Identification and validation of coagulation and fibrinolytic-related diagnostic biomarkers for ulcerative colitis by bioinformatics analysis.

Abnormalities in coagulation and fibrinolytic status have been demonstrated to be relevant to inflammatory bowel disease. Nevertheless, there is no study to methodically examine the role of the coagulation and fibrinolysis-related genes in the diagnosis of ulcerative colitis (UC). UC-related datasets (GSE169568 and GSE94648) were originated from the Gene Expression Omnibus database. The biomarkers related to coagulation and fibrinolysis were identified through combining differentially expressed analysis and machine learning algorithms. Moreover, Gene Set Enrichment Analysis and immune analysis were carried out. A total of 4 biomarkers (MAP2K1, CREBBP, TAF1, and HP) were identified, and biomarkers were markedly enriched in pathways related to immunity, such as T-cell receptor signaling pathway, primary immunodeficiency, chemokine signaling pathway, etc. In total, the infiltrating abundance of 4 immune cells between UC and control was markedly different, namely eosinophils, macrophage M0, resting mast cells, and regulatory T cells. And all biomarkers were significantly relevant to eosinophils. Our findings detected 4 coagulation and fibrinolysis-related biomarkers (MAP2K1, CREBBP, TAF1, and HP) for UC, which contributed to the advancement of UC for further clinical investigation.

Fibrinolysis is impaired in patients with primary immune thrombocytopenia.

BACKGROUND: Patients with primary immune thrombocytopenia (ITP) have an increased risk of thrombosis, which may be due to altered fibrinolysis. OBJECTIVES: To elucidate the clinical impact of delayed fibrinolysis in ITP patients. METHODS: A turbidimetric clot formation and lysis assay and a fluorometric plasmin generation (PG) assay were performed, and levels of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), tPA-PAI-1 complexes, α2-antiplasmin, thrombin activatable fibrinolysis inhibitor, and D-dimer were assessed in 86 adult primary ITP patients and 78 healthy controls (HCs). RESULTS: ITP patients showed significantly delayed clot formation, increased clot density, and prolonged clot lysis time (CLT) compared with HCs, with a median (IQR) CLT of 28.0 (13.7-34.7) minutes in patients and 17.3 (12.0-28.0) minutes in HCs, while in the PG assay, only the lag time was prolonged. In ITP patients compared with controls, PAI-1 was higher (1.2 [0.8-2.6] vs 1.1 [0.6-2.1] U/mL) and tPA antigen and activity were lower (tPA antigen: 2.6 [1.1-4.4] vs 3.7 [3.2-4.7] ng/mL; tPA activity ≤ 0 U/mL: 26% vs 7%). TPA-PAI-1 complex levels were positively associated with CLT in multiple linear regression analysis (ß = 0.241; P = .019), whereas PG parameters were not associated with CLT. Six patients who developed thrombosis during follow-up had higher levels of tPA-PAI-1 complexes. CONCLUSION: Prolonged CLT and delayed onset of PG may indicate a hypofibrinolytic tendency in ITP patients, as also indicated by high PAI-1 and low tPA levels. No association was found between fibrinolytic potential and the bleeding phenotype, whereas higher tPA-PAI-1 complex levels were associated with prolonged CLT and increased in patients with future thrombosis.

Reduced protein carbonylation on hormone therapy is associated with improved fibrinolysis in postmenopausal women: the impact of PAI-1 and TAFI activity.

Hormone therapy (HT) has been reported to reduce protein carbonylation (PC) in postmenopausal women, in whom fibrinolysis is impaired. We investigated whether PC affects fibrinolysis and if HT modulates this effect. We enrolled 150 women aged 55.5 ± 4.7 years in a randomized interventional open-label study, including 50 on standard oral HT, 50 on ultra-low-dose HT, and 50 controls. PC, along with global fibrinolysis (clot lysis time, CLT), fibrinolysis proteins, and prothrombotic markers were determined at baseline and at 24 weeks. Patients with the baseline top quartile PC (> 2.07 nM/mg protein) had 10.3% longer CLT, higher activity (but not antigen) of TAFI (+ 19.9%) and PAI-1 (+ 68.1%) compared to the remainder. No differences were observed in thrombin generation, factor VIII, plasminogen or α2-antiplasmin. On-treatment PC decreased by 16.4% (p < 0.0001), without differences related to the type of HT, compared to baseline and by 30% compared to controls, in whom PC and fibrinolysis markers remained unchanged. Patients with PC > 2.07 nM/mg had shortened CLT during HT compared to baseline, along with lower PAI-1 (-69%) and TAFI (-26%) activity. In this subgroup CLT was 5.8% shorter compared to controls with the highest PC. In postmenopausal women with increased PC, HT was accompanied by PC reduction and faster clot lysis together with decreased PAI-1 and TAFI activity.

