Plasminogen activator inhibitor-1 suppresses endogenous fibrinolysis in a canine model of pulmonary embolism

BACKGROUND. Plasminogen activator inhibitor-1 (PAI-1), the specific, fast-acting inhibitor of tissue-type plasminogen activator (t-PA), binds to fibrin and has been found in high concentrations within arterial thrombi. These findings suggest that the localization of PAI-1 to a thrombus protects that same thrombus from fibrinolysis. In this study, clot-bound PAI-1 was assessed for its ability to suppress clot lysis in vivo. METHODS AND RESULTS. Autologous, canine whole blood clots were formed in the presence of increasing amounts of activated PAI-1 (0-30 micrograms/ml). Approximately 6-8% of the PAI-1 bound to the clots under the experimental conditions. Control and PAI-1-enriched clots containing iodine-125-labeled fibrin (ogen) were homogenized, washed to remove nonbound elements, and delivered to the lungs of anesthetized dogs where the homogenates subsequently underwent lysis by the endogeneous fibrinolytic system. 125I-labeled fibrin degradation products appeared in the blood of control animals within 10 minutes and were maximal by 90 minutes. PAI-1 reduced fibrin degradation product release in a dose-responsive manner at all times between 30 minutes and 5 hours (greater than or equal to 76% inhibition at 30 minutes, PAI-1 greater than or equal to 6 micrograms/ml). PAI-1 also suppressed D-dimer release from clots containing small amounts of human fibrin (ogen). t-PA administration attenuated the effects of PAI-1, whereas latent PAI-1 (20 micrograms/ml) had no effect on clot lysis. Blood levels of PA and PAI activity remained unaltered during these experiments. CONCLUSIONS. The results indicate that PAI-1 markedly inhibits endogenous fibrinolysis in vivo and, moreover, suggest that the localization of PAI-1 to a forming thrombus is an important physiological mechanism for subsequent thrombus stabilization.     

Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments

Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro- UK), purified human thrombin converted pro-UK (scu-PA) to a two-chain form that had no amidolytic activity. The two chains (Mr approximately 33,000 and 22,000) were disulfide linked and resistant to subsequent activation by plasmin. By contrast, thrombin did not inactivate tissue plasminogen activator or two-chain urokinase. The enzyme from snake venom Agkistrodon contortrix, relatively specific for fibrinopeptide B, had an effect similar to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod), specific for fibrinopeptide A, did not. When pro- UK (scu-PA) was present during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA) in the clot supernatant was seen, whereas virtually full recovery (95%) of radioactivity was found. A loss of latent amidolytic activity in the clot supernatant was also found, the extent of which could be correlated with the degree of degradation of the radiolabeled probe. It was concluded that thrombin inactivation of pro-UK (scu-PA) accounts for the loss of amidolytic activity in the clot supernatant, which has been attributed to fibrin binding. Further confirmation was obtained from experiments in which ancrod was used as the clotting agent. Full recovery of both radioactivity and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was obtained under these conditions. These findings indicate that thrombin may introduce an artifact in the results of certain experiments designed to study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).     

Differential binding of plasminogen to crosslinked and noncrosslinked fibrins: its significance in hemostatic defect in factor XIII deficiency

In spontaneous fibrinolysis of an alpha 2-plasmin inhibitor-deficient plasma clot or tissue-type plasminogen activator-induced fibrinolysis in a purified system without alpha 2-plasmin inhibitor, the lysis was faster when factor XIII-mediated crosslinking of fibrin to fibrin did not occur. During the initial period, the binding of plasminogen to fibrin steadily increased with incubation time. The initial level and subsequent increase of the binding, which may be critical for the subsequent development of fibrinolysis, were more remarkable when fibrin was not crosslinked. The amount of glu- or lys-plasminogen bound to noncrosslinked fibrin was around 4 or 1.5 times larger than the amount of the respective plasminogen bound to crosslinked fibrin. Plasmin was also found to be bound to noncrosslinked fibrin twice as much as the amount bound to crosslinked fibrin. Structural changes induced by crosslinking of fibrin alpha-chain may reduce either the affinity or the number of available complementary sites to lysine binding sites of plasmin(ogen), thereby decreasing the binding of plasmin(ogen) to fibrin. These results suggest that an increased affinity of noncrosslinked fibrin for plasmin(ogen) is contributory to the accelerated fibrinolysis observed in factor XIII deficiency, in addition to an absence of crosslinking of alpha 2-plasmin inhibitor to fibrin.     

