Plasma Clot Lysis Time Research Articles

Restenosis is associated with prothrombotic plasma fibrin clot characteristics in endovascularly treated patients with critical limb ischemia

IntroductionHypolysible fibrin clots composed of tightly packed fibers characterize patients with peripheral artery disease (PAD) especially those with critical limb ischemia (CLI). Little is known about the impact of a prothrombotic clot phenotype on restenosis following endovascular revascularization in CLI. The goal of this study was to compare fibrin clot properties and their determinants in CLI patients with restenosis after endovascular treatment (ET) and those free of this complication.Methods85 patients with CLI and restenosis within 1 year after ET on optimal pharmacotherapy and 47 PAD control patients without restenosis were included into the study. Plasma fibrin clot permeability (Ks, a measure of the average pore size in the fibrin network) and clot lysis time (CLT) with its potential determinants were determined. During follow-up, the composite endpoint including re-intervention, amputation and death was assessed.ResultsCompared with the control group, patients with restenosis had reduced Ks (− 9.5%, p < 0.001), prolonged CLT (+ 12.4%, p = 0.003), higher thrombin generation (+ 7.9%, p < 0.001) and elevated von Willebrand factor (vWF) antigen (+ 14.2%, p < 0.001). During a 24 months follow-up the composite endpoint occurred in 54 CLI patients with restenosis (63.5%) and nine control patients (19.1%, p < 0.001) with no association with baseline Ks and CLT.ConclusionThe increased thrombin formation and unfavorable fibrin clot properties occur in patients with CLI who experienced restenosis despite optimal endovascular and pharmacological therapy.

Faster fibrin clot degradation characterizes patients with central pulmonary embolism at a low risk of recurrent peripheral embolism

It is unclear whether thrombus location in pulmonary arteries is associated with particular clot characteristics. We assessed 156 patients following either central or peripheral pulmonary embolism (PE). Plasma clot lysis time, the rate of D-dimer release from plasma clots (D-Drate) with the maximum D-dimer concentration achieved (D-Dmax), as well as fibrin formation on turbidimetry, plasma clot permeation, thrombin generation, and fibrinolytic parameters were measured 3–6 months after PE. Patients following central PE (n = 108, 69.3%) were more likely smokers (38.9% vs 18.8%; p = 0.01), less likely carriers of factor XIII Val34Leu allele (40.7% vs 62.5%, p = 0.01), exhibited 16.7% higher D-Drate and 12.7% higher tissue plasminogen activator antigen (tPA:Ag) compared with peripheral PE (p = 0.02 and p < 0.0001, respectively). Saddle PE patients (n = 31, 19.9%) had 11.1% higher D-Drate and 7.3% higher D-Dmax compared with central PE (both p < 0.05). Twenty-three recurrent PE episodes, including 15 central episodes, during a median follow-up of 52.5 months were recorded. Plasma D-dimer and tPA:Ag were independent predictors for central recurrent PE, whereas D-Drate and peak thrombin predicted peripheral recurrent PE. Plasma clots degradation is faster in patients following central PE compared with peripheral PE and fibrinolysis markers might help to predict a type of recurrent PE.

Reduced plasma fibrin clot permeability is associated with recurrent thromboembolic events in patients with antiphospholipid syndrome.