A challenging issue in COVID-19 infection: The relationship between PA1-1 and TAFI levels in patients with coagulation disorder: A retrospective and observational study.

COVID-19 disrupts the balance between coagulation and fibrinolysis. Especially in the clinical course of serious disease, plasminogen activator inhibitor-1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI), and tissue plasminogen activator levels increase in association with hypercoagulable state and hypofibrinolysis. This explains the increased incidence of thrombosis seen in COVID-19 infection. In this study, we aimed to examine the changes in PAI-1 and TAFI levels of COVID-19 patients. Department of Infectious Diseases and Clinical Microbiology, University of Health Sciences, Diskapi Yildirim Beyazit Training and Research Hospital-Ankara Turkey, between April 1 and May 7, 2021. Patients who were diagnosed with COVID-19 were included in this retrospective study. TAFI and PAI-1 levels were analyzed from the samples that had been stored at -80 °C formerly. One hundred thirty-five patients diagnosed with COVID-19 and followed up in the service or intensive care unit were included in the study. Thirty-four (25.2%) patients required follow-up in the intensive care unit. Mortality rate was 10.4%, the coagulation tests of these patients were also compared. PA1-1 levels were found to be statistically significantly higher in intensive care unit patients (median: 133 pg/mL vs 31 pg/mL; P < .001), and there was no significant difference in TAFI levels (median:7.31 ng/mL vs 9.80 ng/mL; P = .171) between the 2 groups. TAFI levels were found to be higher in patients who died. In COVID-19 infection, as the severity of the disease increases, the coagulation balance deteriorates and eventually a hypercoagulable state occurs with an increase in PAI-1 and TAFI levels. Markers such as PAI and TAFI can be illuminating in further studies in determining prognosis and mortality and developing new treatment options.

Thrombin activatable fibrinolysis inhibitor plasma levels and TAFI Thr325Ile genetic polymorphism in a cohort of Egyptian sickle cell disease patients and impact on disease severity.

BACKGROUND: Thrombin is a critical protease modulating thrombosis as well as inflammation, which are one of the main pathophysiological mechanisms in sickle vasculopathy, and its levels were reported to be high in sickle cell disease (SCD). The thrombin-thrombomodulin complex activates the TAFI inhibitor of fibrinolysis, which acts by reducing plasmin affinity for its substrate thus hindering fibrinolysis. OBJECTIVE: We aimed to determine the influence of the Thr325Ile single nucleotide polymorphism (SNP) on TAFI antigen levels and potential effects on the severity of SCD in a cohort of Egyptian patients. METHODS: Genotyping of Thr325lle polymorphism using Taq-Man SNP genotyping assay and TAFI level measurement using an enzyme-linked immunosorbent assay were performed for 80 SCD patients (45 homozygous HbSS, 16 S/ß0 and 19 Sß+) as well as 80 age- and gender-matched healthy control subjects. RESULTS: Plasma TAFI levels were higher in SCD patients with Thr325Ile polymorphism, yet the difference was not statistically significant (p = .204). SCD patients with polymorphic genotypes had a greater number of hospital admissions (p = .03). Ten patients with acute chest syndrome had the homozygous polymorphic genotype (GG), and all patients with pulmonary hypertension had the polymorphic genotype (six were homozygous [GG] and five were heterozygous [GA]). Patients with SCD complicated with pulmonary hypertension showed significantly higher plasma TAFI levels (p = .044). CONCLUSION: The analysis of Thr325Ile polymorphisms combined with plasma TAFI levels suggests that the analyzed SNP could influence plasma TAFL levels and SCD disease severity and hospitalization rates, which could be predictors for complex disease.

High thrombin-activatable fibrinolysis inhibitor expression in thrombi from stroke patients in elevated estrogen states.

