Activation of blood coagulation and thrombin generation in acute ischemic stroke treated with rtPA.

The impact of thrombolysis with recombinant tissue plasminogen activator (rtPA) on blood coagulation in acute ischemic stroke (AIS) patients is not completely understood. We studied the effect of thrombolysis on the thrombin generation (TG) profile as well as coagulant activity of activated factors IX (FIXa), XI (FXIa) and tissue factor (TF) in AIS patients. In a case-control study, TG parameters as well as FIXa, FXIa and TF levels were assessed in 95 AIS patients, including individuals receiving rtPA treatment within 4.5 h since AIS onset (n = 71, 74.7%) and those ineligible for thrombolysis (n = 24, 25.3%). Blood samples were collected at baseline and after 24 h since admission. The two groups were similar with regard to demographics and clinical factors. In thrombolysed patients, all TG parameters measured after 24 h were markedly decreased, with strongest impact on lag time (LT), when compared with the baseline values (81.3% longer LT, p < 0.0001), as well as when compared to the non-thrombolysed group (86% longer LT, p = 0.002). In non-thrombolysed AIS patients the TG remained unaltered. Logistic regression adjusted for potential confounders showed that high baseline ETP value (the top quartile) was solely predicted by the presence of circulating FIXa, whereas after 24 h FXIa predicted high ETP in the subgroup of thrombolysed and in all AIS patients. Thrombolysis in AIS patients markedly attenuates the TG. Elevated FXIa contributes to thrombin formation capacity after 24 h, highlighting a role of this factor in the regulation of blood coagulation in AIS.

Multi-analyte validation in heterogeneous solution by ELISA.

Enzyme Linked Immunosorbent Assay (ELISA) is a standard assay that has been used widely to validate the presence of analyte in the solution. With the advancement of ELISA, different strategies have shown and became a suitable immunoassay for a wide range of analytes. Herein, we attempted to provide additional evidence with ELISA, to show its suitability for multi-analyte detection. To demonstrate, three clinically relevant targets have been chosen, which include 16kDa protein from Mycobacterium tuberculosis, human blood clotting Factor IXa and a tumour marker Squamous Cell Carcinoma antigen. Indeed, we adapted the routine steps from the conventional ELISA to validate the occurrence of analytes both in homogeneous and heterogeneous solutions. With the homogeneous and heterogeneous solutions, we could attain the sensitivity of 2, 8 and 1nM for the targets 16kDa protein, FIXa and SSC antigen, respectively. Further, the specific multi-analyte validations were evidenced with the similar sensitivities in the presence of human serum. ELISA assay in this study has proven its applicability for the genuine multiple target validation in the heterogeneous solution, can be followed for other target validations.

Correlation between factor (F)XIa, FIXa and tissue factor and trauma severity.

BACKGROUND: It has been observed that trauma patients often display elevated procoagulant activity that could be caused, in part, by tissue factor (TF). We previously observed that trauma patients with thermal, blunt, and penetrating injuries have active FIXa and FXIa in their plasma. In the current study, we evaluated the effect of injury severity, with or without accompanying shock, on the frequency and concentration of TF, FIXa, and FXIa in plasma from trauma patients. METHODS: Eighty trauma patients were enrolled and divided equally into four groups based on their Injury Severity Score and base deficit:Blood was collected at a 0 time-point (first blood draw upon arrival at hospital) and citrate plasma was prepared, frozen, and stored at -80 °C. FXIa, FIXa, and TF activity assays were based on a response of thrombin generation to corresponding monoclonal inhibitory antibodies. RESULTS: The frequency and median concentrations of TF were relatively low in non-severe injury groups (17.5% and 0 pM, respectively) but were higher in those with severe injury (65% and 0.5 pM, respectively). Although FXIa was observed in 91% of samples and was high across all four groups, median concentrations were highest (by approximately fourfold) in groups with shock. FIXa was observed in 80% of plasma samples and concentrations varied in a relatively narrow range between all four groups. No endogenous activity was observed in plasma from healthy individuals. CONCLUSIONS: (1) Frequency and concentration of TF is higher in patients with a higher trauma severity. (2) Concentration of FXIa is higher in patients with shock. (3) For the first time reported, the vast majority of plasma samples from trauma patients contain active FIXa and FXIa. LEVEL OF EVIDENCE: Prognostic/epidemiological study, level II.

