Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

Introduction

Oral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties.

Subjects and Methods

Plasma fibrin clot permeability (K_s) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-D_rate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) - twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n = 25). All participants had no personal history of VTE.

Results

OC discontinuation in FVL(+/−) women resulted in shortened CLT (− 9%), and increased K_s (+ 4%) and D-D_rate (+ 1.4%; all p < 0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1 + 2 (F1 + 2) (− 8%), plasminogen activator inhibitor-1 (PAI-1) antigen (− 11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (− 20%; all p < 0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-D_max, along with lower D-D_rate and K_s. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC.

Conclusion

FVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.     

Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-κB pathways

Introduction

Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.

Materials and Methods

Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.

Results

(1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48 h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p < 0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs ADMA 3435.78 ± 22.33 ng/ml, p < 0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.

Conclusions

ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.     

Unfavorably altered fibrin clot properties in patients with active rheumatoid arthritis

Objective

Altered fibrin clot properties have been reported in cardiovascular diseases (CVD) and inflammatory states. Given increased prevalence of CVD in patients with rheumatoid arthritis (RA), we investigated whether fibrin characteristics are also altered in RA patients.

Patients and methods

We studied 46 consecutive RA patients versus 50 controls matched for age and gender. Ex vivo plasma clot permeability, turbidity, tissue-type plasminogen activator (tPA)-induced fibrinolysis, and scanning electron microscopy (SEM) images of clots were evaluated.

Results

Patients with RA had lower clot permeability, faster clot formation, higher maximum clot absorbancy indicating thicker fibrin fibers, maximum clot mass and prolonged fibrinolysis time than controls. Maximum rates of clot lysis were similar in both groups. SEM images showed formation of dense clots with many projections on fibrin fibers. Clot permeability inversely correlated with fibrinogen, tPA, plasminogen activator inhibitor-1 (PAI-1), CRP, platelet count, disease activity score (DAS28) and a marker of oxidative stress, 8-iso-prostaglandin F_2α (r from -0.44 to -0.79; all, p < 0.0001). Similar positive associations were found for clot lysis time (r 0.44 to 0.69; all, p < 0.01). Multiple regression analysis showed that fibrinogen was the only independent predictor of clot permeability (R² = 0.87, p < 0.0001) and lysis time (R² = 0.80, p < 0.003) in RA. Maximum D-dimer levels released from clots, maximum clot turbidity and the time of clot formation were predicted by PAI-1 (all, p < 0.05).

Conclusion

We showed unfavorably altered plasma fibrin clot structure and function in RA, which might contribute to an increased risk of thrombotic events in this disease.     

Transgenic overexpression of a stable Plasminogen Activator Inhibitor-1 variant

Introduction

Plasminogen Activator Inhibitor-1 (PAI-1) is a member of the Serine Protease Inhibitor (SERPIN) gene family and a key regulator of fibrinolysis. PAI-1 is unique among SERPINs in its spontaneous transition to a latent, inactive state, with a half-life of approximately 2 hours under physiologic conditions. The biologic importance of the PAI-1 transition to latency is unknown. This study aimed to engineer transgenic overexpression of a stable murine PAI-1 variant to examine the physiologic effects in vivo from delayed transition of PAI-1 to latency.

Materials and Methods

Ten independent transgenic lines were generated with expression of a stable PAI-1 variant driven by the hybrid CMV/chicken β-actin promoter.

Results

Plasma PAI-1 levels in the transgenic founders ranged from 3.1 ± 0.1 ng/mL to 1268.8 ± 717.0 ng/mL. Quantitative PCR analysis in 3 transgenic lines demonstrated elevated PAI-1 mRNA in multiple tissues, with the highest increases observed in liver, brain, heart, and kidney. The fold-increase in PAI-1 mRNA over wild-type ranged from 2-fold to > 2000-fold. Immunohistochemistry showed increased PAI-1 in liver, kidney, heart, spleen, and lung. Histologic examination of transgenic mice showed no evidence of thrombosis. The two founders with the highest plasma PAI-1 levels failed to produce any transgenic offspring that survived to weaning, although genotyping of expired pups revealed successful transmission of the transgene.