Activation of the fibrinolytic system

The fibrinolytic system is activated either directly or indirectly by proteins that convert plasminogen to plasmin in the circulation, within the interstitices, and on the surface of fibrin clots, or both. Agents that activate circulating and clot-bound plasminogen comparably induce a systemic lytic state accompanying clot lysis. Agents that activate plasminogen in the domain of fibrin preferentially exhibit clot selectivity. Fibrinolytic activators are assayed by detection of protein, generally immunologically, or of functional activity, generally by visualization of lysis of fibrin or by spectrophotometric determination of amidolytic activity.     

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.     

An Analysis of Mechanisms Underlying the Antifibrinolytic Properties of Radiographic Contrast Agents

Background: Radiographic contrast agents inhibit fibrinolysis, although by poorly defined pathways. The purpose of this study was to define specific mechanisms by which contrast agents inhibit clot lysis. Methods and Results: Diatrizoate (high osmolar ionic agent), ioxaglate (low osmolar ionic), and ioversol (nonionic) were studied in vitro. Diatrizoate inhibited clot lysis by 81.3±0.6% vs. control (p<0.001). Ioxaglate inhibited clot lysis by 41.7±11.9%, which was of borderline significance (p=0.07). Ioversol did not significantly inhibit clot lysis (14.9±11.5% decrease vs. control; p>0.3). Inhibition of fibrinolysis was not explained by the high osmolarities of contrast agents, by their iodine content, or by their effects on the amidolytic activities of t-PA, urokinase, or plasmin. However, plasminogen activation by t-PA, urokinase, or streptokinase was significantly inhibited by contrast agents. Diatrizoate, ioxaglate, and ioversol inhibited plasminogen binding to plasma clots by 51±4% (p<0.001), 30.1±4% (p<0.01), and 19.4±7% (p=0.07), respectively. Plasma clots formed in the presence of contrast agents were resistant to lysis by plasmin. Diatrizoate produced the most potent effect, inhibiting clot lysis by 40±5.7% (p<0.03). Contrast agents did not inhibit plasminogen binding to fibrin or plasmin-mediated fibrinolysis if they were added after clot formation. Contrast agents altered clot turbidity, an index of fibrin structure, if present during clot formation, but not if added to preformed clots. Contrast agents did not affect plasminogen activator inhibitor-1 or ₂-antiplasmin function. Conclusions: Contrast agents inhibit clot lysis by inhibiting plasminogen activation and by disrupting interactions of plasminogen and plasmin with fibrin by altering fibrin structure. Significant variation in antifibrinolytic properties exists between different contrast agents. Abbreviated Abstract. The purpose of this study was to define specific mechanisms by which contrast agents inhibit clot lysis. In both a purified clot lysis system and a plasma clot lysis system, diatrizoate, an ionic agent, produced the most potent inhibition of fibrinolysis. Contrast agents did not inhibit the active sites of plasminogen activators or plasmin, but did inhibit plasminogen activation. Binding of plasminogen to fibrin and lysis of fibrin by plasmin were inhibited by contrast agents if they were present during clot formation, but not if they were added after clot formation was complete. Contrast agents altered clot turbidity, an index of fibrin structure, if present during clot formation, but not if added to preformed clots. Contrast agents did not affect plasminogen activator inhibitor-1 or ₂-antiplasmin function. The effects of contrast agents on fibrinolytic parameters were not explained by their high osmolarities. These results suggest that contrast agents inhibit clot lysis by inhibiting plasminogen activation and by disrupting interactions of plasminogen and plasmin with fibrin by altering fibrin structure.     

Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin-bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu-plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.     