APS is associated with arterial and venous thrombosis. The unfavourable fibrin clot phenotype, including formation of dense and poorly lysable clots, has been reported in thrombotic APS. We investigated whether abnormal plasma clot properties are predictive of recurrent thromboembolism in APS. We followed 126 consecutive patients with thrombotic APS and 105 control subjects, without APS, matched for thrombotic events. Plasma fibrin clot permeability (Ks), turbidity measurements and clot lysis time were evaluated ⩾5 months after a thrombotic event. The primary composite end point was symptomatic recurrent venous thromboembolism, ischaemic stroke and/or myocardial infarction. During follow-up (median, 62 months; range 46-74 months; 1183.2 patient-years), the primary outcome was observed in 33 (26.2%) APS patients and 16 (15.2%) controls, including 25 (19.8%) and 14 (13.3%) subjects with recurrent venous thromboembolism, respectively. Reduced Ks and prolonged clot lysis time predicted recurrent thromboembolic events in APS patients [per 1 × 10-9 cm2: hazard ratio (HR) = 0.37; 95% CI: 0.24, 0.56; and per 10 min: HR = 1.20; 95% CI: 1.01, 1.40, respectively] and in controls (per 1×10-9 cm2: HR = 0.23; 95% CI: 0.11, 0.42; and per 10 min: HR = 1.51; 95% CI: 1.08, 2.16, respectively). A multivariate analysis showed that positive IgG and IgM anti-β2 glycoprotein I antibodies, withdrawal of anticoagulation, lower platelet count and reduced Ks predicted thromboembolic events in APS patients. Formation of denser fibrin networks could be a novel risk factor for recurrent thromboembolism in APS, which highlights the importance of fibrin phenotype in thrombotic disorders.

Fibrin clot properties independently predict adverse clinical outcome following acute coronary syndrome: a PLATO substudy.

AimsTo determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syndrome (ACS).Methods and resultsPlasma samples were collected at hospital discharge from 4354 ACS patients randomized to clopidogrel or ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial. A validated turbidimetric assay was employed to study plasma clot lysis time and maximum turbidity (a measure of clot density). One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were estimated using Cox proportional hazards models. After adjusting for CV risk factors, each 50% increase in lysis time was associated with CV death/spontaneous MI [HR 1.17, 95% confidence interval (CI) 1.05–1.31; P < 0.01] and CV death alone (HR 1.36, 95% CI 1.17–1.59; P < 0.001). Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death (HR 1.24, 95% CI 1.03–1.50; P = 0.024). After adjustment for other prognostic biomarkers (leukocyte count, high-sensitivity C-reactive protein, high-sensitivity troponin T, cystatin C, N-terminal pro B-type natriuretic peptide, and growth differentiation factor-15), the association with CV death remained significant for lysis time (HR 1.2, 95% CI 1.01–1.42; P = 0.042) but not for maximum turbidity. These associations were consistent regardless of randomized antiplatelet treatment (all interaction P > 0.05). Neither lysis time nor maximum turbidity was associated with major bleeding events. ConclusionFibrin clots that are resistant to lysis independently predict adverse outcome in ACS patients. Novel therapies targeting fibrin clot properties might be a new avenue for improving prognosis in patients with ACS.

Effects of ultra-low-dose versus standard hormone therapy on fibrinolysis and thrombin generation in postmenopausal women

ObjectiveTo assess the effects of different doses of oral hormone therapy (HT) on thrombin generation and fibrinolysis. Study designOne hundred fifty postmenopausal women were assigned in a randomized controlled study in which the effect of standard dose (1mg 17β-estradiol/5mg dydrogesterone), ultra-low-dose HT (0.5mg 17β-estradiol/2.5mg dydrogesterone) on fibrinolysis and coagulation was compared to controls. Factors measured included plasma clot lysis time (CLT), fibrinolysis activators and inhibitors, thrombin generation (prothrombin fragments 1+2 [F1+2], endogenous thrombin potential [ETP]), normalized activated protein C sensitivity ratio (nAPCsr), and factor (F)VIII activity and were determined before and after 24 weeks of HT. ResultsIn ultra-low-dose group posttreatment CLT tended to be shorter when compared to standard HT (−7%) and control group (−4%) with reduced plasminogen activator inhibitor-1 (PAI-1) antigen (by −16% vs both groups). Higher mean changes from baseline between ultra-low-HT group and standard group in CLT (−7.4 vs 3.05) and PAI-1 antigen concentration (−5.1 vs 0.9) were observed. Standard HT, compared with the ultra-low-dose HT, led to higher F1+2 (+9%) and FVIII activity (+37%). ConclusionsIn contrast to the standard HT, ultra-low-dose HT may enhance fibrinolysis through reduced PAI-1 levels.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

BβArg448Lys polymorphism is associated with altered fibrin clot structure and fibrinolysis in type 2 diabetes.

