Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.     

Effect of endothelin-1 on some hemostatic parameters in normotensive rats

Some parameters of hemostasis and fibrinolysis were investigated in rats administered with endothelin-1 (ET-1). ET-1 (0.5, 1.0, 5.0 nmol/kg) dose-dependently shortened the bleeding time (BT). Concomitantly significant shortening of the clotting time (CT) was observed. ET-1 produced prolongation of the activated partial thromboplastin time (APTT), whereas prothrombin time (PT) remained unchanged. ET-1 did not influence in vitro platelet aggregation induced by ADP and collagen. The euglobulin clot lysis time (ECLT) was significantly shortened after ET-1 administration. Our results suggest that ET-1 modulates the process of hemostasis and fibrinolysis in the rat.     

The contribution of the three hypothesized integrin-binding sites in fibrinogen to platelet-mediated clot retraction

Fibrinogen is a plasma protein that interacts with integrin alphaIIb beta3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the gamma chain and two Arg-Gly-Asp (RGD) sequences in the Aalpha chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aalpha 95-97, a substitution of RGE for RGD at position Aalpha 572-574, and a triple substitution of RGE for RGD at both Aalpha positions and deletion of AGDV from the gamma chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aalpha D97E fibrinogen or Aalpha D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet alpha-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.     

Pro-haemorrhagic effects of calcium antagonists: a comparison of isradipine and atenolol on ex vivo platelet function in hypertensive subjects

It has been suggested that long term treatment with calcium antagonist drugs might inhibit platelet function and lead to an anti-atheromatous effect. However recent data have also suggested that such an effect might increase mortality due to an increased incidence of gastrointestinal bleeding. We identified 43 subjects from general practice with uncomplicated mild to moderate hypertension to compare the effects of the calcium antagonist isradipine with that of the beta-blocker atenolol on platelet function, plasma beta-thromboglobulin levels, fibrinolysis, and serum lipids in a randomised double-blind parallel group study. After careful evaluation to exclude concomitant aspirin use, only 24 subjects were eligible to enter the study. While isradipine and atenolol produced comparable and clinically significant falls in blood pressure (167 +/- 2/102 +/- 1 to 153 +/- 3/91 +/- 2 mm Hg, and 165 +/- 2/101 +/- 1 to 156 +/- 4/91 +/- 2 mm Hg, respectively), neither drug produced a detectable effect on ex vivo platelet aggregation, platelet retention, or thromboxane generation with adrenaline, collagen, adenosine-di-phosphate, or platelet activating factor. However a decrease in plasma beta-thromboglobulin levels was observed which reached statistical significance (P < 0.05) after 12 weeks treatment in the isradipine but not the atenolol group. A 39% reduction with isradipine compared with 34% following atenolol treatment. Euglobulin clot lysis time was not altered by either drug. Serum cholesterol concentrations were also unaltered by drug treatment. Therapeutic doses of the calcium antagonist isradipine may produce a minor indirect effect on platelet function after several weeks of treatment. However, this is of doubtful clinical importance and may simply reflect an effect of lowered blood pressure on platelet function.     

Primary fibrinolysis during supraceliac aortic clamping

PURPOSE: An increased incidence of bleeding complications has been observed after supraceliac aortic clamping (SCC). This study was performed to identify possible hemostatic abnormalities that contribute to this problem. METHODS: A prospective cohort study over a 3-month period was performed by comparing hemostatic parameters in 10 consecutive patients who required elective SCC with those of eight concurrent randomly selected control subjects who required infrarenal clamping (IRC) for abdominal aortic reconstruction. Measures of coagulation, fibrinolysis, platelet function, temperature, hemodilution, and hepatic function were performed at selected times before, during, and after operation. RESULTS: Aneurysm size, fibrinogen, D-dimers, prothrombin, partial thromboplastin time, platelet counts, bleeding times, hemodilution, and temperature were comparable in both groups. Patients in the SCC group, however, consistently developed a primary fibrinolytic state within 20 minutes after supraceliac clamping, reflected by significantly decreased euglobulin clot lysis times (ECLT; p < 0.0001), elevated tissue plasminogen activator (t-PA) levels (p < 0.0006), elevated t-PA-to-plasminogen activator inhibitor-1 ratios (p < 0.0001), and reduced alpha 2-antiplasmin levels (p < 0.002). SCC produced hepatocellular injury documented by elevations in both aspartate transaminase (p < 0.0001) and lactate dehydrogenase (p < 0.009). CONCLUSIONS: SCC rapidly induces a primary fibrinolytic state manifested by increased circulating t-PA, reduced alpha 2-antiplasmin, and increased fibrinolytic activator-to-inhibitor ratios. These effects may be a result of hepatic hypoperfusion caused by SCC leading to insufficient clearance of t-PA. Antifibrinolytic agents may be of benefit if bleeding develops after aortic procedures that require supraceliac clamping.     

