Thrombin generation and fibrin clot structure

Generation of a hemostatic clot requires thrombin-mediated conversion of fibrinogen to fibrin. Previous in vitro studies have demonstrated that the thrombin concentration present at the time of gelation profoundly influences fibrin clot structure. Clots formed in the presence of low thrombin concentrations are composed of thick fibrin fibers and are highly susceptible to fibrinolysis; while, clots formed in the presence of high thrombin concentrations are composed of thin fibers and are relatively resistant to fibrinolysis. While most studies of clot formation have been performed by adding a fixed amount of purified thrombin to fibrinogen, clot formation in vivo occurs in a context of continuous, dynamic changes in thrombin concentration. These changes depend on the local concentrations of pro- and anti-coagulants and cellular activities. Recent studies suggest that patterns of abnormal thrombin generation produce clots with altered fibrin structure and that these changes are associated with an increased risk of bleeding or thrombosis. Furthermore, it is likely that clot structure also contributes to cellular events during wound healing. These findings suggest that studies explicitly evaluating fibrin formation during in situ thrombin generation are warranted to explain and fully appreciate mechanisms of normal and abnormal fibrin clot formation in vivo.     

经皮腔内激光血管成形术后家兔血管壁修复过程的动态观察

本文动态观察激光烧灼兔主动脉粥样硬化斑块后的修复过程。用带金属帽的光纤传输Nd:YAG激光至主动脉弓烧灼四个部位。术后分别在即刻1、4、7、14、21及28天处死动物,用光镜和电镜观察发现,带金属帽的光纤传输Nd:YAG激光可有效地烧灼粥样斑块,其创面在短期内即可修复,没有明显的并发症。     

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.     

Common variation in the C-terminal region of the fibrinogen beta-chain: effects on fibrin structure, fibrinolysis and clot rigidity

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.     

Ultrasound enhancement of thrombolytic therapy: observations and mechanisms

Fibrinolytic therapy is a proven approach for achieving reperfusion of occluded coronary arteries during myocardial infarction, resulting in reduced mortality and preservation of ventricular function. The amount of myocardial muscle loss is proportional to the duration of ischemia. Bleeding complications are not infrequent. Adjuvant therapy by ultrasound might enhance the rate of fibrinolysis and reduce the concentrations of lytic agents required to achieve an equivalent degree of clot lysis. ties and high frequencies, parameters that potentially could be applied and tolerated in vivo, have been proven to significantly accelerate the rate of fibrinolysis in both in vitro and in vivo models, in pure fibrin as well as whole blood clots. Such enhancement is not drug-specific. These effects were achieved by nonthermal mechanism. Ultrasound exposure did not cause mechanical fragmentation of the clot, did not alter the size of plasmatic derivates and degradation products. Ultrasound caused increased flow rate through thrombi, probably by cavitation-induced changes in fibrin ultrastructure; disaggregation of uncrosslinked fibrin fibers into smaller fibers has been shown. This resulted in increased trans-Noninvasive ultrasound at low intensi- port of the lytic agent into the clot, alteration of binding affinity and increased maximum binding. Presence of echo-contrast agent induced further acceleration of thrombolysis by ultrasound. (Int J Cardiovasc Intervent 2000; 3: 81-89)     

Polyphosphate enhances fibrin clot structure

Polyphosphate, a linear polymer of inorganic phosphate, is present in platelet dense granules and is secreted on platelet activation. We recently reported that polyphosphate is a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation. Because polyphosphate did not alter thrombin clotting times, it appeared to exert all its procoagulant actions upstream of thrombin. We now report that polyphosphate enhances fibrin clot structure in a calcium-dependent manner. Fibrin clots formed in the presence of polyphosphate had up to 3-fold higher turbidity, had higher mass-length ratios, and exhibited thicker fibers in scanning electron micrographs. The ability of polyphosphate to enhance fibrin clot turbidity was independent of factor XIIIa activity. When plasmin or a combination of plasminogen and tissue plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of polyphosphate exhibited prolonged clot lysis times. Release of polyphosphate from activated platelets or infectious microorganisms may play an important role in modulating fibrin clot structure and increasing its resistance to fibrinolysis. Polyphosphate may also be useful in enhancing the structure of surgical fibrin sealants.     

