Altered properties of the fibrin gel structure in patients with IDDM

Summary High plasma fibrinogen levels are associated with vascular complications in the general population. Fibrin, the structural element in a clot, is derived from fibrinogen by activation of thrombin. An abnormal fibrin gel structure has been demonstrated in patients with myocardial infarction and in diabetic patients during poor metabolic control. In the present study the properties of fibrin gel structure were investigated in 20 patients with insulin-dependent diabetes mellitus (IDDM): 10 patients without (age: 30 ± 8; diabetes duration: 7 ± 6 years), and 10 patients (age: 44 ± 7; diabetes duration: 27 ± 9 years) with microangiopathy. Fifteen healthy subjects served as controls (age: 40 ± 8 years). The glycosylated haemoglobin level (HbA_1c) was elevated (p < 0.001) in the patients: 6.5 ± 1.5 % in diabetic patients without, and 7.1 ± 1.0 % in diabetic patients with microangiopathy. C-reactive protein and plasma fibrinogen were similar as compared to healthy control subjects. The properties of the fibrin gel structure; i. e. the permeability coefficient (Ks) and the fibre mass length ratio (μ) formed in recalcified plasma on addition of thrombin were investigated. Ks was decreased in the diabetic patients, with (6.5 ± 2.0 cm²; p < 0.01) and without microangiopathy (6.5 ± 2.7 cm²; p < 0.05), as compared to healthy subjects (10.0 ± 3.4 cm²), while μ was not significantly (p = 0.14) altered. The results indicate a lower fibrin gel porosity in patients with IDDM, despite normal plasma fibrinogen and irrespective of microangiopathy. The abnormal fibrin gel structure may be due to an increased glycosylation of the fibrin (-ogen) molecule caused by long-term hyperglycaemia and may be of importance for the development of angiopathy in diabetic patients. [Diabetologia (1996) 39: 1519–1523] Received: 7 May 1996 and in revised form: 9 September 1996     

N-tosyl-L-phenylalanine chloromethyl ketone inhibits LHRH-degrading activity and increases in vitro LHRH release from the immature rat median eminence

Median eminence (ME) luteinizing-hormone-releasing hormone (LHRH)-degrading activity (LHRH-DA) may play a role in regulating the availability of releasable LHRH. Incubation of LHRH with ME tissue supernatant yields LHRH(1-5) and LHRH(6-10) degradation fragments, as detected by high-performance liquid chromatography (HPLC) analysis, suggesting a 5-6 cleavage of the decapeptide. Since these fragments are also present after incubation of LHRH with alpha-chymotrypsin (alpha-CH), we examined the possibility that the irreversible inhibitor of alpha-CH, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), might inhibit LHRH-DA and affect LHRH release. Irreversible inhibitors of trypsin-like proteases [N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and phenylmethylsulfonylfluoride (PMSF)] were used as controls. LHRH-DA was determined by HPLC estimation of the loss of synthetic LHRH incurred when the peptide was incubated with aliquots of ME supernatant in the presence or absence of the inhibitors. LHRH release from ME fragments was assessed by radioimmunoassay after incubating the tissue with the inhibitors in Krebs-Ringer bicarbonate buffer. The LHRH-DA in both the incubation medium and the ME tissue was determined at the end of the incubation. TPCK (0.5-100 microM) added to ME tissue supernatant inhibited LHRH-DA in a dose-dependent manner. In contrast, when TPCK was added to medium in which intact ME were being incubated to assess LHRH release, the LHRH-DA of these ME was inhibited only at the 25-, 50- and 100-microM doses of TPCK, suggesting a relative inability of the inhibitor to reach endopeptidase pools in intact tissue. These same doses of TPCK increased LHRH release from the incubated ME.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antipain inhibits N-methyl-N''-nitro-N-nitrosoguanidine-induced transformation and increases chromosomal aberrations.

