), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), and tissue factor pathway inhibitor (TFPI) concentrations, or with respect to fasting plasma values of FVII protein, FVII:c, FVIIa, F<em>1</em>+<em>2</em>, or TFPI.

CONCLUSIONS

A background diet rich in olive oil may attenuate the acute procoagulant effects of fatty meals, which might contribute to the low incidence of IHD in Mediterranean areas.