Continuous thrombin infusion leads to a bleeding phenotype in sheep

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo.

Methods

Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n = 5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n = 3 animals; 7 experiments).

Results

Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p < 0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p = 0.006), prolonged thrombin time by 70% (p = 0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p < 0.05) and decreased the activity of the coagulation factors VIII and XIII (p < 0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p = 0.019) and induced a 65% decrease in maximal clot firmness (p < 0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism.

Conclusion

Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.     

Tissue factor-induced Thrombin Generation in the Fasting and Postprandial State among Elderly Survivors of Myocardial Infarction

Introduction

Tissue factor (TF)-induced thrombin generation (TG) ex vivo has been suggested to be an important method to assess thrombotic risk. No studies have investigated the impact of postprandial lipemia on TF-induced TG. Since myocardial infarction (MI) is associated with elevated postprandial levels of triglycerides, we hypothesized a differential impact of postprandial lipemia on coagulation activation in MI-patients and healthy controls.

Material and Methods

Elderly survivors of acute MI (n = 44) and healthy age-and sex matched controls (n = 43) underwent a fat tolerance test (1 gram per kg body weight) to assess coagulation activation during postprandial lipemia.

Results

The incremental area under the curve (AUCi) for serum triglycerides was higher in MI-patients than in healthy age-and sex matched controls (5.64 ± 0.52 mmol/L?h and 3.94 ± 0.39 mmol/L?h, p = 0.012) during the postprandial phase. Subsequent endogenous activation of coagulation, assessed by FVIIa and thrombin generation (F1 + 2), was similar among groups and not related to levels of triglycerides during the postprandial phase. Healthy individuals had a gradual decline in TF-induced thrombin generation ex vivo, assessed by endogenous thrombin potential (ETP) (AUCi = - 542.4 ± 71.4 nM?min?h, p < 0.001), whereas MI-patients retained their ETP (AUCi = 127.4 ± 89.0 nM?min?h, p = 0.47) in plasma during the postprandial phase (p for group difference = 0.005).

Conclusions

MI-patients had elevated postprandial lipemia and retained their ability for TF-induced TG in plasma ex vivo in the postprandial phase, whereas the capacity gradually decreased in healthy individuals. Further studies are warranted to reveal underlying mechanism(s) and clinical implications.     

Endogenous thrombin potential (ETP) in plasma from patients with AMI during antithrombotic treatment

Background

The beneficial impact of warfarin in preventing new events after AMI is well established. Decrease in thrombin generation seems to be the key element in anticoagulant treatment.

Objectives

The aims were to investigate the effect of warfarin and platelet inhibition on thrombin generation, assessed by the endogenous thrombin potential (ETP), and study the relation between coagulation parameters and ETP in patients with AMI.

Patients/Methods

In the present sub-study of the WARIS II trial, patients with AMI were randomly assigned to treatment with aspirin 160 mg/d (n = 57), aspirin 75 mg/d and warfarin (INR 2.0-2.5) (n = 68) or warfarin (INR 2.8-4.2) (n = 61). Fasting blood samples were collected from patients at discharge from hospital and after 6 weeks treatment.

Results

Correlation analyses showed that both ETP and peak thrombin levels were significantly correlated with Factor VII Ag (r = 0.38 and 0.36 respectively, p < 0.01 for both) and with F1+2 (r = 0.26 and 0.23 respectively, p = 0.01 for both) at baseline. Antithrombotic treatment for 6 weeks caused a highly significant inhibition of ETP in patients treated with warfarin (− 28% ± 5%, p < 0.001), and patients treated with aspirin/warfarin (− 24% ± 8%, p = 0.04). Similarly, peak thrombin levels were reduced in patients treated with warfarin (− 18% ± 7%, p = 0.049) and aspirin/warfarin (− 19% ± 5%, p = 0.029), whereas an increase (12% ± 4%, p = 0.029) occurred during aspirin treatment alone. F1+2 levels decreased by 64% and 58% in the warfarin and aspirin/warfarin groups, respectively (p = 0.001 for both).

Conclusions

In patients with AMI, warfarin significantly reduced the endogenous thrombin generation and the potential to generate thrombin in plasma ex vivo, whereas aspirin alone had no effect on thrombin generation in vivo or ex vivo, assessed by ETP.     

Platelet activation and endogenous thrombin potential in pre-eclampsia

Introduction

Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia.

Materials/methods

Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n = 46) and without (n = 46) pre-eclampsia, and in healthy non-pregnant women (n = 42).