The morphologic transformation induced in Syrian hamster embryo cells by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.25 microgram/ml of medium) is inhibited by posttreatment with antipain (6-600 microgram/ml), a protease inhibitor, but is unaffected by pretreatment. DNA replication relative to untreated controls is not affected by MNNG, antipain, or the combination of the two; no synergistic lethality of antipain and MNNG occurred as reflected in the cloning efficiency. Antipain was ineffective in influencing MNNG-induced sister chromatid exchanges, but it increased frequencies of chromosomal aberration (per metaphase) at 10, 26, and 40 hr when cells were treated with MNNG at 0.25 microgram/ml of medium followed by antipain 10 min later, the procedure used in the transformation studies. Antipain also increased the average number of aberrations at the second mitosis (34 hr) when the MNNG concentration was doubled. Chromatid exchanges increased 26 hr posttreatment with the combination of MNNG and antipain used for transformation. No difference in MNNG-induced aberrations was observed when antipain preceded MNNG by 24 hr. Although the mode of actin of antipain is unknown, antipain does not inhibit transformation by suppressing chromosomal rearrangements that could convert recessive mutations to the homozygous state.     

Hypoxia inhibits renal thromboxane and not prostacyclin release

The effect of hypoxia on renal prostanoid synthesis and renal function was studied. The kidneys of male Sprague-Dawley rats were cannulated and perfused in vitro with Krebs-Henseleit buffer with a pO2 of 460 (control) or 60 mm torr (hypoxia). The rate of perfusion was adjusted to maintain perfusion pressure at 100 mm Hg. The renal venous effluent was collected at 15, 30, 60, 120, 150, and 180 min and assayed for basal release of 6-keto-PGF1 alpha, PGE2, and thromboxane B2 by radioimmunoassay and inulin and sodium clearance. Prostanoid release was high at 15 min, decreased to a lower level up to 120 min, and then progressively increased after 150 min of perfusion. Hypoxia significantly decreased TxB2 release at 30, 60, 150, and at 180 min but not PGE2 or 6-keto-PGF1 alpha release. Hypoxia proportionally decreased both sodium and inulin clearances suggesting that the decrease in renal function was from decreased renal flow. The kidney responded to the altered renal function by increasing the ratio of vasodilator to vasoconstrictor prostanoids by inhibition of TxB2 release.     

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Thrombosis is complicated by life-threatening embolic events, caused by parts of an intravascular blood clot breaking off and traveling downstream to block other blood vessels supplying critical organs. Thromboembolism occurs in both the venous and arterial circulation and is associated with life-threatening pulmonary embolism (PE) (1) and ischemic stroke (2). PE occurs when thrombi in the deep veins of the limb embolise and passage with the flowing blood through the inferior vena cava, the right atrium, and ventricle of the heart to the lungs (3), causing pulmonary hypertension and respiratory failure (4). Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and PE, which globally affects over 1 million people each year (1), results in substantial healthcare costs (5) and is a major cause of death worldwide (1, 6). Thromboembolism is clinically challenging to treat. Anticoagulation with vitamin K antagonists or direct oral anticoagulants are used to treat VTE and prophylactically to prevent VTE recurrence (7). In PE, localized thrombolysis with plasminogen activators is challenging and often only used as a last resort to help remove emboli resistant to anticoagulation (8). Improvements in treatment and prevention of thromboembolic disorders are therefore urgently needed.Recent studies indicate that structural and functional properties of the clot could be critical in thromboembolism, and these parameters may offer novel areas for therapeutic intervention. Hypofibrinolysis was reported to increase the risk of a first DVT (9), while changes in clot properties (increased clot formation rate and fiber density, reduced fibrinolysis) promoted the recurrence of DVT (10). Abnormalities in the establishment of clot viscoelastic properties have been shown to increase risk of PE (11), and reduced clot elastic modulus has been associated with VTE recurrence (12). However, the exact mechanisms linking altered clot properties to increased thromboembolic risk are unclear, and therefore, treatment options remain limited and rely on dissolution of fibrin networks and prevention of future clot formation, which carry significant risk of bleeding events.A key regulator of clot mechanical properties is coagulation Factor XIII (FXIII), a protransglutaminase that is converted into the active transglutaminase (FXIIIa) by thrombin (13). FXIIIa catalyzes the formation of γ-glutamyl-ε-lysine isopeptide bonds between adjacent molecules within the fibrin fibers to substantially increase elastic moduli and reduce storage moduli of both individual fibrin fibers and fibrin networks (14–17), therefore making the clot more elastic and less viscous. FXIIIa cross-links fibrin γ-chain residues Q398 and Q399 with K406 (18, 19), and α-chain residues Q221, Q237, Q328, and Q366 with numerous lysine residues (20–22). We previously demonstrated a critical role for γ-chain cross-linking by FXIIIa in generating clot viscoelastic properties, in particular by increasing the elastic or Young’s modulus, using a human recombinant fibrinogen γ-3X (γ-Q398N/Q399N/K406R) mutant of the essential γ-chain cross-linking sites (23–25).The fibrin γ-chain cross-linking sites for FXIIIa are highly conserved, and based on our previous in vitro data (23–25), we have now generated a genetically modified mouse in which the fibrin γ-chain cross-linking sites are mutated (FGG3X) to understand the role of fibrin fiber cross-linking in predisposition to embolic disease. We confirm the importance of γ-chain cross-linking in enhancing clot mechanical properties in vivo. Furthermore, using two protocols to study VTE, we demonstrate that lack of γ-chain cross-linking by FXIIIa increases thromboembolism using advanced whole-body and whole-organ imaging. We further show that fibrin fibers lacking γ-chain cross-linking are more prone to rupture at lower stress. These data indicate that fibrin γ-chain cross-linking enhances the resistance of fibrin fibers to rupture, consequently reducing clot fragmentation and thromboembolism.     

