Effect of renal function on whole blood and fibrin clot formation in atrial fibrillation patients on warfarin

Objective: Atrial fibrillation (AF) brings a risk of thrombosis, requiring oral coagulation, and is associated with renal impairment. The two processes may be linked, as altered fibrin clot structure is present in end-stage renal failure. We hypothesised that progressively deteriorating renal function is linked to altered whole blood and fibrin clot properties and fibrinolysis. Methods: Thrombogenesis and fibrinolysis in 200 warfarinised AF patients was assessed by thromboelastography (TEG), a micro-plate assay (MPA) and the international normalized ratio (INR). Renal function was determined by creatinine clearance and two versions of the estimated glomerular filtration rate (eGFR). Results: Two TEG indices independently reflecting thrombogenesis were linked to creatinine clearance (p?p?p?p?p?=?0.001). The INR was unrelated to any renal function index, and the CHA₂DS₂VASc score was unrelated to any index. Conclusion: In warfarinised AF patients, renal function is linked to whole blood clot and fibrin clot formation, structure and dissolution, but has no effect on the INR.

• Key messages

• Despite oral anticoagulation, patients with atrial fibrillation (AF) still suffer from stroke and venous thromboembolism.

• The effect of renal function in warfarinised patients with AF is unknown and may account for excess thrombosis and/or haemorrhage.

• Using two different laboratory methods, our data point to an effect of renal function on clot structure and function that is independent of an effect of warfarin.

     

Evidence that fibrinogen γ′ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness

Summary. Background: Fibrinogen contains an alternatively spliced γ‐chain (γ′), which mainly exists as a heterodimer with the common γA‐chain (γA/γ′). Fibrinogen γ′ has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown. Objective: We aimed to investigate the molecular mechanism underpinning the influence of γ′ on fibrin polymerization, structure and viscoelasticity. Methods: γA/γA and γA/γ′ fibrinogens were separated using anion exchange chromatography. Cross‐linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel‐point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. Results: γA/γ′ fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ′ clots displayed a non‐homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non‐cross‐linked γA/γA fibrin was approximately 2.7 × stiffer than γA/γ′. Cross‐linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ′). Conclusions: Fibrinogen γ′ is associated with the formation of mechanically weaker, non‐uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ′.     

The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease

BACKGROUND: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. OBJECTIVES: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. METHODS: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k(cat) and K(m) values for plasmin action on fibrin were determined. RESULTS: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 microM and did not increase further as the fibrinogen concentration was raised to 20 microM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 microM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K(m) and k(cat) values for plasmin were 1.1 +/- 0.6 microM and 28 +/- 2 min(-1), respectively. K(m) values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 +/- 0.2 microM in the purified system and 2.1 +/- 0.9 microM in plasma. CONCLUSION: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.     

MTHFR基因型与晚期肾脏病/肾移植受者血浆高半胱氨酸水平及心血管病发生率的关系

目的 探讨血浆高半胱氨酸(Hcy)在肾脏病患者发生心血管疾患中的影响。方法 采用荧光偏振免疫分析、DNA基因型别分析等技术,对50例晚期肾脏病患者及肾移植受者进行了Hcy水平、N5,N10-亚甲基四氢叶酸还原酶(MTHFR)基因多态性分布测定及相关分析,并与肾病综合征患者及正常人进行对照。结果 (1)受检人群中Hcy水平较正常人及肾病综合征患者组明显升高;(2)受检人群中MTHFR的TT突变机率较正常人及肾病综合征组明显升高;(3)TT基因型与Hcy水平升高及心血管疾患的发生机率密切相关。结论 血浆中的Hcy可能是晚期肾脏病患者和肾移植受者体内的一种重要毒性物质。受检人群中MTHFR的高频率TT突变可能与血浆Hcy的水平升高有关。     

Antibodies to tissue-type plasminogen activator (t-PA) in patients with inflammatory bowel disease: high prevalence, interactions with functional domains of t-PA and possible implications in thrombosis