Results

The percentage of, CD62P+ platelets (p = 0.013), CD62P+ platelet microparticles (p = 0.029) and platelet-monocyte aggregates (p = 0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p  < 0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p < 0.001) than the normotensive pregnant controls.

Conclusion

In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.     

Plasma factor and inhibitor composition contributes to thrombin generation dynamics in patients with acute or previous cerebrovascular events

Introduction

More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events.

Materials and Methods

We studied (1) 100 patients with acute ischemic stroke (n = 50) or transient ischemic attack (TIA) (n = 50) within the first 24 hours from symptom onset, and (2) 100 individuals 1 to 4 years following ischemic stroke (n = 50) or TIA (n = 50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI).

Results

The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p ≤ 0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p < 0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced.

Conclusion

Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors.     

Fibrin clot properties in acute ischemic stroke: relation to neurological deficit

Introduction

Hypercoagulable state occurs in patients with acute vascular events. We wondered whether clot structure/function is altered in acute ischemic stroke (AIS), like in acute myocardial infarction.

Patients and methods

In 45 consecutive patients with AIS (24M, 21F), aged 67.4 ± 10.9 years, and 45 healthy controls matched for age and sex, we investigated plasma fibrin clot structure/function by permeation, turbidity, and efficiency of fibrinolysis.

Results

Compared to controls, AIS patients produced clots that had 30.5% less porous network (p < 0.0001), were less susceptible to fibrinolysis (10.8% longer lysis time, p = 0.001), were 20.5% more compact (p < 0.0001), had 17.1% higher clot mass (p < 0.0001), and showed increased (by 10.2%) overall fiber thickness (p < 0.0001) with 8% shorter lag phase of fibrin formation (p = 0.0002). Maximum rate of D-dimer release from clots was similar. Multiple regression analyses for all subjects (n = 90) showed that being a stroke patient (p < 0.0001), fibrinogen (p < 0.0001) and lipoprotein(a) (p = 0.0075) were independent predictors of clot permeability (model R2 0.79). Only fibrinogen (p < 0.0001) and lipoprotein(a) (p = 0.0026) predicted lysis time. All other fibrin parameters were predicted only by being a stroke patient. Clot compaction was associated with neurological deficit on admission (r = -0.81; p < 0.0001) and at discharge (r = -0.69; p < 0.0001). Patients with 0 or 1 point in the modified Rankin scale (n = 19) had 13.3% higher clot permeability compared to the remainder (p = 0.02).

Conclusions

This study is the first to show that AIS is associated with unfavorably altered fibrin clot properties that might correlate with neurological deficit.     

Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis

Introduction

The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery.

Materials and Methods

We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n = 20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (< 50% lumen diameter reduction, n = 14).

Results

The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p = 0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5 s identify patients at high risk (sensitivity = 95%) for luminal narrowing.

Conclusions

Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree in-stent restenosis in the superficial femoral artery.     

Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow

Introduction

Preeclampsia (PE) and fetal growth restriction (FGR) are multifactorial diseases, whose pathogenesis is largely unknown. A significant relationship between haemostasis and angiogenesis in placentae from uneventful pregnancies was previously shown.

Materials and Methods

RNA expression of haemostasis (TF, TFPI, TFPI-2, PAI-2, Anx V, TM) and angiogenesis (Ang-1, Ang-2, PlGF, VEGF) markers in placentae from PE (n = 12), PE+FGR (n = 17) and FGR (n = 20) in respect of placentae from uncomplicated pregnancies (n = 21) were investigated.

Results

Placentae from complicated pregnancies showed a significant lower expression (p ≤ 0.05 Mann-Whitney U test) of TF, TFPI, TFPI-2, Anx V, PAI-2 than those from in uncomplicated ones. VEGF and PlGF were not different in the considered groups; Ang-1 and Ang-2 were significantly higher (p ≤ 0.05 Mann-Whitney U test) in the PE group.Correlations between factors involved in haemostasis and those involved in angiogenesis, observed in placentae from uneventful pregnancies are lacking in those from complicated ones.

Conclusions

Haemostasis factors are reduced in placentae from complicated pregnancies. The relationship between haemostasis and angiogenesis observed in uncomplicated pregnancies is impaired in PE and FGR.     

Evidence of increased platelet reactivity in the first six months after acute ST segment elevation myocardial infarction

Introduction

Platelets play a crucial role in the pathogenesis of acute coronary syndromes. Accordingly, previous studies showed increased platelet reactivity on admission in these patients. In this study we assessed platelet reactivity at short-medium term follow-up in patients with ST-segment elevation acute myocardial infarction (STEMI).