Immunohistochemical quantitation for extracellular matrix proteins in rats with glomerulonephritis induced by monoclonal anti-Thy-1.1 antibody

During clot retraction, platelets interact with fibrin resulting in marked reduction of clot volume. Altered fibrin structure has been reported to affect clot retraction as measured by serum expression. This study was performed to test whether such altered retraction was the result of increased resistance to network collapse or due to decreased force development by platelets. Altered fibrin structure was documented as variation of fibre mass/length ratios (mu) and shifts in clot elastic modulus. The force developed by platelets during clotting was measured directly. Increasing the fibrinogen concentration led to thinner fibre formation (decreased mu), and a linear increase in gel elastic modulus. Over a fibrinogen concentration range of 100 to 400 mg/dl, force development was minimally affected. Force development and clot elastic modulus increased in a linear fashion with increasing platelet concentration. Increasing the calcium concentration from 5 to 20 mM caused a 160% increase in fibrin fibre size (mu), and a 52% decline in clot modulus. Force developed at 1200 s declined by 17%. At 15 mg/ml, dextran and hydroxyethyl starch (HES) also increased mu, and decreased clot modulus; however, both agents markedly reduced force development. Increasing ionic strength or the addition of IgG decreased mu and increased gel elastic modulus. Force development increased modestly with increased ionic strength, did not change with addition of IgG in saline and declined with addition of IgG in maltose. This study indicates that force development is primarily dependent on platelet function while clot modulus depends on both fibrin structure and platelet function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of heparin and alpha 1-acid glycoprotein on thrombin or Activated Thrombofax Reagent-induced platelet aggregation and clot formation

alpha 1-Acid glycoprotein (AAG) obtained from ascites and/or pleural fluids exhibited anti-heparin effects in platelet-poor plasma when evaluated by activated partial thromboplastin (Activated Thrombofax Reagent) and heparin-thrombin clotting time assays. An anti-heparin effect for AAG was also demonstrable in platelet-rich plasma (PRP) challenged with thrombin, but only over a limited range of heparin concentrations; at elevated heparin concentrations, and only in the presence of AAG, both platelet aggregation and clot formation were substantially inhibited. However, no detectable anti-heparin effect was observed following challenge of PRP with Activated Thrombofax Reagent; indeed, the anti-coagulant effect of heparin appeared synergistically amplified in these systems containing AAG, and AAG exhibited platelet pro-aggregate and pro-coagulant properties in the absence of heparin. The platelet pro-aggregating activity of AAG, though independent of heparin, appeared to require the onset of the coagulation cascade prior to the generation of thrombin; in the absence of such initiation, AAG remained a potent inhibitor of platelet activation.     

Follow-up of fibrin clot growth in blood plasma

The authors propose a device for following up the kinetics of the fibrin clot enlargement in the plasma. Coagulation is induced by contact activation with a fragment of an arterial wall put into the plasma. Besides the initial time of clot formation, the device helps assess the rate of clot growth. Two phases of contact-induced clotting may be distinguished. The first is slow activation of the clotting system and formation of minor amounts of fibrin, the other is rapid growth of the clot, with the linear increment of the clot size being constant during 15 min.     

Assessing clot lysis strategies using a simplified mathematical model

This paper attempts to describe lysis of a clot by infusion of lytic agent using a simple geometrical approach, in which the rate of clot lysis is assumed proportional to the exposed surface of the clot and the concentration of lytic agent. Six simple realizations (a)-(f) of this basic model are developed which account for the dependence of clot lysis time on five different clot geometries. In all six cases the clot is initially described as a uniform cylinder which totally occludes a vessel. In model (a) lysis proceeds as an advancing front at the proximal face of the clot. In model (b) lysis proceeds radially outwards from the central axis of the vessel while in model (c) lysis occurs radially inwards from the surface adjacent the wall, of the cylinder. In models (d) and (e) it is assumed that the clot breaks into a uniform spherical and cylindrical fragments, respectively, while model (f) uses the spherical fragment model combined with a lytic agent concentration which decreases with time. The validity of the models was assessed using previously published data from 76 patients in whom lysis time and clot size were recorded. Least squares linear regression analysis based on the six model equations yielded highly significant correlation coefficients r2 of 0.457, 0.412, 0.412, 0.495, 0.469, 0.663 for models (a)-(f), respectively. The results suggest that when a constant lytic agent concentration is assumed, no single geometry accounts for significantly more variation than any other, but that a combination of varying lytic agent concentration and clot geometry significantly influences clot lysis time and accounts for much of the observed variation.     

