Polyphosphate modulates blood coagulation and fibrinolysis

Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing.     

Alteration of fibrin network by activated protein C

The antithrombotic plasma enzyme, activated protein C (APC), may play a role in thrombolysis. In vitro, acceleration of clot lysis by APC depends on its ability to inhibit the activation of prothrombin. The effect of APC on the assembly and dispersion of fibrin network was studied using turbidimetry, plasmin digestion of fibrin, and electron microscopy of plasma clots. The addition of APC before clotting but not after clotting accelerated clot lysis. The rate of increase in the turbidity of clotting plasma was reduced by APC. The turbidity of plasma clots containing APC was directly related to the clot lysis time. Fibrin from plasma clots that were formed in the presence of APC yielded less fibrin degradation products than fibrin from clots without added APC. Furthermore, APC reduced the diameter and relative number of fibrin fibers in plasma clots during gel assembly. We propose that APC may enhance the efficacy of thrombolysis by reducing the relative mass of fibrin within maturing thrombi.     

Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation

Inorganic polyphosphate is widespread in biology and exhibits striking prohemostatic, prothrombotic, and proinflammatory effects in vivo. Long-chain polyphosphate (of the size present in infectious microorganisms) is a potent, natural pathophysiologic activator of the contact pathway of blood clotting. Medium-chain polyphosphate (of the size secreted from activated human platelets) accelerates factor V activation, completely abrogates the anticoagulant function of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. Polyphosphate may have utility as a hemostatic agent, whereas antagonists of polyphosphate may function as novel antithrombotic/anti-inflammatory agents. The detailed molecular mechanisms by which polyphosphate modulates blood clotting reactions remain to be elucidated.     

Homophenotypic Aalpha R16H fibrinogen (Kingsport): uniquely altered polymerization associated with slower fibrinopeptide A than fibrinopeptide B release.

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aalpha R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.     

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.     

Fluid phase coagulation events have minimal impact on plasma fibrin structure

Reports of altered fibrin structure in clots formed from factor VIII-deficient plasma have raised the possibility that plasma clots mediated by activation of the fluid phase coagulation system might differ from clots formed by the direct addition of thrombin to plasma. In this study, turbidity measurements were used to compare the assembly and structure of clots formed from platelet-poor plasma by either the addition of thrombin or the exposure of recalcified plasma to glass. When clotted by recalcification, the lag phase before initial increase in turbidity was 10 to 25 times longer than when clotted by the addition of thrombin. Decreasing the ionic strength or increasing the calcium concentration shortened the lag phase. At high calcium concentrations (greater than 25 mM) polymerization was delayed and precipitation was noted. pH had a minimal impact over the range of 7.0 to 7.4. Fibrin fiber mass-length ratios for plasma gels formed by activation of the intrinsic cascade were virtually identical to those in gels formed by the direct addition of thrombin. These studies indicate that fluid phase coagulation events before the production of thrombin have a minimal impact on plasma fibrin structure.     

Factor XI enhances fibrin generation and inhibits fibrinolysis in a coagulation model initiated by surface-coated tissue factor.

In-vitro studies have shown that thrombin-mediated factor XI activation enhances thrombin and fibrin formation, rendering the clot more thrombogenic and protecting it from lysis by activation of thrombin activatable fibrinolysis inhibitor. These effects of factor XI are only observed when coagulation is initiated by a low concentration of soluble tissue factor. At high concentrations of soluble tissue factor no effects of factor XI are seen on coagulation and fibrinolysis. In vivo, tissue factor is present in large amounts in the vascular wall. This makes it difficult to extrapolate these in-vitro findings on factor XI to the in-vivo situation. To address the question of whether factor XI could play a role in coagulation initiated on a tissue factor-containing surface we devised a static in-vitro coagulation model in which clotting is initiated in recalcified citrated plasma by tissue factor coated on the bottom of microtiter plates. The effect of factor XI was studied with an antibody that blocked the activation of factor IX by activated factor XI. The tissue factor coating strategy produced clotting times similar to those obtained with cultured tissue factor-expressing vessel wall cells (smooth muscle cells, fibroblasts and activated endothelial cells) grown to confluence in the same wells. A factor XI-dependent effect on clot formation and clot lysis was observed depending on the plasma volume used. In clots formed from small amounts of plasma (100 microl) no effect of factor XI was detected. In larger clots (200-300 microl) factor XI not only increased prothrombin activation and the fibrin formation rate but also inhibited fibrinolysis. Effects of factor XI were observed at short clotting times (3-4 min) similar to the clotting times found on cultured tissue factor-expressing vessel wall cells. This is in contrast with earlier studies using soluble tissue factor, in which effects of factor XI were only observed at much longer clotting times using low soluble tissue factor concentrations. We conclude that factor XI not only enhances coagulation initiated by surface bound tissue factor but also protects the clot against lysis once it is formed. On the basis of these results, we propose a coagulation model in which initial clot formation in the proximity of the tissue factor surface is not factor XI dependent. Clot formation becomes dependent on factor XI in the propagation phase when the clot is increasing in size. These findings support a role for factor XI in the propagation of clot growth after tissue factor-dependent initiation.     

