Fladia Phoenix

Effects of Aspirin on Clot Structure and Fibrinolysis Using a Novel In Vitro Cellular System

Objectives—The purpose of this study was to investigate the direct effects of aspirin on fibrin structure/function. Methods and Results—Chinese Hamster Ovary cell lines stably transfected with fibrinogen were grown in the absence (0) and presence of increasing concentrations of aspirin. Fibrinogen was purified from the media using affinity chromatography, and clots were made from recombinant protein. Mean final turbidity [OD(±SEM)] was 0.083(±0.03), 0.093(±0.002), 0.101(±0.005), and 0.125(±0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (P<0.05). Permeability coefficient (Ks cm2×10−8) was 1.68(±0.29) and 4.13(±0.33) comparing fibrinogen produced from cells grown with 0 mg/L and 100 mg/L aspirin respectively (P<0.05). Scanning electron microscopy confirmed a looser clot structure and increased fiber thickness of clots made from aspirin-treated fibrinogen, whereas rheometer studies showed a significant 30% reduction in clot rigidity. Fibrinolysis was quicker in clots made from aspirin-treated fibrinogen. Ex vivo studies in 3 normal volunteers given 150 mg aspirin daily for 1 week demonstrated similar changes in clot structure/function. Conclusion—Aspirin directly altered clot structure resulting in the formation of clots with thicker fibers and bigger pores, which are easier to lyse. This study clearly demonstrates an alternative mode of action for aspirin, which should be considered in studies evaluating the biochemical efficacy of this agent.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Thyroid Dysfunction and Fibrin Network Structure: A Mechanism for Increased Thrombotic Risk in Hyperthyroid Individuals

CONTEXT Hyperthyroidism is associated with increased thrombosis risk, and fibrin clot structure determines susceptibility to vascular thrombotic events. OBJECTIVE Our objective was to investigate clot formation and lysis in hyperthyroidism using observational and interventional studies. DESIGN Ex vivo fibrin clot structure/fibrinolysis and plasma levels of thrombotic/inflammatory markers were investigated in hyperthyroid individuals (n = 24) and matched controls (n = 19), using turbidimetric assays, ELISA, and confocal and electron microscopy. The effects of normalizing thyroid function were analyzed (n = 19) and the role of short-term exogenous hyperthyroidism in healthy volunteers studied (n = 16). RESULTS Hyperthyroid subjects displayed higher clot maximum absorbance compared with controls (0.41 ± 0.03 and 0.27 ± 0.01 arbitrary units, respectively; P < 0.01), and longer clot lysis time (518 ± 23 and 461 ± 18 sec, respectively; P < 0.05), which correlated with free T(4) levels. Plasma levels of fibrinogen and plasminogen activator inhibitor-1 were significantly higher in patients compared with controls. Normalizing thyroid function in 19 subjects was associated with lower maximum absorbance and shorter lysis time, accompanied by reduction in fibrinogen, plasminogen activator inhibitor-1, and D-dimer levels. Complement C3, but not C-reactive protein, levels were higher in hyperthyroid subjects compared with controls (0.92 ± 0.05 and 0.64 ± 0.03 g/liter, respectively; P < 0.01), correlated with clot structure parameters, and decreased after intervention. Confocal and electron microscopy confirmed more compact clots and impaired fibrinolysis during hyperthyroidism. Exogenous hyperthyroidism in healthy volunteers had no effect on any of the clot structure parameters. CONCLUSIONS Endogenous hyperthyroidism is associated with more compact clots and resistance to fibrinolysis ex vivo, related to the degree of hyperthyroidism and C3 plasma levels, and these changes are modulated by achieving euthyroidism. Altered clot structure/lysis may be one mechanism for increased thrombotic risk in hyperthyroidism.

