OBJECTIVE

Patients with unstable coronary disease have changes in the hemostatic system. These patients are often treated with low molecular weight heparin. In patients who are accepted for coronary artery bypass grafting, treatment with low molecular weight heparin is frequently continued until surgery. We hypothesized that in coronary artery bypass grafting, the hypercoagulable state seen in unstable patients persists into the intra- and postoperative phase despite preoperative treatment with low molecular weight heparin. The aim of this study was to explore and describe the perioperative hemostatic process in patients with unstable coronary artery disease undergoing coronary artery bypass grafting.

METHODS

Thirty-two patients with unstable coronary disease treated preoperatively with enoxaparin, and 3<em>2</em> stable control patients not treated with enoxaparin, were included. All patients were taking low dose aspirin until the day before surgery. Before cardiopulmonary bypass, all patients were given tranexamic acid as a bolus injection. Blood samples for analysis of platelet counts, international normalized ratio, activated partial thromboplastin time, fibrinogen, protein S, protein C, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complex, antithrombin, plasmin-antiplasmin complex, D-dimer, neutrophil-activating peptide <em>2</em>, platelet-monocyte complexes, and heparin concentrations were drawn preoperatively, after 30 min on cardiopulmonary bypass, and 30 min, 3 h, and <em>2</em>0 h postoperatively. Heparin was given during cardiopulmonary bypass to maintain an activated clotting time above 480 s.

RESULTS

Patients in the enoxaparin group needed more heparin to maintain an activated clotting time above 480 s, and had higher heparin concentrations and lower antithrombin values compared with control patients. Neutrophil-activating peptide <em>2</em> concentrations were higher in the enoxaparin group.

CONCLUSIONS

Patients treated with enoxaparin before coronary artery bypass grafting showed signs of heparin resistance intraoperatively. Enoxaparin-treated patients also had increased perioperative platelet activation. Reasons for the observed difference in platelet activation remain unclear.

Thrombolytic therapy activates the contact system, and factor XII activation may activate the coagulation cascade and inflammation. It is not known whether an early inflammatory response is induced by thrombolytic therapy in patients with acute myocardial infarction (AMI). We prospectively measured the plasma levels of activated factor XII, cleaved kininogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (as indexes of the contact phase and coagulation activation), and interleukin-6 and C-reactive protein (CRP) (as indexes of inflammation) in 39 patients hospitalized for AMI within <em>1</em><em>2</em> hours of symptom onset: <em>2</em>6 receiving thrombolytic therapy and <em>1</em>3 heparin alone. Blood samples were collected at baseline and after 90 minutes and <em>2</em>4 hours. Patients undergoing thrombolysis had a significant early increase in activated factor XII (from <em>2</em>.<em>2</em> ng/ml at baseline to 4.7 ng/ml after 90 minutes; p = 0.000<em>1</em>), cleaved kininogen (from <em>2</em>6% to 37%; p = 0.00<em>1</em>), and <em>fragment</em> <em>1</em> + <em>2</em> (from <em>1</em>.4 to <em>2</em>.<em>1</em> nmol/L; p = 0.000<em>1</em>), whereas the <em>2</em>4-hour levels were similar to baseline levels. The levels of interleukin-6 significantly increased during the first 90 minutes (from 3.9 to 6.3 microg/ml; p = 0.00<em>1</em>), and were even higher after <em>2</em>4 hours (<em>1</em><em>1</em>.9 ng/ml, p = 0.000<em>1</em>). CRP levels increased only after <em>2</em>4 hours (p = 0.000<em>1</em>). There were no changes in these parameters in patients receiving heparin alone, except for a <em>2</em>4-hour increase in interleukin-6 and CRP levels. Thus, in patients with AMI receiving thrombolytic therapy, early activation of inflammation parallels the activation of the contact system and the coagulation cascade, which might contribute to microvascular obstruction and reperfusion injury.

OBJECTIVE

The purpose of this study was to determine whether the elevated levels of hemostatic markers in the early phase of myocardial infarction may serve as risk factors for subsequent cardiac mortality.

BACKGROUND

Increased plasma hemostatic markers were noted in acute myocardial infarction, indicating that the blood coagulation system is highly activated in those patients. However, there are few clinical data concerning the association between the elevated hemostatic markers and survival in patients with myocardial infarction.

METHODS

Blood samples were obtained from 64 patients (mean age 67 +/- <em>1</em><em>1</em> years; 49 male) with acute myocardial infarction within <em>1</em><em>2</em> h after the onset of symptoms and before the initiation of any antithrombotic treatment. We measured plasma concentrations of fibrinopeptide A (FPA), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complex (TAT) using the enzyme-linked immunosorbent assay method, and examined the associations between the level of these markers and survival with Cox proportional hazards models.

RESULTS

The follow-up time was <em>2</em>7 +/- <em>1</em>7 months, and <em>1</em>9 patients died of cardiac causes during the follow-up. Univariate survival analysis identified Killip class IV (hazard ratio 4.86; 95% confidence interval [CI] <em>1</em>.55-<em>1</em>5.<em>1</em>9), left ventricular ejection fraction (hazard ratio 0.94; 95% CI 0.90-0.99), FPA (hazard ratio <em>1</em>.54; 95% CI <em>1</em>.<em>1</em>3-<em>2</em>.<em>1</em>0), F<em>1</em>+<em>2</em> (hazard ratio <em>2</em>.03; 95% CI <em>1</em>.<em>1</em>7-3.53) and TAT (hazard ratio <em>1</em>.88; 95% CI <em>1</em>.<em>2</em>7-<em>2</em>.79) as significant factors associated with cardiac mortality. In multivariate analyses, only FPA level (hazard ratio <em>1</em>.84; 95% CI <em>1</em>.03-3.30) and left ventricular ejection fraction (hazard ratio 0.93; 95% CI 0.88-0.98) were independent predictors of cardiac mortality.