Platelets interact with fibrin only after activation

Interactions between platelets and fibrin have been visualized by phase contrast, epifluorescence, and scanning electron microscope examination of clots formed with dansylcadaverine-labeled fibrin and gel-filtered platelets. After thrombin activation, the platelets appeared as fluorescent aggregates with bridging strands of fibrin; formaldehyde- fixed platelets were not fluorescent under the same experimental conditions. Scanning electron micrographs demonstrated that thrombin- activated cells had numerous pseudopods to which the fibrin strands adhered; fixed platelets exhibited a smooth discoid appearance and did not interact with the clot. Platelets trapped in clots formed with Batroxobin (Pentapharm) (platelets are not activated by Batroxobin as confirmed by light-scattering aggregometry measurements) remained as nonfluorescent, discoid cells, whereas platelets first activated by adenosine diphosphate formed brightly fluorescent aggregates. Light- scattering data of thrombin activation (0.2 U/mL) indicated that preincubation of platelets with 0.1 mmol/L prostaglandin E1 (PGE1) prior to addition of thrombin decreased the extent and rate of platelet shape change and resulted in 100-fold slower aggregation. Clots formed in the presence of PGE1 revealed decreased fluorescence intensity and fewer platelet-fibrin contacts. Gly-Pro-Arg-Pro, which blocks fibrinogen binding and fibrin assembly, was also effective in blocking platelet-fibrin interactions. These results indicate that platelet activation is a prerequisite for attachment of platelets to fibrin.     

Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting

The effects of varying concentrations of platelets, fibrinogen and thrombin on platelet aggregation and on fibrin clotting were investigated. The results indicated that a threshold thrombin to platelet concentration ratio may be required to cause platelet activation. Above the threshold ratio, platelets exhibited properties which enhanced thrombin action in causing aggregation and fibrin clotting. At T/P ratios below the threshold level, the presence of platelets reduced thrombin activity, in other words, platelets exerted an antithrombin action. Fibrinogen at low concentrations (0.02-1.5 mg/ml) enhanced platelet aggregation induced by thrombin; whereas, at high concentrations of fibrinogen (2.0-4.0 mg/ml), aggregation was markedly inhibited. Continuous mixing of samples of paltelets and fibrinogen at physiological concentrations with thrombin at low concentrations (less than 2.0 U/ml) resulted in platelet aggregation. On the other hand, fibrin clots formed in samples without mixing or with high thrombin concentrations (greater than or equal to 5.0 U/ml). These results suggested that the quantitative relationships between platelets, fibrinogen and thrombin, and the presence or absence of cell contact may be important factors in determining the overall hemostasis.     

High dose factor VIIa improves clot structure and stability in a model of haemophilia B

Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin-activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.     

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.     

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.     