BACKGROUND: The risk of acute ischemic stroke (AIS) associated with high estrogen states, including pregnant patients and those using oral contraceptives, has been well documented. We described the histological composition of thrombi collected in these cases. METHODS: From a prospective tissue registry (STRIP registry) of thrombi retrieved during mechanical thrombectomy for AIS, we identified 5 patients with high estrogen states: 1 post-partum patient, 1 undergoing hormone replacement therapy and 3 consuming oral contraceptive pills. Five male control patients were randomly chosen matched by age. Immunohistochemistry for CD42b (platelets), von Willebrand factor (vWF), thrombin-activatable fibrinolysis inhibitor (TAFI), fibrinogen and plasminogen activator inhibitor-1 (PAI-1) was performed. Expression was quantified using Orbit Image Software. Student's t-test was performed as appropriate. RESULTS: Mean TAFI content for the high estrogen state group was higher than controls (25.6 ± 11.9% versus 9.3 ± 9.0%, p = 0.043*). Mean platelet content for the high estrogen state group was lower than controls (41.7 ± 10.6% versus 61.8 ± 12.9%, p = 0.029*). No significant difference was found in vWF, fibrinogen and PAI-1 expression. Mean time to recanalize was higher in the high estrogen state group compared to the control group (57.8 ± 27.6 versus 22.6 ± 11.4 min, p = 0.0351*). The mean number of passes required was higher in the high estrogen group compared to controls 4.6 versus 1.2, p = 0.0261*). CONCLUSIONS: TAFI expression, a powerful driver of thrombosis, was significantly higher in stroke thrombi among patients with high estrogen states compared to controls.

Comparative analysis of hyperfibrinolysis with activated coagulation between amniotic fluid embolism and severe placental abruption.

Amniotic fluid embolism (AFE) and placental abruption (PA) are typical obstetric diseases associated with disseminated intravascular coagulation (DIC). AFE is more likely to be complicated with enhanced fibrinolysis than PA. AFE may have an additional mechanism activating fibrinolytic cascade. We aimed to compare the coagulation/fibrinolysis factors among AFE, PA, and peripartum controls. We assessed AFE cases registered in the Japanese AFE Registry, and PA cases complicated with DIC (severe PA) and peripartum controls recruited at our hospital. The following factors in plasma were compared: prothrombin fragment 1 + 2 (PF1 + 2), plasmin α2-plasmin inhibitor complex (PIC), tissue factor (TF), tissue plasminogen activator (tPA), annexin A2 (AnnA2), total thrombin activatable fibrinolysis inhibitor (TAFI) including its activated form (TAFIa), and plasminogen activator inhibitor-type 1 (PAI-1). PF1 + 2 and PIC were markedly increased in both AFE (n = 27) and severe PA (n = 12) compared to controls (n = 23), without significant difference between those disease groups; however, PIC in AFE showed a tendency to elevate relative to PF1 + 2, compared with severe PA. AFE had significantly increased tPA and decreased total TAFI levels compared with severe PA and controls, which might be associated with further plasmin production in AFE and underlie its specific fibrinolytic activation pathway.

Brain Expression of CPB2 and Effects of Cpb2 Deficiency in Mouse Models of Behavior.

BACKGROUND: Procarboxypeptidase B2 (proCPB2 or TAFI) is a zymogen that after activation cleaves C-terminal basic residues from peptides or proteins with many identified targets. A splice variant of CPB2 has been found in the brain lacking essential residues for its carboxypeptidase function. The aim was to determine CPB2 expression in the brain and effects of CPB2 deficiency (Cpb2 -/-) on behavior. MATERIALS AND METHODS: Behavioral effects were tested by comparing Cpb2 -/- mice in short-term (open field and elevated zero maze tests) and long-term (Phenotyper) observations with wild-type (WT) controls. RESULTS: Long-term observation compared day 1 (acclimatizing to novel environment) to day 4 (fully acclimatized) with the inactive (day) and active (night) periods analyzed separately. Brain expression of CPB2 mRNA and protein was interrogated in publicly available databases. Long-term observation demonstrated differences between WT and Cpb2 -/- mice in several parameters. For example, Cpb2 -/- mice moved more frequently on both days 1 and 4, especially in the normally inactive periods. Cpb2 -/- mice spent more time on the shelter and less time in it. Differences were more pronounced on day 4 after the mice had fully acclimatized. In short-term observations, no differences were observed between Cpb2 -/- mice and WT mice. Brain expression of CBP2 was not detectable in the human protein atlas. Databases of single-cell RNAseq did not show expression of CPB2 mRNA in either human or mouse brain. CONCLUSION: Continuous observation of home-cage behavior suggests that Cpb2 -/- mice are more active than WT mice, show different day-night activity levels, and might have a different way of processing information.

