Graft Neutrophil Sequestration and Concomitant Tissue Plasminogen Activator Release During Reperfusion in Clinical Kidney Transplantation.

BACKGROUND: Inflammation, coagulation, and fibrinolysis are tightly linked together. Reperfusion after transient ischemia activates both neutrophils, coagulation, and fibrinolysis. Experimental data suggest that tissue plasminogen activator (tPA) regulates renal neutrophil influx in kidney ischemia and reperfusion injury. METHODS: In 30 patients undergoing kidney transplantation, we measured renal neutrophil sequestration and tPA release from blood samples drawn from the supplying artery and renal vein early after reperfusion. tPA antigen levels were measured using a commercial enzyme-linked immunosorbent assay kit. For each parameter, transrenal difference (Δ) was calculated by subtracting the value of the arterial sample (ingoing blood) from the value of the venous sample (outgoing blood). RESULTS: Positive transrenal gradients of tPA antigen occurred at 1 minute [Δ = 14 (3-46) ng/mL, P < .01] and 5 minutes [Δ = 5 (-3 to 27) ng/mL, P < .01] after reperfusion. At 5 minutes after reperfusion, a negative transrenal gradient of neutrophils was observed [Δ = -0.17 (-1.45 to 0.24) x 10E9 cells/L, P < .001]. At 1 minute after reperfusion, neutrophil sequestration into the kidney (ie, negative transrenal neutrophil count) correlated significantly with tPA release from the kidney (ie, positive transrenal tPA concentration), (R = -0.513 and P = .006). CONCLUSIONS: The findings suggest a proinflammatory role for tPA in ischemia and reperfusion injury in human kidney transplantation.

Determining obstetric patient safety indicators: the differences in neonatal outcome measures between different-sized delivery units.

OBJECTIVE: To study the differences in neonatal outcome and treatment measures in Finnish obstetric units. DESIGN: A registry study with Medical Birth Register data. SETTING AND POPULATION: All births (n = 2 94 726) in Finland from 2006 to 2010 with a focus on term, singleton non-university deliveries. METHODS: All 34 delivery units were grouped into small (below 1000), mid-sized (1000-2999) and large (3000 or more) units, and the adverse outcome rates in neonates were compared using logistic regression. MAIN OUTCOME MEASURES: Early neonatal deaths, stillbirths, Apgar scores, arterial cord pH, Erb's paralysis, respirator treatment, the proportion of post-term deliveries (gestational age beyond 42 weeks) and the proportion of newborns still hospitalised 7 days after delivery. RESULTS: From an analysis of term, singleton non-university deliveries, the early neonatal mortality was significantly higher in the small relative to the mid-sized delivery units [odds ratio (OR), 2.07; 95% confidence interval (CI), 1.19-3.60]. The rate of Erb's paralysis was lowest in the large units (OR, 0.65; 95% CI, 0.50-0.84). The use of a respirator was more than two-fold more common in large relative to mid-sized units (OR, 2.38; 95% CI, 2.00-2.83). The proportion of post-term deliveries was highest in the large units (OR, 1.36; 95% CI, 1.31-1.42), where a significantly higher percentage of post-term newborns were still hospitalised after 7 days (OR, 1.50; 95% CI, 1.19-1.89). CONCLUSIONS: There are significant differences in several neonatal indicators dependent on the hospital size. An international consensus is needed on which indicators should be used.

Ischemic intestinal injury during cardiopulmonary bypass does not show an association with neutrophil activation: a porcine study.

