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.     

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.     

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.     

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta- fibrin)

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.     

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.     

Impact of homocysteine-thiolactone on plasma fibrin networks

Epidemiologic studies have shown that hyperhomocysteinemia is an independent risk factor for vascular disease. Homocysteine (Hcy) circulates as different species, mostly protein bound, and approximately 1 % as its reduced form and the cyclic thioester homocysteine-thiolactone (HTL). Despite the level of plasma thiolactone being markedly low, detrimental effects are related to its high reactivity. HTL reacts with proteins by acylation of free basic amino groups; in particular, the epsilon-amino group of lysine residues forms adducts and induces structural and functional changes in plasma proteins. In order to assess the effects of HTL on plasma fibrin networks, a pool of normal plasma incubated with HTL (100, 500 and 1,000 μmol/L, respectively) was evaluated by global coagulation tests and fibrin formation kinetic assays, and the resulting fibrin was observed by scanning electron microscopy. HTL significantly prolonged global coagulation tests in a concentration-dependent manner with respect to control, and increases were up to 14.5 %. Fibrin formation kinetic parameters displayed statistically significant differences between HTL-treated plasma and control in a concentration-dependent way, showing higher lag phase and lower maximum reaction velocity and final network optical density. Electron microscopy analysis of HTL plasma networks revealed a compact architecture, with more branches and shorter fibers than control. We can conclude that HTL induced a slower coagulation process, rendering more tightly packed fibrin clots. Since these features of the networks have been related to impaired fibrinolysis, the N-homocysteinylation reactions would be involved in the prothrombotic effects associated to hyperhomocysteinemia.     

Rapid clot formation and abnormal fibrin structure in a symptomatic patient taking fenfluramine--a case report

A 35-year-old woman experienced symptomatic calf pain while taking a combination of fenfluramine and phentermine. All symptoms resolved when the medications were stopped, but pain returned when fenfluramine was restarted. Laboratory evaluation revealed mild elevations of aspartate aminotransferase and lactate dehydrogenase and a remarkably shortened prothrombin time (6.3 seconds). Additional studies revealed that the clots were composed of very thin fibrin fibers. All laboratory abnormalities, including the abnormal fibrin structure, completely resolved when fenfluramin was stopped. Direct addition of fenfluramine or phentermine to normal plasma did not alter either coagulation kinetics or fibrin structure, supporting the concept that the induced changes may have originated at the hepatic level. Clots composed of thin fibers are much more resistant to fibrinolysis, and could potentially put such patients at risk for thrombotic complications. This is the first report of clotting abnormalities associated with fenfluramine use. Subsequent to its use in this patient, fenfluramine was removed from clinical use due to reports of acquired valvular heart disease.     

The elasticity of an individual fibrin fiber in a clot

A blood clot needs to have the right degree of stiffness and plasticity to stem the flow of blood and yet be digestable by lytic enzymes so as not to form a thrombus, causing heart attacks, strokes, or pulmonary emboli, but the origin of these mechanical properties is unknown. Clots are made up of a three-dimensional network of fibrin fibers stabilized through ligation with a transglutaminase, factor XIIIa. We developed methods to measure the elastic moduli of individual fibrin fibers in fibrin clots with or without ligation, using optical tweezers for trapping beads attached to the fibers that functioned as handles to flex or stretch a fiber. Here, we report direct measurements of the microscopic mechanical properties of such a polymer. Fibers were much stiffer for stretching than for flexion, as expected from their diameter and length. Elastic moduli for individual fibers in plasma clots were 1.7 +/- 1.3 and 14.5 +/- 3.5 MPa for unligated and ligated fibers, respectively. Similar values were obtained by other independent methods, including analysis of measurements of fluctuations in bead force as a result of Brownian motion. These results provide a basis for understanding the origin of clot elasticity.     

Effects of gliclazide on fibrin network

Fibrin network structure is altered by diabetes, peripheral vascular disease, and by some drugs. The antidiabetic drug, gliclazide, increases fibrin fiber thickness but reduces whole network permeability. The networks are, however, more lysable. These effects are further examined in this study using electron microscopy. Changes were observed in protein concentrations in fibrin fibers, in fibrin fiber alignment and in fiber porosity. These results show that gliclazide modifies fibrin monomer polymerization so that the fibrin network is rendered more susceptible to fibrinolysis. This pharmacological action of gliclazide may be useful in the treatment of thromboembolism.     