CONCLUSIONS

Elevated FPA in the early phase of myocardial infarction identifies patients with increased risk for subsequent cardiac death. This association appears to be independent of residual left ventricular function after infarction.

This study examines the evolution of the thrombotic activity in patients with myocardial infarction (MI) treated with aspirin (<em>2</em>00 mg/day) for <em>2</em> years after MI. Plasma samples of <em>1</em>0 patients were collected at 7, 30, 60, 90, <em>1</em><em>2</em>0, <em>1</em>50, <em>1</em>80, 360 and 7<em>2</em>0 days. In all the samples we measured fibrinogen (Fg), high molecular weight Fg (HMW-Fg), fibrinopeptide A (FPA), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), beta-thromboglobulin (beta-TG), von Willebrand factor (vWF), tissue factor (TF) and TF pathway inhibitor (TFPI). The plasma Fg, HMW-Fg, FPA, F<em>1</em>+<em>2</em>, beta-TG and vWF levels were significantly elevated in the patients at the beginning of the study as compared to the normal group. The 95% confidence intervals were Fg <em>2</em>77-333 mg/dl, HMW-Fg <em>2</em>00-<em>2</em>44 mg/dl, FPA 5.3-<em>1</em>6.5 ng/ml, F<em>1</em>+<em>2</em> <em>1</em>.4-<em>1</em>.8 nmol/l, beta-TG <em>1</em><em>1</em>0-<em>1</em><em>1</em>8 IU/ml and vWF <em>1</em>39-<em>1</em>95%. At thirty days Fg and HMW-Fg returned to normal levels, whereas the increase in FPA and F<em>1</em>+<em>2</em> levels persisted throughout the study. At <em>1</em><em>2</em>0 and <em>1</em>50 days, respectively, beta-TG and vWF returned to normal levels. The increase in thrombin generation and activity pointed to a persistent hypercoagulable state <em>2</em> years after MI. Plasma levels of TF and TFPI showed no statistically significant variations with respect to the normal values over the <em>2</em>-year period studied. In conclusion, these results suggest a persistent generation and activity of thrombin and cellular activation in these patients after MI.

Plaque rupture with the exposure of a tissue factor-rich procoagulant surface is considered the common pathogenetic mechanism of unstable angina and myocardial infarction. Activated factor VII, the key enzyme for initiating blood coagulation under resting conditions, is increased in pathological situations associated with tissue factor exposure. We measured the plasma levels of activated factor VII and studied their relation with signs of coagulation enzyme activity in patients with acute coronary syndromes. The plasma levels of activated factor VII, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and fibrinopeptide A were measured on admission in consecutive patients presenting with acute myocardial infarction (n = <em>2</em>8), unstable angina (n = 3<em>2</em>), and stable angina (n = <em>1</em>7) and in age- and sex-matched healthy individuals (n = 33). Plasma determinations of the same markers were also repeated at <em>1</em>5 days and 3 and 6 months. On admission, the patients with unstable angina or myocardial infarction had significantly higher plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (P < .000<em>1</em>) and fibrinopeptide A (P < .000<em>1</em>) than those with stable angina or healthy individuals, whereas no differences were detected in the plasma levels of activated factor VII. During follow-up there was a significant decrease in the plasma levels of fibrinopeptide A both in patients with unstable angina (P < .00<em>1</em>) and in those with myocardial infarction (P < .00<em>1</em>), whereas no changes in plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> or activated factor VII levels were observed. Hence, in the acute and chronic phases of myocardial infarction and unstable angina, heightened coagulation enzyme activity is not accompanied by an increase in activated factor VII.

BACKGROUND

The association between intravascular ultrasound (IVUS) signs of plaque instability and plasma levels of biomarkers was determined in patients with unstable angina and non-ST-segment elevation myocardial infarction (UA/NSTEMI).

METHODS

Fifty-two patients underwent coronary angiography and IVUS 8 +/- 5 hours after the onset of chest pain. IVUS analysis included plaque morphology, disruption, thrombi and eccentricity, lumen, external elastic membrane, and plaque plus media areas of culprit lesion and reference segments and arterial remodeling. Plasma levels of the thrombin activation system (thrombin-antithrombin complex [TAT], tissue factor pathway inhibitor [TFPI], and <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>]) and plasmin activation system (tissue and urokinase-type plasminogen activator [t-PA and u-PA], plasminogen activator inhibitor-<em>1</em> [PAI-<em>1</em>], and D-dimer) were measured with enzyme-linked immunosorbent assay kits before angiography.

RESULTS

Elevated levels of TAT (7.<em>2</em> +/- 6.0 microg/L), F<em>1</em>+<em>2</em> (<em>1</em>.8 +/- <em>1</em>.0 nmol/L), TFPI (<em>1</em>79.<em>1</em> +/- <em>1</em>3<em>1</em>.0 ng/mL), PAI-<em>1</em> (95.4 +/- 54.6 ng/mL), t-PA (<em>1</em>0.6 +/- 8.8 ng/mL), and u-PA (<em>2</em>.6 +/- 0.9 ng/mL) were found in patients with UA/NSTEMI. The serum levels of D-dimer (40.0 +/- 39.5 ng/mL) remained in reference range. Expansive and constrictive remodeling were found in <em>1</em>8 (35%) and <em>1</em><em>2</em> (<em>2</em>3%) patients, respectively. Expansive remodeling of the culprit lesion was associated with significantly higher plasma levels of PAI-<em>1</em> (<em>1</em><em>2</em><em>1</em>.6 +/- 55.0 vs 87.7 +/- 6<em>1</em>.5 and 77.4 +/- 4<em>2</em>.8 ng/ml, P =.039), and u-PA (3.0 +/- <em>1</em>.<em>2</em> vs <em>2</em>.<em>2</em> +/- 0.5 and <em>2</em>.5 +/- 0.7 ng/mL, P =.0<em>2</em>6) as compared with constrictive and neutral remodeling. Increased plasma levels of u-PA were associated with plaque rupture (3.0 +/- 0.7 vs <em>2</em>.5 +/- 0.9 ng/mL, P =.06<em>2</em>). Plasma levels of PAI-<em>1</em> and u-PA correlated positively with plaque plus media (P =.0<em>2</em>97 and P =.0093) and external elastic membrane areas (P =.0<em>1</em>0 and P =.000<em>2</em>).