BACKGROUND: Neutrophil activation and tissue sequestration are crucial events in intestinal ischemia-reperfusion injury, but their role in the gut wall after clinical cardiopulmonary bypass (CPB) remains unclear. We tested whether local post-CPB inflammatory response in the gut wall would be associated with intestinal mucosal perfusion. METHODS: Twenty pigs underwent 60 min of aortic clamping and 75 min of normothermic perfusion. Intestinal biopsies were taken after 120 min of reperfusion. Based on ileal myeloperoxidase activity (MPO), the animals were divided into 2 groups, CPB-induced increase in MPO (MPO+) versus no such increase (MPO-), for comparison of the parameters that measure gut mucosal perfusion. Ileal p(CO)((2)) and intramucosal pH were determined, and arterial gases were analyzed. Additionally, several hemodynamic parameters and blood thrombin-antithrombin complexes (TAT) were measured. RESULTS: Myocyte degeneration, endothelial activation and vasculitis were more pronounced in the MPO+ group (p < 0.05), while the MPO- group showed significantly increased pi(CO)((2)) and lower mucosal pH values during reperfusion. Hemodynamics and TAT levels did not differ between the groups. CONCLUSION: Tissue sequestration of neutrophils was poorly associated with perturbed mucosal perfusion after CPB. Mechanisms of gut wall injury after a low-flow/reperfusion setting can differ from those in reperfusion injury after total ischemia.

Antithrombin reduces pulmonary hypertension during reperfusion after cardiopulmonary bypass in a pig.

BACKGROUND: Antithrombin (AT) may alleviate many cardiopulmonary bypass (CPB) and ischemia-reperfusion (I/R)-related adverse effects. Using a porcine model of clinical cardiac surgery on CPB, we tested the effects of supplementary AT on myocardial and lung I/R injury. METHODS: Twenty pigs undergoing 60-min aortic clamping and 75-min normothermic perfusion were randomized in a blinded setting to receive an intravenous (i.v.) bolus of AT (250 IU/kg) (AT group, n = 10) or placebo (n = 10) 15 min before aortic declamping. An additional group of five animals received 500 IU/kg AT in an open-label setting (AT+). Thrombin-antithrombin complexes (TAT), activated clotting times (ACT), AT and myeloperoxidase (MPO) activities, troponin T, and several hemodynamic parameters were measured before CPB and after weaning from CPB up to 120 min after aortic declamping. After 120 min of reperfusion, myocardial and lung biopsies were taken for histological examination. RESULTS: AT effectively inhibited coagulation as assessed by ACT. In the AT and AT+ groups only, cardiac output (CO) and stroke volume (SV) showed a trend of post-ischemic recovery during the first 15 min after CPB. AT-attenuated reperfusion induced an increase in pulmonary arterial diastolic pressure (PAPD) but did not have significant effects on systemic or pulmonary vascular resistance. The effects of AT on SV, CO, and PAPD were fortified in the AT+ group. AT did not show effects on inflammatory changes in either myocardial or pulmonary tissue specimens. AT did not reduce post-ischemic troponin T release. CONCLUSION: Supplementary AT, in doses with significant anticoagulant effect, did not alleviate myocardial I/R injury in terms of histological inflammatory changes or post-ischemic troponin T release. Instead, however, AT-attenuated reperfusion induced an increase in pulmonary pressure after CPB. Mechanisms and clinical implications of these effects remain to be explored.

Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damage.

BACKGROUND: Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia-reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown. OBJECTIVES: We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage. METHODS: One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma prothrombin fragment F(1+2) and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day. RESULTS: Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F(1+2) during early reperfusion associated with postoperative pulmonary vascular resistance index. F(1+2) at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.001) and TnT (r = 0.44, P < 0.001) at 18 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F(1+2) during reperfusion than others (P < 0.002). Logistic regression models identified F(1+2) during reperfusion to independently associate with postoperative myocardial damage (odds ratios 2.5-4.4, 95% confidence intervals 1.04-15.7). CONCLUSIONS: Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage.

A simple automatized audit system for following and managing practices of platelet and plasma transfusions in a neonatal intensive care unit.