Fibrinopeptides A and B release in the process of surface fibrin formation

Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: "side-on" at low concentrations and "end-on" at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen. The release of fibrinopeptides from surface-adsorbed fibrinogen and from surface-bound fibrinogen-fibrin complexes differed significantly compared with that from fibrinogen in solution. The release of FpB occurred without the delay (lag phase) characteristic of its release from fibrinogen in solution. The amount of FpB released from end-on adsorbed fibrinogen and from adsorbed fibrinogen-fibrin complexes was much higher than that of FpA. FpB is known as a potent chemoattractant, so its preferential release suggests a physiological purpose in the attraction of cells to the site of injury. The N-terminal portions of fibrin β chains including residues Bβ15-42, which are exposed after cleavage of FpB, have been implicated in many processes, including angiogenesis and inflammation.     

Potassium softens vascular endothelium and increases nitric oxide release

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.     

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69-196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.     

Dynorphin A inhibits and naloxone increases the electrically stimulated release of oxytocin but not vasopressin from the terminals of the neural lobe

Oxytocin release from the rat neurohypophysis is under endogenous opioid inhibition. It has recently been established that dynorphin precursor-derived peptides are colocalized with vasopressin (VP) in the secretory granules in nerve terminals of the neural lobe, and that the opiate receptors in the neural lobe are restricted to the kappa-subtype. Therefore, we hypothesized that dynorphin, which is copackaged and thus coreleased with VP, is the endogenous opioid that inhibits release from neighboring oxytocin (OT) terminals. To test this hypothesis we examined the effects of dynorphin-(1-8), dynorphin-(1-17), and naloxone on the electrically stimulated release of OT and VP from isolated rat neurointermediate lobes throughout a range of stimulus frequencies. Both dynorphin-(1-8) and -(1-17) (2 microM) produced a substantial reduction in OT release during a 4-Hz stimulus, and this effect was abolished by naloxone (10 microM). Neither form of dynorphin, however, affected OT secretion at a stimulus frequency of 12 or 30 Hz at concentrations up to 10 microM. Naloxone (10 microM) by itself did not affect OT release during the 4-Hz stimulus, but it produced a substantial increase in OT release at a stimulus frequency of 12 Hz. In contrast, neither form of dynorphin produced inhibition, nor did naloxone augment VP secretion at any frequency tested. Frequency-dependent secretion curves (4, 8, 12, 20, and 30 Hz) for OT and VP in the presence and absence of naloxone indicated that the degree of naloxone augmentation of OT release at a given stimulus frequency was positively correlated with the amount of VP release at that frequency. These data support the hypothesis that dynorphin released in parallel with VP during in vitro stimulations of the rat neurohypophysis simultaneously inhibits stimulated OT release.     

Atrial natriuretic factor inhibits dehydration and hemorrhage-induced vasopressin release

Peptides of cardiac origin, termed atrial natriuretic factors, possess both natriuretic and diuretic properties, actions which physiologically contradict those of the antidiuretic peptide, arginine vasopressin (AVP). In addition to their opposing actions in the kidney, the present results indicate that one of these factors, Atriopeptin III, can inhibit dehydration and hemorrhage-induced AVP release in the rat. 3 days of water deprivation resulted in elevated plasma AVP levels (36.1 +/- 4.7 pg AVP/ml) which were significantly reduced following intravenous infusion of 0.02 (21.4 +/- 3.6), 0.2 (15.6 +/- 1.6), and 2.0 (13.9 +/- 3.8) nmol Atriopeptin III. Furthermore, 2.0 nmol Atriopeptin III significantly reduced post-hemorrhage levels (54.8 +/- 13.7) of AVP to values that approximated resting levels (10.2 +/- 3.7). The results suggest a role for cardiac peptides in the control of AVP release as well as the existence of a counterregulatory system, peptidergic in nature, for the maintenance of fluid and electrolyte homeostasis.     

Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin

Coagulation factor XIIIa is a transglutaminase that catalyzes covalent cross-link formation in fibrin clots. In this report, we demonstrate that factor XIIIa also mediates adhesion of endothelial cells and inhibits capillary tube formation in fibrin. The adhesive activity of factor XIIIa was not dependent on the transglutaminase activity, and did not involve the factor XIIIb-subunits. The adhesion was inhibited by 99% using a combination of monoclonal antibodies directed against integrin alpha(v)beta(3) and beta(1)-containing integrins, and was dependent on Mg(2+) or Mn(2+). Soluble factor XIIIa also bound to endothelial cells in solution, as detected by flow cytometry. In addition, factor XIIIa inhibited endothelial cell capillary tube formation in fibrin in a dose-dependent manner. Furthermore, the extent of inhibition differed in 2 types of fibrin. The addition of 10 to 100 microg/mL factor XIIIa produced a dose-dependent reduction in capillary tube formation of 60% to 100% in gammaA/gammaA fibrin, but only a 10% to 37% decrease in gammaA/gamma' fibrin. These results show that factor XIIIa supports endothelial cell adhesion in an integrin-dependent manner and inhibits capillary tube formation. (Blood. 2000;95:2586-2592)     

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in all cells. AAA+ ATPases in these proteases bind protein substrates and power their mechanical denaturation and subsequent translocation into a secluded degradation chamber where polypeptide cleavage occurs. Here, we show that model unfolded substrates are engaged rapidly by ClpXP and are then spooled into the degradation chamber at a rate proportional to their length. Degradation and competition studies indicate that ClpXP initially binds native and unfolded substrates similarly. However, stable native substrates then partition between frequent release and infrequent denaturation, with only the latter step resulting in committed degradation. During degradation of a fusion protein with three tandem native domains, partially degraded species with one and two intact domains accumulated. These processed proteins were not bound to the enzyme, showing that release can occur even after translocation and degradation of a substrate have commenced. The release of stable substrates and committed engagement of denatured or unstable native molecules ensures that ClpXP degrades less stable substrates in a population preferentially. This mechanism prevents trapping of the enzyme in futile degradation attempts and ensures that the energy of ATP hydrolysis is used efficiently for protein degradation.     

Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells

The stimulation of polyphosphoinositide (PPI) turnover is associated with cellular activation and hormone secretion in numerous systems. GTP-binding proteins appear to couple receptors to phospholipase-C-mediated PPI breakdown. We assessed the effects of fluoride, an activator of GTP-binding proteins, on inositol phosphate accumulation, intracellular free Ca2+ [(Ca2+)i], cAMP content, and PTH release in dispersed bovine parathyroid cells. Sodium fluoride (5-30 mM) produced marked dose-dependent increases in inositol phosphates. With anion exchange HPLC, we confirmed that 30 mM fluoride stimulated a rapid increase in 1,4,5-inositol trisphosphate, a potent Ca2+-mobilizing compound. Using the Ca2+-sensitive probe fura-2, we determined that 30 mM fluoride increased [Ca2+]i from 339 +/- 9 to 650 +/- 39 nM (n = 8) within 30-60 sec at 1 mM extracellular Ca2+. After the depletion of extracellular Ca2+ by the addition of 1 mM EGTA, 30 mM fluoride increased [Ca2+]i 45 +/- 9% (n = 4), indicating that fluoride can mobilize intracellular Ca2+ stores. Fluoride (1-30 mM) also inhibited PTH release in dose-dependent fashion. Fluoride (30 mM) produced 72.8 +/- 4.2% suppression of maximal low Ca2+-stimulated PTH release comparable to the 83.7 +/- 3.7% inhibition by 2.0 mM extracellular Ca2+. Since changes in both [Ca2+]i and cAMP regulate PTH release, we measured the effect of fluoride on intracellular cAMP. Fluoride did not detectably change basal cAMP content, but it reduced forskolin-stimulated increases in cAMP. We conclude that fluoride may activate at least two GTP-dependent processes in parathyroid cells, resulting in PPI breakdown and cAMP accumulation. While both may contribute to the fluoride-induced suppression of PTH release, our findings suggest that the stimulation of PPI turnover leads to inhibition of PTH secretion.     

