Markers of endothelial and platelet activation are associated with high on-aspirin platelet reactivity in patients with stable coronary artery disease

Introduction

Aspirin inhibits the cyclooxygenase-1 (COX-1) mediated thromboxane A2 synthesis. Despite COX-1 inhibition, in patients with coronary artery disease (CAD), platelets can be activated through other mechanisms, like activation by thrombin.

Materials and Methods

At baseline in this cross-sectional substudy of the ASCET trial, 1001 stable CAD patients, all on single aspirin treatment, were classified by the PFA100® method, as having high on-aspirin residual platelet reactivity (RPR) or not. Markers of hypercoagulability, endothelial and platelet activation as related to RPR, were evaluated to explore the potential mechanisms behind high on-aspirin RPR.

Results

Altogether, 25.9% (n = 259) of the patients were found to have high on-aspirin RPR. S-thromboxane B₂ levels were very low and did not differ between patients having high on-aspirin RPR or not. Patients with high on-aspirin RPR had significantly higher levels of von Willebrand Factor (vWF) (124 vs 100%, p < 0.001, platelet count (236 vs 224 × 10⁹/l, p = 0.008), total TFPI (68.4 vs 65.5 ng/ml, p = 0.005) and ß-thromboglobulin (ß-TG) (33.3 vs 31.3 IU/ml, p = 0.041) compared to patients with low on-aspirin RPR. No significant differences between the groups were observed in levels of endogenous thrombin generation (ETP), pro-thrombin fragment 1+2 (F1+2), D-dimer, soluble TF (sTF) or P-selectin (all p > 0.05).

Conclusions

The high on-aspirin RPR as defined by PFA100® seems not to be due to increased thrombin activity as evaluated with ETP, sTF, F1+2 or D-dimer. The elevated levels of platelet count, ß-TG, TFPI and especially vWF might be explained by increased endothelial and platelet activation in these patients.     

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.     

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.     

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.     

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.     

Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults

Introduction

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation.

Materials and Methods

An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated.

Results

STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p < 0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p < 0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent.

Conclusion

Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.     

Circulating tissue factor positive microparticles in patients with acute recurrent deep venous thrombosis

Introduction

Circulating tissue factor positive microparticles (MPTF) were reported in a wide range of diseases with thrombotic tendency. Though D-dimer assay had a high negative predictive value for deep venous thrombosis (DVT) recurrence, there are currently no reliable positive predictors for recurrent DVT. We therefore quantified MPTF in patients with acute recurrent DVT to determine whether MPTF levels could be used to predict recurrent DVT.

Materials and Methods

Microparticles (MPs) were isolated from plasma of initial DVT patients (n = 25), recurrent DVT patients (n = 25) and sex- and age-matched healthy individuals (n = 25), stained with annexin V, cell-specific monoclonal antibodies (MoAbs) and a MoAb directed against tissue factor (TF), and analyzed by flow cytometry. We also determined the plasma procoagulant activity with a Human TF Chromogenic Activity Assay Kit.

Results

We found total MPTF to be elevated in recurrent DVT patients versus normal individuals (P = 0.001). The number of monocyte-derived MPTF in both initial and recurrent DVT was higher than in normal individuals (P < 0.01, respectively). The platelet and endothelial cell derived MPTF in recurrent DVT were significantly increased relative to other MPTF (P < 0.05), although there was no difference between initial DVT patients and normal individuals. We demonstrated elevated procoagulant activity of platelet-free plasma in DVT patients relative to normal individuals, and a positive correlation with MPTF.

Conclusions

The elevated MPTF could be a potentially predictor for DVT recurrence. Further studies are needed to validate its sensitivity and specificity.     

Inhibitory effects of LDL-associated tissue factor pathway inhibitor

Introduction

Tissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI_1–161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays.

Materials and Methods

LDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4 μM phospholipids or 0.5 nM factor Xa (FXa) and 4 μM phospholipids, respectively.

Results

TFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI_1–161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height.

Conclusions

Our results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI_1–161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.     

Plasma factor VII: a potential marker of pre-eclampsia

Introduction

Normal pregnancy is associated with a local hypercoagulable state that becomes more profound in certain obstetric complications such pre-eclampsia (P-EC). Current literature on the levels of individual haemostatic factors in women with P-EC is limited and results are inconsistent. In this study we provide detailed investigation on the tissue factor (TF)-dependent pathway in women with P-EC.

Materials and Methods

Enzyme-linked immunosorbent assays (ELISA) were used to measure plasma factor (F) FVII, FVIIa, TF and tissue factor pathway inhibitor (TFPI) in healthy non-pregnant women (n = 22), normal pregnant women (n = 15), and women with P-EC (n = 20). All subjects were age matched. In addition, pregnant women were matched for gestational age, parity and were all at the third trimester.