Both type 2 diabetes (T2DM) and Bβ448Lys variant of fibrinogen are associated with dense fibrin clots, impaired fibrinolysis and increased cardiovascular risk. It was our objective to investigate whether BβArg448Lys adds to vascular risk by modulating fibrin network structure and/or fibrinolysis in diabetes. The primary aim was to study effects of BβArg448Lys on fibrin network characteristics in T2DM. Secondary aims investigated interactions between gender and BβArg448Lys substitution in relation to fibrin clot properties and vascular disease. Genotyping for BβArg448Lys and dynamic clot studies were carried out on 822 T2DM patients enrolled in the Edinburgh Type 2 Diabetes Study. Turbidimetric assays of individual plasma samples analysed fibrin clot characteristics with additional experiments conducted on clots made from purified fibrinogen, further examined by confocal and electron microscopy. Plasma clot lysis time in Bβ448Lys was longer than Bβ448Arg variant (mean ± SD; 763 ± 322 and 719 ± 351 seconds [s], respectively; p<0.05). Clots made from plasma-purified fibrinogen of individuals with Arg/Arg, Arg/Lys and Lys/Lys genotypes showed differences in fibre thickness (46.75 ± 8.07, 38.40 ± 6.04 and 25 ± 4.99 nm, respectively; p<0.001) and clot lysis time (419 ± 64, 442 ± 87 and 517 ± 65 s, respectively; p=0.02), directly implicating the polymorphism in the observed changes. Women with Bβ448Lys genotype had increased risk of cerebrovascular events and were younger compared with Bβ448Arg variant (67.2 ± 4.0 and 68.2 ± 4.4 years, respectively; p=0.035). In conclusion, fibrinogen Bβ448Lys variant is associated with thrombotic fibrin clots in diabetes independently of traditional risk factors. Prospective studies are warranted to fully understand the role of BβArg448Lys in predisposition to vascular ischaemia in T2DM with the potential to develop individualised antithrombotic management strategies.

Impaired plasma clot lysis and its determinants in patients with obstructive sleep apnea syndrome.

Evidence indicates that hypercoagulability and impaired fibrinolysis have been observed in patients with obstructive sleep apnea syndrome (OSAS). It is unclear which factors determine prolonged fibrin clot lysis in OSAS. One hundred and sixty-five consecutive patients suspected of OSAS underwent overnight polysomnography. Prior to polysomnography, we determined plasma clot lysis time (CLT), plasminogen activator inhibitor (PAI)-1 antigen, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), plasmin, and antiplasmin. OSAS was diagnosed in 110 (66.7%) patients, including 35 (31.8%) with severe OSAS, 26 (23.6%) with moderate OSAS, and 49 (44.6%) mild OSAS. Compared with 55 (33.3%) individuals in whom OSAS was not confirmed, OSAS patients had prolonged CLT (+12.8%), associated with higher PAI-1 antigen (+18.1%) (after adjustment for age, diabetes, and body mass index; both P < 0.01) and similar levels of TAFIa, plasmin, or antiplasmin. PAI-1, TAFIa, and CLT correlated positively with apnea/hypopnea index, which reflects the severity of OSAS (R = 0.66, P < 0.001; R = 0.29, P = 0.002; R = 0.55, P = 0.001, respectively), and with other polysomnography parameters, with the most potent correlations observed for desaturation index. Regression analysis adjusted for potential confounders showed that in OSAS, CLT was independently predicted by apnea/hypopnea index (B = 0.29, P = 0.002), PAI-1 (B = 0.42, P < 0.001), and TAFIa (B = 0.81, P = 0.044), whereas both PAI-1 and TAFIa were predicted only by desaturation index (B = 0.24, P = 0.002; and B = 0.14, P = 0.001, respectively). The severity of OSAS is closely associated with hypofibrinolysis measured in a global plasma-based assay, driven largely by PAI-1. Attenuated fibrinolysis might contribute to high risk of thromboembolic events in this disease.

Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis

Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca++), whereas this shortening was not observed in the presence of Ca++ where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in which tPA is supplemented in excess and PAI-1 activity is mostly lost. On the contrary, thrombin dose-dependently prolonged the tPA-PCLT, which was mostly abolished by inhibitors of carboxypeptidase and activated FXIII, suggesting that the prolongation is TAFI- and Factor XIII-dependent. The s-PCLT was shortened when thrombin generation was boosted by supplementing tissue factor and phosphatidylserine together with Ca++, which was more apparent in the presence of inhibitors of activated FXIII and activated TAFI. Thus, thrombin appeared to express its enhancing effect on fibrinolysis even in plasma, in addition to its inhibiting effect. These bidirectional functions of thrombin on fibrinolysis seem to take place on demand under different environments to maintain adequate vascular blood flow.

Determinants of hypofibrinolysis in patients with digestive tract cancer.

IntroductionRecently, we demonstrated that digestive tract cancer (DTC) is associated with reduced fibrin clot permeability and impaired fibrinolysis.AimWe investigated determinants of fibrinolysis in DTC patients.Material and methodsIn 44 consecutive patients with DTC and 47 controls matched for age, sex, and cardiovascular risk, we evaluated fibrinolysis proteins, platelet activation markers, thrombin formation, together with plasma clot lysis time assays in the absence (CLT) and presence of carboxypeptidase potato inhibitor (CLT CPI) that blocks thrombin activatable fibrinolysis inhibitor (TAFI).ResultsIn the DTC group CLT (by 22.3%) and CLT CPI (by 27.4%) were longer compared with controls. The DTC patients had higher plasma fibrinolysis inhibitors, plasminogen activator inhibitor 1 (PAI-1) (by 18.2%), TAFI activity (by 17.3%), and antigen (by 11.2%). The patients had markedly increased platelet markers – soluble CD40 ligand (by 338%) and P-selectin (by 97%), together with von Willebrand factor (vWF) antigen (by 61%). Thrombin-antithrombin complexes (TAT) (by 48.7%) and soluble thrombomodulin (sTM) (by 17.2%) were also increased in the DTC group (all p < 0.05). Patients with high-grade tumours (n = 26) compared with remainders (n = 18) had longer CLT, higher tissue-type plasminogen activator antigen, both TAFI antigen and activity levels, vWF, and sTM. Multiple regression analysis after adjustment for potential confounders showed that independent predictors of CLT in DTC patients were TAT, TAFI activity, and vWF. The only independent predictor of CLT CPI was TAT.ConclusionsHypofibrinolysis in DTC patients is largely driven by enhanced thrombin generation, TAFI, and endothelial injury.

Proteinuria Severity Is Directly Correlated with Markers of Hypofibrinolysis in a Podocyte Specific Experimental Model of Nephrotic Syndrome

IntroductionNephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury or dysfunction), hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. However, anticoagulation is associated with a substantial risk for adverse bleeding events. Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. We have recently demonstrated that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity),in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Thromboelastometry studies also suggested a resistance to fibrinolysis during rat NS. Thus, the aim of the present study was to further delineate the relationship between proteinuria severity and hypofibrinolysis using a podocyte-specific rat model of NS. We hypothesized that hypofibrinolysis is directly proportional to severity of proteinuria.MethodsUsing a transgenic rat which expresses the human diphtheria toxin receptor (hDTR) on a podocyte specific promoter (podocin), we compared markers of global hemostasis (ROTEM) and fibrinolysis to proteinuria severity. A range of proteinuria severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On Day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Thrombin and plasmin generation assays are currently being performed.ResultsThere were significant differences (P<0.005) between the highest proteinuria hDTR rat group and controls, in both hypercoagulopathic (clot formation time, maximum clot firmness, and clot size (amplitude at 10 & 20 min)), and hypofibrinolytic (amount of lysis at 60 min; LI60) ROTEM parameters. Importantly, there was a significant negative correlation between proteinuria severity and LI60 (R2 =0.362, P=0.02), suggesting that hypofibrinolysis is directly proportional to podocyte injury and therefore disease severity during NS. Preliminary results from the CLA (n=2 control & 2 high proteinuria) also suggest a marked impairment (~50% difference) in plasma clot lysis time in proteinuria rats.ConclusionsThese results demonstrate that proteinuria severity is directly proportional to both hypercoagulability and hypofibrinolytic capacity in a podocyte-specific rodent model of NS, thus confirming our recent findings in two other well-established animal models of NS in a third, more specific, experimental model of glomerular disease. Importantly, these data also strongly suggest a marked impairment in fibrinolysis during NS, which is directly correlated with proteinuria severity. Therefore it appears that severe proteinuria is associated with both hypercoagulopathic and hypofibrinolytic defects in the coagulation system. Future studies will delve into the molecular mechanisms involved in these defects. DisclosuresNo relevant conflicts of interest to declare.

