Markers Of Thrombin Formation Research Articles

Menstrual Phase Affects Coagulation and Hematological Parameters during Central Hypovolemia.

Background: It has been reported that women have a higher number of heart attacks in the “follicular phase” of the menstrual cycle. We, therefore, tested the hypothesis that women in the follicular phase exhibit higher coagulability. As lower body negative pressure (LBNP) has been used previously to assess coagulation changes in whole blood (WB) samples in men and women, effects of menstrual phase on coagulation was assessed during LBNP. Methods: Seven women, all healthy young participants, with no histories of thrombotic disorders and not on medications, were tested in two phases of the menstrual cycle (early follicular (EF) and mid-luteal (ML)). LBNP was commenced at −10 mmHg and increased by −10 mmHg every 5 min until a maximum of −40 mmHg. Recovery up to 10 min was also monitored. Blood samples were collected at baseline, at end of LBNP, and at end of recovery. Hemostatic profiling included comparing the effects of LBNP on coagulation values in both phases of the menstrual cycle using standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and markers of thrombin formation. Results: LBNP led to coagulation activation determined in both plasma and WB samples. During both phases, coagulation was affected during LBNP, as reflected in their decreased partial thromboplastin time (PTT) and elevated coagulation factor VIII FVIII, F1 + 2, and thrombin-antithrombin (TAT) levels. Additionally, during the ML phase, greater PT [%] and shorter time to peak (ttPeak) values (implying faster maximum thrombin formation) suggest that women in the ML phase are relatively hypercoagulable compared to the early follicular phase. Conclusions: These results suggest that thrombosis occurs more during the midluteal phase, a finding with substantial medical implications.

Coagulation changes induced by lower-body negative pressure in men and women.

We investigated whether lower-body negative pressure (LBNP) application leads to coagulation activation in whole blood (WB) samples in healthy men and women. Twenty-four women and 21 men, all healthy young participants, with no histories of thrombotic disorders and not on medications, were included. LBNP was commenced at -10 mmHg and increased by -10 mmHg every 5 min until a maximum of -40 mmHg. Recovery up to 10 min was also monitored. Blood samples were collected at baseline, at end of LBNP, and end of recovery. Hemostatic profiling included comparing the effects of LBNP on coagulation values in both men and women using standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and markers of thrombin formation. LBNP led to coagulation activation determined in both plasma and WB samples. At baseline, women were hypercoagulable compared with men, as evidenced by their shorter "lag times" and higher thrombin peaks and by shorter "coagulation times" and "clot formation times." Moreover, men were more susceptible to LBNP, as reflected in their elevated factor VIII levels and decreased lag times following LBNP. LBNP-induced coagulation activation was not accompanied by endothelial activation. Women appear to be relatively hypercoagulable compared with men, but men are more susceptible to coagulation changes during LBNP. The application of LBNP might be a useful future tool to identify individuals with an elevated risk for thrombosis, in subjects with or without history of thrombosis. NEW & NOTEWORTHY LBNP led to coagulation activation determined in both plasma and whole blood samples. At baseline, women were hypercoagulable compared with men. Men were, however, more susceptible to coagulation changes during LBNP. LBNP-induced coagulation activation was not accompanied by endothelial activation. The application of LBNP might be a useful future tool to identify individuals with an elevated risk for thrombosis, in subjects with or without history of thrombosis.

Evaluation of platelet activation, fibrinolysis, and hemostatic activation during high dose dobutamine stress echocardiography

Acute myocardial infarction during high-dose dobutamine stress echocardiography (DSE) has been reported in patients with no indication of severe obstructive coronary artery disease, but the mechanism of thrombosis remains unclear. We hypothesized that an increase in platelet and hemostatic activation secondary to high dose dobutamine might be a contributing factor. Twelve patients undergoing outpatient DSE and with either a normal DSE results (n = 11) or normal coronaries by follow-up angiography (n = 1) were included. Patients with documented ischemia were excluded to eliminate the possibility of ischemia-induced platelet activation. Three separate samples of blood were drawn at baseline, peak dobutamine infusion and 30 minutes after the end of infusion. Blood was assayed for beta-thromboglobulin (BTG) as a platelet activation marker, D-Dimer as a fibrinolytic marker, and prothrombin fragment 1.2 (F1.2) as a marker of thrombin formation. Using ANOVA analysis, there were no significant differences between BTG, D-Dimer or F1.2 at baseline, peak dobutamine and half-hour into recovery. In this study, high dose DSE did not result in a significant increase in platelet, hemostatic or fibrinolytic activation in patients with low risk for obstructive coronary artery disease.