CONCLUSIONS

Elevated levels of biomarkers of plasmin activation system are associated with signs of plaque instability of culprit lesion in UA/NSTEMI and might therefore serve as non-invasive determinants of the population that is at high risk for subsequent adverse events.

OBJECTIVE

The risk of stroke caused by a symptomatic high-grade carotid stenosis (CS) is high. Disturbed balance between the procoagulant and fibrinolytic activity in blood associated with unfavorable hemorheology could render CS symptomatic. We wanted to assess whether hemostatic and fibrinolytic plasma markers as well as basic indicators of hemorheology differentiate asymptomatic and symptomatic patients with a high-grade CS and whether they are associated with the macroscopic appearance of the plaque and the rate of microembolization.

METHODS

We recruited 9<em>2</em> consecutive consenting patients referred to the neurological or the surgical department of our university teaching hospital for treatment of their high-grade CS. Blood samples were collected before surgery for determination of <em>prothrombin</em> <em>fragments</em> F<em>1</em> and <em>2</em>, thrombin-antithrombin complex, tissue-type plasminogen activator (tPA) activity and antigen, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) activity and antigen, D-dimer, homocysteine, fibrinogen, in plasma, and hematocrit in blood, and the patients underwent transcranial Doppler ultrasonology for evaluation of microembolic signals (MES).

RESULTS

Patients with symptomatic plaques had higher hematocrit levels (p = 0.04), as well as trends for higher tPA antigen and MES rate (p = 0.07). Hematocrit, tPA antigen, and PAI-<em>1</em> antigen and activity were positively correlated with the degree of stenosis. Ulceration was more common in symptomatic plaques but did not reflect variables of hemostasis or fibrinolysis. In multivariate analysis, tPA antigen and hematocrit were risk factors for a symptomatic high-grade stenosis.

CONCLUSIONS

Mediators of fibrinolysis and unfavorable hemorheology may contribute to the development of a symptomatic disease in patients with a high-grade CS.

OBJECTIVE

Acute traumatic coagulopathy is characterised by fibrinolysis and low fibrinogen. It is unclear how much fibrinogenolysis contributes to reduce fibrinogen levels. The study aim was to: investigate in vitro the effects of tissue-plasminogen activator (t-PA) and tranexamic acid (TXA) on coagulation and fibrinolysis.

METHODS

Whole blood was spiked with varying t-PA concentrations. Clauss fibrinogen levels and thrombelastography (TEG, Haemonetics) were performed, including functional fibrinogen level (FLEV). TXA effects were assessed using four TXA concentrations. Recorded parameters from kaolin activated TEG included maximal amplitude (MA), clot strength (G), percentage lysis (LY). Plasmin-antiplasmin complex (PAP), endogenous thrombin potential (ETP), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (PF<em>1</em>+<em>2</em>), factor V and factor VIII levels were all measured.

RESULTS

t-PA induced fibrinolysis: it increased PAP and LY, but decreased MA and G. t-PA induced fibrinogenolysis, with a concentration-dependant decrease in fibrinogen from <em>2</em>.7 (<em>2</em>.6-3.<em>1</em>) to 0.8 (0.8-0.9) g/L with 60 nM t-PA. FLEV and fibrinogen levels were well correlated. High t-PA doses increased PF<em>1</em>+<em>2</em>, decreased ETP of <em>1</em>9% and FVIII of 63% but not FV. TXA had no effect on plasmin generation as evidenced by no change in PAP. It corrected LY, MA and G and partly protected fibrinogen against fibrinogenolysis: 0.03 mg/mL TXA reduced the fibrinogen fall induced by t-PA <em>2</em>0 nM from 43% to <em>1</em>4%. TXA halved the FVIII fall and increased ETP.

CONCLUSIONS

t-PA induced plasminogen activation and fibrinogenolysis in a concentration-dependant manner. TXA did not affect plasmin activation but reduced fibrinogenolysis. These results suggest that TXA given early in bleeding patients may prevent fibrinogenolysis.

OBJECTIVE

Vital exhaustion (VE) predicts a first myocardial infarction (MI) and new cardiac events after a coronary angioplasty. To explore potential underlying pathophysiological mechanisms, we tested whether VE is associated with more pronounced diurnal variations in hemostasis.

METHODS

Blood was drawn from <em>2</em>9 VE and 30 control males, all healthy and nonsmokers, to assess hemostatic measures at 7:00 AM and 6:00 PM.

RESULTS

All measures of fibrinolysis were in their normal range and showed significant diurnal variations. These variations were more pronounced in VE as all fibrinolytic measures were significantly higher in VE at 7:00 AM and similar to those of controls at 6:00 PM, thus supporting our hypothesis with respect to fibrinolysis. Diurnal decreases in tPA and tPA-PAI ranged from <em>1</em>.5 (VE) to <em>1</em>.3 (controls), whereas the diurnal decrease in PAI-<em>1</em> was more than fourfold in VE and <em>2</em>.7-fold in controls. This suggests a decreased fibrinolytic capacity in VE during the early morning. All coagulation measures were in their normal range, and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complexes, and activated factor VII showed significant diurnal variations. These variations were similar in VE and control individuals, thus not supporting our hypothesis with respect to coagulation. Finally, F<em>1</em>+<em>2</em> and fibrinogen were both significantly higher throughout the day in VE.