Materials and methods

Fifty-nine patients (58 ± 11 years, 45 men), treated with primary angioplasty, were studied 1 month after STEMI. Thirty-five patients were retested at 6 months. Twenty matched patients with stable coronary artery disease served as controls. Platelet reactivity was assessed by flow cyometry at rest and at peak exercise, with and without adenosine diphosphate (ADP) stimulation, by measuring monocyte-platelet aggregates (MPAs) and glycoprotein IIb/IIIa (CD41) expression in the MPA gate, and CD41 and fibrinogen receptor (PAC-1) expression in the platelet gate.

Results

Compared to controls, basal MPAs and CD41 in the MPA gate were higher in STEMI patients both at 1 month (p = 0.001 and p = 0.002, respectively) and at 6 months (p = 0.03 and p = 0.01, respectively). Basal CD41 and PAC-1 expression was also higher in STEMI patients at the two assessments compared to controls (P < 0.001 for both). Exercise induced a similar increase in platelet reactivity in patients and controls. ADP induced a higher increase in CD41 platelet expression in STEMI patients compared to controls both at 1 and 6 months (P < 0.001).

Conclusion

Platelet reactivity is increased in the first 6 months after STEMI. The persistence of increased platelet reactivity in this time period may play a role in the early recurrence of coronary events after STEMI.     

Prothrombotic changes with acute psychological stress: combined effect of hemoconcentration and genuine coagulation activation

Introduction

Acute psychosocial stress accelerates blood coagulation and elicits hemoconcentration which mechanisms are implicated in acute coronary thrombotic events. We investigated the extent to which the change in prothrombotic measures with acute stress reflects hemoconcentration and genuine activation of coagulation.

Material and methods

Twenty-one middle-aged healthy men underwent three sessions of a combined speech and mental arithmetic task with one-week intervals. Coagulation and plasma volume were assessed at baseline, immediately post-stress, and 45 min post-stress at sessions one and three. Measures of both visits were aggregated to enhance robustness of individual biological stress responses. Changes in eight coagulation measures with and without adjustment for simultaneous plasma volume shift were compared.

Results

From baseline to immediately post-stress, unadjusted levels of fibrinogen (p = 0.028), clotting factor VII activity (FVII:C) (p = 0.001), FVIII:C (p < 0.001), FXII:C (p < 0.001), and von Willebrand factor (VWF) (p = 0.008) all increased. Taking into account hemoconcentration, fibrinogen (p = 0.020) and FVII:C levels (p = 0.001) decreased, activated partial prothrombin time (APPT) shortened (p < 0.001) and prothrombin time (PT) was prolonged (p < 0.001). Between baseline and 45 min post-stress, unadjusted (p = 0.050) and adjusted (p = 0.001) FVIII:C levels increased, adjusted APTT was prolonged (p = 0.017), and adjusted PT was shortened (p = 0.033). D-dimer levels did not significantly change over time.

Conclusions

Adjustment for stress-hemoconcentration altered the course of unadjusted levels of several prothrombotic factors. After adjustment for hemoconcentration, APPT was shortened immediately post-stress, whereas 45 min post-stress, FVIII:C was increased and PT was shortened. Procoagulant changes to acute stress may reflect both hemoconcentration and genuine activation of coagulation molecules and pathways.     

Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection

Introduction

Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE.

Materials and methods

19 hospital-treated patients were involved. Plasma levels of D-dimer, prothrombin fragments 1 + 2 (F1 + 2), activated partial thromboplastin time (APTT), prothrombin time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 32-54 days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (DIC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH).

Results

APTT was prolonged and D-dimer and F1 + 2 increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F1 + 2 was 3.4-fold and D-dimer even 24-fold higher compared with the recovery phase (median 726 vs 213 pmol/l, and median 4.8 vs 0.2 mg/l, respectively, p < 0.001 for both). Platelet count correlated with AT, PC, and PS (r = 0.73, r = 0.81, and r = 0.71, respectively, p < 0.001 for all) as well as with fibrinogen (r = 0.72, p < 0.001). Only five patients fulfilled the ISTH diagnosis of DIC.

Conclusions

During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.     

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology

Background

Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized.

Objective

To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques.

Methods

We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n = 20) or echogenic (n = 20) carotid plaques, or controls without carotid atherosclerosis (n = 20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression.