Exercise-induced hemostatic activation in patients with dilated cardiomyopathy in sinus rhythm

Abnormalities of baseline hemostatic variables have been related to a hypercoagulable state in patients with chronic heart failure (CHF). Given that physical exercise leads to an activation of coagulation physiologically, this study addressed the question of whether the exercise-induced hemostatic activation is enhanced in patients with CHF. Ten patients with dilated cardiomyopathy (ejection fraction < 40%) and 10 healthy individuals (matched for sex, age and body mass index) were subjected to a maximal exercise test on a bicycle ergometer. Healthy subjects performed a second exercise test at a submaximal intensity level, in which oxygen consumption (VO2) was adjusted to the peak oxygen consumption (VO2peak) of the corresponding patient. Exercise testing had only marginal effects on markers of thrombin formation in patients and healthy individuals alike. In patients with CHF, exercise-induced changes in fibrinopeptide A, an index of fibrin formation, paralleled those observed in controls after submaximal exercise whereas most pronounced changes occurred in healthy subjects after maximal exercise. Plasmin-antiplasmin complexes increased almost three-fold with maximal exercise in both groups, thus indicating a marked formation of plasmin. Maximal physical exercise does not induce an exaggerated formation of thrombin and fibrin in CHF patients. The fibrinolytic response to exercise in terms of plasmin formation is not compromised in patients with dilated cardiomyopathy.

Aprotinin and Hemostasis Monitoring Concerns During Cardiac Surgery

Aprotinin (Trasylol) is a serine protease inhibitor, isolated from bovine lung that initially was marketed for the treatment of pancreatitis. In the mid 1980s, reports of its ability to decrease hemorrhaging after cardiopulmonary bypass surgery introduced the drug to the realm of cardiac surgery. Unfortunately, its introduction into this arena was followed by the publication of multiple studies and case reports that blamed aprotinin for poor outcomes in the form of early graft closure. More than 17 years have passed since the initial article describing the use of aprotinin during cardiopulmonary bypass, and with time there has been a significant increase in scientific knowledge and clinical experience. Interestingly, modern literature does not support the dogma that aprotinin is a procoagulant. Aprotinin increases the activated partial thromboplastin time (aPTT), as well as the kaolin- and celite-activated clotting time (ACT), regardless of heparin. Aprotinin, because of its ability to inhibit kallikrein, has been found to decrease thrombin antithrombin III complexes, fibrin-split products, fibrinopeptide1+2, prothrombin fragments, and all markers of thrombin formation. Some authors have suggested that it may have a synergistic effect with heparin to ensure graft patency. Anticoagulation monitoring during the use of aprotinin also has been developed based on early studies. Aprotinin administration does influence the results of various ACT tests, and consequently different methods of testing anticoagulation have been developed. Researchers have demonstrated that the celite ACT is not “artificially” prolonged in the presence of heparin and aprotinin, rather the kaolin ACT is “artificially” shortened. This article will review the scientific literature with regard to aprotinin’s anticoagulatory effects and review the current recommendations for hemostasis monitoring during the use of aprotinin.

Evaluation of platelet activation, fibrinolysis, and hemostatic activation during high dose dobutamine stress echocardiography

Acute myocardial infarction during high-dose dobutamine stress echocardiography (DSE) has been reported in patients with no indication of severe obstructive coronary artery disease, but the mechanism of thrombosis remains unclear. We hypothesized that an increase in platelet and hemostatic activation secondary to high dose dobutamine might be a contributing factor. Twelve patients undergoing outpatient DSE and with either a normal DSE results (n = 11) or normal coronaries by follow-up angiography (n = 1) were included. Patients with documented ischemia were excluded to eliminate the possibility of ischemia-induced platelet activation. Three separate samples of blood were drawn at baseline, peak dobutamine infusion and 30 minutes after the end of infusion. Blood was assayed for beta-thromboglobulin (BTG) as a platelet activation marker, D-Dimer as a fibrinolytic marker, and prothrombin fragment 1.2 (F1.2) as a marker of thrombin formation. Using ANOVA analysis, there were no significant differences between BTG, D-Dimer or F1.2 at baseline, peak dobutamine and half-hour into recovery. In this study, high dose DSE did not result in a significant increase in platelet, hemostatic or fibrinolytic activation in patients with low risk for obstructive coronary artery disease.

