Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

Clot stability as a determinant of effective factor VIII replacement in hemophilia A.

BACKGROUND: Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. OBJECTIVE: To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties. PATIENTS/METHODS: In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. RESULTS: FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. CONCLUSIONS: Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

Interactions of plasminogen with polymerizing fibrin and its derivatives, monitored with a photoaffinity cross-linker and electron microscopy.

Localization of the plasminogen binding sites on fibrin has been difficult since these interactions occur on polymerizing fibrin, and studies with fragments can be misleading because of multiple carboxyl-terminal lysines that may bind to plasminogen. A hetero-functional photoaffinity cross-linker was used to study these interactions. Following attachment of the cross-linker to plasminogen in the dark, a clot was formed by addition of fibrinogen or fragment X and thrombin, and then the plasminogen was cross-linked to adjacent parts of fibrin by exposure to light. There was more Glu1-plasminogen bound to fibrin than to fibrinogen and more to fragment X polymer than to fibrin. Electron microscopy of rotary shadowed individual molecules reveals that Glu1-plasminogen appears to be more compact than Lys78-plasminogen or Glu1-plasminogen with 6-aminohexanoic acid. Cross-linked complexes from the dissolved clot observed by electron microscopy reveal plasminogen bound to the end of fibrin or bridging the ends of two fibrin molecules; larger complexes were also observed. Analysis of changes in the appearance of negatively contrasted fibers with plasminogen bound also indicates the probable locations of binding sites, yielding results consistent with the cross-linking studies. The photoaffinity probe was also used to study interactions between plasminogen and fibrin or its derivatives in the course of tissue plasminogen activator-mediated fibrinolysis. Samples cross-linked at various times indicate that complexes with fragment X are particularly dominant during the rapid phase of plasminogen activation. In conclusion, these studies indicate that plasminogen binds to the pocket at the end-to-end junction between two fibrin or fragment X molecules in the protofibril; from this position, it can reach all of the sites that are cleaved during fibrinolysis.

Influence of gamma' fibrinogen splice variant on fibrin physical properties and fibrinolysis rate.

OBJECTIVE: A splice variant of fibrinogen, gamma', has an altered C-terminal sequence in its gamma chain. This gammaA/gamma' fibrin is more resistant to lysis than gammaA/gammaA fibrin. Whether the physical properties of gamma' and gammaA fibrin may account for the difference in their fibrinolysis rate remains to be established. METHODS AND RESULTS: Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting gammaA and gamma' fibrinogens (1 mg/mL). gamma'/gamma' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than gammaA/gammaA fibrin (12+/-2 fiber/10(-3) nm3 versus 16+/-2 fiber/10(-3) nm3 and 274+/-38 nm versus 257+/-41 nm for gamma'/gamma' and gammaA/gammaA fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9+/-1.7 10(-9) cm2 versus 5.5+/-1.9 10(-9) cm2, respectively; P=NS). Unexpectedly, gamma' fibrin was found to be 3-times stiffer than gammaA fibrin (72.6+/-2.6 dyne/cm2 versus 25.1+/-2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate. CONCLUSIONS: Fibrinolysis resistance that arises from the presence of gammaA/gamma' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels.

Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

Binding of a fibrinogen mimetic stabilizes integrin alphaIIbbeta3's open conformation.

The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

A monoclonal antibody to the fibrinogen gamma-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles.

BACKGROUND: We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gamma-chain of mouse fibrinogen, inhibits factor (F)XIIIa-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction; in addition, it facilitates thrombolysis. OBJECTIVES: To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. RESULTS: By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25-fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan delta; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab')2, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. CONCLUSIONS: These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.

Simultaneous occurrence of human antibodies directed against fibrinogen, thrombin, and factor V following exposure to bovine thrombin: effects on blood coagulation, protein C activation and platelet function.

We describe a patient with severe epistaxis, prolonged coagulation tests and decreased plasma factor V following exposure to bovine topical thrombin. Patient IgG, but not normal IgG, showed binding to immobilized thrombin (bovine > human) and fibrinogen, and to factor V by Western blotting; the binding to thrombin was inhibited by hirudin fragment 54-65. Electron microscopy of rotary shadowed preparations showed complexes with IgG molecules attached near the ends of trinodular fibrinogen molecules. Patient IgG inhibited procoagulant, anticoagulant and cell-stimulating functions of thrombin demonstrated by inhibition of fibrinogen clotting, protein C activation and platelet aggregation; thrombin hydrolysis of S-2238 was not inhibited. The results suggest that the antibody is targeted against anion-binding exosite and not catalytic site of thrombin. Antifibrinogen antibodies have not been reported in patients exposed to bovine thrombin. There is a pressing need to re-evaluate the role of bovine thrombin as a therapeutic agent.

Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.

Twisting of fibrin fibers limits their radial growth.

Electron microscopy of freeze-dried, shadowed fibrin fibers has demonstrated that these structures are twisted. The pitch and radius of many fibers were measured from the micrographs. Although there is some variability, the average pitch of 1930 +/- 280 (SD) nm is independent of radius. The distribution of observed radii of fibers assembled in vitro is highly skewed, suggesting that individual fibers grow to a maximum radius of about 50 nm, except when both pH and ionic strength are high; fibers aggregate to form thicker fiber bundles under some conditions. The observed twisting may be responsible for limiting the lateral growth of individual fibers. Protofibrils near the surface of a twisted fiber are stretched relative to those near the center. Consequently, the degree to which a protofibril can be stretched limits the radius of a fiber; protofibrils can be added to a growing fiber until the energy required to stretch an added protofibril exceeds the energy of binding. These properties of assembly arise directly from the intrinsic twist of the fibrinogen molecule determined from structural evidence. Simple geometric considerations lead to conclusions regarding the locations of the binding sites for assembly of the protofibril and the flexibility of the fibrin molecule.

Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly.

Calcium ions occupy low (n congruent to 10; Kd congruent to 1 mM) and high (n = 3; Kd congruent to 1 microM) affinity sites on fibrinogen and facilitate fibrin monomer polymerization. We have previously localized two of the three high affinity Ca2+ sites to gamma 311-gamma 336. However, optimal enhancement of fibrin monomer polymerization occurs only at physiological millimolar Ca2+ concentrations which are two orders of magnitude higher than the concentration required for occupancy of the high affinity Ca2+-binding sites. In this study, we show that removal of fibrinogen sialic acid residues results in loss of low affinity Ca2+-binding sites. Clotting of asialofibrinogen appears to be Ca2+-independent and results in fiber bundles thicker in diameter than normal fibrin bundles as determined by turbidometry and scanning and transmission electron microscopy. By using a Ca2+-sensitive electrode, free sialic acid is shown to bind Ca2+ (Kd congruent to 1 mM). These observations suggest that the high affinity fibrinogen D-domain Ca2+-binding sites may play a role in the tertiary structure of the D-domain, whereas, sialic acid residues are low affinity sites whose occupancy by Ca2+ at physiological calcium concentration facilitates fibrin polymerization.

The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy.

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of SolulinTM, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human alpha-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.

Cl- regulates the structure of the fibrin clot.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl- to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl- opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F-. The effect of Cl- is pH dependent and increases at pH > 8.0, whereas fibers grown in the presence of F- remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl- effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl- in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl- but actually reduces the size of the fibers in the presence of F-. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl- is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl- binding to fibrin.

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers.

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.

Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.

Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels.

The effect of fibrin matrix micromorphology on neurite growth was investigated by measuring the length of neurites growing in three-dimensional fibrin gels with well characterized micromorphologies. Dorsal root ganglia (DRGs) from 7-day chick embryos were entrapped and cultured in gels made from varying concentrations of fibrinogen (5-15 mg/mL) or calcium (2-10 mM). The length of growing neurites was measured with light videomicroscopy, and the number and diameter of fibrin fiber bundles were measured from scanning electron micrographs. An increase in fibrinogen concentration caused a decrease in the average fiber bundle thickness, an increase in the number of fiber bundles, and a marked decrease in neurite length. Gels made with different calcium concentrations had a similar range of variation in fibrin fiber bundle number or diameter, but these variations had little effect on neurite and associated nonneuronal cell outgrowth. These results provide insights into the process of neurite advance within fibrin and may be useful in the design of fibrin-based materials used for peripheral nerve regeneration. Furthermore, this study provides the first detailed experimental data on the micromorphology of fibrin matrices made from more than 5 mg/mL of fibrinogen and indicates that existing kinetic models of fibrin polymerization do not accurately predict fibrin structure at these higher concentrations.

The ultrastructure of fibrinogen Caracas II molecules, fibers, and clots.

