Clot Lysis Assay Research Articles

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

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

Optimization of Self-Assembled Chitosan/Streptokinase Nanoparticles and Evaluation of Their Cytotoxicity and Thrombolytic Activity.

In this study, the enzyme streptokinase (thrombolysis agent) and chitosan (Cs) nanoparticles were prepared by self-assembly. Using experimental design, chitosan concentration, solution pH and stirring time were studied as independent variables to identify their effects on size, polydispersity index (PDI) and loading efficiency of nanoparticles. Results showed that pH and concentration have a direct effect on size. Additionally, minimum PDI was observed at lowest values of concentration and highest values of stirring time. pH-5.6 was also necessary to obtain the smallest PDI and highest loading efficiency values. The model predicted that to obtain maximum loading efficiency and minimum size along with low PDI, optimum values are 0.5 mg/mL, 5.18 and 30 min for the Cs concentration, solution pH and stirring time, respectively. The corresponding mean ± SD values for experimentally prepared nanoparticles were 43 ± 10%, 526 ± 121 nm, 0.3 ± 0.2, respectively. MTT and euglobulin clot lysis assays on the optimized nanoparticles showed that chitosan/streptokinase nanoparticles have slightly toxic effect on human fetal lung fibroblast cells (Mrc-5), compared with chitosan and streptokinase alone as a control. Also, thrombolytic activity of encapsulated streptokinase in nanoparticles is decreased slightly in comparison with free streptokinase. However, the preparation keeps a good potency for use as a thrombolytic agent in vivo.

Anticoagulant activity of crude extract of Holotrichia diomphalia larvae

Ethnopharmacological relevanceHolotrichia diomphalia larvae are one classical folk medicinal material in East Asia which has clinically been used to promote blood circulation and dispel blood stasis for several hundred years. Aim of the studyThe anticoagulant activity of crude extract of H. diomphalia larvae (CEHDL) in vitro and in vivo was evaluated to explore its mechanism as antithrombotic medicine. Materials and methodsThe effects of CEHDL on plasma recalcification time, platelet aggregation, bleeding time, hydrolysis of fibrinogen and fibrin were measured with normal human plasma, plasma-rich platelet, transected mouse tails and bovine fibrinogen; the anti-thrombosis activities of CEHDL in vitro and in vivo were analyzed with clots lysis assay and carrageenan-induced mouse tail thrombosis model. ResultsCEHDL was found to contain large numbers of proteins and could inhibit blood coagulation and platelet aggregation in a dose-dependent manner. Furthermore, CEHDL preferentially cleaved α- and β-chains followed by γ-chains of fibrinogen. Besides, CEHDL could directly degrade fibrin rather than activate plasminogen. It has been noted that fibrinogenolytic activity of CEHDL could be unaffected by metal ions such as Mg2+, Ca2+, Zn2+, Fe2+, Fe3+, Cu2+ and buffers with pH 3-10. Moreover, protease inhibitors like TPSI, aprotinin, leupetin, PMSF, DTT and EDTA only slightly or not inhibited fibrinogenolytic activity of CEHDL. However, CEHDL could be completely inactivated at 75°C and 100°C. In addition, CEHDL exhibited anti-thrombosis activities in both blood clot lysis assay and carrageenan-induced thrombosis model. ConclusionCEHDL possessed potent anticoagulant activity and several fibrin(ogen)olytic agents from H. diomphalia larvae were responsible for its antithrombotic effect as medicine.

Abstract 13304: Thrombin-activated Fibrinolysis Inhibitor in Chronic Thromboembolic Pulmonary Hypertension