Gender-Specific Alterations in Fibrin Structure Function in Type 2 Diabetes: Associations with Cardiometabolic and Vascular Markers

CONTEXT Diabetes is associated with increased incidence of atherothrombotic disease. The fibrin network forms the backbone of the arterial thrombus, and fibrin clot structure determines predisposition to cardiovascular events. OBJECTIVES The aim of the study was to investigate fibrin clot structure/fibrinolysis in the largest type 2 diabetes cohort and analyze associations with cardiometabolic risk factors and vascular pathology. DESIGN Clot structure/fibrinolysis was assessed in 875 participants of the Edinburgh Type 2 Diabetes Study [age, 68 (range, 60-75) yr; 450 males] by turbidimetric assays, and clots were visualized by confocal microscopy. Four parameters of clot structure/fibrinolysis were analyzed, and plasma levels of fibrinogen and plasminogen activator inhibitor-1 were studied by Clauss assay and ELISA, respectively. RESULTS Clot maximum absorbance was increased in females compared with males (0.37 ± 0.005 and 0.34 ± 0.005 arbitrary unit, respectively; P < 0.001), and took longer to lyse (803 ± 20 and 665 ± 12 sec, respectively; P < 0.001). These gender differences in clot structure and fibrinolysis were still evident after correcting for fibrinogen and plasminogen activator inhibitor-1 plasma levels. A prothrombotic fibrin structure profile was associated with increased body mass index and low levels of high-density lipoprotein in women and with inadequate diabetes control in men. Clot formation time was related to previous cardiac ischemic events in both men and women after adjusting for traditional risk factors [odds ratio, 1.22 (95% confidence interval, 1.07, 1.38); and 1.33 (1.15, 1.50), respectively], and prothrombotic clots were associated with low ankle brachial index, renal impairment, and smoking, regardless of gender. CONCLUSIONS Women with type 2 diabetes have compact clots with compromised fibrinolysis compared with men. There are gender-specific associations between clotting parameters and cardiometabolic risk factors in this population, whereas vascular abnormalities, impaired renal function, and smoking are associated with prothrombotic clot structure profile regardless of gender.

Platelet P2Y12 Inhibitors Reduce Systemic Inflammation and Its Prothrombotic Effects in an Experimental Human Model

Objective—Clinical studies suggest that platelet P2Y12 inhibitors reduce mortality from sepsis, although the underlying mechanisms have not been clearly defined in vivo. We hypothesized that P2Y12 inhibitors may improve survival from sepsis by suppressing systemic inflammation and its prothrombotic effects. We therefore determined whether clopidogrel and the novel, more potent P2Y12 inhibitor, ticagrelor, modify these responses in an experimental human model. Approach and Results—We randomized 30 healthy volunteers to ticagrelor (n=10), clopidogrel (n=10), or no antiplatelet medication (controls; n=10). We examined the effect of P2Y12 inhibition on systemic inflammation, which was induced by intravenous injection of Escherichia coli endotoxin. Both P2Y12 inhibitors significantly reduced platelet–monocyte aggregate formation and peak levels of major proinflammatory cytokines, including tumor necrosis factor &agr;, interleukin-6, and chemokine (C–C motif) ligand 2. In contrast to clopidogrel, ticagrelor also significantly reduced peak levels of IL-8 and growth colony-stimulating factor and increased peak levels of the anti-inflammatory cytokine IL-10. In addition, ticagrelor altered leukocyte trafficking. Both P2Y12 inhibitors suppressed D-dimer generation and scanning electron microscopy revealed that ticagrelor also suppressed prothrombotic changes in fibrin clot ultrastructure. Conclusions—Potent inhibition of multiple inflammatory and prothrombotic mechanisms by P2Y12 inhibitors demonstrates critical importance of platelets as central orchestrators of systemic inflammation induced by bacterial endotoxin. This provides novel mechanistic insight into the lower mortality associated with P2Y12 inhibitors in patients with sepsis in clinical studies.