CONCLUSIONS

VE is associated with decreased early morning fibrinolysis and increased fibrinogen levels throughout the day. These hemostatic changes may promote thrombus formation and provide a potential pathophysiological mechanism by which VE is related to MI and its circadian variation.

The effect of high-dose aprotinin treatment on hemostatic activation during cardiopulmonary bypass in pediatric patients having cardiac operations was investigated. Sixty patients weighing less than <em>1</em>0 kg undergoing cardiac operations for different types of congenital heart diseases were studied: <em>2</em>0 patients were treated with aprotinin <em>2</em> x <em>1</em>5,000 KIU/kg, <em>2</em>0 patients with <em>2</em> x 30,000 KIU/kg, and <em>2</em>0 patients without aprotinin treatment served as the control group. Different split products of fibrinogen and/or fibrin and the fibrinolytic activity on fibrin plates were measured to assess fibrinolytic activation. F<em>1</em>/F<em>2</em> <em>prothrombin</em> <em>fragments</em>, thrombin-antithrombin III-complex, and fibrin monomers were measured to estimate thrombin activation. There was a significant dose-dependent reduction in fibrin-fibrinogen split product formation during cardiopulmonary bypass: In the high-dose aprotinin group the concentration of the split products at the end of bypass was <em>1</em>.5 +/- 0.6 micrograms/ml, compared with 3.4 +/- 3.0 micrograms/ml in the low-dose aprotinin group and 6.7 +/- 3.5 micrograms/ml in the control group (p < 00.5). Fibrinolytic activation on fibrin plates was also significantly reduced by aprotinin. Fibrin monomer formation was significantly diminished at the end of cardiopulmonary bypass in the high-dose group: 9.<em>2</em> +/- 5.<em>2</em> micrograms/ml compared with <em>2</em><em>1</em>.6 +/- <em>1</em>4 micrograms/ml in the control group (p < 00.5). Elastase in complex with alpha <em>1</em>-protease inhibitor at the end of bypass was increased to the same amount in the three groups: 784 +/- <em>2</em>78 ng/mL (control group), 693 +/- <em>1</em>89 ng/ml (low-dose aprotinin), and 7<em>1</em>9 +/- <em>2</em>70 ng/mL (high dose aprotinin) (no significant difference). Blood loss 6 hours postoperatively was significantly (p < 00.5) less in the high-dose group (99 +/- 3<em>2</em> ml/m<em>2</em>) than in the control group (<em>1</em>64 +/- 87 ml/m<em>2</em>; low-dose group: <em>1</em>60 +/- <em>1</em>06 ml/m<em>2</em>). These observations suggest an attenuation of hemostatic activation during cardiopulmonary bypass with less plasmin formation and, because of inhibition of contact activation, less thrombin generation with aprotinin treatment. Thus the thrombotic-thrombolytic equilibrium is kept more balanced after cardiopulmonary bypass. High-dose aprotinin treatment is recommended for pediatric patients undergoing cardiac operations.

Thrombin is a coagulation protease that activates platelets, endothelial cells, leukocytes and mesenchymal cells. Thrombin signaling is mediated at least in part by protease-activated receptors (PARs). As little is known about the in vivo regulation of PAR<em>1</em>, this study aimed to characterize the effects of systemic thrombin formation during human endotoxemia on the regulation of PAR<em>1</em> and the associated responsiveness of human platelets to thrombin receptor activating peptide (TRAP). Endotoxin (<em>2</em> ng/kg) was infused into 40 healthy men to study the regulation of PAR<em>1</em> in systemic human inflammation. The SPAN<em>1</em><em>2</em> antibody was used to determine the in vivo regulation of PAR<em>1</em>. To measure whether modulation of the PAR<em>1</em> receptor may be associated with altered platelet reactivity, whole blood was stimulated with TRAP ex vivo. Thrombin generation was determined by <em>prothrombin</em> (F(<em>1</em>+<em>2</em>)) <em>fragment</em>. F(<em>1</em>+<em>2</em>) levels increased almost 9-fold from 0.5+/-0.<em>1</em> nmol/L to 4.5+/-<em>1</em>.9 nmol/L at 4 h (p<0.00<em>1</em>). PAR<em>1</em> decreased by approximately 8% (p<0.00<em>1</em>) within <em>2</em> h after endotoxin infusion and stayed at those levels until 6 h. Concomitantly, TRAP induced P-selectin expression maximally decreased by <em>1</em>8% (p<0.00<em>1</em>) at 6 h. In conclusion, PAR<em>1</em> expression is down-regulated on platelets during systemic thrombin formation induced by inflammation in humans which results in decreased responsiveness to subsequent stimulation of the PAR<em>1</em> receptor.

Factor VII-activating protease (FSAP) is involved in haemostasis and inflammation. FSAP cleaves single chain urokinase-type plasminogen activator (scu-PA). The <em>1</em>60<em>1</em>GA genotype of the <em>1</em>60<em>1</em>G/A polymorphism in the FSAP gene leads to the expression of a FSAP variant with reduced ability to activate scu-PA, without affecting the ability to activate coagulation Factor VII (FVII). Previous studies have investigated the association of the <em>1</em>60<em>1</em>GA genotype with incidence and progression of carotid stenosis and deep venous thrombosis (DVT). The present study is the first to evaluate the potential association between the FSAP phenotype and DVT. We studied the association between the <em>1</em>60<em>1</em>G/A polymorphism, FSAP activity, FSAP antigen, Factor VIIa (FVIIa), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), and C-reactive protein (CRP) in plasmas of <em>1</em>70 patients suspected for DVT. FSAP genotypes were equally distributed in patients with (n=64) and without DVT (n=<em>1</em>06), (P=0.94). The <em>1</em>60<em>1</em>GA genotype was associated with significant reduction of FSAP activity (P<0.00<em>1</em>) and FSAP antigen levels (P=0.04). Patients with DVT showed significantly higher FSAP activity (P=0.008), FSAP antigen (P=0.003), and F<em>1</em>+<em>2</em> levels (P<0.00<em>1</em>) than patients without DVT. The association between the FSAP measures and DVT disappeared when adjusted for CRP levels. F<em>1</em>+<em>2</em> correlated positively to FSAP antigen (P=0.0<em>1</em>), while FVIIa-levels were comparable in patients with and without DVT. We conclude that even though FSAP measures are significantly increased in patients with acute DVT, alterations in the scu-PA activating properties of FSAP are presumably not markedly involved in the development of acute DVT, and that the association between FSAP and DVT disappears after adjustment for CRP.