Coagulation and fibrinolytic protein kinetics in cardiopulmonary bypass.

The development of Cardiopulmonary Bypass (CPB) catopulted the field of cardiothoracic surgery into a new dimension--one that changed the lives of individuals with congenital and acquired heart disease worldwide. Despite its contributions, CPB has clear limitations and creates unique challenges for clinicians and patients alike, stemming from profound hemostatic pertubations and accompanying risk for bleeding and possibly thrombotic complications.

Real-time PCR detection of protein analytes with conformation-switching aptamers.

We have developed a novel method that uses conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte and a different secondary structure in the presence of a protein such as thrombin or platelet-derived growth factor (PDGF). The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (because no signal is produced by background binding) and the proximity ligation assay (PLA) (because only one epitope, rather than two epitopes, on a protein surface must be bound).

Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging.

RNA microarrays were created on chemically modified gold surfaces using a novel surface ligation methodology and employed in a series of surface plasmon resonance imaging (SPRI) measurements of DNA-RNA hybridization and RNA aptamer-protein binding. Various unmodified single-stranded RNA (ssRNA) oligonucleotides were ligated onto identical 5'-phosphate-terminated ssDNA microarray elements with a T4 RNA ligase surface reaction. A combination of ex situ polarization modulation FTIR measurements of the RNA monolayer and in situ SPRI measurements of DNA hybridization adsorption onto the surface were used to determine an ssRNA surface density of 4.0 x 10(12) molecules/cm2 and a surface ligation efficiency of 85 +/- 10%. The surface ligation methodology was then used to create a five-component RNA microarray of potential aptamers for the protein factor IXa (fIXa). The relative surface coverages of the different aptamers were determined through a novel enzymatic method that employed SPRI measurements of a surface RNase H hydrolysis reaction. SPRI measurements were then used to correctly identify the best aptamer to fIXa, which was previously determined from SELEX measurements. A Langmuir adsorption coefficient of 1.6 x 10(7) M(-1) was determined for fIXa adsorption to this aptamer. Single-base variations from this sequence were shown to completely destroy the aptamer-fIXa binding interaction.

[Expression evaluation of in loco coagulation system in colorectal cancer]. / Ocena ekspresji in loco czynników ukladu krzepniecia krwi w raku jelita grubego.

Colorectal cancer is the third most often cause of morbidity and mortality due to cancer in Poland. Thromboembolic complications are common events during the course of the disease. It is well known that hemostatic proteins play an important role in cancer progression. The purpose of the study was to evaluate the in loco interactions among colorecatal cancer and coagulation factors. 21 cases of G2 colorectal adenocarcinoma obtained during surgical resection were examined. Immunohistochemical procedures according to ABC method were employed. Tissue factor (TF) and coagulation factors II, VII, X, IX were observed in cancer cells and except factors II and IX--in tumor associated macrophages. TF was also demonstrated in endothelial cells of small blood vessels. Strong expression of fibrinogen was observed among connective tissue at some distance around malignant tumor while weaker expression was found in tumor stroma. Expression of F(1+2), the by-product of thrombin generation, was revealed in cancer cells, macrophages and in the tumor stroma. The results indicate extravascular activation of blood coagulation in loco in colorectal cancer that is TF-dependent.

Quantitation of seminal factor IX and factor IXa in fertile, nonfertile, and vasectomy subjects: a step closer toward identifying a functional clotting system in human semen.