The influence of fibrin crosslinking on the kinetics of urokinase-induced clot lysis

Summary . The effect of fibrin crosslinking on the lysis of plasma clots was investigated with plasma from a patient congenitally deficient in plasma factor XIII (fibrin stabilizing factor). The thrombin-activated plasma factor XIII was found to render clots more resistant to fibrinolysis when urokinase (UK) was used to induce plasminogen activation. Incorporation of UK in the clot by addition to plasma immediately before clotting resulted in a log-log relationship when lysis time was plotted against UK concentration, with greater differences between normal and factor-XIII deficient clots at lower UK concentrations. Addition of UK to the clot externally, after preincubation of the clot, gave linear plots on rectangular coordinates when either plasma or euglobulin fraction was used; and the difference in lysis time between factor-XIII deficient and normal clots was nearly constant over the range of UK concentrations tested. When the fluorescent amine, dansylcadaverine, was used to measure factor-XIII activity quantitatively, plasma clot lysis times were found to be directly proportional to amine-incorporating activity over a wide range of activities at low UK concentrations. The range of proportionality was reduced when UK concentration was increased. Addition of purified factor XIII to the patient's plasma restored the resistance of these clots to within the normal range.     

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.     

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.     

Functional characterization of fibrinogen Bicêtre II: a gamma 308 Asn-->Lys mutation located near the fibrin D:D interaction sites.

The effects of the gamma-308 Asn-->Lys substitution of fibrinogen Bicêtre II on clot formation, structure and properties were determined to elucidate the role of this part of the molecule in fibrin polymerization. This process was followed by measurement of turbidity, and the structure and biophysical characteristics of the clots were studied by permeation, scanning electron microscopy, and rheological techniques. Turbidity studies revealed an increased lag period and greater final turbidity for fibrin BII clots, indicating impaired oligomer formation. By permeation it was found that BII clots had greater network porosity, four times more than that of the control. The clot architecture visualized by scanning electron microscopy was similar to that of control clots with pore size and fiber diameter slightly increased. BII clots had a stiffness decreased by more than half, and an increased loss tangent, a measure of the inelastic deformation of the clot. All these results suggest a disruption of the proper alignment of fibrin monomers during oligomer formation. Consistent with these results, fibrin cross-linking by adding the physiological concentration of factor XIII to the purified protein showed that gamma and alpha chain cross-linking was impaired in BII clots. This amino acid substitution defines distinctive effects on the surface of the D:D interaction sites that are reflected in the clot structure and functional properties.     

The acceleratory effect of thrombin on fibrin clot assembly

Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme:substrate ratio, 1:400 and 1:800) prior to the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin/fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system. Lowering the ionic strength from 0.14 to 0.09 amplified these differences. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions as a cofactor in the fibrin assembly process.     

Zinc inhibits FPA release and increases fibrin turbidity

Physiologic concentrations of Zn(II) (4-40 microM) can increase the rate of thrombin-induced fibrin clot formation (decreased clotting time, CT) and increase the turbidity of the fibrin gel. Both the initial and ultimate turbidity (AbS 600 nm) of fibrin gels are increased in the presence of Zn(II). Two techniques were used to elaborate the mechanisms of Zn+2 procoagulant effect. Analytical ultracentrifugation indicates that Zn(II) does not induce the formation of fibrinogen multimers. Radioimmunoassay for FPA indicates that thrombin activation of fibrinogen is decreased by Zn(II), with 50% inhibition of FPA release observed at 35 microM Zn(II). These experiments indicate that the critical feature of Zn(II) procoagulant effect is not due to the induction of fibrinogen proteolysis by thrombin, which is actually decreased. Rather, it appears that Zn(II) accelerates the polymerization step of fibrin assembly and concomitantly modifies fibrin gel structure.     

Effect of hirudin on tissue plasminogen activator induced clot lysis

The effect of recombinant hirudin in the in vitro tPA fibrinolytic and thrombolytic activity was investigated. The activity was evaluated by following lysis of radiolabelled fibrin or plasma clot formed in the presence of tPA alone or with hirudin. The results obtained indicate that increasing concentrations of hirudin had a potentiating effect, with faster clot lysis rates and reduced time to complete lysis. However, when radiolabelled plasma or whole-blood clots were immersed in autologous plasma in the presence of tPA and hirudin, no significant difference in the lysis rates and time to complete lysis was observed. The findings suggest that hirudin or hirudin-thrombin complex interferes with the forming fibrin, thereby making clots more susceptible to lysis, while the presence of hirudin in the surrounding medium during lysis of formed clots helps to rapidly neutralize active thrombin released during clot lysis, thereby preventing further activation of coagulation. Thus, use of hirudin as an anticoagulant during thrombolytic therapy may prove to be helpful in reducing the incidence of reocclusion.