Activation of thrombosis and fibrinolysis following brain infarction

To clarify the sequence of alterations in the thrombotic and fibrinolytic systems after acute brain infarction, we prospectively examined sequential changes in coagulatory markers in 38 patients suffering from cardioembolic infarcts (CEI), 41 patients with atherothrombotic infarcts (ATI), 58 patients with lacunar infarcts (LI), and 32 age-matched controls. The plasma level of thrombin–antithrombin III complex (TAT), fibrinopeptide A (FpA), D-dimer, fibrin degradation products-E (FDP-E), fibrinogen, α2-plasmin inhibitor–plasmin complex (PIC), and percent activity of antithrombin III (AT-III) were measured within 48 h, at 1 week, and at 3 weeks after the stroke onset. Significantly elevated levels of TAT and FpA, which are both markers of thrombin formation, were observed in CEI patients, and these elevated levels were associated with increasing D-dimer levels for 3 weeks ( P<0.0001). D-Dimer in CEI patients was significantly elevated compared to control, LI and ATI levels within 48 h ( P<0.001). Percent activity of AT-III was significantly decreased in CEI patients for 3 weeks compared to this activity in controls, LI and ATI ( P<0.001). TAT and FpA also increased significantly within 48 h in ATI subjects and declined thereafter. A significant elevation of FDP-E ( P<0.001) and D-dimer ( P<0.05, P<0.01) was detected in parallel with increasing fibrinogen for 3 weeks. However, there was no significant depletion of percent activity of AT-III in ATI. In LI subjects, no significant elevation of TAT, D-dimer or FDP-E were observed within 1 week. PIC increased significantly in three subtypes of brain infarcts, but did not differ significantly among the three subtypes for 3 weeks. An accurate assessment of sequential alterations in thrombotic and fibrinolytic markers in the acute stage of brain infarct should contribute to the clinical diagnosis of brain infarct subtype. Alterations in these markers in response to activation of the coagulatory system are attributable to the different pathogenesis of ischemic stroke.

Effects on Coagulation of Levonorgestrel- and Desogestrel-containing Low Dose Oral Contraceptives: a Cross-over Study

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in 28 healthy volunteers was to assess potential differences between the effects of an OC containing 150 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing 150 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 32% and 12% respectively; p <0.0001; FII: 16% and 12% respectively; p = 0.048), whereas factor V showed a greater decrease (-11% and -3% respectively; p = 0.010). Only one of the markers for ongoing coagulation (prothrombin fragment 1+2) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

THROMBIN ACTIVATION IN CARDIAC SURGICAL PATIENTS RECEIVING PREOPERATIVE HEPARIN INFUSIONS

Introduction: Patients on heparin infusions (H) have a diminished activated coagulation time (ACT) response to the heparin bolus given for cardiopulmonary bypass (CPB), [1] which may place them at risk for inadequate anticoagulation. The High Dose Thrombin Time (HiTT), a specific measure of heparin activity, may be useful for patients on heparin therapy. [2] We compared patients receiving (H) and not receiving (nH) heparin with respect to ACT, HiTT, and thrombin generation during CPB. Methods: With IRB approval, 65 consecutive patients undergoing elective CPB were studied. Using bovine heparin 300 U/kg, ACT>or=to400 sec was maintained. ACT, and HiTT were measured at multiple time points. Plasma levels of F1.2, thrombin-antithrombin III complex (TAT), and fibrin monomer were determined at baseline, 30 min into CPB and after protamine administration. Blood loss and transfusions were recorded. Patients were compared using ANOVA, and Mann Whitney-U test where appropriate. P<0.05 was considered significant. Results: ACT values differed significantly in H (dotted line, n=32) vs. nH (solid line, n=33) patients, (p<0.003). HiTT values did not significantly differ between groups (p=0.33). (Figure 1) The two groups did not differ in any marker of thrombin formation or activity. (n=20, see Figure 2) Neither CTD (722 +/- 520 ml vs. 724 +/- 311 ml) nor transfusion requirements differed between groups.Figure 1Figure 2Discussion: H patients often receive additional boluses of heparin to maintain ACT>480 sec. Our data suggest that thrombin formation and activity are not enhanced in patients receiving H therapy despite a diminished ACT response to heparin. The utility of ACT in patients receiving H therapy and the threshold values perceived as indicative of adequate anticoagulation for CPB are questioned. The use of HiTT is currently under investigation.