Introduction: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. In the present study, we examined whether fibrinolysis capacity is impaired in CTEPH patients with a special reference to thrombin-activated fibrinolysis inhibitor (TAFI) and if so, whether TAFI is also directly involved in the pathogenesis of CTEPH in mice and humans. Methods and Results: We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (PAH, n=22) and controls (non-PH, n=19). Whole blood clot-lysis assay with monteplase (100 ng/ml) showed that thrombus reduction rate was significantly reduced in CTEPH compared with PAH or controls (58.1 vs. 73.5 and 75.1%, both P&lt;0.01). Plasma clot-lysis assay showed that the clot lysis time tended to be prolonged in CTEPH compared with PAH or controls (894 vs. 652 and 720 sec, both P=0.07). Moreover, serum levels of TAFI were significantly higher in CTEPH than in PAH or controls (46.1 vs. 39.1 and 38.7 μg/ml, both P&lt;0.05), which remained unchanged even after hemodynamic improvement by percutaneous transluminal pulmonary angioplasty (49.6 vs. 50.0 μg/ml, P=0.92). The levels of TAFI released from the platelets upon stimulation by thrombin (0.05 U/ml) were significantly higher in CTEPH than in PAH or non-PH (2.7 vs. 1.7 and 1.0 folds, both P&lt;0.05). Additionally, thrombus reduction rate was significantly improved with a carboxypeptidase R (an inhibitor of activated TAFI) (62.0 vs. 73.4%, P&lt;0.01) or prostaglandin E1 (an inhibitor of activation of platelets) (62.2 vs. 70.4%, P&lt;0.01). Importantly, plasma levels of TAFI were correlated with thrombus reduction rate and improved thrombus reduction rate by an activated TAFI-inhibitor (both P&lt;0.05). Finally, we newly developed TAFI-overexpressing mice (TAFI-Tg) and found that intravenous injection of thrombin (100 units/kg) caused accelerated thrombotic obstruction of the pulmonary artery in TAFI-Tg mice compared with littermate controls (5.4±1.2 vs. 1.8±0.6 vessels/lung fields) (n=9, P&lt;0.05). Conclusions: These results provide the first evidence that TAFI plays a crucial role in the pathogenesis of CTEPH.

Cell-Free DNA Modulates Clot Structure and Impairs Fibrinolysis in Sepsis.

Sepsis is characterized by systemic activation of inflammation and coagulation in response to infection. In sepsis, activated neutrophils extrude neutrophil extracellular traps composed of cell-free DNA (CFDNA) that not only trap pathogens but also provide a stimulus for clot formation. Although the effect of CFDNA on coagulation has been extensively studied, much less is known about the impact of CFDNA on fibrinolysis. To address this, we (1) investigated the relationship between CFDNA levels and fibrinolytic activity in sepsis and (2) determined the mechanisms by which CFDNA modulates fibrinolysis. Plasma was collected from healthy and septic individuals, and CFDNA was quantified. Clot lysis assays were performed in plasma and purified systems, and lysis times were determined by monitoring absorbance. Clot morphology was assessed using scanning electron microscopy. Clots formed in plasma from septic patients containing >5 µg/mL CFDNA were dense in structure and resistant to fibrinolysis, a phenomenon overcome by deoxyribonuclease addition. These effects were recapitulated in control plasma supplemented with CFDNA. In a purified system, CFDNA delayed fibrinolysis but did not alter tissue-type plasminogen activator-induced plasmin generation. Using surface plasmon resonance, CFDNA bound plasmin with a Kd value of 4.2±0.3 µmol/L, and increasing concentrations of CFDNA impaired plasmin-mediated degradation of fibrin clots via the formation of a nonproductive ternary complex between plasmin, CFDNA, and fibrin. Our studies suggest that the increased levels of CFDNA in sepsis impair fibrinolysis by inhibiting plasmin-mediated fibrin degradation, thereby identifying CFDNA as a potential therapeutic target for sepsis treatment.

Delivery of tissue plasminogen activator and streptokinase magnetic nanoparticles to target vascular diseases