A number of studies indicate that coagulation proteases play significant roles in cancer biology. Melanoma is a highly metastatic cancer, and there is evidence that thrombin contributes to this aggressive pattern. However, few studies correlate this type of cancer with formation of the <em>prothrombin</em>ase complex, which is responsible for conversion of <em>prothrombin</em> into thrombin in the coagulation system. The aim of this study was to investigate the assembly and regulation of <em>prothrombin</em>ase complex on the murine melanoma cell line, B<em>1</em>6F<em>1</em>0. B<em>1</em>6F<em>1</em>0 cells were unable to activate <em>prothrombin</em> except when previously incubated with factor Xa. This effect was dependent on factor Xa binding to cell membranes, since no activation was detected with Gla-domainless factor Xa. The thrombin formation by B<em>1</em>6F<em>1</em>0-bound factor Xa was enhanced approximately <em>1</em>0 fold in the presence of factor Va, indicating the assembly of <em>prothrombin</em>ase complex. Differently from platelets, B<em>1</em>6F<em>1</em>0-assembled <em>prothrombin</em>ase complex was inhibited by <em>prothrombin</em> <em>fragment</em> <em>1</em> but not by <em>fragment</em> <em>2</em>. In addition, bothrojaracin, a specific ligand of proexosite I on <em>prothrombin</em>, caused a significant decrease in the zymogen activation. Our data demonstrate that B<em>1</em>6F<em>1</em>0 melanoma cells generate thrombin by promoting assembly of the <em>prothrombin</em>ase complex. This ability might be correlated with the increased metastatic potential of this cell line. Moreover, B<em>1</em>6F<em>1</em>0-assembled <em>prothrombin</em>ase complex seems to be modulated in a different way from that found for the physiological complex assembled on platelets.

BACKGROUND

It has been suggested that changes in blood coagulation and fibrinolysis might explain the observed association between depression and coronary artery disease (CAD). So far, only a few coagulation factors have been investigated in this regard, and the results were not consistent.

METHODS

The aim of our study was to analyse a broad range of coagulation and fibrinolytic factors, with emphasis on factors directly involved in clot formation and degradation or reflecting coagulation activation, in patients with CAD and controls without CAD, as assessed by coronary angiography, who also underwent a diagnostic procedure for depression.

METHODS

We screened 306 patients with CAD and controls without CAD for depression using the Hospital Anxiety and Depression Scale and Allgemeine Depressions Skala-L questionnaires. In participants with positive screening result, diagnosis of major depression was confirmed or excluded by a structured interview. We analysed the following coagulation and fibrinolytic factors: fibrinogen, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, factor XIII A-subunit, factor XIII B-subunit, tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em>, thrombin-activable fibrinolysis inhibitor, and D-dimer.

RESULTS

We did not observe significant associations between depression and CAD, nor between depression and cardiovascular risk factors. Coagulation and fibrinolytic factors showed no differences between patients with CAD and controls, but they were associated with several cardiovascular risk factors. Depression was not associated with coagulation and fibrinolytic factors. No associations were found either when both CAD and depression were taken into account.

CONCLUSIONS

Our study gives no evidence that there is a significant relation among depression, CAD, and blood coagulation and fibrinolysis.

OBJECTIVE

This study investigated the influence of early spontaneous intermittent reperfusion on the extent of myocardial damage and its relation to endogenous hemostatic activity.

BACKGROUND

In the early phase of acute myocardial infarction coronary occlusion is often intermittent, even before thrombolytic therapy is administered. The relation between this phenomenon, myocardial damage and hemostatic activity is unknown.

METHODS

Holter ST segment recording and pretreatment plasma tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) antigen, <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> and soluble fibrin levels were measured in 57 patients with acute evolving myocardial infarction. Spontaneous intermittent myocardial reperfusion, defined as two or more episodes of transient resolution of ST segment elevation to within 0.05 mV of baseline, lasting>> or = <em>1</em> min, before the start of recombinant t-PA (rt-PA) treatment was present in <em>2</em>8 patients (group <em>1</em>) and absent in <em>2</em>9 (group <em>2</em>). Left ventriculography and coronary angiography were performed 90 min after intravenous rt-PA administration. Plasma creatine kinase-MB fraction (CK-MB) levels were measured every 6 h for <em>2</em>4 h, and C-reactive protein levels were measured daily for 3 days.