BACKGROUND: Patients with inflammatory bowel disease (IBD) have an increased prevalence of thromboembolic events. The pathogenetic mechanisms of these events include reduced fibrinolysis, which may be caused by antibodies to tissue-type plasminogen activator (t-PA). OBJECTIVES: To evaluate anti-t-PA antibodies in patients with IBD, considering clinical, biochemical and functional characteristics. PATIENTS AND METHODS: We immunoenzymatically measured anti-t-PA antibodies in plasma from 97 consecutive IBD patients and 97 age- and sex-matched healthy controls. We also assessed the antibody interactions with different epitopes of t-PA, the antibody inhibition on t-PA activity and the correlations with clinical features and other serum antibodies. RESULTS: IBD patients had higher median anti-t-PA antibody levels (5.4 U mL(-1) vs. 4.0 U mL(-1); P < 0.0001): 18 patients were above the 95th percentile of the controls (OR 5.3; 95% CI 1.7-16.3; P < 0.003), and the six with a history of thrombosis tended to have high levels (6.9 U mL(-1)). Anti-t-PA antibody levels did not correlate with IBD type, activity, location or treatment, or with age, sex, acute-phase reactants or other antibodies. The anti-t-PA antibodies were frequently IgG1 and bound t-PA in fluid phase; they recognized the catalytic domain in 10 patients and the kringle-2 domain in six. The IgG fraction from the three patients with the highest anti-t-PA levels slightly reduced t-PA activity in vitro. CONCLUSIONS: The prevalence of anti-t-PA antibodies is high in IBD patients. By binding the catalytic or kringle-2 domains of t-PA, these antibodies could lead to hypofibrinolysis and contribute to the prothrombotic state of IBD.     

Elevated plasma fibrin D‐dimer as a risk factor for vascular dementia: the Three‐City cohort study

Summary. Background: Hemostatic biomarkers have been associated with coronary heart disease (CHD) and stroke. However, few studies have investigated these associations in the elderly. Moreover, vascular factors may be involved in dementia. Data on the relationship between hemostatic biomarkers and dementia remain scarce. Objectives: Our study aimed to investigate the relationship between hemostatic biomarkers and the risk of CHD, stroke and dementia in an elderly population. Patients/Methods: In the Three‐City cohort study including men and women aged ≥ 65, we investigated the association of fibrinogen, D‐dimer and von Willebrand factor with the 4‐year incidence of arterial disease (CHD, n = 199; and stroke, n = 111) and dementia (n = 218). Measurements were performed for all cases and for a random sample of the entire cohort (n = 1254). Hazards ratios (HR) compared the last quintile with the first of each parameter’s distribution and P‐values refer to the test for linear trend across quintiles. Results: Elevated fibrinogen was associated with the risk of CHD and myocardial infarction (HR = 2.20, P < 0.05 and 2.45 P < 0.05, respectively). Moreover, high D‐dimer was associated with the risk of CHD among younger subjects (aged < 75, HR = 3.64, P < 0.01) but not older subjects (P for interaction = 0.01). Furthermore, the risk of vascular dementia (VaD) increased with D‐dimer level (HR = 3.05, P < 0.01). Conclusions: In the elderly, elevated fibrinogen and D‐dimer levels were associated with incident arterial disease. In addition, high D‐dimer level could represent a new risk factor for VaD.     

Genome scan of clot lysis time and its association with thrombosis in a protein C‐deficient kindred

Summary. Background: Previously, we found increased clot‐lysis time (CLT), as measured with a plasma‐based assay, to increase the risk of venous thrombosis in two population‐based case–control studies. The genes influencing CLT are as yet unknown. Patients/Methods: We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis. Results: Protein C‐deficient family members had shorter CLTs than non‐deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4‐fold in non‐deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9‐fold. Combining both risk factors yielded a 27.8‐fold increased risk. The heritability of CLT was 42–52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin‐activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. Conclusion: Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13.     