Fibrinogen Caracas II is an abnormal fibrinogen involving the mutation of A alpha serine 434 to N-glycosylated asparagine. Some effects of this mutation on the ultrastructure of fibrinogen Caracas II molecules, fibers, and clots were investigated by electron microscopy. Electron microscopy of rotary shadowed individual molecules indicated that most of the alphaC domains of fibrinogen Caracas II do not interact with each other or with the central domain, in contrast to control fibrinogen. Negatively contrasted Caracas II fibers were thinner and less ordered than control fibers, and many free fiber ends were observed. Scanning electron microscopy of whole clots revealed the presence of large pores bounded by local fiber networks made up of thin fibers. Permeation experiments also indicated that the average pore diameter was larger than that of control clots. The viscoelastic properties of the Caracas II clot, as measured by a torsion pendulum, were similar to those of control clots. Both the normal stiffness and increased permeability of the Caracas II clots are consistent with the observation that subjects with this dysfibrinogenemia are asymptomatic.

Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments.

After fibrin polymerizes to form a clot, the transglutaminase Factor XIIIa cross-links the gamma and alpha chains to stabilize the clot. There has been conflicting evidence on whether the gamma chain isopeptide bonds occur between molecules that are interacting in a longitudinal (end-to-end) manner or transverse (half-staggered) manner between the two strands of the protofibril. Since the topology of the cross-links has important consequences for fibrin structure, as well as for its stability and susceptibility to and pattern of fibrinolysis, cross-linked fibrin fragments were examined by electron microscopy to distinguish between these two possibilities for the arrangement of the ligated molecules. Cross-linked fibrin clots were produced by prolonged incubation of fibrinogen with thrombin and Factor XIII, and then digested with plasmin. The resulting soluble cross-linked fibrin complexes were rotary-shadowed with tungsten and examined by electron microscopy, revealing protofibril-like structures consisting of clusters of globular domains with a repeat of 22.5 nm. Longer plasmin digestion times yielded increasingly shorter structures. Rotary-shadowed cross-linked fibrin fragments, produced by dilution of the complexes into 0.125% acetic acid at pH 3.5 to dissociate all non-covalently linked fragments, showed uniformly single-stranded structures with a characteristic spacing of nodules, consistent with longitudinal cross-linking. Long, thin strands were seen at short digestion times, while shorter strands appeared with longer digestion. The smallest structures observed included two nodules together, and two such nodules with another nodule at a short distance from one or both ends, compatible with fragments DD, DY, and YY. Longer strands had the appearance of fibrin molecules that were linked end-to-end, usually with a fragment D or Y at each end. In conclusion, these results are consistent with previously proposed structures of these derivatives and clearly demonstrate that the interactions between cross-linked gamma chains are longitudinal (end-to-end) and not transverse.

The shape of high molecular weight kininogen. Organization into structural domains, changes with activation, and interactions with prekallikrein, as determined by electron microscopy.

Knowledge of the organization of the kininogen gene and protein structure and function correlations has allowed the development of a model of high molecular weight kininogen. Domains 1-3 on the heavy chain are evolutionarily related to cystatin and the latter two are inhibitors of cysteine proteases. Proteolytic cleavage in domain 4 to release bradykinin causes a conformational change, exposing a surface-binding region (domain 5) on the disulfide-linked light chain. The carboxyl-terminal domain 6 contains a zymogen binding sequence for factor XI and prekallikrein which, with domain 5, accounts for its cofactor activity. To explore further the domain structure, we have determined the shapes of high molecular weight kininogen and prekallikrein by electron microscopy of rotary shadowed preparations and computer image processing. High molecular weight kininogen appears to be a linear array of three linked globular regions about 16 nm long, with the two ends also connected by another thin strand. Both prekallikrein and kallikrein have a compact globular shape, with a subdivision that is sometimes visible. Different functional domains of high molecular weight kininogen were identified by monoclonal antibodies against these regions, as well as ligand binding of prekallikrein. These studies indicate that one end globular region is the prekallikrein-binding domain, the other comprises the cysteine protease inhibitor domains and the smaller central nodule is the surface-binding domain. Cleavage of high molecular weight kininogen with plasma kallikrein to yield two-chain high molecular weight kininogen results in a striking change in conformation: the central surface-binding domain swings out so that it is still adjacent to the prekallikrein-binding domain but no longer in the middle. These structural studies provide insight into the interactions of these proteins and aspects of the mechanisms of their actions.

The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure.

Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (k(cat)/K(m)) of thrombin was 0.5 for FXIII 34Leu and 0.2 (micromol/L)(-1) x sec(-1) for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (micromol/L)(-1) x sec(-1), respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of gamma- and alpha-chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.