The influence of type 2 diabetes on fibrin clot properties in patients with coronary artery disease

Type 2 diabetes mellitus (T2DM) increases the risk of coronary thrombosis and both conditions are associated with altered fibrin clot properties. However, the influence of T2DM on fibrin clot properties in patients with coronary artery disease (CAD) remains unclear. We aimed to investigate the influence of T2DM on fibrin clot properties in patients with CAD. Fibrin clot structure and fibrinolysis were investigated in 581 CAD patients (148 with T2DM) using turbidimetric assays, confocal and scanning electron microscopy. Clots made from plasma and plasma-purified fibrinogen were studied, and plasma levels of inflammatory markers were analysed. T2DM patients had increased clot maximum absorbance compared with non-diabetic patients (0.36 ± 0.1 vs 0.33 ± 0.1 au; p=0.01), displayed longer lysis time (804 [618;1002] vs 750 [624;906] seconds; p=0.03) and showed more compact fibrin structure assessed by confocal and electron microscopy. Fibrinogen levels were elevated in T2DM (p< 0.001), but clots made from purified fibrinogen showed no differences in fibrin properties in the two populations. Adjusting for fibrinogen levels, T2DM was associated with C-reactive protein and complement C3 plasma levels, with the former correlating with clot maximum absorbance (r=0.24, p< 0.0001) and the latter with lysis time (r=0.30, p< 0.0001). Independent of fibrinogen levels, females had more compact clots with prolonged lysis time compared with males (all p-values< 0.001). In conclusion, T2DM is associated with prothrombotic changes in fibrin clot properties in patients with CAD. This is related to quantitative rather than qualitative changes in fibrinogen with a possible role for inflammatory proteins.

Effects of dabigatran on the cellular and protein phase of coagulation in patients with coronary artery disease on dual antiplatelet therapy with aspirin and clopidogrel. Results from a prospective, randomised, double-blind, placebo-controlled study.

There is growing interest in understanding the effects of adding an oral anticoagulant in patients on dual antiplatelet therapy (DAPT). Vitamin K antagonists (VKAs) and clopidogrel represent the most broadly utilised oral anticoagulant and P2Y12 receptor inhibitor, respectively. However, VKAs can interfere with clopidogrel metabolism via the cytochrome P450 (CYP) system which in turn may result in an increase in platelet reactivity. Dabigatran is a direct acting (anti-II) oral anticoagulant which does not interfere with CYP and has favourable safety and efficacy profiles compared with VKAs. The pharmacodynamic (PD) effects on platelet reactivity and clot kinetic of adjunctive dabigatran therapy in patients on DAPT are poorly explored. In this prospective, randomised, double-blind, placebo-controlled PD study, patients (n=30) on maintenance DAPT with aspirin and clopidogrel were randomised to either dabigatran 150 mg bid or placebo for seven days. PD testing was performed before and after treatment using four different assays exploring multiple pathways of platelet aggregation and fibrin clot kinetics: light transmittance aggregometry (LTA), multiple electrode aggregometry (MEA), kaolin-activated thromboelastography (TEG) and turbidimetric assays. There were no differences in multiple measures of platelet reactivity investigating purinergic and non-purinergic signaling pathways assessed by LTA, MEA and TEG platelet mapping. Dabigatran significantly increased parameters related to thrombin activity and thrombus generation, and delayed fibrin clot formation, without affecting clot structure or fibrinolysis. In conclusion, in patients on DAPT with aspirin and clopidogrel, adjunctive dabigatran therapy is not associated with modulation of profiles of platelet reactivity as determined by several assays assessing multiple platelet signalling pathways. However, dabigatran significantly interferes with parameters related to thrombin activity and delays fibrin clot formation.

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.