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.     

Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis.

In this study, we investigated tissue-type plasminogen activator (tPA)-induced lysis of glutamic acid (glu)-plasminogen-containing or lysine (lys)-plasminogen-containing thrombin-induced fibrin clots. We measured clot development and plasmin-mediated clot disintegration by thromboelastography, and used scanning electron microscopy (SEM) to document the structural changes taking place during clot formation and lysis. These events occurred in three overlapping stages, which were initiated by the addition of thrombin, resulting first in fibrin polymerization and clot network organization (Stage I). Autolytic plasmin cleavage of glu-plasminogen at lys-77 generates lys-plasminogen, exposing lysine binding sites in its kringle domains. The presence of lys-plasminogen within the thrombin-induced fibrin clot enhanced network reorganization to form thicker fibers as well as globular complexes containing fibrin and lys-plasminogen having a greater level of turbidity and a higher elastic modulus (G) than occurred with thrombin alone. Lys-plasminogen or glu-plasminogen that had been incorporated into the fibrin clot was activated to plasmin by tPA admixed with the thrombin, and led directly to clot disintegration (Stage II) concomitant with fibrin network reorganization. The onset of Stage III (clot dissolution) was signaled by a sustained secondary rise in turbidity that was due to the combined effects of lys-plasminogen presence or its conversion from glu-plasminogen, plus clot network reorganization. SEM images documented dynamic structural changes in the lysing fibrin network and showed that the secondary turbidity rise was due to extensive reorganization of severed fibrils and fibers to form wide, occasionally branched fibers. These degraded structures contributed little, if anything, to the structural integrity of the residual clot, and eventually collapsed completely during the course of progressive clot dissolution. These results provide new perspectives on the major structural events that occur in the fibrin clot matrix during fibrinolysis.     

The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.     

Thermal Effects In Vivo from Holmium: YAG Lasing in the Intracoronary Setting

While the thermal effect of laser energy does ablate atheromatous plaque, thermal injury to adjacent tissue produces high rates of arterial thrombosis and spasm. Holmium:YAG lasers use a pulsed laser source to maximize photoblative effects while minimizing thermal effects. These lasers have been utilized clinically to ablate thousands of complex coronary lesions with low rates of spasm and thrombosis, suggesting that little or no thermal injury occurs with these devices. However, we have been able to detect thermal injury in patients angioscopically in coronary arteries after holmium:YAG lasing. Here we report the use of directional coronary atherectomy (DCA) to òbiopsyó arteries in patients following holmium:YAG laser treatment, allowing direct histologic examination of lased tissue. Thirty such lased DCA samples were matched for patient age, gender, target vessel, and lesion characteristics with thirty control DCA samples obtained from patients undergoing DCA without prior lasing. Blinded pathologic examination correctly identified 27/30 control samples but only 18/30 lased samples. Subsequent unblinded analysis, sometimes with recutting and restaining of tissue blocks, resulted in the detection of thermal effects in 27/30 lased samples. The thermal effects seen included edge disruption, charring, coagulation necrosis, and most commonly, vacuolization. We conclude that holmium:YAG lasing does produce detectable thermal effects in tissue in most patients. These effects can be quite subtle or can be extensive, but do not predict poor patient outcome.     