During neonatal intensive care, blood components are often used in clinical situations where both their efficacy and safety lack solid justification. A practical system to continuously analyse actual transfusion practices is a prerequisite for improvements of quality in transfusion therapy. We hypothesized that such a system would reveal inappropriate variations in clinical decision making and offer a means for staff education and quality improvement and assurance. The study consisted of three 120-152-day periods (P I, P II and P III) between January 2000 and October 2001 and involved 543 new patient admissions (141 patients with birth weight < 1501 g) and 6227 days of patient care at a single tertiary level NICU. P I was a control with no intervention, P II was after technically introducing the computer system and, the last period, P III was after presenting and discussing the results of P I and P II at a staff meeting. Upon an order of platelet or fresh frozen plasma (FFP) unit from the blood bank, a computer-based audit system compared the last platelet count or prothrombin time [expressed as percentage of normal clotting activity, prothrombin time (PT-%)] to predefined criteria. In the case of exceeding the preset thresholds, the system required additional information and recorded the pretransfusion laboratory values for later analysis. Thirty-two per cent of platelet transfusions were given with pretransfusion platelet count >49 x 10(9) L(-1), and 60% of these transfusions (19% of all platelet transfusions) could not be clinically justified in retrospective chart review. There was no significant change in this practice from P II to P III. FFP transfusions were given with significantly different pretransfusion PT-% values during P II and P III. The proportions of FFP transfusions with pretransfusion PT-% > 49% were 7.8% and 0.9% during P II and P III, respectively (P < 0.0001). In chart review, none of the FFP transfusions with pretransfusion PT-% > 49% could be justified by clinical grounds. Inappropriate transfusions of both platelets and plasma remain a significant challenge for quality assurance of neonatal intensive care. Automated recording of pretransfusion platelet count and prothrombin time reliably identified the poorly justified transfusions and thus offered a practical resource-saving tool for quality assurance of transfusion in the NICU. A significant shift towards more appropriate use of plasma was demonstrated after implementation of the audit system.

Fresh frozen plasma reduces thrombin formation in newborn infants.

BACKGROUND: Newborn infants undergoing intensive care are at risk of bleeding and thrombotic complications. Fresh frozen plasma (FFP) is used in hope of preventing these complications, despite poorly defined effects on the coagulation system and lack of proven clinical efficacy. OBJECTIVES AND METHODS: We prospectively evaluated coagulopathy and the effect of standardized amount of FFP transfusion (10 mL kg-1 + 4 mL in 2 h) on various coagulation markers in 33 newborn infants during the first 24 h of intensive care. RESULTS: Increased levels of prothrombin fragment F1+2, thrombin-antithrombin complexes (TAT), and d-dimer were found prior to the transfusion in 97%, 81%, and 100% of the patients, respectively. FFP transfusion was associated with a decrease in F1+2 level in 26/32 (81%) of the patients. The extent of F1+2 decrease correlated with the pretransfusion F1+2 level (R = 0.65, P < 0.0001). The patient series was divided into two groups according to increasing pretransfusional F1+2 level: Group 1 (preFFP F1+2 > or = 2.35 nm, n = 16), Group 2 (F1+2 <2.35 nm, n = 16). In Group 1, F1+2 decreased on average 1.58 nm (P < 0.01) from the baseline during FFP transfusion but no significant change in the level of F1+2 during the transfusion was observed in Group 2. Pretransfusional levels of individual factors or prothrombin time (PT) did not correlate with the FFP-associated decrease in F1+2 level. CONCLUSIONS: In the patients with the highest pretransfusional thrombin formation, FFP had an acute thrombin-reducing effect. Pretransfusion thrombin generation markers, rather than PT or individual pro- and anticoagulants, may be helpful in identifying the patient who will have measurable coagulational effects induced by FFP.

Activated protein C and inflammation in human myocardium after heart surgery.

To assess possible interactions between inflammation and activation of the anticoagulant protein C system during post-ischemic reperfusion protein C, APC, neutrophil L-selectin expression and myocardial myeloperoxidase activity (MPO) were measured in 19 patients undergoing cardiopulmonary bypass. After reperfusion for 10 min, APC to protein C ratio (APC/PC) increased from pre-reperfusion value 1.43 +/- 0.12 (mean +/- SEM) to 2.25 +/- 0.29, p = 0.015. Negative correlations were observed between APC/PC and MPO activity (Spearman r -0.64, p = 0.007) and APC/PC and neutrophil L-selectin expression (r = -0.7, p = 0.007, demonstrating that post-ishemic protein C activation was associated with decreased neutrophil tissue sequestration. Thus, physiological protein C activation may be involved in regulation of the inflammatory injury during reperfusion of human ischemic coronary circulation.