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.     

Ultrathin self-assembled fibrin sheets

Fibrin polymerizes into the fibrous network that is the major structural component of blood clots and thrombi. We demonstrate that fibrin from three different species can also spontaneously polymerize into extensive, molecularly thin, 2D sheets. Sheet assembly occurs in physiologic buffers on both hydrophobic and hydrophilic surfaces, but is routinely observed only when polymerized using very low concentrations of fibrinogen and thrombin. Sheets may have been missed in previous studies because they may be very short-lived at higher concentrations of fibrinogen and thrombin, and their thinness makes them very difficult to detect. We were able to distinguish fluorescently labeled fibrin sheets by polymerizing fibrin onto micro-patterned structured surfaces that suspended polymers 10 μm above and parallel to the cover-glass surface. We used a combined fluorescence/atomic force microscope system to determine that sheets were ≈5 nm thick, flat, elastic and mechanically continuous. Video microscopy of assembling sheets showed that they could polymerize across 25-μm channels at hundreds of μm²/sec (≈10¹³ subunits/s·M), an apparent rate constant many times greater than those of other protein polymers. Structural transitions from sheets to fibers were observed by fluorescence, transmission, and scanning electron microscopy. Sheets appeared to fold and roll up into larger fibers, and also to develop oval holes to form fiber networks that were “pre-attached” to the substrate and other fibers. We propose a model of fiber formation from sheets and compare it with current models of end-wise polymerization from protofibrils. Sheets could be an unanticipated factor in clot formation and adhesion in vivo, and are a unique material in their own right.     

Influence of homocysteine on fibrin network lysis.

To elucidate some of the links between homocysteine and vascular disease, we have evaluated the effect of the amino acid on the formation (by kinetics studies), structure (by electron microscopy) and lysis of the fibrin network, using tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have studied whether homocysteine could alter the activity of the components involved in fibrinolysis (by amidolytic and thrombolytic methods). The results showed that homocysteine-associated networks were more compact and branched than controls (52 +/- 6 vs 44 +/- 5 fibers/field, P = 0.008), and were formed by shorter and thicker fibers. This clot proved to be more resistant to fibrinolysis with u-PA than control [lysis time 50%: 257 +/- 16 (homocysteine) vs 187 +/- 6 min (control); P < 0.004], but there were no differences with t-PA. Homocysteine did not affect the biological activities of plasmin, or plasminogen activation by t-PA and u-PA. Defective fibrinolysis with u-PA was therefore associated with homocysteine-fibrin structural alterations rather than the homocysteine effect on the biological activities of the fibrinolytic components evaluated. Results suggest that hyperhomocysteinemic patients could produce tight clots, were more resistant to lysis, and generated a procoagulant environment in situ. We believe that our findings may contribute to understanding the mechanisms involved in the homocysteine harmful effect.     

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.     

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.     

Structural Studies of Fibrinolysis by Electron Microscopy

Fibrin is degraded by the fibrinolytic system in which a plasminogenactivator converts plasminogen to plasmin, a serine protease thatcleaves specific bonds in fibrin leading to solubilization. Toelucidate further the biophysical processes involved in conversion ofinsoluble fibers to soluble fragments, fibrin was treated with eitherplasmin or the combination of plasminogen and plasminogen activator,and morphologic changes were observed using scanning electronmicroscopy. These changes were correlated with biochemical analysis andwith characterization of released, soluble fragments by transmissionelectron microscopy. Initial changes in the fibrin matrix includedcreation of many free fiber ends and gaps in the continuity of fibers.With more extensive digestion, free fiber segments associatedlaterally, resulting in formation of thick fiber bundles. Supernatantsof digesting clots, containing soluble derivatives, were negativelycontrasted and examined by transmission electron microscopy. Large,complex fragments containing portions of multiple fibers were observed,as were pieces of individual fibers and smaller fragments previouslyidentified. Some large fragments had sharply defined ends, indicatingthat they had been cleaved perpendicularly to the fiber direction.Other fibers showed splayed ends or a lacy meshwork of surroundingprotofibrils. Longer times generated more small fragments whosemolecular composition could be inferred from their appearance. Theseresults indicate that fibrinolytic degradation results in larger piecesthan previously identified and that plasmin digestion proceeds locallyby transverse cutting across fibers rather than by progressive cleavageuniformly around the fiber.     

Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin.

Following proteolytic conversion of fibrinogen to fibrin, clot assembly commences with formation of double-stranded fibrils that subsequently branch extensively in forming a three-dimensional network. Plasmin digests of fibrin clots that had first been covalently crosslinked by plasma transglutaminase (factor XIIIa) contained multimeric proteolytic fragments composed of crosslinked outer (D) domains of neighboring fibrin molecules. Two of these were larger than the well-known "D dimer" fragment and corresponded to D trimers and D tetramers, respectively. Whereas D dimers originate from crosslinked D domains at bimolecular junctions within two-stranded fibrils, D trimers and D tetramers evidently arise through crosslinking of contiguous D domains at trimolecular and tetramolecular junctions or at fibril branch points, respectively. Measurement of the widths of fibrils comprising trifunctional branches in thin fiber networks revealed tetramolecular branch points, which are formed by bifurcation of two double-stranded fibrils. In addition, another type of trifunctional structure, which we term the trimolecular branch point, was composed of three double-stranded fibrils. Crosslinking of D domains to form trimers may occur at this type of junction. These findings add to our understanding of the crosslinking arrangements that stabilize fibrin clot structure and the ways that fibrin molecules polymerize to form branches in the clot matrix.     

Tensile destruction test as an estimation of partial proteolysis in fibrin clots

We report a technique devised to evaluate the effects of partial proteolysis on the mechanical characteristics of acellular non-cross-linked fibrin clots. The destruction technique applies coaxial tension on mechanically preconditioned cylindrical molded clots and measures the number of mechanical failures vs the total number of samples at a given load (2, 3, and 4 grams force). We used different plasmin concentrations (0, 0.01, 0.02, 0.04, and 0.08 U/mL) in the bathing medium to cause partial proteolysis. We monitored the fibrinolysis process by measuring the amount of protein released in the bathing medium. Our results showed no difference in the creep function in all the groups studied. We compare our technique with compaction, a commonly used mechanical technique that compresses the sample by centrifugation, and found that our technique is capable of detecting minor changes of fibrinolysis (the results of the least square fit for the destruction test at 2 grams force, as a function of plasmin concentration, has a coefficient of determination of R(2) = 0.55), while compaction did not show a statistically significant difference in the same conditions, suggesting that each individual fibrin fiber bears load only under tension. Our findings suggest that when the fibers are cleaved their capacity to withstand stress is seriously challenged; thus, in principle, tensile destruction test can detect a minimal degree of proteolysis.     

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.     

Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability

Previous studies have shown that a heterozygous mutation in the fibrinogen Aalpha chain gene, which results in an Aalpha R16C substitution, causes fibrinolytic resistance in the fibrin clot. This mutation prevents thrombin cleavage of fibrinopeptide A from mutant Aalpha R16C chains, but not from wild-type Aalpha chains. However, the mechanism underlying the fibrinolytic resistance is unclear. Therefore, this study investigated the biophysical properties of the mutant fibrin that contribute to fibrinolytic resistance. Fibrin clots made from the mutant fibrinogen incorporated molecules containing fibrinopeptide A into the polymerised clot, which resulted in a 'spiky' clot ultrastructure with barbed fibrin strands. The clots were less stiff than normal fibrin and were cross-linked slower by activated FXIII, but had an increased average fiber diameter, were more dense, had smaller pores and were less permeable. Protein sequencing showed that unclottable fibrinogen remaining in the supernatant consisted entirely of homodimeric Aalpha R16C fibrinogen, whereas both cleaved wild-type alpha chains and uncleaved Aalpha R16C chains were in the fibrin clot. Therefore, fibrinolytic resistance of the mutant clots is probably a result of altered clot ultrastructure caused by the incorporation of fibrin molecules containing fibrinopeptide A, resulting in larger diameter fibers and decreased permeability to fibrinolytic enzymes.     

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.