Conclusion

These results suggest that high expression of a stable variant of PAI-1 may be lethal in mice, while more moderate expression is generally well tolerated and produces no apparent thrombosis.     

Tissue plasminogen activator (tPA) activity is a novel and early marker of asymptomatic LEAD in type 2 diabetes

Introduction

Lower extremity arterial disease (LEAD) is often one of the first signs of a generalized atherosclerotic disease in type 1 and type 2 diabetic subjects.

Materials and methods

We studied 143 diabetic subjects at 30-70 years of age, M/F 69/74, 74 with type 1 and 69 with type 2 diabetes, without previously known or suspected lower extremity arterial disease. The relationship between early asymptomatic lower extremity arterial disease and blood levels of HbA1c, lipids and fibrinolysis markers (tPA-activity, tPA mass, PAI-1 activity, tPA-PAI-1 complex) was assessed. In parallel, a group with non-diabetic subjects (n = 80) was studied.

Results

35 (24%) diabetic subjects were classified as having sign(s) of LEAD, defined as having at least one reduced peripheral blood pressure measurement, 28% in type 1 vs 20% in type 2 diabetic subjects (p = NS). In univariate logistic regression analyses age, glycemic level (HbA1c), male gender (only in type 1 diabetic subjects), hypertension and tPA activity (only in type 2 diabetic subjects) were positively associated with LEAD. When markers of fibrinolysis were entered into a multivariate model adjusting for age, hypertension, and HbA1c, only tPA activity remained independently associated with LEAD (p = 0.01) and this was also found in type 2 diabetic subjects (p = 0.05). In type 1 diabetic subjects the increase in odds ratio was non-significant.

Conclusions

Tissue plasminogen activator (tPA) activity may be an independent and early marker for asymptomatic lower extremity arterial disease in diabetic subjects, particularly in type 2 diabetes. Thus an altered fibrinolytic activity could be an early marker of atherosclerosis development in the lower extremities but the cause-effect relationship remains unclear.     

Increased thrombin generation in inflammatory bowel diseases

Background

Inflammatory bowel diseases (IBD) are characterized by an increased thrombotic risk of uncertain etiology. Endogenous thrombin potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders.

Aims

To study ETP in IBD patients and to correlate the results with clinical and biochemical features.

Methods

Seventy-four IBD patients (37 ulcerative colitis and 37 Crohn's disease) and 74 sex- and age-matched healthy individuals. ETP was measured upon activation of coagulation with small amounts of tissue factor and phospholipids in the presence or absence of thrombomodulin; results were expressed as nM thrombin·minutes.

Results

Mean±SD ETP values were significantly higher in patients (1,499 ± 454) than controls (1,261 ± 385) (p < 0.001) only when the test was performed in the presence of thrombomodulin. ETP evaluated as ratio (with/without thrombomodulin), taken as an index of hypercoagulability, was significantly higher in patients (0.69 ± 0.14) than controls (0.62 ± 0.18) (p < 0.006). Patients with increased C-reactive protein (CRP) had significantly higher mean ETP (1,721 ± 458) than those with normal CRP (1,357 ± 394) or controls (1,261 ± 385) (p < 0.001). Patients who at the time of blood sampling were classified as having a clinically active disease had ETP higher than those who were quiescent (1,655 ± 451 versus 1,388 ± 427, p < 0.001) or controls (1,261 ± 385, p < 0.001).

Conclusions

ETP measured in the presence of thrombomodulin or as ratio (with/without thrombomodulin) is increased in IBD patients, mainly in those with increased CRP or active disease. It may be considered as a candidate test for prospective studies aimed at assessing the risk of thrombosis in IBD patients.     

