Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. METHODS: We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. RESULTS: We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. CONCLUSIONS: Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Tranexamic Acid Does Not Influence Cardioprotection by Ischemic Preconditioning and Remote Ischemic Preconditioning.

Prior studies have suggested that the antifibrinolytic drug aprotinin increases the infarct size after ischemia and reperfusion (I/R) and attenuates the effect of ischemic preconditioning (IPC). Aprotinin was replaced by tranexamic acid (TXA) in clinical practice. Here, we investigated whether TXA influences I/R injury and/or cardioprotection initiated by IPC and/or remote ischemic preconditioning (RIPC). Anesthetized male Wistar rats were randomized to 6 groups. Control animals were not further treated. Administration of TXA was combined with and without IPC and RIPC. Estimated treatment effect was 20%. Compared to control group (56% ± 11%), IPC reduced infarct size by 46% (30% ± 6%; mean difference, 26%; 95% confidence interval, 19-33; P < .0001), and RIPC reduced infarct size by 29% (40% ± 8%; mean difference, 16%; 95% confidence interval, 9-24; P < .011). Additional application of TXA had no effect on I/R injury and cardioprotection by IPC or RIPC. TXA does not abolish infarct size reduction by IPC or RIPC.

[Advances in differential diagnosis and treatment of patients with sarcoidosis]. / Postepy w róznicowaniu i leczeniu chorych na sarkoidoze.

The implementation of treatment in patients with sarcoidosis (SA) must be associated with the certainty of diagnosis, which is difficult due to the lack of unambiguous criteria. Finding the presence of noncaseating granulomas in bioptic material is not always indicative of SA. The main point of SA's diagnosis is the level of its activity, because only patients in the active stage should be qualified for treatment. In therapy, glucocorticosteroids or second-line drugs - methotrexate or azathioprine are still recommended. Introduced monoclonal antibodies (infliximab, etanercept, adaluimumab, golimumab, rituximab), tested for efficacy in other pathologies associated with the formation of granulomas, have a limited application in patients with SA. In contrast, anti-fibrotics (pirfenidone and nintedanib) are in clinical trials. The latest method of controlling the fibrosis of the parenchyma in the course of SA is the use of mesenchymal cells obtained from umbilical cord blood. Preliminary results indicate a real possibility of using this therapy in patients with SA.

Discovery and Evaluation of Anti-Fibrinolytic Plasmin Inhibitors Derived from 5-(4-Piperidyl)isoxazol-3-ol (4-PIOL).

Inhibition of plasmin has been found to effectively reduce fibrinolysis and to avoid hemorrhage. This can be achieved by addressing its kringle 1 domain with the known drug and lysine analogue tranexamic acid. Guided by shape similarities toward a previously discovered lead compound, 5-(4-piperidyl)isoxazol-3-ol, a set of 16 structurally similar compounds was assembled and investigated. Successfully, in vitro measurements revealed one compound, 5-(4-piperidyl)isothiazol-3-ol, superior in potency compared to the initial lead. Furthermore, a strikingly high correlation (R2 = 0.93) between anti-fibrinolytic activity and kringle 1 binding affinity provided strong support for the hypothesized inhibition mechanism, as well as revealing opportunities to fine-tune biological effects through minor structural modifications. Several different ligand-based (Freeform, shape, and electrostatic-based similarities) and structure-based methods (e.g., Posit, MM/GBSA, FEP+) were used to retrospectively predict the binding affinities. A combined method, molecular alignment using Posit and scoring with Tcombo, lead to the highest coefficient of determination (R2 = 0.6).

In vitro effects of an extracorporeal membrane oxygenation circuit on the sequestration of ϵ-aminocaproic acid.

OBJECTIVE: To assess the in vitro effects of drug sequestration in extracorporeal membrane oxygenation (ECMO) on ϵ-aminocaproic acid (EACA) concentrations. METHODS AND DESIGN: This in vitro study will determine changes in EACA concentration over time in ECMO circuits. A pediatric dose of 2,500 mg was administered to whole expired blood in the simulated pediatric ECMO circuit. Blood samples were collected at 0, 30, 60, 360 and 1440-minute intervals after initial administration equilibration from three different sites of the circuit: pre-oxygenator (PRE), post-oxygenator (POST) and PVC tubing (PVC) to determine the predominant site of drug loss. The circuit was maintained for two consecutive days with a re-dose at 24 hours to establish a comparison between unsaturated (New) and saturated (Old) oxygenator membranes. Comparisons between sample sites, sample times and New versus Old membranes were statistically analyzed by a linear mixed-effects model with significance defined as a p-value <0.05. RESULTS: There were no significant differences in EACA concentration with respect to sample site, with PRE and POST samples demonstrating respective mean differences of 0.30 mg/ml and 0.34 mg/ml as compared to PVC, resulting in non-significant p-values of 0.373 [95% CI (-0.37, 0.98)] and 0.324 [95% CI (-0.34, 1.01)], respectively. The comparison of New vs. Old ECMO circuits resulted in non-significant changes from baseline, with a mean difference of 0.50 mg/ml, 95% CI (-0.65, 1.65), p=0.315. CONCLUSION: The findings of this study did not show any significant changes in drug concentration that can be attributed to sequestration within the ECMO circuit. Mean concentrations between ECMO circuit sample sites did not differ significantly. Comparison between New and Old circuits also did not differ significantly in the change from baseline concentration over time. Sequestration within ECMO circuits appears not to be a considerable factor for EACA administration.