Denser plasma clot formation and impaired fibrinolysis in paroxysmal and persistent atrial fibrillation while on sinus rhythm: Association with thrombin generation, endothelial injury and platelet activation

IntroductionFormation of compact and poorly lysable fibrin clots have been demonstrated in patients following ischemic stroke. Recently, it has been shown that denser fibrin networks and impaired fibrinolysis occurs in subjects with permanent atrial fibrillation (AF). Fibrin clot phenotype in other types of AF remains to be established. We evaluated fibrin clot properties in paroxysmal (PAF) and persistent AF (PsAF). Material and MethodsWe studied 88 non-anticoagulated patients with AF on sinus rhythm and free of stroke (41 with PAF, 47 with PsAF) versus 50 controls. Ex-vivo plasma fibrin clot permeability (Ks) and clot lysis time (CLT) were evaluated along with von Willebrand factor (vWF), peak thrombin generation (TG), platelet factor 4 (PF4) and fibrinolytic proteins. ResultsCompared with control subjects, clots obtained from plasma of patients with PAF and PsAF had similarly lower Ks (−7.7%, P=0.01; −8.6%, P=0.005, respectively) and prolonged CLT (+10.8%, P=0.006; +7.8% P=0.04, respectively). No associations of Ks and CLT with CHA2DS2-VASc and HAS-BLED score were observed. Patients with AF had higher TG, vWF, PF4 and plasminogen activator inhibitor-1 (PAI-1) antigen compared with controls. Multiple linear regression adjusted for age, gender, body mass index and fibrinogen showed that TG (β=−0.41), vWF (β=−0.29) and PF4 (β=−0.28) are the independent predictors of Ks (R2=0.78), while CLT was independently predicted by TG (β=0.37), PAI-1 antigen (β=0.29) and vWF (β=0.26) in the AF group (R2=0.39). ConclusionsPatients with PAF and PsAF while on sinus rhythm display unfavorably altered fibrin clot properties, which might contribute to thromboembolic complications.

Altered plasma fibrin clot properties in essential thrombocythemia

Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 103/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (−38%, p < 0.0001) and prolonged CLT (+34%, p < 0.0001) were observed in ET. The differences remained significant after adjustment for fibrinogen and platelet count. ET was associated with a slightly shorter lag phase (−5%, p = 0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p < 0.001). The ET patients had higher plasma P-selectin by 193% (p < 0.00001) and platelet factor 4 (PF4) by 173% (p < 0.00001), with higher P-selectin observed in 19 (44%) patients with JAK-2 gene V617F mutation. Higher t-PA (+20%, p < 0.001), 23% higher plasminogen activator inhibitor-1, PAI-1 (+23%, p < 0.01) and unaltered thrombin-activatable fibrinolysis inhibitor, plasminogen and α2-antiplasmin activity were found in the ET group. Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.

Aspirin therapy is associated with less compact fibrin networks and enhanced fibrinolysis in patients with abdominal aortic aneurysm