Modeling premature occurrence of acute coronary syndrome with atherogenic and thrombogenic risk factors and gene markers in extended families

BACKGROUND: The interaction between genetic and environmental risk factors in determining premature cardiovascular events has been largely investigated in case-control association studies. By contrast, few family based analyses have been performed so far. PATIENTS/METHODS: From a series of 2936 subjects aged 45-64, we selected probands who died for a premature (<50 years) ischemic heart disease (IHD) and with at least one family member with a history of IHD also occurring under the age of 50. Ninety-four families from 32 pedigrees including 296 subjects were identified. In this population, we analyzed the relationship between background risk factors [age, gender, the G1691A polymorphisms of factor V gene, the C677T polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene, the 844ins68bp polymorphisms of the cystathionine-beta-synthase (CBS) gene, and the apolipoprotein E (APOE) polymorphisms] and environmental risk factors, both atherogenic (smoke, hypertension, diabetes, dyslipidemia, obesity) and thrombogenic (smoke, homocysteine, fibrinogen) by a Markov block-recursive modeling approach. Results: None of the studied polymorphisms had an independent direct effect on the risk of IHD. As opposed to atherogenic factors, thrombogenic factors (homocysteine and fibrinogen) turned out to be possible mediators of the indirect effect of the MTHFR gene on IHD risk (OR: 1.30; 95% CI: 1.01-1.69, for every 8 mm increase in plasma levels of homocysteine in TT-carriers compared with CT/CC-carriers (OR: 1.11; 95% CI: 1.01-1.22), for every 20 g L(-1) increase in plasma levels of fibrinogen in TT-carriers compared with CT/CC-carriers). Conclusion: Plasma levels of homocysteine and fibrinogen may be interpreted as intermediate factors mediating the effect of predisposing genes on the risk premature cardiovascular disease.     

Role of clotting factors and fibrin structure in predisposition to atherothrombotic disease

Atherothrombotic disease is a multifactorial disorder that develops secondary to a complex gene–environment interaction. The formation of an obstructive thrombus represents the final stage of the atherothrombotic process, and understanding the mechanisms involved in clot formation is essential in order to develop new preventive and therapeutic strategies aimed at decreasing mortality and morbidity from the disease. Studies have demonstrated an important correlation between final clot structure and predisposition to atherothrombotic disease. Both genetic and environmental factors contribute to the final ultrastructure of the clot, which, in turn, influences an individual’s risk of the disease. This paper reviews the factors involved in determining clot structure. The role of commonly used therapeutic agents in modulating clot structure will also be discussed.     

Inflammation, thrombosis and atherosclerosis: results of the Glostrup study

Summary.  Inflammation and thrombosis are important mechanisms in cardiovascular disease, as illustrated by the consistent association between inflammatory and hemostatic variables and the risk of cardiovascular events in epidemiological studies. However, the relationship between plasma concentrations of inflammatory and hemostatic markers and the severity of atherosclerosis is not yet well studied. We have evaluated 325 men and 370 women of 60 years, participating in the Danish Glostrup study. We diagnosed atherosclerosis by ultrasonographic measurement of intima-media thickness (IMT) of the right carotid artery and the assessment of plaque occurrence. Plasma samples were analyzed for the concentration of C-reactive protein (CRP), fibrinogen, d -dimer, plasminogen activator inhibitor type-1 (PAI-1) antigen and activity, tissue-type plasminogen activator (t-PA) antigen and activity, factor VII (FVII) antigen, FVII coagulant activity (FVII:C) and activated FVII (FVIIa). DNA variations were determined for fibrinogen, PAI-1, t-PA, FVII, factor XIII and methylene tetrahydrofolate reductase (MTHFR). Subjects with high IMT (upper 10% of distribution, n  = 63) had higher CRP levels [2.2 mg L⁻¹ (SE 0.3)] than subjects with IMT in the lowest tertile ( n  = 217) [1.7 mg L⁻¹ (SE 0.1), P  = 0.04], whereas there was no association between the hemostatic variables and IMT. There was an association between fibrinogen and d -dimer concentrations and number of plaques ( P  < 0.01), whereas there were no associations between CRP and the other hemostatic variables and the number of plaques. Genetic variation in the t-PA and MTHFR gene was associated with IMT. In conclusion, in the Glostrup population study, thrombosis and inflammation are associated with the severity of atherosclerosis, as reflected by IMT and plaque occurrence.     