Circulating platelet-derived microparticles and endothelium-derived microparticles may be a potential cause of microthrombosis in patients with osteonecrosis of the femoral head

Introduction

To test the hypothesis that the platelet microparticle (PMP) and endothelial microparticle (EMP) may contribute to the hypercoagulability associated with microvascular thrombosis in patients with nontraumatc osteonecrosis of the femoral head (ONFH).

Materials and methods

The study comprised 46 patients who had been diagnosed with ONFH and 20 control subjects. The plasma was ultracentrifuged, and then PMPs and EMPs were examined by the flow cytometry. The thrombotic and fibrinolytic disorders were investigated.

Results

The numbers of PMPs expressing P-selectin and CD42a and EMPs expressing E-selectin and CD31 in the ONFH patients were significantly higher than those in the controls (P < 0.001). The number of MPs was correlated with the level of the serum C-reactive protein (CRP) (r = 0.661, P < 0.001), but there was a poor correlation between the MPs counts and the risk factors for ONFH (P > 0.05). The mean levels PAI-1, F1 + 2, and TAT were higher in the patients with ONFH than in the controls (P < 0.05).

Conclusions

The elevated numbers of PMPs and EMPs may contribute to hypercoagulability in the ONFH patients. This may provide important pathophysiological insights into the hypercoagulability associated with nontraumatic ONFH and have implications for pharmacological prevention and treatment of ONFH.     

Plasma fibrin clot properties in arterial hypertension and their modification by antihypertensive medication

Background

We sought to determine plasma fibrin clot properties in hypertensive subjects and to evaluate potential effects of antihypertensive therapy on these parameters.

Patients and Methods

Sixty-one patients (30 men, 31 women) with essential arterial hypertension stage 1 or 2 (aged 46.6 ± 14.4 years), free of clinically evident vascular disease, were randomly allocated for monotherapy with one of the 5 antihypertensive agents, i.e. quinapril, losartan, amlodipine, hydrochlorothiazide, or bisoprolol. Plasma fibrin clot permeability, turbidimetry and efficiency of fibrinolysis were investigated at baseline and after 6 months of therapy.

Results

Baseline systolic blood pressure in a 24-hour ambulatory monitoring was correlated with clot permeability (r = − 0.37, p < 0.05), lysis time (r = 0.42, p < 0.05) and maximal D-dimer concentration released from clots (r = 0.45, p < 0.05). Antihypertensive treatment resulted in reduction of systolic/diastolic blood pressure in office measurements and 24-hour monitoring (all p < 0.001), accompanied by an increase in clot permeability, reduction in clot lysis time and lower maximal D-dimer concentration released from fibrin clots (all p < 0.05). No changes were observed in turbidimetric variables. Posttreatment changes in plasma fibrin clot properties were related to reductions in systolic blood pressure, complement component C3 and total cholesterol.

Conclusions

Reduction in systolic blood pressure during antihypertensive treatment leads to increased plasma fibrin clot permeation and susceptibility to lysis, which might be a novel antithrombotic mechanism of blood pressure lowering therapy.     

Ambient temperature affects thrombotic potential at rest and following exercise

Introduction

During exercise, ischemic risk increases, possibly due to changes in coagulation and fibrinolytic activity. Previous research suggests ambient temperature affects resting thrombotic potential, but the effect of heat and cold on hemostasis during exercise is unknown. The purpose of this study was to assess changes in coagulation and fibrinolysis during maximal exercise in hot and cold temperatures, and to compare those responses to exercise under temperate conditions.

Materials & Methods

Fifteen healthy men completed maximal exercise tests in hot (30 °C), temperate (20 °C) and cold (5° - 8 °C) temperatures. Blood samples were obtained before and immediately after exercise and analyzed for concentrations of thrombin-antithrombin III (TAT), active tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Results were analyzed by ANOVA.