Results

TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p = 0.007), and positive association between serum apoB and monocyte surface TFPI expression (p = 0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio.

Conclusions

Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.     

Increased thrombin generation in splanchnic vein thrombosis is related to the presence of liver cirrhosis and not to the thrombotic event

Introduction

In recent years there have been increasing evidence associating liver disease with hypercoagulability, rather than bleeding. The aim of the study was to evaluate the haemostatic potential in patients with liver disease.

Patients and methods

We measured thrombin generation in the presence and absence of thrombomodulin in patients with portal vein thrombosis (PVT, n = 47), Budd-Chiari syndrome (BCS, n = 15) and cirrhosis (n = 24) and compared the results to those obtained from healthy controls (n = 21). Fifteen patients with PVT and 10 patients with BCS were treated with warfarin and were compared to an equal number of patients with atrial fibrillation matched for prothrombin time-international normalized ratio. We assessed resistance to thrombomodulin by using ratios [marker measured in the presence/absence of thrombomodulin].

Results

There were no differences in thrombin generation between patients on warfarin treatment and their controls. Cirrhotic patients generated more thrombin in the presence of thrombomodulin and exhibited thrombomodulin resistance compared to controls [p = 0.006 for endogenous thrombin potential (ETP) and p < 0.001 for peak thrombin and both ratios ETP and peak] and patients with non-cirrhotic PVT (p = 0.001, p = 0.006, p < 0.001, p < 0.001 for ETP, peak, ratio ETP, ratio peak, respectively). The patients with cirrhotic PVT exhibited higher ETP (p = 0.044) and peak (p = 0.02) in the presence of thrombomodulin than controls, as well as thrombomodulin resistance (ETP and peak ratios: p = 0.001).

Conclusions

Hypercoagulability and thrombomodulin resistance in patients with cirrhosis were independent of the presence of splanchnic vein thrombosis. The hypercoagulability in patients with cirrhotic PVT could have implications for considering longer or more intensive treatment with anticoagulants in this group.     

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation

Introduction

Z4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5.

Methods

We investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay.

Results

In vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC₅₀ values of 0.46 ± 0.05 μM (n = 10), 0.23 ± 0.05 μM (n = 10) and 0.21 ± 0.02 μM (n = 10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC₅₀ values of 0.48 ± 0.07 μM (n = 8) and 0.63 ± 0.12 μM (n = 6), respectively. Z4A5 failed to inhibit α_Vβ₃ integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls.

Conclusions

Z4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin α_Vβ₃.     

Activated factor XI and tissue factor in chronic obstructive pulmonary disease: links with inflammation and thrombin generation

Introduction

Increased cardiovascular mortality and risk of venous thromboembolism are serious extra-pulmonary complications of chronic obstructive pulmonary disease (COPD). Previously, circulating active tissue factor (TF) and factor XIa (FXIa) have been reported to be associated with acute coronary syndromes.

Objective

To measure plasma FXIa and active TF, prothrombin fragment 1.2 (F1.2), and markers of systemic inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], tumor necrosis factor α [TNFα] and matrix metalloproteinase 9 [MMP-9]) in 60 patients with documented stable COPD free of previous thromboembolic events.

Methods

In-house clotting assays using inhibitory monoclonal antibodies against FXIa and TF.

Results

FXIa was detected in 9 (15%) and TF activity in 7 (11.7%) COPD patients. Subjects positive for FXIa and/or TF (n = 10; 16.7%) had higher F1.2 (median [interquartile range], 398 [216] vs 192 [42] pM, p < 0.000001), fibrinogen (5.58 [2.01] vs 3.97 [2.47] g/L, p = 0.0007), CRP (14.75 [1.20] vs 1.88 [2.95] mg/L, p < 0.000001), IL-6 (8.14 [4.74] vs 2.45 [2.24] pg/mL, p = 0.00002), and right ventricular systolic pressure (47 [15] vs 38 [12] mmHg, p = 0.023), and lower vital capacity (66 [15] vs 80 [17] % predicted, p = 0.04) than COPD patients without detectable FXIa and TF. COPD severity was not associated with the presence of circulating FXIa and active TF.

Conclusions

This is the first study to show that active FXIa and TF are present in stable COPD patients, who exhibit enhanced systemic inflammation and thrombin generation. Our findings suggest a new prothrombotic mechanism which might contribute to elevated risk of thromboembolic complications in COPD.     