Perforation complications of percutaneous central venous catheters in very low birthweight infants.

OBJECTIVE: To prospectively survey perforation complications of consecutively inserted percutaneous central venous catheters (PCVC) in very low birthweight (VLBW) infants over a 2 year period. METHODOLOGY AND RESULTS: Three serious perforation complications were encountered in a series of 100 consecutive PCVC. One infant (birthweight 685 g) developed pericardial effusion and fatal cardiac tamponade during the use of a polyurethane PCVC. At autopsy, the pericardial sac contained 8 mL fluid with a glucose concentration of 109 mmol/L and the catheter tip was embedded in the right ventricular wall. The second infant (birthweight 1380 g) showed pleural effusion and transient immobility of the right diaphragmatic leaf after perforation of a similar PCVC into the right pleural cavity. The third perforation, causing subcutaneous oedema, occurred in a 655 g infant who had a silastic PCVC. CONCLUSIONS: The data suggest a 3% incidence for PCVC-associated symptomatic perforation complications and a 1% incidence for fatal perforations, despite a policy of careful placement. The data also indicate that perforation complications occur regardless of the size or material of the PCVC. Proper visualization of the PCVC and vigilant attention to its location is required to prevent these rare but potentially fatal complications.

Increased risk of intraventricular hemorrhage in preterm infants with thrombophilia.

The multifactorial etiology of cerebral intraventricular hemorrhage (IVH) may involve coagulation disturbances and venous infarction. We tested whether coagulation abnormalities associated with adult venous thrombosis would constitute a risk factor for IVH in newborn infants. In 22 infants (gestational age 24.3--39.9 wk, median 28.0 wk) with neonatal IVH grade II to IV, the frequencies of congenital resistance to activated protein C due to a point mutation in the factor V gene (Gln506-FV) and a polymorphism in the prothrombin gene (G20210A-FII) were assessed and compared with those observed in 29 premature newborn infants without IVH and in 302 (Gln506-FV) or 526 (G20210A-FII) healthy adults. In infants with IVH, four (18%) heterozygous carriers of Gln506-FV and one (5%) heterozygous carrier of G20210A-FII were found. One infant without IVH was heterozygous for Gln506-FV (3%). When compared with the frequency of Gln506-FV in the general population, the odds ratio for being a carrier of Gln506-FV for patients with IVH was 5.9 (95% confidence interval 1.7--20.3, p = 0.013) and for patients without IVH 0.9 (95% confidence interval 0.1--7.6, p > 0.99). The absolute risk of IVH in a newborn infant with heterozygous Gln506-FV and born before 30 wk of gestation was estimated at 80%, whereas the corresponding risk for all infants born before 30 wk was 14%. Gln506-FV was more common in newborn infants with IVH than in the general population, whereas there was no difference in the frequencies of Gln506-FV in infants without IVH and in the general population. Thus, Gln506-FV may be a risk factor of IVH. The risk of IVH in a premature infant with Gln506-FV or other established thrombophilic coagulation abnormality may be considerable.

Neonatal exchange transfusion with heparinised whole blood or citrated composite blood: a prospective study.

Heparinised blood should be kept as an available option for neonatal exchange transfusions.

Cardiolipin enhances protein C pathway anticoagulant activity.

The anticoagulant activity of activated protein C (APC) was studied using factor Xa-1-stage assays of both the procoagulant and anticoagulant activities of phospholipid vesicles containing phosphatidylserine or cardiolipin as active phospholipids. In the absence of APC, phosphatidylserine vesicles showed higher procoagulant activity than cardiolipin vesicles whereas cardiolipin vesicles supported APC-dependent anticoagulant activity better than phosphatidylserine vesicles. Enhancement of APC anticoagulant activity in plasma by cardiolipin was markedly stimulated by the APC cofactor protein S. In purified reaction mixtures, cardiolipin in phospholipid vesicles dose-dependently enhanced APC anticoagulant activity. This effect of cardiolipin was partially dependent on protein S, and immunoblotting studies showed that cardiolipin enhanced the APC-mediated cleavage of the factor Va heavy chain at Arg506 and Arg306. In solid-phase binding assays, increasing amounts of cardiolipin in multicomponent phospholipid vesicles increased the affinity for protein S and to a lesser extent APC. These data are consistent with the hypothesis that cardiolipin stimulates the anticoagulant protein C pathway by increasing the affinity of phospholipid surfaces for protein S:APC and by enhancing inactivation of factor Va by APC due to cleavages at Arg506 and Arg306 in factor Va. Based on this, it is further hypothesized that anti-cardiolipin or anti-oxidized cardiolipin antibodies may be thrombogenic because they inhibit phospholipid-dependent expression of the anticoagulant protein C pathway.