Thrombolytic therapy for acute myocardial infarction standardly makes use of the medications streptokinase (SK) and tissue plasminogen activator (tPA). In this study, the potential of silica-coated magnetic nanoparticles (SiO2-MNPs) as nanocarriers clinical thrombolytic therapy was investigated. SiO2-MNPs for use in targeted therapeutic delivery of tPA and SK were prepared using a combined technique incorporating controlled precipitation and hydrothermal methods. Response surface methodology (RSM) was employed to evaluate the efficiency of the SiO2-MNPs. The production of SK secreted from Streptococcus equi was enhanced using random mutagenesis. The tPA and SK A were encapsulated by means of a silanizing agent with a surface rich in 3-aminopropyltrimethoxysilane layered around the SiO2-MNPs. Blood clot lysis assays and fibrin-containing agarose plates were used to carry out in vitro thrombolysis testing. The optimum conditions for producing MNPs were found to be at pH=13 and at a temperature of 75°C for 45min. Culture conditions of 2.75% NaCl concentration at initial pH=7.5 for 90s under UV resulted in maximum SK activity. The tPA/SK-conjugated SiO2-MNPs (SiO2-MNP-tPA-SK) increased operating stability in whole blood and storage stability in a buffer by 92%. More effective thrombolysis using magnetic targeting was indicated by a 38% reduction in blood clot lysis time achieved with SiO2-MNP-tPA-SK compared to administering the SiO2-MNPs without guidance. The silica-coated magnetic nanocarriers developed in this study show potential for improved clinical thrombolytic therapy.

The Occurrence of Thrombosis in Inflammatory Bowel Disease Is Reflected in the Clot Lysis Profile.

The occurrence of thromboembolic events (TE) is an important extraintestinal manifestation in patients with inflammatory bowel disease (IBD). The aim of this study was to compare fibrinolysis and clot lysis parameters between (1) patients with IBD and healthy controls and (2) patients with IBD with TE (IBD + TE) and without TE (IBD - TE). One hundred thirteen healthy controls and 202 patients with IBD, of which 84 patients with IBD + TE and 118 patients with IBD - TE, were included in this case-control study. Three clot lysis parameters (area under the curve, 50% clot lysis time, and amplitude) were determined using a clot lysis assay. Plasminogen activator inhibitor 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor concentrations were determined by enzyme-linked immunosorbent assay. PAI-1 antigen, active PAI-1, and intact thrombin activatable fibrinolysis inhibitor concentrations, as well as 50% clot lysis time and area under the curve, were significantly associated with the presence of IBD (all P < 0.05). The median time between TE and plasma collection was 5.0 (1.8-11.0) years. Comparing IBD + TE versus IBD - TE, active to total PAI-1 ratio (0.36 [0.24-0.61] versus 0.24 [0.13-0.40]), area under the curve (31 [24-49] versus 22 [13-31]), 50% clot lysis time (110 [64-132] versus 95 [70-126] minutes), and amplitude (0.295 [0.222-0.436] versus 0.241 [0.168-0.308]) were significantly higher in IBD + TE (all P <0.05) and remained higher after adjustment for age, gender, C-reactive protein, type of disease, presence of comorbidities, and disease activity. Patients with IBD have an altered clot lysis profile compared with healthy controls. Clot lysis parameters differ significantly between patients with IBD with and without a history of TE and should be included in the risk assessment.

The Fibrinolytic Status in Liver Diseases.

The liver is the main site of synthesis and/or clearance of proteins involved in fibrinolysis. Therefore, chronic liver disease, including cirrhosis, leads to altered plasma levels of fibrinolytic proteins. Historical studies using in vitro clot lysis assays suggested that patients with chronic liver disease had accelerated fibrinolysis. Subsequent studies measured levels of individual pro- and antifibrinolytic proteins and showed that levels of tissue-type plasminogen activator are elevated. Plasma levels of plasminogen activator inhibitor-1 may also be altered, which leads to a shift in balance in the fibrinolytic system. Despite the fact that a more recent study using a plasma clot lysis assay challenged the existence of hyperfibrinolysis, other recent studies detected hyperfibrinolysis in a considerable number of patients with cirrhosis. Therefore, it is now recognized that hyperfibrinolysis may occur in 30 to 50% of patients with end-stage liver disease. A causal role of hyperfibrinolysis in bleeding is difficult to establish because also other concomitant changes in hemostasis occur. Treatment of hyperfibrinolysis consists of the use of fibrinolysis inhibitors, such as tranexamic acid. In this review we summarize current insights of the role of the liver in fibrinolysis, changes in fibrinolytic proteins, the potential clinical implications, and management of hyperfibrinolysis in liver disease.