Rationale for the selective administration of tranexamic acid to inhibit fibrinolysis in the severely injured patient.

Postinjury fibrinolysis can manifest as three distinguishable phenotypes: 1) hyperfibrinolysis, 2) physiologic, and 3) hypofibrinolysis (shutdown). Hyperfibrinolysis is associated with uncontrolled bleeding due to clot dissolution; whereas, fibrinolysis shutdown is associated with organ dysfunction due to microvascular occlusion. The incidence of fibrinolysis phenotypes at hospital arrival in severely injured patients is: 1) hyperfibrinolysis 18%, physiologic 18%, and shutdown 64%. The mechanisms responsible for dysregulated fibrinolysis following injury remain uncertain. Animal work suggests hypoperfusion promotes fibrinolysis, while tissue injury inhibits fibrinolysis. Clinical experience is consistent with these observations. The predominant mediator of postinjury hyperfibrinolysis appears to be tissue plasminogen activator (tPA) released from ischemic endothelium. The effects of tPA are accentuated by impaired hepatic clearance. Fibrinolysis shutdown, on the other hand, may occur from inhibition of circulating tPA, enhanced clot strength impairing the binding of tPA and plasminogen to fibrin, or the inhibition of plasmin. Plasminogen activator inhibitor -1 (PAI-1) binding of circulating tPA appears to be a major mechanism for postinjury shutdown. The sources of PAI-1 include endothelium, platelets, and organ parenchyma. The laboratory identification of fibrinolysis phenotype, at this moment, is best determined with viscoelastic hemostatic assays (TEG, ROTEM). While D-dimer and plasmin antiplasmin (PAP) levels corroborate fibrinolysis, they do not provide real-time assessment of the circulating blood capacity. Our clinical studies indicate that fibrinolysis is a very dynamic process and our experimental work suggests plasma first resuscitation reverses hyperfibrinolysis. Collectively, we believe recent clinical and experimental work suggest antifibrinolytic therapy should be employed selectively in the acutely injured patient, and optimally guided by TEG or ROTEM.

The antifibrinolytic function of factor XIII is exclusively expressed through α2-antiplasmin cross-linking.

Factor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α2-antiplasmin (α2AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α2AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α2AP depleted thrombi. Addition of PNP to FXIII or α2AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α2AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α2AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α2AP.

Binding and activation of plasminogen at the surface of Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, anaerobic bacterium, which has been suggested to be a normal inhabitant of the oral flora. In rare cases, it can invade the tonsils and deeper tissues, causing the serious condition Lemièrre's syndrome. Recruitment of host plasminogen is a well-known bacterial virulence mechanism, and plasmin activity at the bacterial surface is thought to be important for bacterial invasion. Herein we show that plasminogen can be recruited to the surface of F. necrophorum, that surface-bound plasminogen is more easily converted to active plasmin than plasminogen in buffer, and that bound plasminogen is protected against inactivation by α2-antiplasmin. These findings add to the understanding of the pathogenesis of Lemièrre's syndrome.

The plasmin-antiplasmin system: structural and functional aspects.

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α(2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α(2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α(2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α(2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.

Tranexamic acid integrated into platelet-rich fibrin produces a robust and resilient antihemorrhagic biological agent: a human cohort study.