A bleeding model in rabbits demonstrate fresh clot selectivity for a genetically engineered variant of tissue-type plasminogen activator and for streptokinase

The way in which three thrombolytic agents, tissue-type plasminogen activator (t-PA), streptokinase (SK) and a genetically engineered variant of t-PA composed of the second kringle and the protease domain (K2P), cause the dissolution of haemostatic plugs of differing ages was investigated in a novel rabbit model. Standardized incisions were made on the rabbit ear and the wounds were left to heal for 0.5 h or 24 h, before the thrombolytic agents were infused. In the absence of heparin, t-PA showed little discrimination between clots of different ages (36% and 28% lysis of the 0.5 h and 24 h wounds, respectively). In contrast, K2P and SK showed a pronounced fresh clot selectivity since they were significantly more effective in lysing fresh clots than old ones (68% and 4% lysis for K2P and 72% and 36% for SK, respectively). In the presence of heparin the potency of t-PA on fresh clots was considerably increased whilst the effect on old clots was not affected, a fresh clot selectivity for t-PA (64% lysis of fresh clots, 24% lysis of old clots) was thus effected. Heparin did not significantly affect the fresh clot selectivity of K2P or SK, although lysis of old clots was increased (from 4% to 36%) when it was given together with K2P. Furthermore, heparin did not affect the time to onset of bleeding nor was the bleeding time prolonged by its addition. The bleeding time observed for t-PA (20-25 min) was markedly shorter than that found for K2P or SK (40-50 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraventricular and brachial artery thrombosis in nephrotic syndrome

PURPOSE: To investigate the influence of hyperthermia up to 45 degrees C on fibrinolysis with recombinant tissue-type plasminogen activator (rt-PA). METHODS: Standardized fibrin clots were incubated in a water bath for 5 hr with either rt-PA (test group) or 0.9% sodium chloride (control group) and blood plasma at temperatures of 30-45 degrees C. Concentrations of D-dimer and time to complete clot lysis were measured. RESULTS: The activity of fibrinolysis with rt-PA rose with increasing temperature: time to lysis approximately halved from 30 degrees C to 40 degrees C and the concentration of D-dimer tripled. In the control group clot size did not change. CONCLUSIONS: Activity of rt-PA-induced fibrinolysis rises distinctly with higher temperatures. Since even healthy subjects show a physiologic decline in body temperature in the extremities, in patients with occlusive arterial disease decreased activity of fibrinolysis with rt-PA can be expected. Controlled hyperthermia may improve fibrinolysis with rt-PA and should be investigated in vivo.     

A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.     

Assessment of DNAse activity by the rivanol clot method

A method for assessing DNAse activity in various biological substrata is offered. It is based on the capacity of rivanol to form a clot with DNA inversely proportionate to depolymeraization of DNAse under the effect of nucleases of different origin. The sensitivity of the method is more than 10 times higher than of viscosimetry and the alcohol clot formation test. In addition, the new method permits quantitative assessment of the clot, with the detection performed by any colorimetric or fluorimetric method. The method is adapted to measurement of the activities of commercial DNAse preparations, serum and immunoglobulin DNAse, and bacterial nuclease activities.     

Excessive fibrinolysis: the coagulopathy following Merrem''s hump-nosed viper (Hypnale hypnale) bites

In 56 patients with proven hump-nosed viper (Hypnale hypnale) bites, 12 (21.4%) developed continued oozing of blood from the site of the bite and a prolonged clotting time. Further investigations showed low fibrinogen levels and increased fibrinogen degradation products in plamsa. The bleeding time, platelet count, prothrombin time, and partial thromboplastin time with kaolin were normal. The bite of this snake can be complicated with a coagulopathy in which excessive fibrinolysis seems to be the main abnormality.     