Results

A main effect of time was observed for TAT (temperate = 1.71 ± 0.82 - 2.61 ± 0.43 ng/ml, hot = 1.81 ± 0.73 - 2.62 ± 0.67 ng/ml, cold = 2.33 ± 0.65 - 2.89 ± 0.81 ng/ml, PRE to POST, respectively) and tPA activity (temperate = 0.72 ± 0.44 - 2.71 ± 0.55 IU/ml, hot = 0.72 ± 0.38 - 2.64 ± 0.61 IU/ml, cold = 0.86 ± 0.45 - 2.65 ± 0.77 IU/ml, PRE to POST, respectively). A trend was observed for the PAI-1 response to exercise (temperate = 14.5 ± 23.7 - 12.3 ± 20.2 IU/ml, hot = 15.1 ± 26.5 - 10.0 ± 15.1 IU/ml, cold = 10.5 ± 10.4 - 7.9 ± 9.7 IU/ml, PRE to POST, respectively, p = 0.08). TAT concentrations were significantly higher in cold compared to temperate and hot conditions.

Conclusion

Coagulation potential is elevated during exposure to cold temperatures. These data suggest that risk of an ischemic event may be elevated in the cold.     

The role of IL-6, IL-8 and MCP-1 and their promoter polymorphisms IL-6 -174GC, IL-8 -251AT and MCP-1 -2518AG in the risk of venous thromboembolism: a case-control study

Introduction

Cytokines increased the risk of venous thromboembolism (VTE) in some case-control studies, but not in a prospective study. Data concerning the role of cytokines in the risk of VTE are limited. We examined in a case-control study the association of VTE and levels of interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) and assessed whether promoter polymorphisms (IL-6 -174GC, IL-8 -251AT, MCP-1 -2518AG) would affect the thrombotic risk and cytokine levels.

Materials and methods

The study included 119 patients (94 women) with a first event of VTE aged between 18-60 years, and 126 healthy controls (100 women) matched for age (± 5 years). Blood was collected > 7 months after the thrombotic event. Odds ratios (ORs) were calculated per increase of cytokines levels by 1 pg/mL.

Results

ORs adjusted for age and sex were 1.520 [95% Confidence Interval (CI) 1.177 - 1.962] for IL-6, 1.095 (95% CI 1.002 - 1.196) for IL-8 and 1.000 (0.988 - 1.012) for MCP-1. With additional adjustment for ethnic composition, body mass index (BMI) and high sensitive C-reactive protein (hs-CRP), risk estimates remained significant for IL-6 and became of borderline statistical significance for IL-8. Polymorphisms did not influence the thrombotic risk and the cytokine levels in study participants.

Conclusion

VTE was associated with IL-6 and IL-8 levels, and for IL-6 this association was independent of BMI and hs-CRP. Thus far, a causal relationship between inflammation and VTE remains to be clarified and more prospective data are warranted.     

Residual vein thrombosis and onset of post-thrombotic syndrome: Influence of the 4G/5G polymorphism of plasminogen activator inhibitor-1 gene

Background

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of plasminogen activator. The functional 4G/5G polymorphism of the gene coding for PAI-1 may affect PAI-1 plasmatic activity, influencing the imbalance between coagulation and fibrinolysis cascades.In this prospective cohort analytic study, we investigated the role of this single nucleotide polymorphism in the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome.

Patients/Methods

In a group of 168 patients with post-surgical deep vein thrombosis of the legs, we analyzed the 4G/5G polymorphism in the promoter of PAI-1 gene and plasmatic PAI-1 activity.Enrolled patients were divided in two groups: patients with 4G/5G polymorphism and increased PAI-1 activity (n = 85) and patients without 4G/5G polymorphism and normal PAI-1 activity (n = 83). All patients were treated according to current protocols and re-examined after 3, 12 and 36 months in order to evaluate the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome.

Results

We found a significantly increased PAI activity in carrier of the 4G allele, who experienced much more frequently a persistence of thrombosis after 3, 12 and 36 months and/or the development of post-thrombosis syndrome, in spite of the anticoagulant treatment.