The fibrinogen Aalpha R16C mutation results in fibrinolytic resistance

The fibrinogen Aalpha R16C mutation is a common cause of dysfibrinogenaemia and has been previously associated with both bleeding and thrombosis. However, the mechanism underlying the thrombotic phenotype has not yet been elucidated. This report characterises the defect in fibrinolysis seen as a result of the Aalpha R16C mutation. A young patient with dysfibrinogenaemia (fibrinogen Hershey III) was found to be heterozygous for the Aalpha R16C mutation. Functional assays were performed on the purified fibrinogen to characterise clot formation and lysis with plasmin and trypsin. Consistent with previous results, clot formation was diminished. Unexpectedly, fibrinolysis was also delayed. Plasminogen activation was normal, ruling out decreased plasmin generation as the mechanism behind the fibrinolytic resistance. Western blot analysis showed no difference in the amount of bound alpha2-antiplasmin or albumin. When clot lysis was assayed with trypsin substituted for plasminogen, a significant delay was also observed, indicating that defective binding to plasminogen could not explain the fibrinolytic resistance. These results suggest that the defective fibrinolysis is due to increased proteolytic resistance, most likely reflecting changes in clot structure.     

Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis

Recently, an alternative pathway for factor XI activation has been described in which factor XI is activated by thrombin. Patients with a factor XI deficiency bleed mostly from tissues with high local fibrinolytic activity. Therefore, the role of thrombin-mediated factor XI activation in both fibrin formation and fibrinolysis was studied in a plasma system. Clotting was induced by the addition of tissue factor or thrombin to recalcified plasma in the presence or absence of tissue- type plasminogen activator, after which clot formation and lysis were measured using turbidimetry. Thrombin-mediated activation of factor XI was found to take place in plasma under physiologic conditions in the absence of a dextran sulfate-like cofactor. At high tissue factor concentrations, no effect of factor XI was seen on the rate of fibrin formation. Decreasing amounts of tissue factor resulted in a gradually increasing contribution of factor XI to the rate of fibrin formation. In addition, thrombin-mediated factor XI activation resulted in an inhibition of tissue-type plasminogen activator-induced lysis of the clot. This inhibition occurred even at tissue factor concentrations at which no effect of factor XI was observed on fibrin formation. Trace amounts of activated factor XI (1.25 pmol/L, representing 0.01% activation) were capable of completely inhibiting fibrinolysis in our system. The inhibitory effect was found to be mediated by thrombin that is additionally generated in a factor XI-dependent manner via the intrinsic pathway and is capable of protecting the clot against lysis. We also observed that formation of additional thrombin continued after the clot had been formed. We conclude that thrombin-mediated factor XI activation can take place in plasma. The presence of factor XI during coagulation results in the formation of additional thrombin within the clot capable of protecting this clot from fibrinolytic attack. The large amounts of thrombin that are formed by the intrinsic pathway via factor XI may play an important role in the procoagulant and thrombogenic state of clots and may therefore have important clinical and therapeutic implications.     

Fibrinolysis during normal human pregnancy: complex inter-relationships between plasma levels of tissue plasminogen activator and inhibitors and the euglobulin clot lysis time

Although it has been previously considered that blood fibrinolytic capacity is reduced during pregnancy, this has been disputed. Also the mechanisms underlying any change in fibrinolysis in pregnancy require clarification. We have therefore measured the plasma activity of tissue plasminogen activator (t-PA) and inhibitors (t-PAi) and the concentration of the pregnancy specific inhibitor (PA12) antigen, as well as the euglobulin clot lysis time (ECLT) during normal pregnancy. Plasma concentrations of fibrinogen, plasminogen, fibrin(ogen) degradation products (FDP) and cross-linked products (D-dimer) were also monitored. We confirm a marked reduction of the fibrinolytic activity of the plasma euglobulin fraction from the second trimester, and a parallel reduction in t-PA and increase in t-PAi activities, with rapid return to non-pregnant levels post-partum. In contrast, PAI2, whilst undetectable in non-pregnant control plasma, was already measurable in the first trimester, increased through pregnancy, and remained at a high concentration up to at least 48 h post-partum. Fibrinogen and plasminogen concentrations rose progressively through pregnancy and FDP and D-dimer were frequently detectable in late pregnancy plasma. Changes in the ECLT and plasma t-PA and t-PAi activities in pregnancy cannot therefore be directly related to the concentration of PAI2 antigen. Also, despite the apparent marked reduction in fibrinolytic capacity fibrin(ogen) breakdown products are frequently present in increased plasma concentrations in late pregnancy.     