Evaluation of the overall haemostatic effect of recombinant factor VIIa by measuring thrombin generation and stability of fibrin clots

Summary. It has been reported that thrombin generation test (TGT) may be a useful tool to monitor recombinant factor VIIa (rFVIIa). However, TGT does not reflect the stability of fibrin clot and its resistance to fibrinolysis which are crucial. Using whole‐blood thromboelastography (TEG) and tissue plasminogen activator (tPA), we developed an in‐vitro model to assess fibrin clot stability. Fibrin fibres were thicker in haemophiliacs compared with controls (P < 0.0001). After addition of rFVIIa 90 μg kg^?1, the diameter of fibrin fibres was dramatically decreased (P = 0.006). TEG‐tPA assay showed a dose‐dependent improvement of clot stability in the presence of rFVIIa. These data demonstrate a significant correlation between fibrin clot structure and its stability (P = 0.001). We also showed a correlation between thrombin generating capacity and clot resistance to fibrinolysis. Despite this overall correlation, a relatively large spreading around a general trend was observed, suggesting that the two assays bring complementary information on the haemostatic effect of rFVIIa.     

The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis

The functions of the alphaC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aalpha chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of alpha251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of alpha251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the alphaC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of alpha251 fibrin clots differed markedly from control clots; alpha251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the alphaC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked alpha251 and control clots showed that gamma chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of alpha251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the alphaC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.     

Fibrin-associated plasminogen activation in alpha 2-plasmin inhibitor deficiency

The clot formed from the plasma of a patient with congenital deficiency of alpha 2-plasmin inhibitor underwent a spontaneous extensive fibrinolysis. Radiolabeled fibrinogen was added to the plasma before clotting, and the whole process of the fibrinolysis was followed by measuring the release of radiolabels. Plasminogen activation was also followed by measuring the amidolytic activity that developed. There was an initial latent period, followed by an exponential increase of fibrinolytic activity. During the latent period, there was little or no release of radiolabels and no development of amidolytic activity. During the latent period, the clot was washed thoroughly to remove unbound proteins from fibrin and was incubated in buffered saline. The washed clot still underwent fibrinolysis, similar to the original plasma clot, suggesting that the plasminogen/plasminogen activators bound to fibrin during the initial latent period are responsible for fibrinolysis. The amount of plasminogen bound to fibrin during the latent period was close to the amount of plasminogen activated during the whole process of fibrinolysis. When the amount of plasminogen bound to fibrin was decreased by epsilon aminocaproic acid, the extent of fibrinolysis was decreased in parallel with the decrease of the amount of the bound plasminogen. This suggests that the amount of plasminogen bound to fibrin is one of the determinants of the rate of the fibrinolytic process.     

A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.     

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.     

Pharmacological manipulation of gastric juice: thrombelastographic assessment and implications for treatment of gastrointestinal haemorrhage.

The impairment of formation and maintenance of a formed fibrin clot contributes to the prolonged bleeding and high incidence of rebleeding in upper gastrointestinal haemorrhage. To investigate the basis for the use of drug therapy in gastric bleeding, this study used thrombelastography to determine the effects of pharmacological manipulation of gastric juice on coagulation and fibrinolysis. The thrombelastograph is a mechanical device that provides a visual assessment of all stages of coagulation and fibrinolysis. The effects of fresh and pharmacologically changed gastric juice was assessed after its addition to fresh whole blood in the thrombelastograph cuvette. Pharmacological manipulation was achieved through alkalisation or through addition of tranexamic acid, aprotinin, or sucralfate. Fresh gastric juice delayed clot formation, decreased maximum clot amplitude, and stimulated clot lysis. Alkalisation inhibited the lytic effects of fresh gastric juice and improved the induced abnormalities in coagulation. Tranexamic acid partially inhibited gastric juice induced clot lysis but did not exhibit a beneficial effect on coagulation. Sucralfate, and to a lesser extent aprotinin significantly inhibited fibrinolysis but exacerbated the detrimental effect of gastric juice on the parameters of coagulation. Alkalisation of gastric juice reduces the adverse effect on coagulation and fibrinolysis. Tranexamic acid, aprotinin, and sucralfate can all reduce or inhibit clot lysis, but the adverse effects on clot formation may outweigh any potential benefit in the treatment of gastrointestinal bleeding.     

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ(42) specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ(42)-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ(42), and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ(42). Our results indicate the existence of 2 mechanisms of Aβ(42) involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)-fibrin binding.     

Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor

The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C- dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.     

Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (< 20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.     

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.