Plasma clot properties in patients with a mild-to-moderate bleeding tendency of unknown cause.

In a large proportion of patients with mild bleeding disorders (MBDs) no diagnosis can be established by routine coagulation tests. We investigated whether alterations in plasma clot properties account for MBDs of unknown cause. Ninety-five patients with MBDs of unknown origin and 98 age- and sex-matched healthy controls were investigated. Furthermore, data of 25 patients with a deficiency of factor VIII were analyzed. Plasma clot characteristics in the absence and presence of recombinant tissue plasminogen activator (rtPA) represented by the lag phase, rate of protofibril formation (Vmax), fibrin structure (ΔAbs), time to peak (TTP), half lysis time (t50 and area under the curve (AUC) were measured in turbidometric clot formation and lysis assays. In the fibrinolysis assay, Vmax was lower in patients than in healthy controls. No differences in the other parameters of clot formation and lysis were detected between the groups. There was no clear association of plasma clot properties with the clinical severity of bleeding in patients with MBDs. Patients with known decreased factor VIII levels also showed a lower Vmax. Fibrinogen levels were positively associated with each of the assessed parameters in both groups, with the strongest association with ΔAbs, indicating altered fibrin structure. Factor VIII activity correlated with altered clot characteristics similar to fibrinogen, especially in patients, with the strongest positive correlation to Vmax. This cohort of patients with MBDs of unknown origin showed a lower rate of fibrin formation in the fibrinolysis assay, but otherwise similar plasma clot properties compared to healthy controls.

Assessing the anticoagulant effect of dabigatran in children: An in vitro study

ObjectivesAge-related changes in the hemostatic system result in variation in response to anticoagulants and coagulation assays over childhood. This study used in vitro methods to determine i) optimum coagulation assays for dabigatran in children and ii) anticoagulant effect of dabigatran across pediatric age groups. Materials and MethodsPooled plasma samples from healthy children aged 0 to <1, 1 to <5, 5 to <10, 10 to <17years and adults were spiked with increasing concentrations of dabigatran and the effect was assessed in five coagulation assays. The samples were also assessed for overall hemostasis potential using a fibrin clot formation and lysis assay. ResultsIn all five coagulation assays, there were no differences in responses to dabigatran over all pediatric age groups. The international normalized ratio was the least sensitive measure. Activated partial thromboplastin time showed moderate sensitivity and a nonlinear response curve. Thrombin time (TT), dilute TT (dTT) and ecarin clotting time were linearly correlated with dabigatran concentrations; however, the ecarin time and TT were overly sensitive. In the overall hemostasis potential assay, increasing dabigatran concentrations delayed the initiation of clot formation and reduced the time to 50% clot lysis. The responses to initiation of clot formation and clot lysis were consistent across all pediatric groups and comparable to responses in adults. ConclusionThe dTT is the most suitable assay for measuring dabigatran concentrations in children. Fibrin clot generation and lysis assay responses to dabigatran in children over all ages were consistent and comparable to those of adults.

Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.

Synergistic Effect in Bleed Reduction By superfva and Recombinant Human FVIIa in Vivo Suggests a Novel Bypassing Strategy for Hemophilia Patients with Inhibitors