Thrombotic changes in fibrin networks contribute to increased cardiovascular risk in patients with abdominal aortic aneurysm (AAA). Given that aspirin modulates the fibrin network, we aimed to determine if aspirin therapy is associated with changes in ex-vivo fibrin clot characteristics in AAA patients and also conducted an exploratory analysis of 5-year mortality in these individuals. We recruited 145 male patients, divided into controls (aortic diameter <3cm, n=49), AAA not taking aspirin (AAA-Asp, n=50) and AAA on 75mgday(-1) aspirin (AAA+Asp, n=46), matched for aneurysm size. Characteristics of clots made from plasma and plasma-purified fibrinogen were investigated using turbidimetric analysis, permeation studies, and confocal and electron microscopy. Plasma fibrinogen, D-dimer and inflammatory marker levels were also measured. Maximum absorbance (MA) of plasma clots from controls was lower than that of AAA patients not on aspirin (AAA-Asp) at 0.30±0.01 and 0.38±0.02au, respectively (P=0.002), whereas aspirin-treated subjects had MA similar to controls (0.31±0.02 P=0.9). Plasma clot lysis time displayed an identical pattern at 482±15, 597±24 and 517±27s for control, AAA-Asp and AAA+Asp (P=0.001 and P=0.8). The lysis time of clots made from purified fibrinogen of AAA-Asp was longer than that of AAA+Asp patients (756±47 and 592±52s, respectively; P=0.041). Permeation studies and confocal and electron microscopy showed increased clot density in AAA-Asp compared with the AAA+Asp group. Mortality in AAA-Asp and AAA+Asp was similar, despite increased cardiovascular risk in the latter group, and both exhibited higher mortality than controls. Aspirin improves fibrin clot characteristics in patients with AAA, which may have important clinical implications.

Increased Oxidation as an Additional Mechanism Underlying Reduced Clot Permeability and Impaired Fibrinolysis in Type 2 Diabetes.

Aims. We sought to investigate whether enhanced oxidation contributes to unfavorable fibrin clot properties in patients with diabetes. Methods. We assessed plasma fibrin clot permeation (K s, a measure of the pore size in fibrin networks) and clot lysis time induced by recombinant tissue plasminogen activator (CLT) in 163 consecutive type 2 diabetic patients (92 men and 71 women) aged 65 ± 8.8 years with a mean glycated hemoglobin (HbA1c) of 6.8%. We also measured oxidative stress markers, including nitrotyrosine, the soluble form of receptor for advanced glycation end products (sRAGE), 8-iso-prostaglandin F2α (8-iso-PGF2α), oxidized low-density lipoprotein (oxLDL), and advanced glycation end products (AGE). Results. There were inverse correlations between K s and nitrotyrosine, sRAGE, 8-iso-PGF2α, and oxLDL. CLT showed a positive correlation with oxLDL and nitrotyrosine but not with other oxidation markers. All these associations remained significant for K s after adjustment for fibrinogen, disease duration, and HbA1c (all P < 0.05), while oxLDL was the only independent predictor of CLT. Conclusions. Our study shows that enhanced oxidative stress adversely affects plasma fibrin clot properties in type 2 diabetic patients, regardless of disease duration and glycemia control.

Coronary thrombus composition: Links with inflammation, platelet and endothelial markers

Objectives: We investigated whether markers of platelet, neutrophil and endothelial activation, plasma fibrin clot properties and patient clinical profile may characterize coronary thrombus composition in ST-segment elevation myocardial infarction (STEMI) patients. Methods: A total of 40 intracoronary thrombi obtained 4.0–16.5 h since chest pain onset by manual aspiration during primary coronary intervention (PCI) were assessed using scanning electron microscopy. Plasma fibrin clot permeation coefficient (Ks) and clot lysis time (CLT), together with platelet and endothelial activation, fibrinolysis, and inflammation markers, were measured ex vivo in 16 patients on admission (pre-PCI group) and on the next morning in 24 patients (post-PCI group). Results: Fibrin, erythrocyte, platelet and white blood cell content in the thrombi were estimated at 49.1%, 24.2%, 11.6% and 3.7% respectively. In the pre-PCI group, in addition to fibrinogen, P-selectin and plasminogen activator inhibitor-1 were positively correlated with thrombus fibrin content. In the post-PCI group, in addition to von Willebrand factor antigen (vWF:Ag), soluble CD40 ligand and myeloperoxidase (MPO) were positively correlated with thrombus fibrin content. After adjustment for fibrinogen and onset-to-thrombectomy time circulating vWF:Ag in both groups, and MPO and P-selectin in the pre-PCI group were the independent predictors of fibrin-rich intracoronary thrombus presence. Other predictors were renal impairment, arterial hypertension and time from symptom onset to thrombus aspiration in all patients. Conclusions: In STEMI patients coronary thrombus composition is partly characterized by plasma markers of platelet, neutrophil and endothelial activation, with a varying contribution of these factors over time.