Results

Plasma FVII levels were significantly higher in women with P-EC compared to the healthy non-pregnant (P < 0.001) or the normal pregnant groups (P < 0.001). No such significant trends were observed for plasma FVIIa, TF or TFPI levels. Plasma FVII levels can distinguish women with P-EC from healthy non-pregnant women or normal pregnant women at the third trimester, with high sensitivity (90%), specificity (80%), positive and negative predictive values (86%).

Conclusions

Plasma FVII levels are significantly elevated in women with P-EC, in the absence of comparable changes in other TF-dependent pathway factors (FVIIa, TF and TFPI). We propose the use of plasma FVII as a marker for P-EC.     

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.     

Impaired glucose metabolism and type 2 diabetes are associated with hypercoagulability: potential role of central adiposity and low-grade inflammation--the Hoorn Study

Introduction

Type 2 diabetes (DM2) is associated with greater risk for cardiovascular disease (CVD), which may, at least partially, be explained by prothrombotic alterations. We therefore investigated; first, the extent to which individuals with impaired glucose metabolism (IGM) and/or DM2 had greater levels of thrombin generation than those with normal glucose metabolism (NGM); and second, whether any differences were independent of other cardiovascular risk factors, such as smoking, hypertension, dyslipidaemia, (micro)albuminuria, glycemic control and (central) adiposity, and/or were potentially ‘mediated’ by low-grade inflammation (high-sensitivity C-reactive protein (hsCRP)).

Materials and methods

We studied 744 individuals from the Hoorn Study (275 NGM, 176 IGM and 293 DM2, mean age 68.6 ± 7.1 years). Thrombin generation in platelet-poor plasma was measured using the Calibrated Automated Thrombogram and three parameters were derived: lag time, peak height and endogenous thrombin potential (ETP). Data were analyzed with multiple linear regression analyses.

Results

After adjustment for age, sex, prior CVD and smoking status, individuals with IGM or DM2 had a longer lag time [ß = 0.14 min (95% CI: 0.02; 0.26)], higher peak height [ß = 7.29 nM (− 1.33; 15.91)] and ETP [ß = 35.65nM*min (0.97; 70.34)] than those with NGM. These differences were attenuated to ß = 0.06 min (− 0.07; 0.19), 3.82 nM (− 5.46; 13.10) and 16.34 nM*min (− 20.92; 53.59), respectively, when further adjusted for waist circumference and hsCRP.

Conclusion

Individuals with IGM or DM2 had up to 4% higher thrombin generation compared with NGM, which may be explained, to a great extent, by the greater levels of central adiposity and related low-grade inflammation characterizing these individuals.     

Thrombophilic screening in young patients (< 40 years) with idiopathic ischemic stroke: a controlled study

Introduction

Despite extensive clinical and laboratory investigations, the etiology of ischemic stroke remains unknown in approximately one third of patients.

Materials and Methods

Thirty-four consecutive patients less than 40 years old (Males 13, Females 21, mean age 26.6 years, range 2-39) with documented ischemic stroke underwent, one year after the acute event, laboratory evaluation of antithrombin, protein C, free and total protein S, activated protein C resistance, fibrinogen, factor VII:C, homocysteine levels and antiphospholipid antibodies (APA). Moreover, prevalence of F5 R506Q, F2 G2021A and homozygosis for thermolabile variant C677T of the methylenetetrahydrofolate reductase (MTHFR) were also evaluated and compared to the results obtained in 120 normal controls.

Results

Antithrombin and protein C levels resulted normal in all cases. One patient (2.9%) showed free protein S deficiency and 3 patients (8.8%) had activated protein C resistance. Homocysteine levels above 15 μmol/L were found in one patient (2.9%). APA were found in 21 patients (61.7%) and in only 2 out of 120 (1.66%) controls (OR = 95.31; 95% C.I.: 18.22-667.81). The multivariate analysis selected that the presence of APA was significantly associated with an increased risk of stroke (OR = 156.60; 95% C.I.: 25.99-943.47) in this cohort of patients. The combination between APA and cardiovascular risk factors determined a risk of 29-fold (OR = 29.31; 95% CI: 3.28-261.69).

Discussion

Our data suggest that the presence of APA is associated with an increased risk of idiopathic ischemic stroke in young patients. Furthermore, also the combination of APA and cardiovascular risk factors is significantly associated with development of idiopathic ischemic stroke.     

An experimental model to study isolated effects of thrombin in vivo

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo.

Methods

Thrombin (0.05-0.9 U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n = 38). The study consisted of three parts: dose escalation (n = 21), dose verification (n = 5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n = 12).

Results

A thrombin dose of 0.9 U/kg/min decreased platelet counts by 70% compared to the control group (median 230 × 10^9/L vs. 752 × 10^9/L; p = 0.041). In accordance, infusion of 0.9 U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56 mg/dl vs. 220 mg/dl; p = 0.046). Cumulative thrombin doses of ≥ 0.1 U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥ 0.15 U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9 U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306 pg/ml vs. 47 pg/ml, p < 0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia.