Biochemical and physiologic principles of tissue-type plasminogen activator

After wound healing the protective fibrin clot is removed by the fibrinolytic system. In addition fibrinolysis is one of the most important counter-reactions of blood coagulation. Fibrinolysis is controlled by activation and inhibition processes. Tissue type plasminogen activator (t-PA) and Pro-urokinase (single chain urokinase; scu-PA) hold a key position in physiological plasminogen activation. Plasmin itself is a rather unspecific protease capable of degrading a great variety of proteins besides fibrin. In vivo however--except for certain pathological situations--the fibrinolytic process is restricted to its actual target the fibrin clot. This surprising situation in terms of structure function interrelation is physiologically managed by N-terminal modules in the protein structure of the essential factors providing fibrin affinity. Free plasmin will be immediately inactivated by alpha 2-antiplasmin. Therefore fibrin plays a central role as cofactor in the fibrinolytic system in determining initiation and localization of the fibrinolytic process. Because of the superior properties of t-PA and scu-PA with respect to fibrin specificity both activators must be regarded as the future thrombolytic agents for therapy.     

Effects of pro-urokinase and urokinase on the fibrinolytic system in man

The fibrinolytic and fibrinogenolytic properties of kidney cell pro-urokinase (PUK) were compared with those of natural urinary urokinase in human volunteers. Comparable degrees of fibrinolysis were obtained at a concentration of 500,000 U UK and a concentration of 500,000 IU natural urokinase. Natural urokinase showed a strong activation of the fibrinolytic system in plasma, evidenced by plasminogen activation, alpha 2-antiplasmin consumption, and the rise in fibrinogen-fibrin degradation products. In contrast to UK, there was no fall in plasminogen, no consumption of alpha 2-antiplasmin, and only a slight amount of fibrinogen-fibrin degradation products produced with PUK. These findings with PUK reveal a high affinity for fibrin and may result in better clot selectivity.     

LACK OF EVIDENCE FOR ABNORMAL FIBRINOLYSIS IN CHRONIC LOW BACK PAIN

It has been reported that plasma fibrinolytic activity is abnormalin some patients with chronic low back pain. In an attempt toconfirm this finding we studied 22 patients with chronic mechanicallow back pain and compared them with 18 healthy controls whodenied symptoms of back pain. Factors known to interfere withplasma fibrinolysis such as age, weight, seasonal and diurnalvariation, exercise, smoking and drugs were controlled as faras possible. Plasma fibrinogen was significantly higher (2.8versus 2.3 g/l, P<0.005) in patients than in controls, butthere were no significant differences in the median plasma concentrationsof euglobulin clot lysis time, fibrin plate lysis area, plasminogen,-2-antiplasmin, tissue plasminogen activator activity, and antigen,tissue plasminogen activator inhibition and plasminogen activatorinhibitor-1 antigen level. The results fail to confirm abnormalitiesof plasma fibrinolytic activity in a group of unselected casesof chronic low back pain. KEY WORDS: Impaired fibrinolysis, Backache     

Ascites fluid as a possible origin for hyperfibrinolysis in advanced liver disease

OBJECTIVES: Advanced liver disease is associated with both exaggerated fibrinolysis and with ascites. This study was undertaken to determine whether fibrinolytic activity exists in the ascites fluid of patients with liver disease and to see whether such activity is associated with evidence of plasma fibrinolysis. METHODS: Both the ascites fluid and plasma from 15 patients with cirrhotic ascites (group A) were evaluated for markers of fibrinolysis: fragment D-dimer, plasminogen, fibrinogen, and fibrin split products. In addition, the euglobulin lysis time, a test highly specific for fibrinolysis, was evaluated in the ascites fluid samples. As a control group, the plasma from 15 cirrhotic patients without ascites (group B) was evaluated for markers of fibrinolysis. RESULTS: In group A, elevated fragment D-dimer and fibrin split products were uniformly found in ascites fluid in concentrations that would be considered pathologically elevated if in plasma. Ascites fluid was also depleted, compared with plasma, of both plasminogen and fibrinogen. These results, along with the short euglobulin lysis time in 83% of the patients, suggest that increased fibrinolytic activity is present in ascites fluid. In 93% of these patients, plasma D-dimer was elevated. The mean plasma plasminogen was also low in these patients. In group B, only 33% of patients had elevated plasma D-dimer. CONCLUSIONS: Ascites fluid has fibrinolytic activity. Because ascites fluid reenters the systemic circulation via the thoracic duct, via a natural peritoneovenous shunt, ascites fluid warrants serious consideration as a pathological fluid that contributes to the systemic fibrinolytic state found in the majority of our patients with ascites.     