Disaggregation of in vitro preformed platelet-rich clots by abciximab increases fibrin exposure and promotes fibrinolysis

The glycoprotein IIb/IIIa receptor inhibitor abciximab has been shown to facilitate the rate and the extent of pharmacological thrombolysis with recombinant tissue plasminogen activator (rtPA) in patients with acute myocardial infarction. However, the underlying mechanisms remain not fully determined. We sought to demonstrate that this facilitating effect of abciximab could be related to its potential to modify the clot architecture and the clot physical properties. Compared with fibrin-rich clots, platelets dramatically modified the in vitro properties of the fibrin network, leading to a significant increase of the permeability (K(s)) and the viscoelasticity (G') indexes but also leading to the appearance of platelet aggregates (surface area [S.ag]). These modifications resulted in a 2.6-fold decrease of the fibrinolysis rate when rtPA (1 nmol/L) was added before the initiation of clotting. Adding aspirin (100 microgram/mL) or abciximab (0.068 micromol/L) before the clotting of platelet-rich clots (PRCs) lowered K(s) by 50% and 70%, respectively (P<0.01), G' by 41% and 66%, respectively (P<0.01), and S.ag by 32% and 61%, respectively (P<0.01). As a consequence, the lysis speed was increased by 21% with aspirin (P<0.01) and 45% with abciximab (P<0.01). However, unlike aspirin, permeation of preformed PRCs with abciximab (0.068 micromol/L) decreased G' (37%, P<0.01), K(s) (35%, P<0.001) and S.ag (25%, P=NS) and resulted in a 27% (P<0.01) increase of the lysis speed when abciximab and rtPA (0.2 micromol/L) were simultaneously permeated. This effect was found to be time dependent and was observed only with early permeation, starting within the first 10 minutes of clotting. These changes in the physical properties of the PRC architecture suggest that fibrin is removed from the platelet-fibrin aggregates and reexposed into the surrounding fibrin network, increasing rtPA access to fibrin and therefore the fibrinolysis rate. The superiority of abciximab over aspirin in accelerating fibrinolysis of forming and preformed PRCs is related to its ability to modulate the interactions of fibrinogen and fibrin with platelets. These findings provide new mechanistic information on reperfusion therapy.     

Anomalous mechanics of Zn2+-modified fibrin networks

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn²⁺; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn²⁺ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn²⁺, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn²⁺ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