The clinical significance of differences between point-of-care and laboratory INR methods in over-anticoagulated patients

Introduction

Patients receiving warfarin are at increased risk of bleeding when their International Normalised Ratio (INR) > 4.5. Although not standardised above 4.5 the INR is measured in over-anticoagulated patients, consequently we have examined the reliability of INR results ≥ 4.5. We assessed: the relationship between different prothrombin time systems for INRs > 4.5; the relationships between the INR and levels of vitamin K-dependent coagulation factors (VKD-CF) and thrombin generation test (TGT) parameters; and the impact that variation in results would have on warfarin dosing.

Methods

INRs were performed using a CoaguChek XS Plus point-of-care (POC) device (measuring range 0.6-8.0). For POC INRs ≥ 4.5, laboratory INRs were also measured using a recombinant tissue factor (rTF) and a rabbit brain (RBT) thromboplastin.

Results

There was good correlation between POC (INR ≥ 4.5, < 8.0) and Lab INRs (rTF n = 154, rs = 0.87, p < 0.0001; RBT n = 102, rs = 0.76, p < 0.0001); and significant correlations between each of the VKD-CF and the INR, the strongest being with FVII (POC INR rs = -0.53 p < 0.0001; Lab rTF-INR rs = -0.70 p < 0.0001). TGT peak thrombin and ETP also showed good correlations with INR values (R² > 0.71). Using POC and Lab rTF-INR, 109/154 (71%), or POC and Lab RBT-INR 75/102 (74%) results exhibited dosage concordance and/or were within 0.5 INR units. In the remaining patients variation in warfarin dosing was generally slight.

Conclusions

Our data suggest that CoaguChek XS Plus INRs > 4.5 and < 8.0 are comparable to laboratory INRs (both methods) and it is probably unnecessary to perform laboratory INRs for clinical management of patients with INRs > 4.5 including those > 8.0.     

Coagulation activation and ultrasound characteristics in patients with carotid artery disease

Introduction

Elevated levels of markers for thrombin activation are associated with plaque echogenicity and degree of stenosis in patients with carotid artery stenosis. The Activated Protein C-Protein C Inhibitor (APC-PCI) complex reflects activation of the Protein C system and is a measure of thrombin generation. The aim of the present study was to examine APC-PCI complex in patients undergoing thrombendartherectomy for carotid artery stenosis, and to relate the findings to clinical characteristics and plaque morphology as determined by ultrasound.

Materials and Methods

Blood was obtained from 125 patients (39 female, median age 71 years) with carotid artery stenosis admitted from September 2005 to May 2007. The APC-PCI complex was measured using a sandwich immunofluorometric method and compared to an age- and sex-matched healthy control-group. Clinical and demographic characteristics, routine laboratory markers and ultrasound characteristics were analysed using univariate and multivariate analysis.

Results

APC-PCI complex concentration was significantly increased in patients with carotid artery stenosis (median 0.21 µg/L; 10th to 90th percentile 0.15-0.36) compared to a healthy control-group (0.19 µg/L; 0.11-0.31; P = .009). There was no significant difference in APC-PCI-values between asymptomatic (n = 48) and symptomatic (n = 77) patients with carotid artery stenosis (0.22 vs. 0.20 µg/L; p = 0.626). Patients with minor stroke (n = 31) had a higher median APC-PCI-concentration (0.27 µg/L; 0.15-0.63) than patients with amaurosis fugax (0.19 µg/L; 0.15-0.36) or transient ischemic attack (0.21 µg/L; 0.12-0.36) (p = 0.016). No association was found between APC-PCI-values and the degrees of carotid artery stenosis or the time from the latest neurological symptoms to blood sampling. Patients with echolucent plaques had significantly lower APC-PCI concentrations (0.20 µg/L; 0.14-0.35 vs. 0.24 µg/L; 0.15-0.60; p = 0.043), according to the Gray-Weale classification.

Conclusions

Patients with carotid artery disease exhibit increased concentrations of APC-PCI compared to a healthy control-group, particularly those patients with echogenic plaques, who have significantly higher APC-PCI levels than patients with echolucent plaques.     

Plasma homocysteine level and left ventricular thrombus formation in acute anterior myocardial infarction patients following thrombolytic therapy with t-PA

Aims

The aim of this study was to evaluate the relationship between homocysteine levels and the development of left ventricular thrombus in acute anterior myocardial infarction patients directed to thrombolytic therapy.

