Porosity, Mineralization, Tissue Type and Morphology Interactions at the Human Tibial Cortex

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

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Fibrin-specificity of a Plasminogen Activator Affects the Efficiency of Fibrinolysis and Responsiveness to Ultrasound: Comparison of Nine Plasminogen Activators In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614533 ◽

1999 ◽

Vol 81 (04) ◽

pp. 605-612 ◽

Cited By ~ 35

Author(s):

Dmitry V. Sakharov ◽

Marrie Barrett-Bergshoeff ◽

Rob T. Hekkenberg ◽

Dingeman C. Rijken

Keyword(s):

Thrombolytic Therapy ◽

Plasminogen Activator ◽

Tissue Type ◽

Bolus Administration ◽

High Frequency Ultrasound ◽

Single Chain ◽

Response Curves ◽

Maximal Speed ◽

Frequency Ultrasound

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

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Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614532 ◽

1999 ◽

Vol 81 (04) ◽

pp. 601-604 ◽

Cited By ~ 55

Author(s):

Hiroyuki Matsuno ◽

Osamu Kozawa ◽

Masayuki Niwa ◽

Shigeru Ueshima ◽

Osamu Matsuo ◽

...

Keyword(s):

Plasminogen Activator ◽

Thrombus Formation ◽

Endothelial Injury ◽

Fibrinolytic System ◽

Tissue Type ◽

Clot Lysis ◽

Wild Type ◽

Type Plasminogen Activator ◽

Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

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Natriuretic Peptides Regulate the Expression of Tissue Factor and PAI-1 in Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614861 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1497-1503 ◽

Cited By ~ 20

Author(s):

Hajime Tsuji ◽

Hiromi Nishimura ◽

Haruchika Masuda ◽

Yasushi Kunieda ◽

Hidehiko Kawano ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Plasminogen Activator ◽

Natriuretic Peptide ◽

Culture Media ◽

Tissue Type ◽

Dependent Manner ◽

Ang Ii ◽

Plasminogen Activator Inhibitor 1 ◽

Pai 1

SummaryIn the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay.Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.

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Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

Nuklearmedizin ◽

10.1055/s-0038-1628893 ◽

1987 ◽

Vol 26 (05) ◽

pp. 224-228 ◽

Cited By ~ 2

Author(s):

Y. Isaka ◽

H. Etani ◽

K. Kimura ◽

S. Yoneda ◽

T. Kamada ◽

...

Keyword(s):

Plasminogen Activator ◽

Abdominal Aorta ◽

Half Life ◽

Balloon Catheter ◽

Biochemical Properties ◽

Tissue Type ◽

Fibrin Deposition ◽

Tissue Type Plasminogen Activator ◽

High Affinity ◽

Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

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A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642777 ◽

1988 ◽

Vol 59 (02) ◽

pp. 310-315 ◽

Cited By ~ 64

Author(s):

P W Koppert ◽

E Hoegee-de Nobel ◽

W Nieuwenhuizen

Keyword(s):

Enzyme Immunoassay ◽

Degradation Products ◽

Blood Clot ◽

Tissue Type ◽

Fibrin Degradation Products ◽

Type Plasminogen Activator ◽

Strong Positive Reaction ◽

Sandwich Type ◽

Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

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Deep Vein Thrombosis and Fibrinolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646432 ◽

1991 ◽

Vol 66 (04) ◽

pp. 426-429 ◽

Cited By ~ 16

Author(s):

Marcel Levi ◽

Anthonie W A Lensing ◽

Harry R Büller ◽

Paolo Prandoni ◽

Gerard Dooijewaard ◽

...

Keyword(s):

Deep Vein Thrombosis ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Controlled Study ◽

First Episode ◽

Control Group ◽

Vein Thrombosis ◽

Type Plasminogen Activator ◽

Deep Vein

SummaryIn the present study 57 consecutive patients with a first episode of venographically proven deep vein thrombosis were investigated to evaluate the release of tissue-type plasminogen activator (t-PA) and of urokinase-type plasminogen activator (u-PA) in response to DDAVP stimulation as well as the resting plasminogen activator inhibitor (PAI) concentration, comparing this to the results obtained in 66 similar patients with a clinical suspicion of thrombosis but with a normal venogram. All assays were performed without knowledge of the patient's status.Four patients in the deep vein thrombosis-group (7%) had an absent u-PA antigen response upon DDAVP infusion, while a normal response was observed in all control subjects. Patients and controls showed similar increases in t-PA antigen level upon DDAVP. High resting PAI antigen levels were encountered in 5 patients in the deep vein thrombosis-group (9%) and in 6 subjects in the control group (9%).The results from this controlled study indicate that a defective release of u-PA may occur in patients with deep vein thrombosis and may have pathogenetic significance. Furthermore it is concluded that elevation of PAI levels cannot be considered as a specific risk factor for venous thrombosis.

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Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647055 ◽

1988 ◽

Vol 60 (02) ◽

pp. 328-333 ◽

Cited By ~ 44

Author(s):

N J de Fouw ◽

Y F de Jong ◽

F Haverkate ◽

R M Bertina

Keyword(s):

Plasminogen Activator ◽

Protein C ◽

Blood Platelets ◽

Plasminogen Activator Inhibitor ◽

Protein S ◽

Tissue Type ◽

Clot Lysis ◽

Activator Inhibitor ◽

Pai 1

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

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The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647041 ◽

1988 ◽

Vol 60 (02) ◽

pp. 255-261 ◽

Cited By ~ 81

Author(s):

A Hotchkiss ◽

C J Refino ◽

C K Leonard ◽

J V O'Connor ◽

C Crowley ◽

...

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Sodium Periodate ◽

Glycosylation Site ◽

Tissue Type ◽

Carbohydrate Structure ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

High Mannose ◽

Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

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