Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products.

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.     

The lysis of crosslinked human fibrin by plasmin yields initially a single molecular complex, D dimer-E

Physicochemical and immunological comparisons between plasmin mediated crosslinked (XL) fibrin digests made in buffer, serum and buffered Trasylol systems have shown that all the D dimer released from XL fibrin is associated with the fibrin E fragment in a strong, non-covalent and electrophoretically stable complex which has the empirical formula, 2D-E. The complex dissociates and reassociates in the presence and absence of detergent. The subunits of D dimer moiety of the complex are made up of A chain remnants (MW 12, 000), Bβ chain remnants (42,000) and crosslinked γ chain remnants (MW about 80, 000). A diagram showing the locations of the three domains of the complex in three distinct fibrin subunits is presented. Aggregates of the complex were found in fibrin lysates made in human serum and their significance to fibrin lysis in vivo is discussed.     

Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma

Carboxypeptidase U (CPU,TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this study was to investigate the time course of the generation of CPU activity during coagulation and fibrinolysis using an in vitro clot lysis model in human plasma. A first peak of CPU activity appeared after initiation of the coagulation phase and a second rise in CPU activity was observed during the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation during coagulation but not during fibrinolysis. Addition of the plasmin inhibitor aprotinin during fibrinolysis resulted in a decrease in CPU activation during the lysis phase. These results demonstrate that proCPU was activated during coagulation by thrombin and during fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.     

Enhancement of clot lysis in vitro and in vivo with a bispecific monoclonal antibody directed against human fibrin and against urokinase-type plasminogen activator.

A hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent Mr 150,000 on nonreduced SDS-PAGE and had an affinity for both human fibrin (Ka = 2 x 10(7) M-1) and for u-PA (Ka = 10(8) M-1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively. In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per microgram/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FU1-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA. It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.     

Influence of polymorphonuclear leukocytes on the plasma clot formation as evaluated by thromboelastometry (ROTEM)

Objective

It has been emphasized that polymorphonuclear leukocytes (PMN) participate in the regulation of coagulation. However, the mechanisms of action are not clear. Besides a procoagulant activity, anticoagulant or fibrinolytic properties are attributed to these cells. To explore their global effect, we have studied their involvement in the clot formation with thromboelastometry, which gives global view over the clotting process, in particular on the structure of the clot and on the kinetic of its formation.

Methods

PMN were isolated from healthy blood donors and resuspended into autologous platelet-free plasma. The ROTEM® device was used. Coagulation was triggered only by adding calcium chloride. Thromboelastometric profiles of PMN-rich plasma (PMN-RP) were compared with autologous platelet-rich (PRP) and platelet-poor plasma (PPP). The inhibition of both tissue factor and intrinsic pathways was also studied.

Results and conclusions

The procoagulant activity of resting PMN was demonstrated as the initiation of fibrin formation with PMN-RP was significantly faster compared with both PRP and PPP. The kinetic of plasma clotting was remarkably improved with PMN-RP compared with PPP. However, the clot with PMN-RP had the same poor viscoelastical properties as PPP. Thromboelastometry gives a new point of view in the involvement of PMN in coagulation, in the absence of any PMN pre-activation. Their impact was centred on the kinetic and the facilitation of the clot formation.     

The activation of plasminogen by T-PA-PAI-1 complex in the absence of fibrin.

The activity of tissue plasminogen activator-plasminogen activator inhibitor-1 (t-PA-PAI-1) complex to convert plasminogen to plasmin was examined by using fibrin autography and casein zymography where plasminogen was added or removed for casein layer. Fibrin autography was more sensitive to the activity of t-PA even in the complex with PAI-1, but casein zymography was more sensitive to plasmin. The inhibitory activity of PAI-1 toward t-PA diminished when plasma was clotted, thus in the presence of fibrin. These results indicate that t-PA-PAI-1 complex has a catalytic activity even in the absence of fibrin, but the activity is enhanced in its presence.     

Glycosylation of human fibrinogen and fibrin in vitro. Its consequences on the properties of fibrin(ogen)

As observed for many proteins, glucose has been shown to bind non enzymatically to fibrinogen and to fibrin. The in vitro degree of glycosylation depends upon the concentration of glucose added to fibrinogen and also on the incubation period. This glycosylation induces a decrease in fibrin lysis by plasmin. On the contrary, it does not influence either fibrinogen activity as cofactor in ADP-induced platelet aggregation or the binding of thrombin onto a clot. Despite the 4 day half life of fibrinogen, since clearance of fibrinogen is exponential, it is assumed that very small quantities of fibrinogen may remain for a long time in the circulation leading to the presence of a low level of a highly glycosylated form of fibrinogen. Consequently, poorly degradable fibrin might be derived from this highly glycosylated fibrinogen and thus be responsible for capillary occlusion and also for atherosclerotic complications in the diabetic patient.     