Introduction: In Hemophilia patients with inhibitors, recombinant human (rh)FVIIa-based bypassing strategy does not always achieve hemostasis indicating a clinical need for improved treatment strategies. We engineered an activated FVa variant (superFVa) with mutations at 3 activated protein C cleavage sites (R506, R306 and R679) and an engineered interdomain disulfide bond (H609C-E1691C) connecting the A2 and A3 domains. SuperFVa resisted inactivation and showed superior hemostatic properties in FVIII-deficient plasma and in mouse models of Hemophilia A compared to wild type rhFVa. Therefore, the hemostatic effects of superFVa alone and in combination with rhFVIIa in hemophilia with inhibitors were determined in more detail.Materials and Methods: Procoagulant and clot stabilizing properties of superFVa, rhFVIIa, and combinations thereof were studied in thrombin generation (endogenous thrombin potential (ETP) and peak height) and clot lysis assays in hemophilia A and normal plasma with or without high titer inhibitors. In vivo efficacy of superFVa alone and in combination with rhFVIIa for bleed reduction in hemophilia was tested in FVIII-deficient mice using a tail clip model.Results: Extensive dose-response titrations of superFVa, rhFVIIa, and combinations thereof in hemophilic or normal plasma with inhibitors indicated synergistic responses for normalization of thrombin generation or clot stabilization. For instance, at 0.4 nM rhFVIIa (1/100 of the therapeutic plasma level), the concentration of superFVa required to normalize thrombin generation was reduced 10-fold. Vice versa, rhFVIIa tested up to 40 nM only marginally improved thrombin generation, however, rhFVIIa in the presence of a low concentration of superFVa (3 nM) normalized thrombin generation at concentrations 50-fold below the therapeutic plasma level. Saturation of the synergistic effect between superFVa and rhFVIIa was evident as thrombin generation reached a plateau at ~110% of normal plasma at higher concentrations of superFVa and rhFVIIa. Similar synergistic normalization of clot lysis time was observed when superFVa and rhFVIIa were used together at low concentration.Beneficial effects of superFVa and rhFVIIa combinations were also observed in 5 individual plasma samples of hemophilia A patients with inhibitors (41-280 BU/ml). ETP and peak height improved ~1.5-3 fold when both superFVa (3 nM) and rhFVIIa (2 nM) were present compared to the individual 10 to 20-fold higher concentrations of superFVa (30 nM) or rhFVIIa (40 nM). Also, a therapeutic dose of rhFVIIa administered to 2 hemophilia patients with inhibitors (32 & 64 BU/ml) provided unremarkable improvements of thrombin generation in plasma taken before and 10 minutes post-infusion, whereas thrombin generation was fully restored upon titration of superFVa ex-vivo.In-vivo tail bleed analysis of FVIII-deficient mice showed a dose dependent reduction of bleeding by superFVa (mean blood loss was 25.9 µL/g at 10 U/kg; 9.7 µL/g at 40 U/kg and 2.5 µL/g at 200 U/kg of superFVa compared to 26.3 µL/g for saline). At the highest dose of superFVa (200 U/kg), bleed reduction was indistinguishable from the bleed reduction by rhFVIII (200 U/kg; 2.9 µL/g). Injection of 1 or 3 mg/kg rhFVIIa, reduced mean blood loss from 25.9 µL/g (saline control) to 16.8 (p=0.1) or 7.2 µL/g (p=0.003), respectively, but rhFVIIa’s effects did not reach the blood loss reduction achieved with rhFVIII (2.9 µL/g, p=0.03). However, combination of rhFVIIa (1 mg/kg) with 10 U/kg superFVa, a concentration that did not reduce bleeding by itself, significantly reduced bleeding from 16.7 to 10.2 µL/g (p=0.05). Combination of rhFVIIa (3 mg/kg) with superFVa (40 U/kg) decreased bleeding even further (1.6 µL/g; p=0.01), similar to what was observed with rhFVIII.Conclusion: The engineeredsuperFVa variant showed synergistic procoagulant effects with rhFVIIa in vitro in hemophilic and normal plasma with inhibitors in thrombin generation and clot lysis assays. Notably, in vivo, superFVa reduced bleeding similar to rhFVIII in FVIII-deficient mice in a dose-dependent fashion, and it was able to enhance further bleed reduction when administered in combination with rhFVIIa. These results warrant further characterization of the potential therapeutic benefits of superFVa as a novel bypassing strategy either alone or in combination with rhFVIIa-based therapy in hemophilia patients with inhibitors. Disclosuresvon Drygalski:University of California, San Diego: University of California, San Diego Patents & Royalties. Gale:University of California, San Diego: University of California, San Diego Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. Mosnier:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties.

Lp(a) is not associated with diabetes but affects fibrinolysis and clot structure ex vivo.