血浆同型半胱氨酸及颈动脉斑块对老年女性冠心病的预测价值

目的 探讨血浆同型半胱氨酸（homocysteine,Hcy）水平及颈动脉内-中膜厚度（intima-media thickness,IMT）对老年女性冠心病的预测价值。方法 年龄≥60岁拟诊冠心病女性患者100例,根据冠状动脉造影结果分为冠心病组67例和非冠心病组33例,冠心病组再分为单支病变组33例,2支病变组26例和多支病变组8例,比较各组血浆Hcy水平、颈动脉IMT及Crouse积分差异;计算IMT预测老年女性冠心病的特异性,敏感性及阳性预测值。结果 冠心病组血浆Hcy（（17.24±7.86）μmol/L）、颈动脉IMT（（0.96±0.32）mm）、颈动脉Crouse积分（12.59±1.83）均高于非冠心病组（（12.92±3.30）μmol/L、（0.75±0.07）mm、4.90±4.50）（P〈0.05）;冠心病各亚组血浆Hcy水平、颈动脉IMT及Crouse积分为多支病变组〉2支病变组〉单支病变组,组间比较差异均有统计学意义（P〈0.05）;以IMT=0.96mm为诊断界点,IMT预测冠心病的特异性、敏感性、阳性预测值分别为78.8%（26/33）、70.1%（47/67）、87.0%（47/54）。结论 血浆Hcy水平及颈动脉IMT与老年女性冠心病发生及冠状动脉病变严重程度相关;血浆Hcy水平联合颈动脉IMT可预测老年女性是否发生冠心病及其严重程度。     

Evidence that α2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation

Summary.  Background : Plasma alpha₂-antiplasmin (α₂AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital α₂AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin-activated factor XIII (FXIIIa), α₂AP becomes covalently ligated to the distal α chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that α₂AP is covalently linked to plasma fibrinogen . That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross-linking of fibrinogen in the presence of calcium ions, can also incorporate α₂AP into fibrinogen in the circulation. Results and Conclusions : We now provide evidence that FXIII incorporates I¹²⁵-labelled α₂AP into the Aα-chain sites on fibrinogen or fibrin. We also measured the content of α₂AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2–1.8 moles per mole fibrinogen). We propose that α₂AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.     

Hcy、vWF及D-D联合检测对下肢深静脉血栓形成的诊断价值

目的探讨血浆同型半胱氨酸(Hcy)、血管性血友病因子(vWF)和D-二聚体(D-D)的水平及联合检测对下肢深静脉血栓(LEDVT)患者的诊断价值。方法选取118例LEDVT患者和60例同期健康体检者,分别检测样本中Hcy、vWF和D-D水平,使用ROC曲线确定此3项结果诊断LEDVT的临界值。结果 LEDVT组Hcy、vWF和D-D水平分别为(23.9±8.6)mol/L、(197.8±58.8)%、(3.21±2.82)g/mL,正常对照组分别为(17.7±4.7)mol/L、(125.9±35.6)%、(0.49±0.35)g/mL。LEDVT组的3项检测指标均明显高于正常对照组,差异有统计学意义(P0.05)。Hcy、vWF、D-D的ROC曲线下面积(AUC)为0.747、0.857、0.892。3项指标联合检测对LEDVT诊断的灵敏度为95.8%。结论Hcy、vWF、D-D是LEDVT的敏感指标,三者对LEDVT的早期诊断有着重要的临床价值。     

血浆同型半胱氨酸水平与早发冠心病患者冠脉病变特点的相关性分析

目的分析血浆同型半胱氨酸(Hcy)水平与早发冠心病患者冠脉病变特点的相关性。方法将209例早发冠心病患者设为早发冠心病组,同时选取100例非早发冠心病患者做对照组。根据冠脉病变支数将早发冠心病组分为单支病变组、双支病变组、三支病变组。分析早发冠心病患者的病变程度与Hcy水平的相关性。结果早发冠心病组血浆Hcy水平高于对照组(P<0.05)。早发冠心病组三个亚组的血浆Hcy水平随着病变累及支数增加呈上升趋势(P<0.05)。早发冠心病组的冠脉病变程度与血浆Hcy水平呈正相关(P<0.01)。结论早发型冠心病患者的血浆Hcy水平与其冠状动脉血管病变严重程度呈正相关。     