Fibrin is the major component of blood clots, which stops bleeding from wound sites of blood vessels (1, 2). Upon injury, blood clots form when fibrinogen is converted to fibrin monomers, which polymerize into a fibrous gel that can withstand the pressure from the flowing blood and can therefore stop further blood loss (3–6). The mechanical properties of fibrin gels determine the performance of blood clots during hemostasis (7, 8): They must be mechanically strong enough to withstand the pressure of arterial blood; otherwise, the clots will not stop the loss of blood (9). They must also be strong enough to withstand the viscous forces; otherwise, parts of the gel may break off and be carried in the blood, where they may lodge in a vessel in the brain or the heart, which can cause a stroke or a heart attack (10, 11). The pressure of the blood is not constant; instead, it varies over a wide range, depending on locations in the body (12). Thus, the mechanical response of fibrin gels to the extent of pressure, or stress, is also crucial in determining the success of hemostasis. Fibrin gels exhibit a stress-dependent mechanical response (13, 14), similar to the gel networks formed from many other biopolymers, including actin, vimentin, neurofilaments, and collagen (15). Under small stresses, fibrin gels exhibit linear elasticity with the applied stress linearly proportional to the strain. By contrast, under large stresses, fibrin gels exhibit stress stiffening with the applied stress increasing nonlinearly with the strain.The mechanical property of the fibrin gel depends on the concentration of fibrinogen, as well as on many other factors (16–20). For example, the stiffness increases with the concentration of divalent cations such as Ca²⁺, which effectively acts as an additional cross-linker leading to the formation of a network from the filaments (17). Intriguingly, however, another divalent cation, Zn²⁺, seems to have the opposite effect: The stiffness of clots decreases with increasing concentration of Zn²⁺ (17); furthermore, the permeability of clots increases with increasing concentration of Zn²⁺ (21). This has important consequences as Zn²⁺, the second most abundant trace metal ion in the body (22, 23), is released from activated platelets during hemostasis, which can locally change its concentration (18, 19). Furthermore, Zn²⁺ deficiency in the blood is associated with abnormal blood clotting (24, 25). Nevertheless, the origin of effects and the impact of Zn²⁺ on the structure and properties of blood clots remain unclear. The effect of the addition of Zn²⁺ is correlated with the formation of a sparser network in the fibrin gel, as observed with two-dimensional (2D) scanning electron microscopy (SEM) (21). However, the observed network morphology is likely altered by drying during sample preparation (26), and 2D images cannot provide complete information about the network morphology; thus, the effects of the addition of Zn²⁺ on the three-dimensional (3D) structure of the gel network remains unknown. To understand the origin of the unusual decrease in stiffness upon addition of Zn²⁺, the mechanics of the fibrin gel must be correlated with Zn²⁺-induced changes in its network structure and properties.In this paper, we correlate the 3D structure of fibrin networks formed in the presence of Zn²⁺ with their mechanical properties to determine the consequences of the structure on the mechanical properties of fibrin gels. We use confocal microscopy to probe the 3D structure of the gel in its hydrated state and rheological measurements to probe its mechanics. We focus on the fully gelled structure, where the network has reached its steady state; thus, we can measure the confocal microscopy and rheology on separate samples whose structure and properties will nevertheless be identical. We find that as the Zn²⁺ concentration increases, the diameter of the fibrin fibers in the gel becomes measurably thicker while the total length of the fibrin fibers in the network becomes shorter; these results are explained by an increase in the number of protofibrils that are bundled together to form each fiber in the network. Bulk rheological measurements of the small-stress, linear elastic modulus of these gels are consistent with this structural packing of the protofibrils in the fibers; moreover, these results show that the protofibrils are not strongly coupled to one another in the fibers, which explains why the network becomes softer as the concentration of Zn²⁺ increases. At intermediate applied stresses, pronounced stress-stiffening is observed. Remarkably, at large applied stresses, the data from all the networks can be scaled together, indicating that the elastic modulus of the fibrin gels results from stretching of the individual fibrin protofibril that forms the bundles that make up the network. These results show that the mechanics of a fibrin network is correlated with its microscopic structure and provide important insight into the effect of Zn²⁺ on the mechanics of blood clots.     

Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels

A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific ‘bystander cells’ in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311–322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting␣the experimental parameters of a given in vitro angiogenesis model.     

RNA干扰抑制内皮细胞韦伯潘力氏小体的释放

目的探讨利用RNA干扰方法抑制内皮细胞韦伯潘力氏小体(WPB)释放的效果和意义,为防治心血管病和开发小分子RNA药物奠定基础。方法设计腺病毒介导的针对调节WPB释放的关键蛋白N-乙基顺丁烯二酰亚胺敏感因子(NSF)N端功能区的小发卡RNA(shRNA),筛选鉴定收获病毒,使用NSF shRNA转染人主动脉内皮细胞为实验组、阴性对照病毒感染为阴性组、不加任何干扰为空白组,RT-PCR和Western blot法观察对NSFmRNA及蛋白表达的抑制作用,免疫荧光染色观察对WPB释放的影响。结果用携带NSF shRNA的腺病毒感染内皮细胞后,实验组NSF mRNA表达与空白组(P=0.02)及阴性组(P=0.035)比较,差异有统计学意义;实验组NSFmRNA表达随时间延长持续下降,24、48及72 h明显下降,差异有统计学意义(P=0.048)。实验组NSF蛋白表达,与空白组(P=0.031)及阴性组(P=0.004)比较.差异有统计学意义;而空白组与阴性组差异无统计学意义(P=0.249)。免疫荧光染色显示,NSF-shRNA腺病毒感染,明显抑制凝血酶诱导的WPB释放。结论携带NSF-shRNA的腺病毒感染人主动脉内皮细胞,能明显抑制NSF mRNA及蛋白表达,抑制凝血酶诱导的WPB释放,对未来动脉粥样硬化及急性冠状动脉综合征的防治有一定的参考价值。