Coagulation factor (F) IX is a zymogen of the plasma serine proteases, one that plays an essential role in the regulation of normal blood coagulation. Congenital defects of FIX synthesis or function cause hemophilia B (originally called hemophilia C). Factor IX is activated by Tissue Factor (TF):FVII/FVIIa complex and FXIa. Subsequent to its activation, FIXa combines with FVIIIa on the platelet surface and activates FX to FXa. Human semen forms a semi-solid gelatinous coagulum, which then liquefies within 5-20 minutes in vitro. In spite of evidence demonstrating the importance of the seminal coagulation and liquefaction process in terms of global fertility and despite the fact that the seminal coagulum is composed of fibrin-like material, it has always been addressed from the perspective of High Molecular Weight Seminal Vesicle (HMW-SV) proteins (Semenogelin I and II) and their cleavage by prostate-specific antigen rather than the conventional hemostatic factors. In this study and as part of our continuing investigation of human seminal clotting factors, we report here on seminal FIX and FIXa in normal, subfertile, and vasectomized subjects. Factors IX and FIXa were studied in a total of 119 semen specimens obtained from subfertile (n=18), normally fertile (n=34), and fertile sperm donors (n=27) and vasectomy subjects (n=40). Seminal FIX and FIXa levels were also measured in a group defined by normality in several parameters derived from the World Health Organization fertility criteria and termed "pooled normal semen parameters." Both FIX and FIXa were quantifiable in human semen. There was a wide individual variation in FIX and FIXa levels within groups. Despite the group size, statistically significant associations with fertility-related parameters were infrequent. There is a positive correlation between FIX and its activation product, FIXa (n=36; r=0.51; P <.05). Factor IXa elevation in the high sperm-clump group was significant (P <.05), and days of abstention correlated with FIXa levels (n=63; r=0.3; P <.05). The key finding of the present study is that both FIX and FIXa are present in concentrations that are not dissimilar to plasma levels and that are apparently functional, as the activated form is also present. This fact, taken with other reports of coagulation factors in semen, raises the likelihood that a functional set of hemostatic coagulation proteins exists in semen, potentially to interact with the HMW-SV proteins and the prostate-specific antigen system.

Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa.

The autolysis loop (residues 143-154 in chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation proteases. This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the protease specificity of factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant activated factor X normally both in the absence and presence of factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with antithrombin (AT) in both the absence and presence of the cofactor, heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the autolysis loop are recognition sites for FX independent of factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the serpin is in the heparin-activated conformation.

Activation of factor IX by erythrocyte membranes causes intrinsic coagulation.

As an extension of our earlier work, procoagulant activity of erythrocyte [red blood cell (RBC)] membrane was examined using biochemical and rheological techniques. Western blot analysis of coagulation factors incubated with erythrocytes (RBCs) showed that only factor IX (FIX) was activated by RBC membranes in the presence of calcium ions. A fluorogenic assay suggested that activated FIX is capable of activating factor X. A preliminary crude extraction of the substance from RBC membranes suggested that a FIX-activating enzyme may be located on the RBC membrane. The initiation of FIX activation by RBCs was enhanced by an elevation in hematocrit. Moreover, the rate of FIX activation by RBCs from normal pregnant women and diabetic patients was much faster than that from normal subjects. In addition, glucose treatment of normal RBCs resulted in the increase in procoagulant activity. It is suggested that FIX activation by RBC membranes may serve as a triggering mechanism for blood coagulation, although further study will be required to clarify the putative FIX activating enzyme on the RBC membrane and to permit more extensive physiological experiments to be performed.

Comparison of novel hemostatic factors and conventional risk factors for prediction of coronary heart disease.