RESULTS

Group <em>1</em> had lower peak plasma CK-MB (<em>1</em>4<em>1</em>.9 +/- <em>2</em>8.3 vs. <em>2</em>03.8 +/- <em>2</em>3.3 IU/liter [mean +/- SEM], p < 0.0<em>1</em>4) and C-reactive protein levels (<em>1</em>6 +/- 4 vs. <em>2</em>8 +/- 4 mg/liter on day <em>1</em>; <em>2</em>6.6 +/- 5.5 vs. 6<em>1</em>.8 +/- <em>1</em>4.4 mg/liter on day <em>2</em>; <em>1</em>9.6 +/- 4.<em>2</em> vs. 40.6 +/- 6.5 mg/liter on day 3, p < 0.0<em>1</em><em>2</em>) and a higher left ventricular ejection fraction (6<em>2</em>.9 +/- 4% vs. 5<em>1</em>.<em>1</em> +/- 5%, p < 0.04) than group <em>2</em>. Group <em>1</em> had lower plasma t-PA antigen levels (<em>1</em>5.6 vs. <em>2</em>7 micrograms/liter, p < 0.006) but higher <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> (<em>1</em>.8 vs. <em>1</em>.<em>1</em> nmol/liter, p < 0.003) and soluble fibrin levels (66.8 vs. 3<em>1</em> nmol/liter, p < 0.0<em>1</em>). Coronary patency at 90 min was similar.

CONCLUSIONS

Early spontaneous intermittent reperfusion during acute myocardial infarction is associated with augmented thrombogenic activity and less subsequent myocardial damage. This finding is consistent with a protective effect of intermittency on the myocardium and a procoagulant effect of spontaneous lysis on blood. It may also reflect a different rate of evolution of coronary thrombosis and myocardial infarction in patients with and those without spontaneous intermittent myocardial reperfusion.

BACKGROUND

Patients with acute coronary syndrome and concomitant atrial fibrillation may require antithrombotic triple therapy but clinical evidence of safety and efficacy is poor. We have therefore studied the combination of different antithrombotic medicines for coagulation activation in an in vivo model in the skin microvasculature.

RESULTS

Platelet activation (β-thromboglobulin [β-TG]) and thrombin generation (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> [F<em>1</em>+<em>2</em> ], thrombin-antithrombin complex [TAT]) were studied in an open-label, randomized, parallel group trial in 60 healthy male subjects (n = <em>2</em>0 per group) who received ticagrelor and acetylsalicylic acid (ASA) in combination with dabigatran (<em>1</em>50 mg bid), rivaroxaban (<em>2</em>0 mg od) or phenprocoumon (INR <em>2</em>.0-3.0). Coagulation biomarkers in shed blood were assessed at 3 h after monotherapy with the medicines under study, at 3 h after triple therapy dosing and at steady state trough conditions. Single doses of ticagrelor, dabigatran or rivaroxaban caused comparable decreases in shed blood β-TG and were more pronounced than phenprocoumon at an INR of <em>2</em>.0-3.0. In contrast, thrombin generation was more affected by rivaroxaban and phenprocoumon than by dabigatran. During triple therapy a similarly sustained inhibition of platelet activation and thrombin generation with a maximum decrease of β-TG, F<em>1</em>+<em>2</em> and TAT at 3 h post-dosing was noted, which remained below pre-dose levels at trough steady state.

CONCLUSIONS

A triple therapy at steady state with ticagrelor plus ASA in combination with dabigatran or rivaroxaban is as effective as a combination with phenprocoumon for platelet activation and thrombin generation in vivo.

OBJECTIVE

To examine possible adverse effects on haemostasis from prolonged exposure to inhaled nitric oxide (iNO).

METHODS

Blinded, randomised, experimental animal study in a university animal laboratory.

METHODS

Anaesthetised and intubated piglets received central venous, arterial, and transabdominal urinary catheters. Twelve piglets were studied with triggered pressure support ventilation breathing with an air-oxygen mixture for 30 h with nitric oxide (NO), 40 parts per million (ppm) (n = 6) or without NO gas (n = 6) added. The tests of platelet function were assessed in a separate <em>1</em>-h experiment in which <em>1</em><em>2</em> additional animals were blindly randomised to receive intravenous acetylsalicylic acid (ASA) (n = 7) or placebo (n = 5).

RESULTS

All <em>1</em><em>2</em> animals were clinically stable during the study period of 30 h. Haemostasis was assessed in terms of bleeding time and platelet function by Adeplat-S, reflecting platelet adhesion. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, fibrin D-dimer, tissue plasminogen activator and prothrombin complex were measured to investigate whether inhaled NO (iNO) had any effects on thrombin formation, fibrin formation, fibrinolysis or coagulation. All parameters including bleeding time and Adeplat-S were unaffected by iNO. ASA significantly increased bleeding time, but did not affect Adeplat-S. Nitrate in plasma and NOx (nitrate and nitrite) in urine increased significantly in pigs receiving iNO compared with controls.

CONCLUSIONS

Prolonged exposure to iNO at 40[Symbol: see text]ppm did not affect bleeding time or coagulation parameters in healthy piglets. The findings do not support the hypothesis that iNO increases the risk of bleeding in humans.

Thrombin activity is increased in the setting of acute myocardial infarction (AMI) and has been shown to increase further after the administration of thrombolytic therapy for acute infarction. This increase in thrombin activity may play an important role in the <em>1</em>5% to <em>2</em>5% rate of failure to achieve initial reperfusion and in the 5% to <em>1</em>5% rate of early reocclusion after initially successful thrombolysis. To investigate potential mechanisms of thrombin formation in vivo, to understand better the balance of coagulation and fibrinolysis during treatment with recombinant tissue-type plasminogen activator (rt-PA), and to investigate the role of hemostatic markers as predictors of clinical events, we measured 3 markers of procoagulant activity: fibrinopeptide A (FPA), thrombin-antithrombin III complexes (TAT), and <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>), and a marker of fibrinogenolytic activity (B beta <em>1</em>-4<em>2</em>) in patients enrolled in the Thrombolysis in Myocardial Infarction (TIMI)-5 study. This trial was a randomized, dose-ranging, pilot trial of hirudin versus heparin as adjunctive antithrombotic therapy with rt-PA administered to patients with AMI. Correlation of markers at <em>1</em> hour with clinical outcomes revealed that increased FPA and TAT levels were associated with increased mortality and TIMI grades 0, <em>1</em>, or <em>2</em> flow at 90 minutes; increased F<em>1</em>.<em>2</em> levels were associated with TIMI grade 0 or <em>1</em> flow at 90 minutes; and increased levels of all 3 procoagulant markers were associated with hemorrhagic events. Late (<em>1</em><em>2</em> to <em>2</em>4 hours) increases in F<em>1</em>.<em>2</em>, TAT, and B beta <em>1</em>-4<em>2</em> may be predictive of recurrent ischemia. These results suggest that selected markers of procoagulant and fibrinogenolytic activity may be useful in predicting clinical outcomes in patients treated with thrombolytic therapy for AMI.

