A Comparison of Thromboelastography with Heparinase or Protamine Sulfate Added In Vitro during Heparinized Cardiopulmonary Bypass

1997 ◽  
Vol 78 (02) ◽  
pp. 820-826 ◽  
Author(s):  
Bruce D Spiess ◽  
Michael H Wall ◽  
Bruce S Gillies ◽  
Jane C K Fitch ◽  
Louise O Soltow ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Postoperative Hemorrhage ◽  
Sensitive Indicator ◽  
Blood Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Heparin Neutralization

SummaryThromboelastography (TEG) has been used after cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage. Conventional TEG during CPB is not possible due to the sensitivity of the TEG to even small amounts of heparin, which produces a nondiagnostic tracing. The purpose of this study was to compare heparin neutralization using heparinase or protamine in TEG blood samples obtained during CPB. TEG testing was performed on 48 patients before, during and after CPB. Tissue plasminogen activator activity and antigen were measured on a subset of 32 patients. We found: 1) heparinase neutralized at least 10 IU/ml heparin while 1.6 ug/ml protamine neutralized up to 7 IU/ml heparin, 2) in samples with complete heparin neutralization by both methods, there was no significant difference in the R values, 3) while there was good correlation for other TEG parameters between heparinase and protamine treated samples, heparinase treatment produced shorter K values and higher angle, MA and A60, 4) while fibrinolysis was detected using both methods, heparinase treatment suppressed fibrinolysis in the TEG in both samples from patients and after in vitro addition of tissue plasminogen activator, 5) TEG was not a sensitive indicator of t-PA activity, detecting only 21% of samples with increased t-PA activity during bypass, and 5) heparinase was at least 100 times more expensive than protamine. We conclude that while both heparinase and protamine can be used to neutralize heparin in TEG samples obtained during CPB, protamine neutralization is more sensitive to fibrinolysis and less expensive, but the protamine dose must be carefully selected to match the heparin level used at individual institutions.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

Tissue plasminogen activator in chronic subdural hematomas as a predictor of recurrence

2006 ◽  
Vol 104 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Hiroyuki Katano ◽  
Ken Kamiya ◽  
Mitsuhito Mase ◽  
Motoki Tanikawa ◽  
Kazuo Yamada
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Venous Blood ◽  
Angiogenic Factors ◽  
Subdural Hematomas ◽  
Initial Surgery ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Endothelial Growth Factor

Object Chronic subdural hematomas (CSDHs) recur in 7 to 18% of cases. The present study was conducted to determine whether serum or lesion concentrations of coagulofibrinolytic and angiogenic factors, which have been reported to be potential markers of CSDH development, might predict such recurrences. Methods Sixty consecutive patients (mean age 71.5 years) with CSDHs (74 affected sides) were studied. Samples of serum in preoperative peripheral venous blood and of hematomas (obtained during surgery) were collected and analyzed. The CSDH recurred in six (8.1%) of the 74 affected sides in six patients. None of the values of the coagulative factors or tests in serum showed significant variation between cases with and those without recurrence. Among coagulofibrinolytic factors, tissue plasminogen activator (TPA) in hematomas demonstrated significantly greater levels in recurrent than in nonrecurrent cases; a similar tendency was noted for α2-plasmin inhibitor–plasmin complex in hematomas. Both factors were greater in the lesions than in the serum. Among the angiogenic factors, levels of hepatic growth factor (HGF) and vascular endothelial growth factor (VEGF) in hematomas were significantly greater than in serum, whereas those of basic fibroblast growth factor were rather lower. Note that comparisons between recurrent and nonrecurrent cases revealed no significant difference. Conclusions Patients harboring CSDHs with high TPA concentrations on sampling at the initial surgery have a relatively high probability of recurrence and require follow up with computerized tomography scanning. Angiogenic factors, such as HGF and VEGF, might be candidate markers of CSDH enlargement but are not useful as predictors of recurrence.

Determination of tissue plasminogen activator and its "fast" inhibitor in plasma.

1986 ◽  
Vol 32 (3) ◽  
pp. 482-485 ◽  
Author(s):  
J Chmielewska ◽  
B Wiman
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasma Samples ◽  
Inhibitor Concentration ◽  
Blood Samples ◽  
Analytical Recovery ◽  
Specific Determination ◽  
Tissue Plasminogen ◽  
Coupled Assay

Abstract We describe efficient, accurate methods for specific determination of tissue plasminogen activator (t-PA, EC 3.4.21.31) and its "fast" inhibitor in plasma. In this coupled assay, a sample containing t-PA is incubated with plasminogen, a plasmin (EC 3.4.21.7) substrate of low Km and high Kcat, and fibrin as a stimulator. The inhibitor of t-PA is determined by incubating the sample with a known amount of t-PA in excess, then determining the residual t-PA. Both t-PA and t-PA inhibitor can be determined in many samples simultaneously within a few hours. These assays are modifications of procedures described by us (Clin Chim Acta 1983;127:279-88 and Thromb Res 1983;31:427-36). Their accuracy as assessed by analytical recovery of pure t-PA added to blood samples (91 +/- 4%) or of partly purified inhibitor added to plasma samples (102 +/- 10%) is satisfactory, as is their precision. For the t-PA assay the CV was 1.6% (within run) or 4.1% (between run). The corresponding values for the inhibitor assay were 4.5% (within run) or 8.4% (between run) if the inhibitor concentration exceeded 3 arb. units/mL.

Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

Tissue Plasminogen Activator Expression in Endothelial Cells Exposed to Cyclic Strain in Vitro

1992 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Toshiaki Iba ◽  
Bauer E. Sumpio
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cyclic Strain ◽  
Neutral Position ◽  
Messenger Ribonucleic Acid ◽  
Tissue Plasminogen ◽  
Pai 1

The effects of cyclic strain on the production of tissue plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured endothelial cells (EC) were examined. Human saphenous vein EC were seeded in selective areas of culture plates with flexible membrane bottoms (corresponding to specific strain regions) and grown to confluence. Membranes were deformed by vacuum (-20 kPa) at 60 cycles/min (0.5 s strain alternating with 0.5 s relaxation in the neutral position) for 5 days. EC grown in the periphery were subjected to 7-24% strain, while cells grown in the center experienced less than 7% strain. The results show a significant increase in immunoreactive tPA production on days 1, 3 and 5 compared to day 0 in EC subjected to more than 7% cyclic strain. There was no significant elevation of tPA in the medium of EC subjected to less than 7% strain. tPA activity could only be detected in the medium of EC subjected to more than 7% cyclic strain. PAI-1 levels in the medium were not significantly different in either group. In addition, immunocytochemical detection of intracellular tPA and messenger ribonucleic acid (mRNA) expression of tPA (assessed by the reverse transcriptase polymerase chain reaction utilizing tPA specific sense and antisense primers) was significantly increased in EC subjected to more than 7% cyclic strain. We conclude that a 60 cycles/min regimen of strain that is greater than 7% can selectively stimulate tPA production by EC in vitro and may contribute to the relative nonthrombogenicity of the endothelium in vivo.

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