Conclusions

These data not only confirm the role played by PAI-1 activity and by the 4G/5G SNP of the PAI-1 gene, but also suggest that current therapeutic protocols, recommending the administration of low weight molecular heparin and oral anticoagulant for the treatment of deep vein thrombosis, could be non sufficient for patients genetically predisposed to a less efficient clot lysis.     

Catheter-directed ultrasound-accelerated thrombolysis for the treatment of acute pulmonary embolism

Background

Systemic thrombolysis rapidly improves right ventricular (RV) dysfunction in patients with acute pulmonary embolism (PE) but is associated with major bleeding complications in up to 20%. The efficacy of low-dose, catheter-directed ultrasound-accelerated thrombolysis (USAT) on the reversal of RV dysfunction is unknown.

Materials and methods

We performed a retrospective analysis of 24 PE patients (60 ± 16 years) at intermediate (n = 19) or high risk (n = 5) from the East Jefferson General Hospital who were treated with USAT (mean rt-PA dose 33.5 ± 15.5 mg over 19.7 hours) and received multiplanar contrast-enhanced chest computed tomography (CT) scans at baseline and after USAT at 38 ± 14 hours. All CT measurements were performed by an independent core laboratory.

Results

The right-to-left ventricular dimension ratio (RV/LV ratio) from reconstructed CT four-chamber views at baseline of 1.33 ± 0.24 was significantly reduced to 1.00 ± 0.13 at follow-up by repeated-measures analysis of variance (p < 0.001). The CT-angiographic pulmonary clot burden as assessed by the modified Miller score was significantly reduced from 17.8 ± 5.3 to 8.7 ± 5.1 (p < 0.001). All patients were discharged alive, and there were no systemic bleeding complications but four major access site bleeding complications requiring transfusion and one suspected recurrent massive PE event.

Conclusions

In patients with intermediate and high risk PE, low-dose USAT rapidly reverses right ventricular dilatation and pulmonary clot burden.     

TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells

Introduction

Elevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques.

Materials and methods

The effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy.

Results

TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells.

Conclusions

The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.     

Acute cigarette smoke exposure reduces clot lysis -- association between altered fibrin architecture and the response to t-PA

Background

Enhanced thrombolysis is a proposed mechanism for reduced mortality in cigarette smokers with STEMI (“smoker's paradox”). The mechanisms remain unclear but studies suggest fibrin architecture (FA) may affect thrombolysis. Our group has previously shown that acute cigarette smoke exposure (CSE) alters FA. This study was done to evaluate the association between FA, thrombolysis and CSE.

Methods and Results

Otherwise healthy smokers (n = 22) were studied before and after smoking two cigarettes. Non-smokers (n = 22) served as controls. Two ex-vivo models were used to evaluate clot lysis of venous blood and these data were compared to FA as determined by SEM. In the first model, clot lysis in a glass tube at 60 minutes after addition of t-PA was measured. The second model quantified lysis utilizing thromboelastography. With the latter, after a clot reached maximum strength, t-PA was added and clot lysis at 60 min was noted. SEM studies were performed on platelet poor plasma mixed with thrombin and FA was examined at 20 K.Clot lysis was similar in both groups except that post-smoking, TEG showed a significantly lower lysis compared to pre- and non-smoking clots. SEM analysis showed significantly thinner fibers and denser clots post-smoking.

Conclusions

Venous clots from smokers failed to show an enhanced lysis when exposed to t-PA. In fact, acute CSE was associated with changes in FA and increased resistance to thrombolysis. These findings in part may explain enhanced thrombogenicity but suggest that mechanisms other than enhanced fibrinolysis are likely to be responsible for “smoker's paradox.”     

Microscopic clot fragment evidence of biochemo-mechanical degradation effects in thrombolysis

Introduction

Although fibrinolytic treatment has been used for decades, the interactions between the biochemical mechanisms and the mechanical forces of the streaming blood remain incompletely understood. Analysis of the blood clot surface in vitro was employed to study the concomitant effect of blood plasma flow and recombinant tissue plasminogen activator (rt-PA) on the degradation of retracted, non-occlusive blood clots. Our hypothesis was that a faster tangential plasma flow removed larger fragments and resulted in faster overall thrombolysis.