The molecular structure of antibodies associated with autoimmune thrombosis is beginning to be understood. We describe the binding specificities and sequence analysis of anti-beta<em>2</em>-glycoprotein-I (anti-beta<em>2</em>GP-I) or anti-<em>prothrombin</em> (anti-PT) antibody <em>fragments</em> generated by phage display from a patient with primary antiphospholipid syndrome (APS). We obtained 39 positive clones, two that had the correct size reacted with beta<em>2</em>GP-I (Beta <em>1</em> and Beta <em>2</em>). Ten clones with the same restrictive pattern recognized PT (Prot <em>1</em>) and cross-reacted with beta<em>2</em>GP-I. All three clones recognized anionic and zwitterionic phospholipids. The V(H) regions of both anti-beta<em>2</em>GP-I clones are members of the VH4 family. Prot <em>1</em> has a V(H) segment of the VH3 family. The Beta <em>1</em> J(H) segments are J(H)5b and J(H)4b for Beta <em>2</em> and Prot <em>1</em>. V(L) genes are V(lambda)<em>1</em>, 3 and <em>1</em>, respectively. No J(L) was identified for Beta <em>1</em>, while Beta <em>2</em> and Prot <em>1</em> carry J(lambda)3b genes. Beta <em>1</em> and Beta <em>2</em> carry highly conserved germ-line V(H) and V(L) genes. Mutations of the Prot <em>1</em> gene appear to be antigen-dependent, most are hotspot mutations located in the CDR <em>1</em> and <em>2</em> regions. Our work suggests that some anti-beta<em>2</em>GP-I from patients with primary APS are natural autoantibodies. Our work may also help to explain the frequent coexistence of anti-beta<em>2</em>GP-I and anti-PT in the same patient.

OBJECTIVE

A prothrombotic state is associated with organ damage in hypertensive patients. Carotid intima-media thickness (IMT) is an early marker of vascular damage that anticipates the development of atherosclerotic plaques. The aim of the present study was to investigate the relationships between subclinical carotid damage and markers of the prothrombotic state in hypertension.

METHODS

In <em>2</em>58 essential hypertensive patients who were consecutively recruited at a hypertension clinic an ultrasound carotid scan was performed with assessment of the IMT and plasma levels of C-reactive protein, fibrinogen, fibrin D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, homocysteine, and lipoprotein(a) were measured.

RESULTS

Patients with an IMT above the median of the distribution (800 µm) were older and had greater BMI, pulse pressure, duration of hypertension, and prevalence of coronary heart disease than patients with an IMT below the median. Patients with higher IMT had also greater levels of C-reactive protein, fibrinogen, fibrin D-dimer, and homocysteine. Regression analysis showed a direct relationship of IMT with age, waist circumference, pulse pressure, fibrinogen, fibrin D-dimer, and number of cigarettes smoked per day, and an inverse relationship with creatinine clearance. On multivariate analysis, age, pulse pressure, and fibrin D-dimer were independently related with IMT.

CONCLUSIONS

In hypertensive patients, subclinical carotid damage is related with evidence of activated coagulation system suggesting a prothrombotic state. This might contribute to the development of hypertensive arterial damage even in the earliest stages.

BACKGROUND

Plasma aldosterone concentration is an independent determinant of left ventricular (LV) mass in hypertensive patients, and is related to some hemostatic variables along with it may contribute to a prothrombotic state. We conducted a study to investigate the relationship between hemostatic variables, plasma aldosterone, and cardiac morphology and function in essential hypertension.

METHODS

In <em>2</em>05 patients with untreated essential hypertension, we measured components of the renin-angiotensin-aldosterone system, plasma levels of fibrinogen, D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), and assessed cardiac characteristics with standard echocardiography. For statistical purpose, patients were divided into two groups according to whether their median value of plasma aldosterone was>> <em>1</em><em>2</em>5 pg/ml or < <em>1</em><em>2</em>5 pg/ml.

RESULTS

Left ventricular mass index (LVMI) was significantly greater in patients with a high >> 5<em>1</em> ± <em>1</em>3 g/m(<em>2</em>.7)) than with a low plasma aldosterone level (< 4<em>1</em> ± <em>1</em><em>1</em> g/m(<em>2</em>.7); P < 0.0<em>1</em>) only in patients with fibrinogen levels in the highest tertile. Significant interaction was observed between plasma fibrinogen and aldosterone in the association with LVMI (P = 0.04). Left ventricular mass index was significantly and directly related to age, systolic blood pressure (BP), body mass index (BMI), plasma aldosterone, fibrinogen, and D-dimer levels, whereas no relationships were observed between echocardiographic parameters and other hemostatic variables. Fibrinogen was directly related to age, systolic BP, plasma aldosterone, LVMI, relative wall thickness, and left atrial diameter. Multivariate analysis indicated that LVMI was related to plasma fibrinogen and aldosterone (both P < 0.0<em>1</em>) independently of age, BP, and BMI.

CONCLUSIONS

Plasma fibrinogen levels interact with plasma aldosterone in its association with left ventricular mass in patients with hypertension.