Reduced plasma fibrin clot permeability and susceptibility to fibrinolysis are associated with increased intima-media thickness in patients with primary antiphospholipid syndrome

IntroductionFormation of denser fibrin networks displaying impaired lysability has been reported in subjects at an increased risk of atherosclerosis. Given recent data on prothrombotic fibrin clot phenotype reported in patients with antiphospholipid syndrome (APS), we tested the hypothesis that altered fibrin clot properties are associated with increased intima-media thickness (IMT) observed in PAPS. Materials and methodsWe studied 30 consecutive patients with PAPS and 30 controls matched for age, sex and the type of previous thromboembolism. We assessed plasma fibrin clot permeability (Ks) and clot lysis time (CLT) with their potential determinants. The IMT was measured in 3 segments of the carotid arteries. ResultsPatients with APS had 15.2% lower Ks (p=0.002) and 9.7% prolonged CLT (p=0.039) compared with controls. The IMT in the APS group was greater in the common carotid artery (5.7%; p=0.002), at the bifurcation (17.46%; p<0.001), and the internal artery (9.26%; p=0.015). Patients with triple positivity in the antiphospholipid antibody profile (n=9; 30%) had lower Ks and greater IMT (both, p<0.05), compared with those with single positivity (n=13; 43.3%). Multivariate analysis adjusted for potential confounders showed that in APS patients, oxidized low-density lipoproteins (p=0.019) were the only independent predictor of Ks, while thrombin activatable fibrinolysis inhibitor activity (p<0.001) predicted CLT. Plasminogen activator inhibitor-1 (PAI-1) was found to be the independent predictor of the IMT in the common carotid artery (p=0.004), and in the internal carotid artery (p<0.001). ConclusionsReduced Ks and susceptibility to lysis are associated with greater IMT in PAPS, which might contribute to the early atherosclerosis in this disease.

In Black South Africans from Rural and Urban Communities, the 4G/5G PAI-1 Polymorphism Influences PAI-1 Activity, but Not Plasma Clot Lysis Time

Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT.

Abnormalities in blood coagulation, fibrinolysis, and platelet activation in adult patients after the Fontan procedure

Thrombosis occurs in up to 30% of patients with various forms of congenital single ventricle after the Fontan procedure. We investigated hemostatic abnormalities in adult Fontan patients. Forty-eight Fontan patients between ages 18 and 40 years, including 10 (21%) patients with previous thromboembolism 5 to 15 years after surgery, and 35 control subjects matched for age and sex were studied. Coagulation factors and inhibitors, together with markers of fibrinolysis, platelets, and endothelial activation, were determined in peripheral venous blood plasma. Compared with control subjects, Fontan patients showed lower, although mostly within normal ranges, values of all coagulation factors, as well as reduced free protein S, in association with higher antithrombin and free tissue factor pathway inhibitor levels. Thrombin generation, reflected by prothrombin fragment 1.2, and platelet activation markers were increased in Fontan patients. The plasma clot lysis time was prolonged in Fontan patients, which was associated with an increased activity of thrombin-activatable fibrinolysis inhibitor. Fontan patients with previous thromboembolism had lower oxygen saturation, coagulation factors V and VIII, and free protein S, and increased von Willebrand factor, soluble CD40 ligand, and P-selectin. Other laboratory or clinical parameters were not associated with prior thrombotic episodes. Adult Fontan patients are characterized by enhanced platelet activation and endothelial injury, heightened thrombin formation, and impaired fibrinolysis. Patients showed reduced free protein S levels, increased platelet activation, and endothelial damage after thromboembolic events observed late after Fontan surgery. Our findings indicate novel prothrombotic mechanisms in adult Fontan patients, which might help to optimize thromboprophylaxis.