Prolonged Prothrombotic Effects of Antecedent Hypoglycemia in Individuals With Type 2 Diabetes

OBJECTIVE Hypoglycemia has been linked to persistent increases in cardiovascular (CV) mortality in type 2 diabetes after the event. Our aim was to examine acute and downstream effects of hypoglycemia on markers of thrombosis risk and inflammation in type 2 diabetes. RESEARCH DESIGN AND METHODS Twelve individuals with type 2 diabetes with no history of CV disease and 11 age- and BMI-matched volunteers without diabetes underwent paired hyperinsulinemic-euglycemic (glucose 6 mmol/L for two 60-min periods) and hypoglycemic (glucose 2.5 mmol/L for two 60-min periods) clamps on separate occasions on day 0. Fibrin clot properties, platelet reactivity, and inflammatory markers were measured at baseline, end of and after recovery from the initial clamp, day 1, and day 7 using validated assays and electron microscopy. RESULTS Euglycemic hyperinsulinemia reduced platelet reactivity, decreased fibrin clot density, and improved fibrinolytic efficiency in both groups. Platelet reactivity and aggregation increased during acute hypoglycemia in both groups, resolving at recovery. In type 2 diabetes, clot lysis times and clot maximum absorbance increased up to day 7 (P = 0.002 and 0.001 vs. euglycemia, respectively), but clots from control subjects without diabetes showed limited changes. Fibrin network density increased Δ 1.15 ± 0.28 fibers/μm2 at day 7 after the hypoglycemic clamp (P < 0.01 for glycemic arm), whereas fibrinogen and complement C3 increased after hypoglycemia up to day 7 in type 2 diabetes only. CONCLUSIONS Antecedent hypoglycemia has acute and persistent prothrombotic effects, lasting at least 7 days, that were enhanced in individuals with type 2 diabetes. These findings identify mechanisms by which hypoglycemia might increase short- and medium-term risk of CV mortality.

Potential mechanism for increased risk of premature vascular disease in long-term survivors of primary brain tumours with established hormone deficits

Abstract Background Long-term survivors of childhood-onset brain tumours have increased mortality rates, even after excluding deaths related to recurrence or regression of the original tumour. In particular, there is an increased risk of premature vascular disease by mechanisms that remain unclear. We tested our hypothesis that a thrombotic fibrin network profile is one mechanism for the increased vascular risk in this population. Methods We undertook a cross-sectional study in 33 patients (19 men, mean age 31·5 years [SD 14·2]) with previous history of primary brain tumours of either childhood or adulthood onset and 33 age-matched and sex-matched healthy controls. We performed clot structure analysis using a validated turbidimetric assay and also assessed plasma concentrations of thrombotic and inflammatory vascular markers including fibrinogen, C-reactive protein (CRP), and complement C3. The mean time from brain tumour diagnosis to the time of the study was 8·9 years (SD 6·2). Findings All patients had cranial radiotherapy, whereas 25 (76%) and 13 (39%) had additional surgery and chemotherapy, respectively. 31 patients (94%) had growth hormone deficiency, five (15%) had deficiences in luteinising hormone and follicle-stimulating hormone, four (12%) in adrenocorticotropic hormone, and two (6%) in thyroid-stimulating hormone. Patients had raised clot maximum absorbance (a measure of clot density) compared with controls (mean 0·412 arbritary units [SD 0·10] vs 0·277 [0·09], p vs 2720 [636], p vs 906 [704], p vs 0·78 [1·08], p=0·006) with similar findings for C3 (0·75 mg/mL [0·13] vs 0·67 [0·13], p=0·027). Interpretation We demonstrate, for the first time to our knowledge, that survivors of brain tumours with hypopituitarism display a thrombotic fibrin network profile, providing one mechanism for the increased vascular risk in this population. These changes are not related to altered fibrinogen concentrations suggesting that other plasma proteins or qualitative changes in the proteins might contribute to the observed differences. Further longitudinal studies are required to clarify whether optimisation of the hormonal profile results in amelioration of the thrombotic environment in this population. Funding Pfizer (investigator-initiated research grant).