An increase in levels of plasma plasminogen activator inhibitor type <em>1</em> (PAI-<em>1</em>) is one of the main hemostatic alterations in patients with coronary heart disease. Despite growing interest in the fibrinolytic system, few studies have been undertaken to determine the effect exerted on it by the different dietary fatty acids. We investigated the effect of a monounsaturated fat (MUFA)-rich diet in comparison with a low-fat diet (National Cholesterol Education Program step <em>1</em> diet) (NCEP-<em>1</em>) on factors involved in blood coagulation and fibrinolysis. We also determined the effect of dietary cholesterol on these blood parameters. Twenty-one young, male, healthy volunteers followed two low-fat/high-carbohydrate diets (< 30% fat, < <em>1</em>0% saturated fat, <em>1</em>4% MUFA) for <em>2</em>4 days each, with <em>1</em><em>1</em>5 or <em>2</em>80 mg of cholesterol per <em>1</em>000 kcal per day, and two oleic acid-enriched diets (38% fat, <em>2</em>4% MUFA) with the same dietary cholesterol as the low-fat/high-carbohydrate diets. Plasma levels of fibrinogen, thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, plasminogen, alpha <em>2</em> antiplasmin, and tissue plasminogen activator were not significantly different among the experimental diets used in this study. Consumption of the diet rich in MUFA resulted in a significant decrease in both PAI-<em>1</em> plasma activity (P < .005) and antigenic PAI-<em>1</em> (P < .04) compared with the carbohydrate-rich diet (NCEP-<em>1</em>). The addition of dietary cholesterol to each of these diets did not result in any significant additional effect. Changes in insulin levels and PAI-<em>1</em> activity were positively correlated (r = .4<em>2</em>5; P < .0<em>2</em>). In conclusion, consumption of diets rich in MUFAs decreases PAI-<em>1</em> plasma activity, which is accompanied by a parallel decrease in plasma insulin levels.

To estimate the degree of coagulopathy in abdominal sepsis, we measured the plasma levels of <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 plasmin-alpha <em>2</em>-plasmin inhibitor complex (PIC) by the enzyme-linked immunosorbent assay in 38 patients with disseminated intravascular coagulation (DIC). In <em>2</em>0 patients with DIC due to abdominal sepsis, plasma levels of F<em>1</em> + <em>2</em>, TAT and PIC were <em>2</em>.6 nmol/l, <em>2</em>7.9 micrograms/l and <em>1</em>.5 micrograms/ml, respectively, with a mean antithrombin III (AT III) activity of 4<em>1</em>.7%. F<em>1</em> + <em>2</em>, TAT, PIC and AT III levels were 4.7 nmol/l, 75.8 micrograms/l, 8.8 micrograms/ml and 70.9% in <em>1</em>8 patients with DIC as the result of malignancy. Though AT III levels in DIC due to sepsis were lower than those in DIC due to malignancy, the levels of F<em>1</em> + <em>2</em>, TAT and PIC in the former were not significantly more increased than those in the latter. The plasma levels of F<em>1</em> + <em>2</em> were positively correlated with TAT and PIC in DIC patients with malignancy; however, there was no correlation between F<em>1</em> + <em>2</em> and TAT or PIC in DIC patients with sepsis. In addition, the levels of serum albumin in the two groups were similar. These results suggest that activation of coagulation and fibrinolytic systems may not be so prominent in cases of DIC due to abdominal sepsis, compared to related events in DIC due to malignancy. It is also suggested that the depletion of AT III in cases of sepsis is not only caused by a consumption related to intravascular coagulation or to an alternate distribution of protein.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

BACKGROUND

The potential hemostatic effect of infusible platelet membranes (IPM; Cyplex, Cypress Bioscience) prepared from outdated human platelets is investigated.

METHODS

Increasing concentrations of IPM were added to blood samples anticoagulated with low-molecular-weight heparin, in which platelets and WBC counts had been experimentally reduced by a filtration procedure. Thrombocytopenic blood with IPM was circulated in a perfusion chamber at various shear rates (300, 600, and <em>1</em><em>2</em>00/sec(-<em>1</em>)), and platelet and fibrin deposition on the surface of a damaged vessel was measured. <em>Prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> (F<em>1</em>+<em>2</em>) levels were also monitored.

RESULTS

Under conditions of severe thrombocytopenia (<6000 platelets/microL) IPM did not increase platelet deposition. However, a dose-dependent increase in fibrin deposition was observed with concentrations of IPM ranging from 0.5 to <em>2</em> mg per kg in perfusions at 300 and 600 per sec(-<em>1</em>) (p<0.05 vs. thrombocytopenic blood). Experimental studies performed under conditions of moderate thrombocytopenia and higher shear rates (<em>2</em>5, 000-30,000 platelets/microL; at 600 and <em>1</em><em>2</em>00/sec(-<em>1</em>)) showed that IPM concentrations equivalent to 0.5 or <em>1</em> mg per kg improved fibrin deposition (33.5 +/- 9.5% and 37.7 +/- <em>1</em><em>2</em>.8%, respectively, vs. <em>2</em><em>2</em>.7 +/- 5.<em>2</em>% in controls) and also promoted a moderate increase in platelet deposition, with a concomitant significant increase in the size of platelet aggregates (p<0.05). Exposure of thrombocytopenic blood to a damaged vessel resulted in an increase of F<em>1</em>+<em>2</em> levels from 0.8 +/- 0.<em>1</em>5 to <em>1</em>.7 +/- 0.<em>2</em><em>2</em> nM at 300 per sec(-<em>1</em>) and <em>1</em>.94 +/- 0.46 nM at 600 per sec(-<em>1</em>). Postperfusion levels of F<em>1</em>+<em>2</em> after the addition of IPM were always similar to levels in untreated controls.

CONCLUSIONS

IPM promotes local procoagulant activity at sites of vascular damage under conditions of severe and moderate thrombocytopenia. IPM also appears to facilitate platelet cohesive functions under conditions of moderate thrombocytopenia.