Methods and Results

Seventy-nine patients presenting with ST elevated acute anterior myocardial infarction and treated with thrombolytic agent, t-PA, were included in the study. Two-dimensional echocardiography was used to divide patients into 2 groups according to the presence (n = 14) or absence (n = 65) of thrombus in the left ventricle following myocardial infarction. The levels of fasting plasma total homocysteine, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, vitamin B12 and folic acid were assessed. There were no significant differences between two groups in terms of age, gender, hyperlipidemia and smoking. History of diabetes mellitus (28.57% versus 6.15%, p = 0.04), peak creatine phosphokinase levels (4153.54 ± 1228.41 U/L versus 2456.92 ± 1421.36 U/L, p < 0.001), mean left ventricular wall motion score index (2.21 ± 0.18 versus 1.83 ± 0.23, p < 0.001) and total fasting homocysteine levels (18.24 ± 5.67 mmol/L versus 12.31 ± 3.52 mmol/L, p < 0.001) were significantly higher in patients with left ventricular thrombus. In multivariate analysis; only diabetes mellitus (p = 0.03), higher wall motion score index (p = 0.001) and higher homocysteine levels (p = 0.04) were independent predictors of left ventricular thrombus formation.

Conclusion

Our results suggest that; diabetes mellitus, higher wall motion score index and hyperhomocysteinemia independently increases the risk for the development of left ventricular thrombus formation in patients with acute anterior myocardial infarction following thrombolytic therapy.     

The procoagulant properties of purified fibrinogen concentrate are enhanced by carbon monoxide releasing molecule-2

Introduction

Fibrinogen concentrate has been demonstrated to enhance coagulation in vitro and in several clinical settings of coagulopathy. We have recently demonstrated that carbon monoxide releasing molecule-2 (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances fibrinogen as a substrate for thrombin via an attached heme. The objective of this study was to determine if CORM-2 modified fibrinogen concentrate would enhance coagulation more effectively than CORM-2 naïve fibrinogen concentrate.

Materials and Methods

In the first series of experiments, fibrinogen concentrate (final concentration 300 mg/dl) was exposed to 0, 50 or 100 μM CORM-2 for 5 min at 37 °C prior to being added to citrated, fibrinogen depleted plasma. In another series of experiments, citrated plasma obtained from 12 normal subjects was 50% diluted with crystalloid to which was added fibrinogen concentrate (final concentration 300 mg/dl) exposed to 0 or 100 μM CORM-2. Coagulation was activated with tissue factor (n = 8 per condition). Thrombus growth was monitored with thrombelastography for 15 min.

Results and Conclusions

CORM-2 modification of fibrinogen concentrate significantly enhanced the velocity of clot formation (30-50%) and strength (15-31%) in fibrinogen deficient plasma. Similarly, while diluted plasma-derived thrombi demonstrated a marked decrease in velocity of formation (54%) and strength (61%), fibrinogen concentrate significantly enhanced velocity (217%) and strength (171%); however, CORM-2 modified fibrinogen concentrate significantly increased velocity (303%) and strength (205%) to a greater extent. Additional in vitro investigation and in vivo preclinical assessments of the hemostatic efficacy of CORM-2 modified fibrinogen concentrate are warranted.     

Rapid decrease in depressive symptoms with an N-methyl-d-aspartate antagonist in ECT-resistant major depression

Background

Ketamine rapidly improves depressive symptoms in patients with treatment-resistant major depressive disorder (MDD) who do not respond to multiple standard antidepressants. However, it remains unknown whether ketamine is equally effective in patients with MDD who previously also did not respond to electroconvulsive therapy (ECT).

Methods

This study compared 17 patients with treatment-resistant MDD who previously did not respond to ECT and 23 patients with treatment-resistant MDD who had not previously received ECT. All subjects received a single open-label infusion of ketamine (0.5 mg/kg). Patients were evaluated using the Montgomery-Asberg Depression Rating Scale (MADRS) at baseline (60 min before the infusion), as well as at 40, 80, 120, and 230 min after infusion.

Results

Depressive symptoms were significantly improved in the ECT-resistant group at 230 minutes with a moderate effect size (p < .001, d = 0.50, 95% C.I.: 0.21-0.80). At 230 minutes, the non-ECT exposed group showed significant improvement with a large effect size (p < .001, d = 1.00, 95% C.I.: 0.71-1.29).

Conclusion

Ketamine appears to improve depressive symptoms in patients with MDD who had previously not responded to ECT. These preliminary results encourage further investigation with a larger sample size to determine effectiveness compared to other treatment-resistant patients with MDD.