OBJECTIVE: To investigate the incorporation of the antifibrinolytic agent tranexamic acid (TA) during platelet-rich fibrin (PRF) formation to produce a robust fibrin agent with procoagulation properties. STUDY DESIGN: Blood from healthy volunteers was collected. Into 3 tubes, TA was immediately added in 1-mL, 0.4-mL, and 0.2-mL volumes, and the fourth tube was without additions. After PRF preparation, the clots were weighed in their raw (clot) and membrane forms. PRF physical properties were analyzed using a universal testing system (Instron). Protein and TA levels in the PRF were analyzed using a bicinchoninic acid assay and a ferric chloride assay, respectively. RESULTS: The addition of TA to PRF led to a robust weight compared with sham control. PRF weight was greater in females in all tested groups. The addition of TA also led to greater resilience to tears, especially at 1-mL TA addition to the blood. Furthermore, TA addition led to a greater value of total protein within the PRF and entrapment of TA in the PRF. CONCLUSIONS: Addition of TA to a PRF preparation leads to robust PRF with greater protein levels and the amalgamation of TA into the PRF. Such an agent may enhance the beneficial properties of PRF and attribute procoagulation properties to it.

Factor VII activating protease (FSAP) is not essential in the pathophysiology of angioedema in patients with C1 inhibitor deficiency.

BACKGROUND: Excessive bradykinin (BK) generation from high molecular weight kininogen (HK) by plasma kallikrein (PK) due to lack of protease inhibition is central to the pathophysiology of hereditary angioedema (HAE). Inadequate protease inhibition may contribute to HAE through a number of plasma proteases including factor VII activating protease (FSAP) that can also cleave HK. OBJECTIVE: To investigate the interaction between FSAP and C1 inhibitor (C1Inh) and evaluate the potential role of FSAP in HAE with C1Inh deficiency. MATERIALS AND METHODS: Plasma samples from 20 persons with HAE types 1 or 2 in remission were studied and compared to healthy controls. We measured and compared antigenic FSAP levels, spontaneous FSAP activity, FSAP generation potential, activation of plasma pre-kallikrein (PPK) by FSAP, and the formation of FSAP-C1Inh and FSAP-alpha2-antiplasmin (FSAP-α2AP) complexes. Furthermore, we measured HK cleavage and PK activation after activation of endogenous pro-FSAP and after addition of exogenous FSAP. RESULTS: In plasma from HAE patients, there is increased basal FSAP activity compared to healthy volunteers. HAE plasma exhibits decreased formation of FSAP-C1Inh complexes and increased formation of FSAP-α2AP complexes in histone-activated plasma. Although exogenous FSAP can cleave HK in plasma, this was not seen when endogenous plasma pro-FSAP was activated with histones in either group. PK was also not activated by FSAP in plasma. CONCLUSION: In this study, we established that FSAP activity is increased and the pattern of FSAP-inhibitor complexes is altered in HAE patients. However, we did not find evidence suggesting that FSAP contributes directly to HAE attacks.

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa.

BACKGROUND: The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α(2)-antiplasmin (α(2)AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α(2)AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α(2)AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. RESULTS: Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H(6)NQEQVSPLTLLAG(4)Y (designated XL1); H(6)DQMMLPWAVTLG(4)Y (XL2); H(6)WQHKIDLPYNGAG(4)Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α(2)AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α(2)AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA. CONCLUSIONS: Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.

Early detection of venous thromboembolism in patients with neuroepithelial tumor: efficacy of screening with serum D-dimer measurements and Doppler ultrasonography.

The efficacy of combined serum D-dimer level measurement and Doppler ultrasonography of the lower extremity was investigated for screening of venous thromboembolism (VTE) in patients with neuroepithelial tumor. Eighty-one patients with neuroepithelial tumor were prospectively studied. All patients underwent measurement of serum D-dimer levels and Doppler ultrasonography of the lower extremity. The serum D-dimer level was measured every week, and Doppler ultrasonography was performed two and two weeks after surgery, then every two weeks until discharge, or every two weeks for patients who did not undergo surgery. If the serum D-dimer level increased over 10.0 µg/ml, Doppler ultrasonography or computed tomography was performed to detect VTE. VTE occurred in 12 (14.8%) patients (seven males and five females; age 34-75, mean 59.0 years). Only one patient was symptomatic, whereas 11 patients identified by the screening were without symptoms. Five patients were treated with anticoagulant therapy, one with prophylactic inferior vena cava filter placement with anticoagulant therapy, and the other six were closely followed up without medication. No patient died of pulmonary embolism. Serial Doppler ultrasonography showed thrombus regression or organization and no thrombus extension. The maximum serum D-dimer value was significantly higher in patients with VTE than in those without VTE (mean 14.5 vs. 3.46 µg/ml, P < 0.001). The D-dimer cutoff value of 5.4 µg/ml could be used to identify VTE with 83% sensitivity and 84% specificity. The combination of sequential serum D-dimer measurement and Doppler ultrasonography of the lower extremity is an efficient and non-invasive procedure for identifying asymptomatic VTE in patients with neuroepithelial tumor.

D-dimer plasma levels in NSTE-ACS patient.