Lipoprotein (a) [Lp(a)] is a low density lipoprotein (LDL) with one apolipoprotein (a) molecule bound to the apolipoprotein B-100 of LDL. Lp(a) is an independent risk factor for cardiovascular disease (CVD). However, the relationship of Lp(a) to diabetes and metabolic syndrome, both known for increased CVD risk, is controversial. In a population based study on type two diabetes mellitus (T2DM) development in women, Lp(a) plasma levels showed the well known skewed distribution without any relation to diabetes or impaired glucose tolerance. A modified clot lysis assay on a subset of 274 subjects showed significantly increased clot lysis times in T2DM subjects, despite inhibition of PAI-1 and TAFI. Lp(a) plasma levels significantly increased the maximal peak height of the clot lysis curve, indicating a change in clot structure. In this study Lp(a) is not related to the development of T2DM but may affect clot structure ex vivo without a prolongation of the clot lysis time.

Abstract 38: Identification of Thrombomodulin Binding Sites on Thrombin Activatable Fibrinolysis Inhibitor that Mediate Accelerated Activation by Thrombin

Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM), or plasmin to generate the enzyme activated TAFI (TAFIa). TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade. Accumulating evidence from several studies suggests that TM and TAFI make direct contacts such that the enhancement of TAFI activation by TM is through binding at sites remote from the activation site (Arg92-Ala93). The elements of TAFI structure that allow accelerated activation of thrombin by TM are largely unknown. Therefore, alanine scanning mutagenesis of surface-exposed charged residues on TAFI was used to identify sites that mediate acceleration of activation by TM and which thus may indicate sites where TM binds to TAFI directly. The rates of activation by thrombin of the variants was measured in the presence or absence of TM. The variants R12A, E28A and R15A exhibited the lowest enhancement of catalytic efficiency in the presence of TM with decreases of 3.0-fold, 3.2-fold and 2.6-fold, respectively, compared to wild-type. The variants D75A/E77A/D78A, E106A, E112A/E116A, R12A/R15A and D54A/D56A showed about half the extent of rate enhancement of wild-type. On the other hand, the variants E99A and E116G exhibited approximately 2.0 to 2.5- fold increases in rate enhancement which may indicate increased binding between TAFI and TM. We determined the antifibrinolytic potential of each variant using an in vitro plasma clot lysis assay. The variants that showed reduced activation by thrombin/TM correspondingly showed impaired stimulation of antifibrinolytic potential by TM. Overall, the data indicate that the variants that showed the strongest TM dependence were focused around the activation peptide which would be near where the C-loop of EGF-3 of TM would contact TAFI. These residues contribute to the increased efficiency of TAFI activation by thrombin.

Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein-Protein Interaction.

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 μM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Abstract T MP20: A Rapid Clot Lysis Assay to Predict Response to IV rt-PA in Stroke

Background: The cornerstone of acute stroke therapy is intravenous rt-PA, with the goal of rapid thrombolysis and restoration of blood flow. Unfortunately, less than half of patients treated have a favorable outcome. Since recanalization is closely correlated with the clinical response, endogenous resistance to rt-PA may inhibit thrombolysis and neurologic improvement. The primary objective of this research study was to evaluate whether an in vitro clot lysis assay and thrombolytic biomarkers could predict response to IV rt-PA in acute ischemic stroke patients Methods: Ischemic stroke patients who received IV rt-PA were recruited from two sites (Augusta, GA and Udine, Italy). Blood samples were collected prior to rt-PA administration and a favorable clinical response to rt-PA was specified as an NIHSS score of two or less at 24 hours. The time required for 50% platelet-poor clot lysis (C50%) was measured by an in vitro turbidimetric assay. The endogenous major fibrinolytic inhibitors (tissue plasminogen inhibitor, PAI-1; and α2-antiplasmin, α2-AP), D-dimer, fibrinogen and pro-thrombin time (PT), were assessed and correlated with clinical outcome. Results: We enrolled 54 rt-PA recipients (63±15 years; 72% male, and 85% white). The mean NIHSS was 11.2±5.1 at admission and 8.2±8.1 at 24 hours; 36% of patients had a favorable clinical response. There was a moderate reduction in C50% among patients with a favorable clinical outcome compared to those with an unfavorable outcome (669.5±46.3 vs. 704.7±91.6; p=0.075). There was no significant difference in PT (13.6±0.8 sec vs. 13.8±1.2 sec; p=0.444), PAI-1(28.2±34.1 pg/mL vs. 19.8±25.9 pg/mL; p=0.321), α2-AP (85.1±7.2 % vs. 88.8±10.1 %; p=0.153), D-dimer (269.5±232.2 ng/mL vs. 368.2±444.6 ng/mL; p=0.303) or fibrinogen (318.1±71.4 mg/dL vs. 343.0±173.1 mg/dL; p=0.465) levels between subjects with favorable and unfavorable response to rt-PA. Summary: Lack of response to IV rt-PA in stroke patients may reflect endogenous resistance to fibrinolysis reflected by prolonged clot lysis in vitro. The utility of clot lysis to predict rt-PA unresponsiveness should be tested in a larger clinical study.