慢性肺源性心脏病患者心功能分级与HCY、NT-proBNP、Fbg、D-二聚体的相关性分析

目的探讨慢性肺源性心脏病(CHPD)患者心功能分级与同型半胱氨酸(HCY)、氨基末端B型脑钠肽前体(NTproBNP)、纤维蛋白原(Fbg)、D-二聚体(D-D)的相关性。方法 135例CHPD患者依据美国纽约心脏病协会(NYHA)心功能分级进行分级:心功能Ⅰ级(A组33例)、心功能Ⅱ级(B组39例)、心功能Ⅲ级(C组36例)、心功能Ⅳ级(27例),同期选择50名健康体检者作为对照组,比较各组血清HCY、NT-proBNT及血浆Fbg、D-D水平。同时,绘制ROC曲线,计算曲线下面积(AUC),以评价各指标对CHPD的诊断价值,并对心功能分级、HCY、NT-proBNP、Fbg、D-D进行相关性分析。结果在A组中HCY、Fbg、D-D升幅不显,与对照组相比差异无统计学意义(P0.05),而B、C、D组HCY、NT-proBNP、Fbg、D-D与对照组比较,差异均有统计学意义(P0.05)。NT-proBNP在疾病组中升幅较大,并且随着心功能分级增加而明显升高,差异有统计学意义(P0.05)。HCY、NT-proBNP、Fbg、D-D诊断CHPD的AUC分别为0.710、0.919、0.782、0.864。CHPD心功能分级与HCY、NTproBNP、Fbg、D-D具有明显的相关性(P0.01)。结论 HCY、NT-proBNP、Fbg、D-D可用于CHPD的监测和早期诊断。     

慢性阻塞性肺疾病患者血气分析与血浆纤维蛋白原的相关性研究

目的：探讨慢性阻塞性肺疾病患者血浆纤维蛋白原（FIB）水平及与血气分析相关性。方法：对30例慢性阻塞性肺疾病患者治疗前后血气分析、血浆纤维蛋白原及30例对照组的血浆纤维蛋白原分别进行测定。结果：血浆纤维蛋白原水平在慢性阻塞性肺疾病加重期患者明显升高，与对照组相比有显著性差异；治疗后血浆纤维蛋白原明显下降，与对照组相比无显著性差异；血浆纤维蛋白原与动脉血氧分压（PaO2）呈负相关。血浆纤维蛋白原与动脉血二氧化碳分压（PaCO2）呈正相关。结论：慢性阻塞性肺疾病患者血浆纤维蛋白原增高，可导致血液高凝状态，并易导致肺小动脉血栓形成、低氧血症和高碳酸血症。临床应改善缺氧、通气及适当的抗凝治疗对控制病情有益。     

Activated thrombin activatable fibrinolysis inhibitor levels are associated with the risk of cardiovascular death in patients with coronary artery disease: the AtheroGene study

Summary.  Background:  Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis. Results on the association between TAFI levels and the risk of coronary artery disease (CAD) are inconsistent. Objectives:  We investigated the association between TAFI levels and the risk of cardiovascular events in CAD. Patients/Methods:  1668 individuals with angiographically proven CAD at baseline were followed for a median of 2.3 years, as part of the prospective Athero Gene cohort. Fifty-six deaths from cardiovascular (CV) causes and 35 non-fatal CV events were observed. Results:  At baseline, three TAFI measurements were available: one evaluating the total amount of TAFI (t-TAFI), one measuring the TAFIa/TAFIai amount, and the last the released activated peptide (TAFI-AP). TAFIa/TAFIai levels were associated with increased risk of CV death [hazard ratio (HR) for one tertile increase, 2.38 (1.56–3.63); P  < 10⁻⁴]. This association remained significant after adjustment for conventional risk factors, CRP levels, white blood count and markers of thrombin generation and fibrinolysis [HR = 1.69 (1.07–2.67); P  = 0.01]. In addition, CPB2 gene polymorphisms explained 12%, 6%, and 3% of t-TAFI, TAFIa/TAFIai and TAFI-AP levels, respectively, but none was associated with CV events. Conclusions:  The amount of activated TAFI, measured by TAFIa/TAFIai ELISA, but not of the t-TAFI is independently associated with the risk of CV death.