BACKGROUND: Acute Coronary Syndrome (ACS) occurs when a vulnerable plaque ruptures and induces platelet aggregation and coagulation process at the rupture. Thrombogenesis is the final process that forms a clot in the coronary lumen causing myocardial injury. Plasma D-dimer, a primary degradation product and circulating marker offibrin turnover, serves as a direct marker of ongoing fibrinolysis in site of coronary artery occlusion. OBJECTIVE: To determine the correlation between plasma D-dimer levels and severity of coronary artery obstruction based on angiographic data that is composed of the number of coronary arteries affected and the percentage of maximum stenosis of coronary artery lumen in non-ST elevation ACS (NSTE-ACS) patients. MATERIAL AND METHOD: NSTE-ACS patients who admitted in Siriraj hospital duringJune 2009 and March 2010 were enrolled. Conditions that increased plasma D-dimer other than NSTE-ACS were excluded. Demographic characteristics were assessed by a standardized questionnaire. Plasma D-dimer was measured and coronary angiography was performed to evaluate severity of coronary artery stenosis. RESULTS: Total of 74 NSTE-ACS patients were enrolled (29 in unstable angina and 45 in non-ST elevation myocardial infarction). Mean age of these patients (54.1% in female and 45.9% in male) were 66 years. D-Dimer was significantly increased with the number of coronary arteries affected (p = 0.03). In non-significant and single coronary artery disease (CAD) patients, median D-dimer was 406 (178-2,788) mcg/L. In multivessel CAD, median D-dimer was 941 (131-7,110) mcg/L. D-dimer levels had a trend to be increased with percentage of maximum stenosis of coronary artery lumen; atheromatosis, (p = 0.30). In mild and moderate atheromatosis (coronary artery stenosis < 70%), median D-dimer was 479 (182-5902) mcg/L while median D-dimer was 789 (131-7110) mcg/L in severe atheromatosis (coronary artery stenosis > 70%). Moreover plasma D-dimer levels correlated with complication of NSTE-ACS (Congestive heartfailure; p < 0.001, arrhythmia; p = 0.007 and death; p = 0.009) and was increased in patients who underwent treatment with CABG more often than those who received PCI and medication treatment alone. D-dimer also correlated with serum creatinine (r = 0.517, p < 0.001), creatinine clearance (r = -0.463, p < 0.001), troponin-T level (r = 0.381, p < 0.001) and left ventricular ejection fraction (r = -0.368, p = 0.002). CONCLUSION: D-dimer is useful coagulation marker use to evaluate extent of coronary affected and may predict in-hospital CV complication. However, other conditions that increased plasma D-dimer also excluded.

Vitamin K in parenteral nutrition.

Vitamin K (as phylloquinone and menaquinones) is an essential cofactor for the conversion of peptide-bound glutamate to gamma-carboxy glutamic acid (Gla) residues in a number of specialized Gla-containing proteins. The only unequivocal deficiency outcome is a bleeding syndrome caused by an inability to synthesize active coagulation factors II, VII, IX, and X, although there is growing evidence for roles for vitamin K in bone and vascular health. An adult daily intake of about 100 microg of phylloquinone is recommended for the maintenance of hemostasis. Traditional coagulation tests for assessing vitamin K status are nonspecific and insensitive. Better tests include measurements of circulating vitamin K and inactive proteins such as undercarboxylated forms of factor II and osteocalcin to assess tissue and functional status, respectively. Common risk factors for vitamin K deficiency in the hospitalized patient include inadequate dietary intakes, malabsorption syndromes (especially owing to cholestatic liver disease), antibiotic therapy, and renal insufficiency. Pregnant women and their newborns present a special risk category because of poor placental transport and low concentrations of vitamin K in breast milk. Since 2000, the Food and Drug Administration has mandated that adult parenteral preparations should provide a supplemental amount of 150 microg phylloquinone per day in addition to that present naturally, in variable amounts, in the lipid emulsion. Although this supplemental daily amount is probably beneficial in preventing vitamin K deficiency, it may be excessive for patients taking vitamin K antagonists, such as warfarin, and jeopardize their anticoagulant control. Natural forms of vitamin K have no proven toxicity.

Biochemical importance of glycosylation in thrombin activatable fibrinolysis inhibitor.

Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) exerts an antifibrinolytic effect by removing C-terminal lysines from partially degraded fibrin. These lysines are essential for a rapid conversion of plasminogen to plasmin by tissue type plasminogen activator. TAFI is heavily glycosylated at Asn22, Asn51, Asn63, and Asn86. Although the glycans occurring at the glycosylation sites have previously been identified, the biochemical role of these glycans is not known yet. Therefore, we have determined the biochemical importance of the glycosylation in TAFI. Four single, 6 double, 4 triple, and 1 quadruple mutant, in which asparagine was replaced by glutamine, were constructed and transfected into HEK293T cells. Based on the determination of antigen and activity levels on conditioned medium, 4 single and 1 triple mutant were purified and their biochemical properties were determined. The glycosylation knockout mutants did neither reveal an altered fragmentation pattern nor differences in TAFIa stability, but TAFI-N51Q, TAFI-N63Q, and TAFI-N22Q-N51Q-N63Q revealed a decreased TAFIa activity, an increased intrinsic catalytic activity of the zymogen, and a decreased antifibrinolytic potential compared with TAFI-wild-type, whereas TAFI-N22Q and TAFI-N86Q revealed an increased antifibrinolytic potential probably because of an increased catalytic efficiency toward the physiological substrate. From these data it can be concluded that mainly the glycosylation at Asn86 contributes to the biochemical characteristics of TAFI. Furthermore we provide evidence that the activation peptide stays in close proximity to the TAFIa moiety after activation.

Novel hemostatic factor levels and risk of ischemic stroke: the Atherosclerosis Risk in Communities (ARIC) Study.

BACKGROUND AND OBJECTIVE: The role of hemostatic factor levels in cerebral infarction remains uncertain. We studied the association of levels of several under-studied hemostatic factors with ischemic stroke in a population-based cohort. METHODS: The Atherosclerosis Risk in Communities (ARIC) study includes 15,792 individuals aged 45-54 years at intake. Hemostatic factors II, V, IX, X, XI, XII, plasminogen and alpha(2)-antiplasmin were measured on frozen citrate plasma samples from 1990 to 1992. A case-cohort design was used, including all incident ischemic strokes (n = 89) over a median of 7.5 years and a stratified cohort random sample (n = 412). To determine the association of hemostatic factors with incident ischemic stroke, we computed hazard ratios (HRs) using multivariate proportional hazard regression analyses adjusted for demographic and other cardiovascular risk factors. RESULTS: The cohort random sample had a mean age (SD) of 56.9 (5.4) years and 42% were men. The age-, sex- and race-adjusted HRs for highest versus lowest quartiles were: factor XI (2.74, 95% CI 1.42-5.29), factor IX (1.92, 95% CI 0.99-3.73), and alpha(2)-antiplasmin (2.24, 95% CI 1.16-4.33). Correspondingly, the HRs of ischemic stroke per SD increment of factors XI, IX, and alpha(2)-antiplasmin were 1.64, 1.46 and 1.52, respectively (all p < 0.05). After multivariate adjustment including other clinical variables, the standardized HR remained significant for factor XI (1.50, 95% CI 1.10-2.05), but no other factor. CONCLUSION: A greater level of factor XI was associated with an increased risk of ischemic stroke. Higher factor XI levels might help identify patients at elevated ischemic stroke risk.

Role of plasminogen activation in neuronal organization and survival.

We characterized the interactions between plasminogen and neurons and investigated the associated effects on extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding of plasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA) degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from the matrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysine residues and the proteolytic activity of plasmin. Surprisingly, 24h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinase phosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting that prolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival by plasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/or extravasate during vascular accidents or inflammatory processes.

The role of serum D-dimer level in the diagnosis of patients admitted to the emergency department complaining of chest pain.

This study investigated D-dimer levels in 241 patients admitted to the emergency department with sudden-onset chest pain. The patient group included those diagnosed with acute coronary syndrome (ACS; i.e., unstable angina pectoris [USAP], non-ST elevated myocardial infarction [NSTEMI], ST-elevated myocardial infarction [STEMI]); the control group included those diagnosed with non-cardiac chest pain. Mean serum levels of D-dimer, creatine kinase-MB (CK-MB) and troponin I (TPI) were compared between the groups. Levels of D-dimer, CK-MB and TPI in the patient group were significantly higher than in the control group. There were also significantly higher D-dimer, CK-MB and TPI levels in the STEMI and NSTEMI patient subgroups compared with the control group. Only the D-dimer level was significantly higher in the USAP subgroup versus the control group. The sensitivity and specificity of D-dimer for ACS were 83.7% and 95.4%, respectively, suggesting that evaluating D-dimer levels might be useful in the emergency room for diagnosing ACS and predicting mortality in patients presenting with acute chest pain.

Fibrinolysis Inhibitors: Potential Drugs for the Treatment and Prevention of Bleeding.

Hyperfibrinolytic situations can lead to life-threatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, ε-aminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4-amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.