Thrombin-activatable fibrinolysis inhibitor and sex modulate thrombus stability and pulmonary embolism burden in a murine model.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) levels are positively correlated with the risk of thrombosis. The mechanism of how TAFI affects venous thromboembolism (VTE) remains uncertain. In addition, the role of sex on the risk of VTE has also been studied. However, their association also remains unclear. OBJECTIVES: To investigate how TAFI and/or sex affect venous thrombus stability and consequent pulmonary embolism (PE). METHODS: Ferric chloride-induced thrombi were formed within the femoral veins of male and female wild-type (WT) or TAFI-knockout (Cpb2-/-) mice. Thrombi were imaged over 2 hours using intravital videomicroscopy to quantify embolization and thrombus size over time. Lungs were examined by immunohistochemistry to quantify (a) emboli and (b) fibrin composition of these emboli. RESULTS: Embolization events in female mice were higher than in males (7.9-fold in WT and 3.1-fold in Cpb2-/- mice). Although the maximal thrombus sizes were not different across groups, Cpb2-/- mice had thrombi that were, on average, 24% smaller at the end of the 2-hour experiment than WT mice. Loss of TAFI led to a 4.0- and 2.8-fold increase in PE burden in males and females, respectively, while sex had no influence. Pulmonary emboli in Cpb2-/- mice had higher fibrin composition compared with WT mice. CONCLUSION: Female mice had less stable venous thrombi than male mice, suggesting a higher risk of PE in females with deep vein thrombosis. Mice lacking TAFI had more thrombus degradation and higher PE burden than WT mice. These results confirm the role of TAFI in venous thrombosis.

Serum Proteomics Identified TAFI as a Potential Molecule Facilitating the Migration of Peripheral Monocytes to Damaged White Matter During Chronic Cerebral Hypoperfusion.

Neuroinflammation is assumed as the critical pathophysiologic mechanism of white matter lesions (WMLs), and infiltrated peripheral monocyte-derived macrophages are implicated in the development of neuroinflammation. This study sought to explore the blood molecules that promote the migration of peripheral monocytes to the sites of WMLs. The serum protein expression profiles of patients and Sprague-Dawley rat models with WMLs were detected by data-independent acquisition (DIA) proteomics technique. Compared with corresponding control groups, we acquired 62 and 41 differentially expressed proteins (DEPs) in the serum of patients and model rats with WMLs respectively. Bioinformatics investigations demonstrated that these DEPs were linked to various Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms involved in neuroinflammation. Afterward, we identified thrombin-activatable fibrinolysis inhibitor (TAFI) as a shared and overexpressed protein in clinical and animal serum samples, which was further verified by enzyme-linked immunosorbent assay. Additionally, an upregulation of TAFI was also observed in the white matter of rat models, and the inhibition of TAFI impeded the migration of peripheral monocytes to the area of WMLs. In vitro experiments suggested that TAFI could enhance the migration ability of RAW264.7 cells and increase the expression of Ccr2. Our study demonstrates that neuroinflammatory signals can be detected in the peripheral blood of WMLs patients and model rats. TAFI may serve as a potential protein that promotes the migration of peripheral monocytes to WMLs regions, thereby providing a novel molecular target for further investigation into the interaction between the central and peripheral immune systems.

Plasma thrombin-activatable fibrinolysis inhibitor and the 1040C/T polymorphism are risk factors for diabetic kidney disease in Chinese patients with type 2 diabetes.