Materials and Methods

Retracted model blood clots were prepared in an optical microscopy chamber and connected to an artificial perfusion system with either no-flow, or plasma flow with a velocity of 3 cm/s or 30 cm/s with or without added rt-PA at 2 µg/ml. The clot surface was dynamically imaged by an optical microscope for 30 min with 15 s intervals.

Results

The clot fragments removed during rt-PA mediated thrombolysis ranged in size from that of a single red blood cell to large agglomerates composed of more than a thousand red blood cells bound together by partly degraded fibrin. The average and the largest discrete clot area change between images in adjacent time frames were significantly higher with the faster flow than with the slow flow (14,000 μm² and 160,000 μm² vs. 2200 μm² and 10,600 μm²).

Conclusions

On the micrometer scale, thrombolysis consists of sequential removal of clot fragments from the clot surface. With increasing tangential plasma flow velocity, the size of the clot fragments and the overall rate of thrombolysis increases.     

Reduced clot-stability during the first 6 hours after aneurysmal subarachnoid haemorrhage--a prospective case-control study

Introduction

Early rebleeding is an important cause of death and disability following aneurysmal subarachnoid haemorrhage (SAH). Recent studies have shown that 50-90% of the rebleedings occurred within the first 6 hours after the primary bleeding. The mechanism leading to rebleeding remains to be established. In the present prospective case-control study we hypothesize that patients with SAH develop a coagulopathy characterized by reduced clot stability during the early period after the initial bleeding.

Methods

Patients with aneurysmal SAH was studied with a dynamic clot lysis assay and markers of fibrinolysis and clot stabilizers in blood samples taken within and after 6 hours after onset of bleeding. Results were compared with blood samples from age and gender matched healthy controls.

Results

36 patients were enrolled, 26 patients had blood samples collected within 6 hours after the initial bleeding whereas 10 patients had blood samples taken later than 6 hours after the initial bleeding. Patients demonstrated significantly reduced clot stability during the first 6 hours after initial bleeding. Fibrinolytic activity was increased during the first 6 hours along with the inhibitors of fibrinolysis whereas the modulators of fibrinolysis were reduced or inactivated.

Conclusion

During the first 6 hours after SAH patients exhibit reduced clot-stability. Probably a consequence of activated fibrinolysis in combination with reduced or inactivated factor XIII and thrombin-activable fibrinolysis inhibitor.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

Direct microscopic monitoring of initial and dynamic clot lysis using plasmin or rt-PA in an in vitro flow system

Introduction

Plasmin is a direct-acting thrombolytic agent with a favorable safety profile upon intra-arterial delivery in pre-clinical and phase I studies. However, the thrombolytic efficacy of plasmin, relative to that of rt-PA, remains to be established. We have compared the dynamics of clot lysis with plasmin or rt-PA in an in vitro perfusion system, in which thrombolytic agent is administered locally, allowed to induce lysis for short intervals, then washed with plasma in a re-circulation circuit.

Materials and Methods

Whole blood human clots were prepared in observation chambers, exposed to plasmin or rt-PA at equimolar concentrations (1.2/1.0, 1.8/1.5 and 2.4/2.0 mg/ml) for measured intervals of time, followed by perfusion with human plasma. Clot size was monitored by digital analysis of sequential photographs obtained through an optical microscope.

Results

Plasma perfusion after incubation with thrombolytic agent rapidly removed superficial clot fragments. This initial decrease in clot size was greater with plasmin than with rt-PA when tested at the highest concentrations of agent (0.63 ± 0.11 vs. 0.30 ± 0.11, p = 0.001 for clots with non-cross-linked fibrin and 0.53 ± 0.15 vs. 0.14 ± 0.15, p = 0.02, for clots with cross-linked-fibrin). Subsequent clot lysis during plasma flow was greater after prior incubation with rt-PA. Longer incubation times of plasmin resulted in larger portions of the clot being washed free. Repeated plasmin incubations and plasma perfusions of a clot successfully induced stepwise reductions in clot size.