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models: sensitivity to the inhibitory action of thrombin activatable fibrinolysis inhibitor (TAFI)

In this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect of TAFI was examined for each PA by neutralising TAFIa with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5-10 mug/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.     

A rapid monoclonal antibody-based enzyme immunoassay (EIA) for the quantitative determination of soluble fibrin in plasma.

Soluble fibrin is considered as a molecular marker for intravascular fibrin formation, and impending thrombotic events. Most of the existing assays are less suitable for routine clinical applications and their specificity may be limited. We have developed a sandwich-type EIA with a fibrin-specific MoAb described by us before (Proc Natl Acad Sci 1989; 86: 8951) as the capture antibody. An other MoAb (G8; Thromb Haemostas 1988; 60: 145) with an epitope in the carboxyl-terminal sections of the fibrin alpha-chains, was labeled with peroxidase and used as the tagging antibody. The EIA is calibrated against plasma spiked with known concentrations of soluble fibrin. The time-to-result of the EIA is only 1.5 h. Concentrations as low as 0.5 micrograms soluble fibrin/ml plasma are readily measureable. Heparin has no effect on the results. Fibrinogen and fibrin(ogen) degradation products are not detected. The values of fibrinopeptide A and soluble fibrin values found with the "COA-SET soluble fibrin" assay correlated well with the soluble fibrin values found with our EIA i.e. r = 0.998 and 0.984, respectively. The run-to-run variabilities were 7.9% and 6.6% for samples with low and high soluble fibrin concentrations, respectively. The within-run variabilities were 2.5, 1.8, 4.0 and 4.6% for samples with 1, 0.5, 0.25 and 0.125 micrograms soluble fibrin/ml, respectively. The sensitivity, specificity, accuracy and short time-to-result make our EIA suitable for routine clinical applications and the monitoring of the effectivity of heparinization.     

缺血性脑血管病患者载脂蛋白B基因多态性与血脂关系探讨

应用聚合酶链反应（ＰＣＲ），检测了１０６例正常汉族人及５５例缺血性脑血管病（ＩＣＶＤ）病人的ａｐｏＢ基因ｘｂａⅠ酶切位点限制性片段长度多态性（ＲＦＬＰｓ）及其与血脂的关系。结果表明ＩＣＶＤ组ｘｂａⅠ酶切位点上Ｘ＋的等位基因频率明显高于正常对照组（Ｐ＜０．００５）；ＩＣＶＤ组中具Ｘ＋Ｘ－基因型者的血浆ＨＤＬ－Ｃ较Ｘ－Ｘ－基因型者明显降低（Ｐ＜０．０５），而ＬＰ（ａ）和ＴＣ明显增高（Ｐ＜０．０５～０．００５）。提示ａｐｏＢ基因多态分析结合血浆脂蛋白测定更能有效地检测ＩＣＶＤ易患人群。     

Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein.

NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane. The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins. Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor). A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1). A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts. DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD. Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus. We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins.     

A simple, quick one-step ELISA assay for the determination of complex plasma factor XIII (A2B2)

A new highly sensitive sandwich ELISA assay was developed for the determination of plasma factor XIII (FXIII). Plasma FXIII is a tetrameric complex of two types of subunits (A2B2). A biotinylated monoclonal capture-antibody against the B-subunit and a peroxidase-labelled monoclonal tag-antibody against the A-subunit were added to the plasma dilution and the amount of the complex attached to streptavidin-coated microplate was quantitated by measuring peroxidase activity. Only the tetrameric plasma FXIII reacted in the assay, non-complexed A or B subunits showed no reaction. The assay is linear up-to 40 microg/L of FXIII in the assay mixture. It is a quick one-step assay which can be performed within two hours. At normal and low FXIII concentration within batch reproducibility was 2.0% and 3.3%, day to day variation was 5.5% and 8.7%, respectively. Its high sensitivity allows reliable measurement at FXIII concentrations below 1% of normal average. Plasma samples can be stored for the assay at -20 degrees C for at least one month. Plasma levels of healthy individuals were normally distributed and no gender difference was observed. A reference interval of 14-28 mg/L (67-133%) was established.     