BACKGROUND: This study sought to assess whether novel markers of hemostatic activity are predictive of coronary heart disease (CHD) and improve risk assessment. METHODS AND RESULTS: Conventional CHD risk factors, the activation peptides of factor IX and factor X, factor VII activity and antigen, activated factor XII, prothrombin fragment 1+2, fibrinopeptide A, and fibrinogen were measured in 1153 men aged 50 to 61 years who were free of myocardial infarction at recruitment. Activated factor VII (VIIa) was measured in 829 men. During 7.8 years of follow-up, 104 had a CHD event. Baseline status was related to outcome by logistic regression by using a modified nested case-control design. Screening performance was judged from receiver operating characteristic curves. A high activated factor XII was associated with increased CHD risk, but low levels were not protective. Plasma VIIa and factor X activation peptide were independently and inversely related to risk. Plasma factor IX activation peptide and fibrinogen were positively associated with risk, but the relations were no longer statistically significant after adjustment for other factors, including VIIa and apoA-I. Other hemostatic markers were not associated with CHD risk. CONCLUSIONS: Hemostatic status did not add significant predictive power to that provided by conventional CHD risk factors yet was able to substitute effectively for these factors.

Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.

Autoplex-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII inhibitors. While Autoplex-T has a demonstrated record of clinical effectiveness, the procoagulant composition of this material has not been reported. This absence of composition data is a reflection of the lack of techniques appropriate for accurately measuring an individual protease such as factor IXa in complex mixtures of similar proteases. The development of Colorimetric Active Site-Specific ImmunoAssay technology (CASSIA) has permitted the accurate analysis of the coagulant enzymes present in Autoplex-T. Ten lots of Autoplex-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone and biotinylated glutamylglycylarginine chloromethylketone. Only activated forms of the clotting factors present in Autoplex-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex-T by adsorption onto streptavidin. The individual proteins bound to streptavidin were then detected with specific antibodies. Mean results from the analysis of ten lots of Autoplex-T (mean values) are as follows: factor Xla, 5.9 nM or 0.8 microg/ml; factor Xa, 46.5 nM or 2.1 microg/ml; factor IXa, 177.8 nM or 11.7 microg/ml; factor VIIa, 68.6 nM or 3.3 microg/ml and factor IIa, 5.3 nM or 0.2 microg/ml. These results are discussed with respect to the mechanism of action of Autoplex-T in the treatment of factor VIII inhibitor patients.

High sensitivity detection of activated factor IX: application to the analysis of different therapeutical factor IX concentrates and prothrombin complexes.

A very sensitive and highly reliable test system for the detection of activated coagulation factor IX (FIXa) has been established. This assay system is based on the cleavage of a fluorogenic substrate by activated factor X (FXa) which is generated by FIXa. This assay can be used to process a large number of samples at a time and, being based on the convenient microtiter plate format, can easily be adapted to automated processing for routine screening of large sample numbers. With this assay at hand we determined the FIXa content of different commercially available therapeutic FIX sources, such as high purity FIX (HPFIX) and prothrombin complex concentrates (PCC). Here we demonstrate that PCC from several suppliers do not contain significantly higher levels of FIXa as compared to HPFIX from the same supplier. In fact, there is a tendency for HPFIX to contain more FIXa than PCC. Moreover, HPFIX from certain manufacturers who do not produce PCC are characterized by an exceptionally high content of FIXa. Therefore, the higher thrombogenic potential of PCC which is well documented clinically cannot be explained solely -- if at all -- by an increased content of FIXa. Rather, it will be necessary to identify other components responsible for this phenomenon.

Collaborative study on assays of activated FIX (FIXa). On behalf of the factor VIII and factor IX subcommittee of the ISTH. International Society on Thrombosis and Haemostasis.

A collaborative study has been organised by NIBSC to examine the performance of FIXa assays between laboratories, and to investigate the need for a standard. Ampoules of 3 materials, one monocomponent concentrate (coded C) and 2 different preparations of purified human FIXa (one proposed reference preparation, coded A and a test material coded B), have been assayed in 11 laboratories for FIXa using either the NIBSC method or a local method, with local standards (if available, coded D) to determine their potencies. The data showed high between assay variability; with the exception of one laboratory, most of the between assay variation expressed as %geometric coefficients of variation (gcv) were over 15%. The interlaboratory gcv when preparation B was assayed against the local standard was over 1700%, suggesting that most of the local standards are poorly calibrated. The %gcv was improved to 80% when reference A was used as the standard. These data clearly show that an international reference standard for FIXa would help to standardise FIXa preparations and would also improve in house assays for FIXa. However, an accelerated degradation study has shown that reference A is not suitable as a long term standard and another material with suitable stabilizers has to be established as an international standard for FIXa.