Heightened Thrombin Generation in Individuals with Resistance to Activated Protein C

We chose to evaluate whether or not a state of biochemical hypercoagulability was present in 74 individuals (69 heterozygotes and 5 homozygotes) resistant to activated protein C (APC) due to the Arg506 --> Gln mutation in the factor V gene. To this end, plasma levels of two markers of thrombin formation, prothrombin fragment 1 + 2 (F1+2) and thrombin-antithrombin complexes (TAT) were measured. High levels of F1+2 and TAT were found in 32% and 23% of APC-resistant individuals vs 4% in controls. The levels of these markers tended to be particularly elevated in three homozygous subjects. A significant positive correlation between F1+2 and TAT was present in APC-resistant individuals. No relationship between marker values and the previous occurrence of thrombotic episodes was found. Therefore, by measuring F1+2 and TAT a state of biochemical hypercoagulability has been identified in about one-third of APC-resistant individuals. This frequency is similar to that previously observed in comparable individuals with inherited deficiencies of protein C and protein S, which are usually associated with a stronger thrombotic tendency than APC-resistant individuals.

Markers of Hemostatic Activation in Affected Coronary Arteries during Angioplasty

To characterize the extent of early activation of the hemostatic system following angioplasty, we obtained blood samples from the involved coronary artery of 11 stable angina patients during the procedure and measured sensitive markers of thrombin formation (fibrinopeptide A, prothrombin fragment 1.2, and soluble fibrin) and of platelet activation (beta-thromboglobulin). Levels of hemostatic markers in venous blood obtained from 14 young individuals with low pretest probability for coronary artery disease were not significantly different from levels in venous blood or intracoronary samples obtained prior to angioplasty. Also, there was no translesional (proximal and distal to the lesion) gradient in any of the hemostatic markers before or after angioplasty in samples obtained between 18 and 21 min from the onset of the first balloon inflation. Furthermore, no significant difference was noted between angioplasty and postangioplasty intracoronary concentrations. We conclude that intracoronary hemostatic activation does not occur in the majority of patients during and immediately following coronary angioplasty when high doses of heparin and aspirin are administered.

Low‐dose warfarin decreases coagulability without affecting prothrombin complex activity

To assess the efficacy of a fixed, low dose of warfarin in lowering factor VII coagulant activity (FVII:C) and to investigate the effects on the plasma coagulation cascade. An open pilot study with two dose levels of warfarin: 1.25 and 2.5 mg day-1 during two consecutive 4-week periods. All subjects received aspirin 75 mg day-1. Prothrombin fragment 1 + 2 (F(1 + 2)), protein C, protein S, FVII:C, factor X and P-prothrombin complex activity (P-PT) were measured at baseline, at 2-week intervals and 4 weeks after end of treatment. Coagulation activation peptide F(1 + 2) was used as a marker of thrombin formation. Twelve male patients with a history of myocardial infarction. Inclusion was made through a written questionnaire. Warfarin 1.25 mg day-1 lowered FVII:C from 113 U dl-1 to 107 U dl-1 (P = 0.025) and F(1 + 2) from 1.60 nmol l-1 to 1.27 nmol l-1 (P = 0.013) but had no effect on protein C or P-PT. A dose of 2.5 mg day-1 induced further lowering of FVII:C (91 U dl-1, P = 0.0042), and also of protein C from 116% to 99% (P = 0.034) and P-PT from 107% to 81% (P = 0.0096) mean values. Warfarin 1.25 mg day-1 seems to exert an anticoagulant effect without reduction in PT or the natural anticoagulant protein C and is suggested, in combination with aspirin, to be a safe and simple therapy against arterial thrombotic disease, making regular PT controls unnecessary.