Background: Inflammatory and hemostatic disorders in diabetic microangiopathy (DMA) can be linked to thrombin-activatable fibrinolysis inhibitor (TAFI) and its own gene polymorphisms. Thus, the study aimed to investigate the associations of plasma TAFI and gene polymorphisms with DMA in Chinese patients with type 2 diabetes (T2D). Methods: Plasma TAFI of 223 patients with T2D was measured, and the genotypes and alleles of the 1040C/T, 438G/A, and 505G/A polymorphisms of the TAFI gene were analyzed. A ROC curve was constructed to evaluate the identifying power of TAFI between patients with T2D and DMA, and logistic regression analysis was used to observe the correlation of plasma TAFI and gene polymorphisms with the risk for DMA. Results: Plasma TAFI was higher in patients with DMA than in patients with only T2D (p < 0.05). TAFI exhibited the largest area under ROC in identifying diabetic kidney disease (DKD) from only T2D (0.763, 95% CI [0.674-0.853], p < 0.01), and adjusted multivariate analysis showed a high odds ratio (OR: 15.72, 95% CI [4.573-53.987], p < 0.001) for DKD. Higher frequencies of the CT genotype and T allele of the 1040C/T polymorphism were found in DKD compared with only T2D (respectively p < 0.05), and the CT genotype exhibited a high OR (1.623, 95% CI [1.173-2.710], p < 0.05) for DKD. DKD patients with the CT genotype had higher plasma TAFI levels, while T2D and DKD patients with CC/TT genotypes had lower plasma TAFI levels. Conclusion: Plasma TAFI and the CT genotype and T allele of the 1040C/T polymorphism are independent risk factors for DKD in Chinese T2D patients.

Profibrinolytic effects of rivaroxaban are mediated by thrombin-activatable fibrinolysis inhibitor and are attenuated by a naturally occurring stabilizing mutation in enzyme.

Rivaroxaban is a direct factor Xa inhibitor, recently implemented as a favorable alternative to warfarin in anticoagulation therapy. Rivaroxaban effectively reduces thrombin generation, which plays a major role in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) to TAFIa. Based on the antifibrinolytic role of TAFIa, we hypothesized that rivaroxaban would consequently induce more rapid clot lysis. In vitro clot lysis assays were used to explore this hypothesis and additionally determine the effects of varying TAFI levels and a stabilizing Thr325Ile polymorphism (rs1926447) in the TAFI protein on the effects of rivaroxaban. Rivaroxaban was shown to decrease thrombin generation, resulting in less TAFI activation, thus enhancing lysis. These effects were also shown to be less substantial in the presence of greater TAFI levels or the more stable Ile325 enzyme. These findings suggest a role for TAFI levels and the Thr325Ile polymorphism in the pharmacodynamics and pharmacogenomics of rivaroxaban.

[Relationship between plasma levels of thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor-1 and tissue-type plasminogen activator and deep venous thrombosis in patients with systemic lupus erythematosus].

Objective: To investigate the plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in patients with systemic lupus erythematosus (SLE), and their relationship with deep venous thrombosis of the lower limbs. Methods: A case-control study was conducted to retrospectively select 32 SLE patients with deep venous thrombosis of the lower extremities (thrombus group) admitted to Liaocheng People's Hospital in Shandong Province from June 2018 to June 2021, including 4 males and 28 females, with a mean age of (49.7±5.5) years. Meanwhile, 64 SLE patients without deep venous thrombosis of the lower extremities (control group) were also selected, including 11 males and 53 females, with a mean age of (50.8±5.5) years. The plasma levels of TAFI, PAI-1 and t-PA of the two groups were compared. A logistic regression model was used to analyze the correlation of TAFI, PAI-1 and t-PA with SLE in patients. Results: The plasma levels of TAFI, PAI-1 and t-PA were (32.77±5.17) mg/L, (29.43±5.51) µg/L and (6.58±1.40) µg/L in the thrombotic group, while the plasma levels of TAFI, PAI-1 and t-PA in the control group were (23.56±4.40) mg/L, (19.00±4.40) µg/L and (9.40±2.23) µg/L. The levels of TAFI and PAI-1 in the thrombotic group were higher than those in the control group, while the level of t-PA was lower than that in the control group (all P<0.05). The results of logistic regression model showed that higher TAFI levels (OR=1.75, 95%CI: 1.05-2.90, P=0.043), higher PAI-1 levels (OR=1.85, 95%CI: 1.04-3.29, P=0.046), and lower t-PA levels (OR=0.72, 95%CI: 0.52-0.99, P=0.048) were related factors for the occurrence of deep venous thrombosis of the lower limbs in SLE patients. Conclusion: The plasma levels of TAFI and PAI-1 in SLE patients with deep venous thrombosis of the lower extremities increase, while the t-PA level decreases, which are related factors for the occurrence of deep venous thrombosis of the lower extremities in SLE patients.