Conclusion

Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.     

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.     

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.     

Prior simvastatin treatment is associated with reduced thrombin generation and platelet activation in patients with acute ST-segment elevation myocardial infarction

Background

It has been reported that statin therapy produces additional effects including impaired activation of blood coagulation. It is not clear whether statins can affect hemostasis in patients with acute coronary syndrome. The aim of this study was to investigate the effect of prior statin treatment on thrombin generation and platelet activation in patients with ST-segment elevation myocardial infarction (STEMI).

Methods

We studied 53 consecutive STEMI patients admitted within 12 hours of pain onset, including 19 treated with simvastatin (40 mg/d) (simvastatin group) at least one month prior to STEMI, and 34 not receiving any statins (no-statin group) on admission. Thrombin-antithrombin (TAT) complexes generation and soluble CD40 ligand (sCD40L) release were determined in 60-second blood samples collected at the site of microvascular injury.

Results

There were no significant intergroup differences with respect to clinical and laboratory variables, including plasma TAT and sCD40L levels, except lower total cholesterol and low-density lipoprotein cholesterol in the simvastatin group. The mean maximum rate of TAT generation was 47.5% lower (p = 0.0002) and sCD40L release 33.3% lower (p = 0.0006) in the simvastatin group. Total amounts of TAT (p < 0.0001) and sCD40L (p = 0.002) collected within 5 minutes of bleeding were lower in simvastatin-pretreated patients. By multivariate regression analysis, variables describing local TAT and sCD40L profiles in the whole group were independently associated with simvastatin pretreatment (p < 0.0001), but not with cholesterol, platelet count or troponin levels.

Conclusions

Prior simvastatin use is associated with lower thrombin generation and platelet activation following vascular injury in the early phase of STEMI.     

Persistent high factor VIII activity leading to increased thrombin generation - a prospective cohort study

Introduction

A persistently elevated level of factor VIII (FVIII) is an independent risk factor for venous thromboembolism (VTE). Although the pathophysiology of VTE is unclear, the involvement of thrombin generation (TG) has been postulated. Consequently this study was designed to (i) investigate the relationships between FVIII, Thrombin generation test (TGT) parameters and D-dimer in VTE patients, (ii) determine whether elevated levels of FVIII and increased TG in these patients are transient or sustained.

Patients and Methods

After an initial period of anticoagulation had been completed 91 VTE patients and 52 healthy controls were recruited. FVIII levels were determined by one-stage clotting (FVIII:C) and chromogenic (FVIII:Ch) assays. The potential to generate thrombin was measured using the Calibrated Automated Thrombogram (CAT) and D-Dimer was by immuno-turbidometric assay.

Results

Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs = 0.94; p < 0.0001), although FVIII:C exhibited a mean bias of -6%. FVIII:Ch show a significant correlation with TGT Peak Thrombin (rs = 0.30; p = 0.004) and Peak Thrombin was found to be significantly higher (p = 0.04) in patients with FVIII > 200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time.

Conclusion

Our data suggests that high FVIII leading to increased TG confers a significant risk of recurrent VTE and therefore we speculate that these patients may benefit from prolonged anticoagulation therapy.     

Mean platelet volume (MPV) increase during acute non-lacunar ischemic strokes

Introduction

Mean platelet volume (MPV) has been associated with the prognosis in stroke patients. However, its spontaneous variability during the acute phase of the disease is unknown. Materials and Methods — One hundred and thirty-seven patients with ischemic stroke, aged 75.4 ± 11.0 (SD) years, were classified according to several criteria: National Institutes of Health Stroke Scale (NIHSS) score, maximum lesion diameter on CT scan, Oxfordshire Community Stroke Projects (OCSP) and Trial of ORG 10172 in Acute Stroke Treatment (TOAST) categories. Platelet parameters were determined 1.2 days after the onset of symptoms, and after 3.0 further days.

Results

The initial MPV was higher in non-lacunar than lacunar strokes (8.30 ± 1.10 vs. 7.95 ± 0.79 fl, P = 0.04), and correlated with the sampling delay with respect to the onset of symptoms, especially in the strokes with lesions > = 4 cm (r = 0.39, P = 0.009), NIHSS > = 11 (r = 0.35, P = 0.02) and of cardioembolic origin (r = 0.35, P = 0.01). Subsequently a late MPV increment was observed in the remaining categories: from 8.20 to 8.38 fl (P = 0.02) in the strokes with lesions < 4 cm, from 8.11 to 8.31 fl (P = 0.01) in the presence of an NIHSS < 11 and from 8.20 to 8.61 fl (P = 0.03) when the occlusion of a large artery was involved.

Conclusions

Platelet volume is not stable during the acute phase in non-lacunar ischemic strokes, as it increases early in the most severe forms, and later in the remaining subtypes. The release of large and more reactive platelets may contribute to the thrombophilic state associated with ischemic events.     

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.