The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs.

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.     

Mini-plasminogen: a mechanism for leukocyte modulation of plasminogen activation by urokinase

Urokinase activation of blood fibrinolysis involves polymorphonuclear leukocytes. To determine if a leukocyte proteinase can modulate plasminogen activation, plasminogen was digested with leukocyte elastase. A major product was a small, approximately 34,000 dalton fragment (mini-plasminogen), without lysine-binding function, but with fibrin-binding activity. After urokinase activation, the resulting mini- plasmin had amidolytic activity for a tripeptide plasmin substrate and fibrinolytic activity. By 125I-fibrin assay, activities of mini-plasmin and plasmin (12 nmole/liter) were 38 and 20 ng fibrin lysed/min, respectively. Lysis times of fibrin clots containing urokinase, and mini-plasminogen or plasminogen (800 nmole/liter), were 282 and 290 sec, respectively. Mini-plasmin and plasmin were inhibited similarly by epsilon-aminocaproic acid and normal plasma, but differed in responses to gel filtration fractions of plasma containing alpha 2-antiplasmin and alpha 2-macroglobulin, the primary and secondary plasmin inhibitors. With purified inhibitors, mini-plasmin required higher concentrations of, or longer preincubation with, alpha 2-antiplasmin, and lower concentrations of, or shorter preincubation with, alpha 2- macroglobulin, to produce inhibition equivalent to that observed with plasmin. Leukocyte elastase digests plasminogen to generate a mini- plasminogen which, when activated by urokinase, has a novel pattern of response to the major plasmin inhibitors in plasma.     

Influence of cider on the fibrinolytic enzyme system

Ingestion of 750 ml cider inhibited plasma fibrinolytic activity: unfermented apple juice appeared to have some inhibitory effect, but this did not reach statistical significance. Cider inhibited urokinase-induced clot lysis in a concentration-dependent manner; the inhibitory activity was heat-stable and non-dialysable. The fibrinolytic activities of plasmin, urokinase and, more markedly, tissue activator on fibrin plates were inhibited by cider in a concentration-dependent manner. The amidolytic activity of plasmin was also inhibited in the presence of cider.     

Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.     

Hyperhomocysteinemia in patients with pulmonary embolism is associated with impaired plasma fibrinolytic capacity

Hyperhomocysteinemia (HHcy) affects haemostasis and shifts its balance in favour of thrombosis. In vitro and in vivo studies suggested that HHcy may impair fibrinolysis either by influencing the plasma levels of fibrinolytic factors or by altering the fibrinogen structure. We investigated the influence of mild HHcy levels on plasma fibrinolytic potential by using clot lysis time (CLT) and fibrin susceptibility to plasmin-induced lysis in 94 patients with previous pulmonary embolism and no pulmonary hypertension. CLT was measured as lysis time of tissue factor induced clots exposed to exogenous tissue plasminogen activator (t-PA). The rate of in vitro plasmin-mediated cleavage of fibrin β-chain was assessed over a 6-h period on fibrin clots, which were obtained by exposition to thrombin of purified fibrinogen. Homocysteine plasma levels were measured by Abbott Imx immunoassay and we considered as altered the values above 15 μmol/L according to the literature. In 68 patients homocysteine levels were below 15 μmol/L (NHcy) and in 26 they were above (HHcy). Significant differences were observed between the two groups regarding plasma fibrinolytic potential (p = 0.016), TAFIact (expressed as clot lysis ratio) (p = 0.02), t-PA (0.008) and PLG (0.037), but not for the other assessed components. The HHcy-patients had a threefold higher risk to have an impaired fibrinolysis. Instead, a multivariate logistic regression analysis adjusted for significances of univariate showed that HHcy (OR 5.2 95 % CI 1.7–15.9; p = 0.003) and BMI (OR 5.0 95 % CI 1.6–15.9; p = 0.006) resulted independently associated with impaired fibrinolytic activity. HHcy affects TAFI-mediated hypofibrinolysis but not fibrin(ogen) structure or function as documented by fibrin degradation analysis.     

A novel simultaneous clot-fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0·63 μg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1-deficient (-def) and α2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.