The rectal tube: an excellent catheter for severe clot retention

PURPOSE: We describe the use of an alternative catheter for clot irrigation. MATERIALS AND METHODS: Six patients with severe clot retention that could not be treated successfully with irrigation using large bore urethral catheters were subsequently treated with a fenestrated 28 to 32F Rusch red rubber rectal catheter. RESULTS: All clots were successfully evacuated. Cystoscopic evaluation the following day in 4 patients with hematuria confirmed the absence of clots. The other 2 patients did not undergo cystoscopy but continuous bladder irrigation was completely clear for the next 24 hours and the catheters were removed without further sequelae. CONCLUSIONS: Irrigation through a rectal tube can provide immediate relief of clot retention and consequently avoid emergency endoscopic intervention in the operating room.     

Bleeding in portal hypertensive gastropathy evaluated in terms of gastric mucosal microcirculation and coagulation-fibrinolysis system

Gastric intramucosal bleeding in portal hypertensive gastropathy was investigated in terms of gastric mucosal microcirculation, coagulation-fibrinolysis factors, and local fibrinolysis in patients with liver cirrhosis. The gastric mucosa was examined by endoscopy, and the patients were classified into two groups with or without bleeding. Gastric mucosal blood flow was measured simultaneously with coagulation-fibrinolysis factors or local fibrinolysis in both groups. As gastric mucosal blood flow, the gastric mucosal blood volume (IHb) and the oxygenated hemoglobin concentration (ISO2) were determined by the organ reflection spectrum method. Coagulation-fibrinolysis factors were measured in the blood. For evaluation of local fibrinolysis, gastric biopsy specimens were placed on a standard fibrin plate, and the fibrinolysis area was measured. Compared with the non-bleeding group, the bleeding group showed increased IHb and decreased ISO2 (p < 0.05), suggesting marked congestion of blood flow. Gastric intramucosal bleeding was frequently observed in patients with marked congestion of blood flow and markedly abnormal values of coagulation-fibrinolysis factors. Gastric local fibrinolysis was also significantly enhanced in the bleeding group (p < 0.05). In addition, local fibrinolysis was correlated positively with the gastric mucosal blood volume (r = 0.68, p < 0.05) and negatively with the oxygenated hemoglobin concentration (r = -0.58, p < 0.05). These results suggest the following mechanism of gastric mucosal bleeding in liver cirrhosis and portal hypertension. Congestion of gastric mucosal blood flow is present in liver cirrhosis and portal hypertension. An increase in the microvascular pressure and hypoxia cause release of tissue plasminogen activators from gastric mucosal cells and vascular endothelial cells. As a result, gastric local fibrinolysis is enhanced, causing gastric mucosal bleeding.     

New method of studying the release of fibrinolysis activators in tissue cultures

Tissue cultivation in the presence of standardized fibrin clot containing plasminogen permitted to reveal and to study quantitatively the relase of the fibrinolysis activators into the medium (by the amount of the fibrin-fibrinogen degradation products). A possibility of in vitro study of the regulation of fibrinolysis activators release by the tissues was offered by the method described.     

Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator-induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.     

What causes prostate cancer? A brief summary of the epidemiology

Epoetin alfa is the cornerstone of anemia therapy in patients with end-stage renal disease. In addition to stimulating erythropoiesis, Epoetin alfa has been demonstrated to affect hemostasis. Such effects may be important because patients with chronic renal failure have a bleeding diathesis that is multifactorial in origin. Therefore, a computer literature search on the relationship between Epoetin alfa therapy for anemia in patients with end-stage renal disease and platelets, coagulation, coagulation inhibitors, and fibrinolysis was performed. All articles and abstracts reporting original data in the English language on Epoetin alfa and its effect on hemostasis were reviewed. The literature suggests that the effects of Epoetin alfa on the coagulation cascade are of minimal clinical importance. However, Epoetin alfa transiently increases the number of circulating platelets and improves platelet function, and these effects are associated with a return of the bleeding time towards normal.     

Subcutaneous administration of interleukin-2 in ambulatory treatment of patients with metastatic renal cancer. Three-year results of the SCAPP I program

Angina, myocardial infarction, and sudden cardiac death are associated with thrombus formation in coronary arteries. It is generally believed that these conditions benefit from long-term exercise. However, the evidence for such amelioration or prevention is inconclusive and so is the mechanism through which long-term exercise exerts its beneficial effect on various ischaemic conditions. Because of the thrombotic events, the effects of exercise on platelet reactivity, blood coagulation and fibrinolysis have been extensively studied, but the findings are not consistent (1-9). This is mainly due to methodological problems. Analysis of platelet response to a variety of agonists and the dozen of coagulation and fibrinolysis variables makes the assessment of the overall platelet function, coagulation and fibrinolysis status extremely difficult.