Serum tumor marker CA 125 is an early and sensitive indicator of veno-occlusive disease in children undergoing bone marrow transplantation.

Veno-occlusive disease (VOD) is a potentially lethal complication of patients undergoing bone marrow transplantation (BMT). The diagnosis of VOD is currently based on clinical signs and unspecific laboratory findings. CA 125 is an oncofetal antigen used as a tumor marker in various malignancies, especially in those originating from the female reproductive tract or gastrointestinal organs, whereas serum CA 125 levels are not increased in hematological malignancies. Several pathophysiological alterations occurring in VOD may lead to elevations in serum CA 125 levels. Therefore, we explored the behavior of this marker as a diagnostic tool in VOD. Twenty-nine pediatric transplant patients were studied. Eight patients (28%) developed clinical VOD, and a significant increase in serum CA 125 was noted in all of them. During the 7 days preceding the diagnosis of VOD, an increase of at least 57% in serum CA 125 from the pre-BMT value was observed in 6 (86%) of 7 of the evaluable patients with VOD. In contrast, a similar increase was noted in only 6 of the 21 non-VOD patients during the post-BMT period of 30 days. Accordingly, the sensitivity and specificity of serum CA 125 for predicting or detecting VOD were 86% and 71%, respectively. The serum levels of CA 125 were not affected by the presence of Graft-versus-Host Disease (GvHD) or a septic infection. In conclusion, serum CA 125 is of value as an early marker of VOD in children undergoing BMT.

Dermatan sulfate and LMW heparin enhance the anticoagulant action of activated protein C.

Unfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

Cardiopulmonary bypass and activation of antithrombotic plasma protein C.

OBJECTIVE: We hypothesized that antithrombotic plasma-activated protein C plays a defensive antithrombotic role during coronary ischemia and postischemic reperfusion. METHODS AND RESULTS: We evaluated protein C activation during cardiopulmonary bypass and coronary reperfusion in 20 patients undergoing coronary bypass surgery. During cardiopulmonary bypass and during the 10 minutes after aortic unclamping, the plasma levels of protein C (mean +/- standard error of the mean) decreased from 123% +/- 7% to 74% +/- 5% of normal mean. In contrast, the levels of activated protein C in plasma increased from 122% +/- 8% to 159% +/- 21%, and the activated protein C/protein C ratio increased from 1.04 +/- 0.08 to 2.29 +/- 0. 31 (P =.006, 2-tailed Wilcoxon signed rank test). Patients were stratified on the basis of the increase in activated protein C in the coronary sinus plasma at 10 minutes after reperfusion by means of the arbitrary value of 1.5 for the activated protein C/protein C ratio. Within 24 hours, the patients with low increases in activated protein C (ratio < 1.5, n = 8) had a significantly (P <.05) lower cardiac output and mean pulmonary artery pressure, as well as a higher systemic vascular resistance, than patients (n = 11) with high increases in activated protein C (ratio > 1.5). The rapid increase in activated protein C during the first 10 minutes after aortic unclamping indicated protein C activation in the reperfused vascular beds. CONCLUSIONS: The antithrombotic protein C pathway was significantly activated during cardiopulmonary bypass mainly during the minutes after aortic unclamping in the ischemic vascular beds. Suboptimal protein C activation during ischemia may impair the postischemic recovery of human heart and circulation.