Inhibition of thrombin-activatable fibrinolysis inhibitor via DS-1040 to accelerate clot lysis in patients with acute pulmonary embolism: a randomized phase 1b study.

BACKGROUND: The optimal treatment of intermediate-risk pulmonary embolism (PE) in hemodynamically stable patients remains unknown. Fibrinolytics reduce the risk of hemodynamic deterioration but increase bleeding risk. DS-1040, an inhibitor of thrombin-activatable fibrinolysis inhibitor, enhanced endogenous fibrinolytic activity without increasing bleeding risk in preclinical studies. OBJECTIVES: To evaluate the tolerability and explore the efficacy of DS-1040 in patients with acute PE. METHODS: In this multicenter, randomized, double-blind, placebo-controlled study, ascending doses of intravenous DS-1040 (20-80 mg) or placebo were added to enoxaparin (1 mg/kg twice daily) in patients with intermediate-risk PE. The primary endpoint was the number of patients with major or clinically relevant nonmajor bleeding. The percentage change in thrombus volume and right-to-left ventricular dimensions, assessed using quantitative computed tomography pulmonary angiography, at baseline and after 12 to 72 hours were used to explore the efficacy of DS-1040. RESULTS: Of 125 patients with all available data, 38 were randomized to placebo and 87 to DS-1040. The primary endpoint occurred in 1 patient in the placebo group (2.6%) and 4 patients who received DS-1040 (4.6%). One subject experienced major bleeding (DS-1040 80 mg group); no fatal or intracranial bleeding occurred. Thrombus volume was 25% to 45% lower after infusion, with no differences between the DS-1040 and placebo groups. There was no difference in the change from baseline right-to-left ventricular dimensions between the DS-1040 and placebo groups. CONCLUSION: In patients with acute PE, adding DS-1040 to standard anticoagulation was not associated with an increase in bleeding but did not improve thrombus resolution or right ventricular dilation.

Glycemic Control and Plasma Levels of Pro-Inflammatory and Pro-Thrombotic Biomarkers in Diabetic Patients Presenting with Acute Pulmonary Embolism.

Type I and type II diabetes are closely associated with a pro-inflammatory state and to a pro-thrombotic state. The role of glycemic control in pulmonary embolism (PE) is poorly understood and requires additional investigation. The aim of this study is to investigate the relationship between glycemic control and thrombo-inflammatory biomarkers in a PE patient cohort compared to normal samples. Demographic and clinical information for 86 diabetic patients and 106 non-diabetic patients presenting with acute PE was collected via retrospective chart review. Plasma levels of pro-inflammatory (C-reactive protein [CRP], tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6]) and pro-thrombotic (d-dimer, plasminogen activator inhibitor-1 [PAI-1], tissue plasminogen activator [tPA], thrombin activatable fibrinolysis inhibitor [TAFI], von-Willebrand factor [vWF], endogenous glycosaminoglycans [GAGs]) biomarkers were drawn within 24 hours of diagnosis of acute PE. Data was also obtained for a population of healthy adult controls. All the pro-inflammatory and pro-thrombotic biomarkers were elevated in diabetic PE patients in comparison to healthy controls. None of the biomarkers were elevated in diabetic PE patients when compared to non-diabetic PE patients. There was no difference in the levels of the pro-inflammatory biomarkers according to glycemic control. The plasma level of TAFI was elevated in diabetic patients with poor glycemic control. Diabetic patients were more likely to have a more severe PE. These studies demonstrate that thrombo-inflammatory biomarkers are elevated in diabetic PE patients with associated comorbidities in comparison to normal individuals. However, there is no difference between the PE cohort alone in comparison to PE with diabetes. The role of TAFI within the continuum of diabetic vascular disease warrants additional investigation.

Atorvastatin downregulates plasma procarboxypeptidase U concentrations and improves fibrinolytic potential dose-dependently in hyperlipidemic individuals.