Conclusions

Initial clot lysis is greater with direct exposure using plasmin than rt-PA. During washout and circulation with plasma, rt-PA induced continued clot lysis, while plasmin lysis was curtailed, presumably because of plasmin inhibition.     

Similarities in Thromboelastometric (ROTEM®) Findings between Humans and Baboons

Introduction

Interest in visco-elastic testing in different clinical scenarios has increased but few data are available on thromboelastometric findings in primates.

Materials and Methods

Blood cell count (hemoglobin, hematocrit, platelet count), coagulation parameters (prothrombin time, International Normalized Ratio, fibrinogen), and ROTEM® (Tem International GmbH, Munich, Germany) variables were analyzed using blood from 25 anesthetized male baboons and 21 non-anesthetized healthy volunteers. The platelet component of the clot was calculated as the difference in maximum clot elasticity (MCE) between the whole blood clot (EXTEM test) and the fibrin-based clot (FIBTEM test). In subgroups of each species, 10 μg abciximab was added to the regular FIBTEM reagent (cytochalasin D) for additional platelet inhibition.

Results

Blood cell count was comparable between humans and primates. Both fibrinogen concentration (p < 0.0001) and maximum clot firmness (MCF) in FIBTEM assays were significantly lower in baboons (p > 0.0001, and p = 0.006, respectively). PT, INR, and clotting time in NATEM assays were significantly prolonged in humans compared with baboons. MCF in NATEM, EXTEM and INTEM assays was not different between baboons and humans. Clot lysis in NATEM, EXTEM and INTEM assays was significantly higher in humans (p < 0.0001). In contrast FIBTEM clot lysis was significantly higher in baboons (p = 0.01). Addition of abciximab into the FIBTEM assay resulted in a significant reduction in MCF and MCE (p < 0.001) and, consequently, the platelet component increased similar in both humans and baboons (p < 0.001).

Conclusion

Activated ROTEM® tests revealed broad similarities between humans and baboons. ROTEM® assays developed for use in humans can also be used in baboons.     

Long-term stability of fibrinolytic factors stored at -80 °C

Introduction

Blood samples in epidemiological studies are often stored for several years and analysed at different occasions. The reagent kits are continually modified for better precision and accuracy. Our hypothesis was that epidemiological studies are affected by long-term storage and/or modifications of reagent kits.

Materials and Methods

Plasma samples stored at -80 ^°C from two populations were used: A case-referent study with samples collected from 1985 to 2000 and analysed 2005 (n = 1598) were used to study influence of long-term storage. A cross-sectional study analysed 1990 (n = 1558) and re-analysed 2001 (n = 78) and 2005 (n = 828) was used to study influence of reagent kit modifications. Fibrinolytic analyses included immunoassays of tPA, PAI-1 and tPA-PAI-1 complex and chromogenic substrate assays of the activities of tPA and PAI-1.

Results

Long-term storage for a median time of 11.6 years (range 5 to 20) showed an effect of time on tPA antigen R² = 0.01, PAI-1 antigen R² = 0.01 and tPA-PAI-1 complex R² = 0.02. Modifications in reagent kits affected the levels of fibrinolytic factors; for tPA antigen the slope coefficients were between 0.72 and 0.95 (R² 0.47 - 0.75), whereas tPA activity showed an agreement with slope coefficients 1.06 to 1.09 (R² 0.67 - 0.93).

Conclusions

This study showed that long-term storage affects fibrinolytic variables to a negligible extent, but modifications in reagent kits introduced an element of bias. We conclude that analysis of samples on a single occasion is preferable to multiple occasions, as storage has negligible effect.