Upregulation of the antithrombotic protein C pathway at birth.

Serious thrombotic complications occur in sick neonates, while healthy infants have a very low risk of thrombosis. To better understand the regulation of physiological anticoagulation at birth, components of the protein C pathway were measured in cord plasma samples from 14 full-term healthy newborns and in samples from 10 adult controls. Although zymogen protein C was significantly reduced in cord plasma (mean +/- SEM in cord vs. adult sample 37 +/- 1.4% vs. 90 +/- 5.5%, p < 0.0001), levels of the active enzyme, activated protein C (APC), were not (119 +/- 20% vs. 75 +/- 12%, p = 0.0762). Relative to the protein C level, cord plasmas had a 5.2-fold higher APC level (p < 0.01). The APC increase was partially due to slower inactivation of APC in cord plasma (half-life for APC 50 min in cord plasma vs. 27 minutes in adult plasma). Increased sensitivity of factor V to inactivation by APC in cord plasma was observed since the activated partial thromboplastin time-based APC sensitivity ratio was significantly increased for cord vs. adult plasma samples (2.28 +/- 0.09 versus 1.97 +/- 0.03, p < 0.01). Thus, despite low zymogen protein C, the protein C pathway in newborns seems to be functionally well developed and at an activated stage at birth.

Total and lipid-bound serum sialic acid in children with malignancy or infections.

Increased levels of sialic acid-containing glycoconjugates in serum have been observed in malignancies. In this study, significantly elevated serum concentrations of total sialic acid (TSA), TSA/total protein (TSA/TP) and lipid-bound sialic acid (LASA) were observed at diagnosis of children with various malignancies compared to healthy children. Serum TSA and TSA/TP were superior to LASA in differentiating solid tumors (p < 0.005) and leukemias (p < 0.05) from normal controls. In neuroblastoma and yolk sac tumors, a descending trend in TSA and TSA/TP was noted after successful treatment of the malignancy. Children with simultaneous malignancy and infection had significantly higher serum TSA (p < 0.05) and TSA/TP (p < 0.01) levels compared to patients with infectious diseases only, the corresponding areas under the curve were 0.83 and 0.88. The measurement of serum TSA and TSA/TP could be useful adjuncts in exclusion, diagnosis and follow-up of malignancies in children.

Circulating activated protein C in subjects with heterozygous Gln506-factor V.

Congenital resistance to activated protein C due to a point mutation in the factor V gene (Gln506-FV) is the most common genetic risk factor for familial venous thrombosis. Considering the central role of activated protein C as a physiological anticoagulant, the question of why the thrombotic risk associated with Gln506-FV was not more pronounced was asked. We hypothesized that in Gln506-FV heterozygotes, enhanced thrombin formation might preferentially activate protein C and thereby constitute a compensatory antithrombotic effect. We compared the circulatory level of activated protein C in twelve heterozygous carriers of Gln506-FV mutation with that in eighteen noncarriers in same families, and used prothrombin fragment 1+2 as a measure of thrombin generation. The circulating level of activated protein C was higher but not significantly different in heterozygotes compared with normal relatives. Activated protein C levels correlated strongly and positively with protein C antigen levels in both carriers (Spearman R 0.684, p < 0.05) and controls (Spearman R 0.642, p < 0.01). Correlation between activated protein C and prothrombin fragment 1+2 levels was of borderline significance (Spearman R 0.354, p = 0.055). In the current study, thrombin formation assessed by prothrombin fragment 1+2 levels was not significantly enhanced in subjects with heterozygous Gln506-FV compared with family members without the mutation. In conclusion, enhanced thrombin formation is not present in all healthy Gln506-FV heterozygotes in basal conditions. It seems that enhanced protein C activation by thrombin does not constitute a compensatory anticoagulant feedback loop in heterozygous carriers of Gln506-FV. However, the positive correlation between prothrombin fragment 1+2 and activated protein C suggests that, in healthy subjects and in basal conditions, thrombin upregulates the anticoagulant protein C pathway. Thus, it is questionable whether prothrombin fragment 1+2 can directly be used as an indicator of a hypercoagulable state.