BACKGROUND: Statins efficiently lower cholesterol and also exert pleiotropic effects that extend beyond lipid lowering. In a recent pilot study, valuable information on the carboxypeptidase U (CPU) system in hyperlipidemia and the effect of statin therapy was collected. It was shown that proCPU levels are increased in hyperlipidemic patients. Statins significantly decreased proCPU levels and improved plasma fibrinolysis. Furthermore, it was suggested that patients with high baseline proCPU levels are most likely to benefit from statin therapy. OBJECTIVES: We aimed to further substantiate the effect of hyperlipidemia and statin therapy on CPU-related parameters in a larger cohort of hyperlipidemic and statin-treated individuals. METHODS: Blood was collected from 141 individuals treated with different dosages of atorvastatin (10-80 mg), 38 normolipidemic, and 37 hyperlipidemic controls. Lipid parameters and markers of fibrinolysis (proCPU and clot lysis time) were determined and compared between the groups. RESULTS: Pilot study results of high proCPU concentrations in hyperlipidemic patients and the proCPU-reducing effect of atorvastatin were confirmed. Accordingly, an improvement in plasma fibrinolytic potential was seen under the influence of atorvastatin. High interindividual variation in proCPU concentrations was observed in the hyperlipidemic cohort, with up to 80% higher proCPU levels compared with normolipidemic controls. Furthermore, proCPU concentration and the dosage of atorvastatin were inversely correlated. CONCLUSIONS: This study clearly shows that plasma proCPU concentrations and its expected effect on the fibrinolytic rate (as measured by clot lysis time) are increased in hyperlipidemic patients and that these effects can be normalized (and even further reduced compared with normolipidemic patients) by atorvastatin treatment.

ProCPU Is Expressed by (Primary) Human Monocytes and Macrophages and Expression Differs between States of Differentiation and Activation.

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.

Elucidating the molecular mechanisms of fibrinolytic shutdown after severe injury: The role of thrombin-activatable fibrinolysis inhibitor.

BACKGROUND: The mechanisms underlying trauma-induced coagulopathy remain elusive. Hyperfibrinolysis has been linked to increased plasminogen activation and antiprotease consumption; however, the mechanistic players in its counterpart, fibrinolysis shutdown, remain unclear. We hypothesize that thrombin-activatable fibrinolysis inhibitor (TAFI) plays a major role in fibrinolytic shutdown after injury. METHODS: As part of this observational cohort study, whole blood was collected from trauma activation patients at a single, level 1 trauma center. Citrated rapid thrombelastography and the following enzyme-linked immunosorbent assays were conducted: thrombin, antithrombin, thrombin-antithrombin complex, TAFI, plasminogen, antiplasmin, plasmin-antiplasmin (PAP), tissue plasminogen activator, plasminogen activator inhibitor 1, and tissue plasminogen activator-plasminogen activator inhibitor 1 complex. Univariate and cluster analysis were performed. RESULTS: Overall, 56 patients (median age, 33.5 years; 70% male) were included. The majority (57%) presented after blunt mechanism and with severe injury (median New Injury Severity Score, 27). Two clusters of patients were identified: Group 1 (normal fibrinolysis, n = 21) and Group 2 (fibrinolysis shutdown, n = 35). Group 2 had significantly lower fibrinolysis with a median LY30 of 1.1% (interquartile range [IQR], 0.1-1.9%) versus 2.1% (IQR, 0.5-2.8%) in Group 1; while the median LY30 was within physiologic range, 45% of patients in Group 2 were in shutdown (vs. 24% in Group 1, p = 0.09). Compared with Group 1, Group 2 had significantly higher PAP (median, 4.7 [IQR, 1.7-9.3] vs. 1.4 [1.0-2.1] µg/mL in Group 1; p = 0.002) and higher TAFI (median, 152.5% [IQR, 110.3-190.7%] vs. 121.9% [IQR, 93.2-155.6%]; p = 0.04). There was a strong correlation between PAP and TAFI ( R2 = 0.5, p = 0.0002). CONCLUSION: The presented data characterize fibrinolytic shutdown, indicating an initial plasmin burst followed by diminished fibrinolysis, which is distinct from hypofibrinolysis (inadequate plasmin burst and fibrinolysis). After an initial thrombin and plasmin burst (increased PAP), fibrinolysis is inhibited, mediated in part by increased TAFI.