Measurement of activated factor IX in factor IX concentrates: correlation with in vivo thrombogenicity.

Current in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15-1.2 U/1000 in FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25-4. There was a significant positive correlation (r = 0.55, p < 0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

Large-scale preparation and biochemical characterization of a new high purity factor IX concentrate prepared by metal chelate affinity chromatography.

Metal chelate affinity chromatography on copper-charged Chelating Sepharose has been used to purify a factor IX concentrate from 4,000- to 5,000-kg pools of human plasma, with an overall yield of 194 IU/kg. Unwanted proteins and solvent-detergent reagents added to inactivate lipid-enveloped viruses were removed during the chromatographic step. The freeze-dried product was > 80% pure factor IX with a mean specific activity of > 160 IU/mg protein. The concentrate showed no evidence of clotting factor activation by in vitro tests for potential thrombogenicity or by direct assay for activated factor IX. The concentrate did not exhibit proteolytic activity against a range of synthetic peptide chromogenic substrates. Full functional factor IX activity was retained and there was no evidence of protein degradation. Metal chelate affinity chromatography therefore appears to present less physicochemical challenge to the protein than other factor IX purification methods, while allowing the preparation of a clinical factor IX concentrate at a large scale.

Microplate coagulation assays.

This report describes the development of microplate-based blood coagulation assays. The assays require a kinetic microplate reader to follow changes in absorbance at 405 nm caused by the coagulating plasma. Procedures for performing prothrombin time and activated partial thromboplastin time tests are described with intra- and inter-assay variability of a few percentage points. The prothrombin time of normal plasma was 64.5 +/- 3.6 s, and the activated partial thromboplastin time was 69.8 +/- 3.2 s. Clotting times were prolonged when normal plasma was mixed with plasmas deficient in particular coagulation factors, as expected. These assays take advantage of the microplate format (small sample size and multiple simultaneous assays) and can be customized for specific purposes, such as quantifying purified factor IX or assessing protein C activity in plasma.

Activation of coagulation after administration of tumor necrosis factor to normal subjects.

Tumor necrosis factor has been implicated in the activation of blood coagulation in septicemia, a condition commonly associated with intravascular coagulation and disturbances of hemostasis. To evaluate the early dynamics and the route of the in vivo coagulative response to tumor necrosis factor, we performed a controlled study in six healthy men, monitoring the activation of the common and intrinsic pathways of coagulation with highly sensitive and specific radioimmunoassays. Recombinant human tumor necrosis factor, administered as an intravenous bolus injection (50 micrograms per square meter of body-surface area), induced an early and short-lived rise in circulating levels of the activation peptide of factor X, reaching maximal values after 30 to 45 minutes (mean +/- SEM increase after 45 minutes, 34.2 +/- 18.2 percent; tumor necrosis factor vs. saline, P = 0.015). This was followed by a gradual and prolonged increase in the plasma concentration of the prothrombin fragment F1+2, peaking after four to five hours (mean increase after five hours, 348.0 +/- 144.8 percent; tumor necrosis factor vs. saline, P less than 0.0001). These findings signify the formation of factor Xa (activated factor X) and the activation of prothrombin. Activation of the intrinsic pathway could not be detected by a series of measurements of the plasma levels of factor XII, prekallikrein, factor XIIa-C1 inhibitor complexes, kallikrein-C1 inhibitor complexes, and the activation peptide of factor IX. The delay between the maximal activation of factor X and that of prothrombin amounted to several hours, indicating that neutralization of factor Xa activity was slow. We conclude that a single injection of tumor necrosis factor elicits a rapid and sustained activation of the common pathway of coagulation, probably induced through the extrinsic route. Our results suggest that tumor necrosis factor could play an important part in the early activation of the hemostatic mechanism in septicemia.