Relationship between elevated plasma levels of crosslinked fibrin degradation products (XL-FDP) and the clinical presentation of patients with myocardial infarction

To assess whether the intense thrombotic state known to occur early after the onset of acute myocardial infarction is further exacerbated by impaired intrinsic fibrinolysis, we compared the intensity of fibrinolysis as measured by the level of crosslinked fibrin degradation products (XL-FDP) in plasma with the intensity of thrombosis as assessed by fibrinopeptide A (FPA) in 98 patients with transmural and 14 patients with non-Q wave infarction. Patients without complications of infarction such as shock, mural thrombi, or malignant arrhythmias requiring countershock generally had normal plasma levels of XL-FDP, less than or equal to 300 ng/ml (81% of those presenting less than 8 hours after onset and 66% of those presenting greater than 8 hours after onset) on admission despite elevated FPA indicative of ongoing thrombosis. In contrast, patients with complications generally had elevated levels of XL-FDP greater than 300 ng/ml (80% of those presenting early and 62.5% of those presenting late) and 50% of these patients had marked elevations to greater than 1000 ng/ml. FPA was markedly elevated in patients with complications whether they presented early or late after onset of infarction. Our direct measurements at the time of infarction support previous data indicating that intrinsic fibrinolysis is impaired in patients with acute infarction, despite marked thrombin activity, when complications are not present. However, when complications are present initially, a more exuberant fibrinolytic response is observed perhaps due to thrombosis associated with the complications themselves.     

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.     

Determination of factor XII in human plasma with arginine proesterase (prekallikrein) II. Studies on the method

When kaolin-activated human plasma was incubated with partially purified plasma prekallikrein, a straight-linear relationship was established between the amount of activator and the initial rate of activation of the prekallikrein substrate. An almost identical regression line was obtained after incubation of the substrate with kaolin-activated dilutions of normal plasma in factor XII deficient plasma. A very close correlation existed between the plasma prekallikrein-activating capacity and the clot-promoting effect in factor XII deficient plasma, and it is concluded that determination of the prekallikrein-activating capacity may be well suited for determination of the concentration of factor XII in plasma.     

Determination of factor XII in human plasma with arginine proesterase (prekallikrein) I. Preparation and properties of the substrate

An arginine proesterase was purified from human plasma by ion exchange chromatography and was concentrated on hydroxylapatite. Upon activation the preparation yielded 0.96 BAEe esterase units per mg protein. The preparation contained traces of factor XI, but was devoid of factor XII. One major arginine proesterase peak was observed after analytical gel filtration on Sephadex G-100 Superfine and disc electrophoresis at pH 4.3.The proesterase was activated by incubation with kaolin-treated plasma and was identified as plasma prekallikrein. Upon incubation with kaolin-treated plasma the activation proceeded at a constant rate to about 30% consumption of the proesterase. It is concluded that the preparation may serve as a substrate for determination of factor XII in plasma.     

Characterization of the human superior olivary complex by calcium binding proteins and neurofilament H (SMI-32)

This study provides a morphologic characterization of the human superior olivary complex as revealed by immunohistochemistry by using antibodies against the calcium binding proteins parvalbumin, calbindin, calretinin, and the nonphosphorylated neurofilament H SMI-32. By combining these markers, it was possible to establish the neuronal architecture and details of the morphologic organization (including axonal terminals) of the different nuclei. The medial superior olivary nucleus is formed by a sheet of parallel-oriented cells. A clear segregation of axon terminals was noticed on the medially and laterally oriented dendrites of the mostly bipolar neurons. The lateral superior olivary nucleus lacked a distinct nuclear shape but was formed by several patches of rather irregularly arranged neurons. Calretinin or parvalbumin immunoreactive afferent terminals were observed which contacted somata or dendrites of these neurons. The immunolabeling also revealed the boundaries of the dorsal periolivary nucleus and morphologic detail of its neurons. A coherent nuclear structure that could be addressed as the medial nucleus of the trapezoid body was not identified by any single one or by combinations of the markers used. The data were also used to establish a three-dimensional-reconstruction of the three major subnuclei of the superior olivary complex. The results are discussed with respect to the possible role of the superior olivary complex in the processing of spatial acoustic information in the azimuthal plane.