Identification of a novel, nanobody‐induced, mechanism of TAFI inactivation and its in vivo application

Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates. We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg⁻¹) to 4.4 U mg⁻¹ (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37 °C. Incubation of mTAFI with VHH-mTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice. The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.

Cloning and expression of hybrid streptokinase towards clot-specific activity

Streptokinase (SK) is a thrombolytic agent that is widely used to treat myocardial infarction and pulmonary embolism. The lack of fibrin specificity of SK for the clot lysis is one of the limitations of SK. In this study, we have incorporated the finger and Kringle 2 domains from the human tissue type plasminogen activator gene (t-PA) at the 5′ end of the SK gene. These domains are responsible for specific binding to fibrin. We have used the pRSETB vector in an attempt to express the hybrid streptokinase possessing specificity for fibrin. On this regard, three hybrid streptokinase were constructed and expressed in Escherichia coli BL21 (DE3): the finger domain with SK (FSK), the Kringle 2 domain with SK (KSK) and the finger domain+Kringle 2 with SK (FKSK). The activities of the hybrid SKs were assessed by caseinolytic assay and clot lysis assay. All hybrid SKs were found to activate plasminogen in the caseinolytic plate assay. In the clot lysis assay, KSK and FSK were able to dissolute human blood and artificial clots in a fibrin-dependent manner unlike the SK and FKSK proteins.

Hemostatic, milk clotting and blood stain removal potential of cysteine proteases from Calotropis gigantea (L.) R. Br. Latex.

Introduction:Plant latex is a natural source of biologically active compounds and several hydrolytic enzymes responsible for their diverse health benefits. Recent past has witnessed substantial progress in understanding their supplementary industrial and pharmaceutical utility. Calotropis gigantea is one of the important latex producing plants belonging to asclepediaceae family with wide ethnopharmacological applications and is rich in proteolytic enzymes. Present study investigates hemostatic, milk clotting and blood stain removal potential of C. gigantea latex proteases.Materials and Methods:The protease activity of crude enzyme (CE), obtained by centrifugation followed by ammonium sulphate precipitation and dialysis, was assayed using casein as the substrate. Effect of pH, temperature and specific inhibitors on protease activity was determined. Native PAGE and in gel protease activity of CE was performed. Hemostatic (Fibrinogen polymerization, fibrinogen agarose plate and blood clot lysis assays), milk clotting and blood stain removal efficacies of CE were determined.Results:CE exhibited high caseinolytic activity. Enzyme activity was optimum at 37-50ºC and pH 8.0. Fibrinogen polymerization assay showed concentration dependent increase in turbidity indicating thrombin like activity which was further confirmed by fibrinogen agarose plate assays. Clot lysis assay indicated 92.41% thrombolysis by CE in 90 min. CE also revealed significantly high ratio of milk clotting to protease activity (Milk Clotting Index, MCI = 827.59 ± 1.52). Complete destaining of blood stained fabric was observed when incubated with 1% detergent incorporated with 0.1mg/ml CE. The study highlights and validates the compound application potential of latex cysteine proteases from C. gigantea.

In vitro and in vivo characterisation of the profibrinolytic effect of an inhibitory anti-rat TAFI nanobody

One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.