Different Concentrations Of Fibrinogen Research Articles

Forces of interaction of red blood cells and endothelial cells at different concentrations of fibrinogen: Measurements with laser tweezers in vitro.

Blood microrheology depends on the constituents of blood plasma, the interaction between blood cells resulting in red blood cell (RBC) and platelets aggregation, and adhesion of RBC, platelets and leukocytes to vascular endothelium. The main plasma protein molecule -actuator of RBC aggregation is fibrinogen. In this paper the effect of interaction between the endothelium and RBC at different fibrinogen concentrations on the RBC microrheological properties was investigated in vitro. Laser tweezers were used to measure the RBC-endothelium interaction forces. It was shown for the first time that the interaction forces between RBC and endothelium are comparable with the RBC aggregation forces, they increase with fibrinogen concentration and reach the saturation level of about 4 pN at the concentration of 4 mg/ml. These results are important for better understanding the mechanisms of RBC and endothelium interaction and developing the novel therapeutic protocols of the microrheology correction in different pathologies.

Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure.

Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1-3mgmL-1 ), thrombin (0.01-0.15UmL-1 ), sodium chloride (40-120mm), and calcium chloride (1-10mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first-order growth kinetic, an increase of the growth constant k (0.015-0.04min-1 ) is found to correlate to a decrease in fibril diameter (1-0.65µm) and pore diameter (11-5µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.

Main Factors Influencing the Platelet Spreading

To explore the main factors of platelet spreading and provide the foundation for related research. Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.

Biophotonic sensors with integrated Si3N4-organic hybrid (SiNOH) lasers for point-of-care diagnostics

Early and efficient disease diagnosis with low-cost point-of-care devices is gaining importance for personalized medicine and public health protection. Within this context, waveguide-(WG)-based optical biosensors on the silicon-nitride (Si3N4) platform represent a particularly promising option, offering highly sensitive detection of indicative biomarkers in multiplexed sensor arrays operated by light in the visible-wavelength range. However, while passive Si3N4-based photonic circuits lend themselves to highly scalable mass production, the integration of low-cost light sources remains a challenge. In this paper, we demonstrate optical biosensors that combine Si3N4 sensor circuits with hybrid on-chip organic lasers. These Si3N4-organic hybrid (SiNOH) lasers rely on a dye-doped cladding material that are deposited on top of a passive WG and that are optically pumped by an external light source. Fabrication of the devices is simple: The underlying Si3N4 WGs are structured in a single lithography step, and the organic gain medium is subsequently applied by dispensing, spin-coating, or ink-jet printing processes. A highly parallel read-out of the optical sensor signals is accomplished with a simple camera. In our proof-of-concept experiment, we demonstrate the viability of the approach by detecting different concentrations of fibrinogen in phosphate-buffered saline solutions with a sensor-length (L-)-related sensitivity of S/L = 0.16 rad nM−1 mm−1. To our knowledge, this is the first demonstration of an integrated optical circuit driven by a co-integrated low-cost organic light source. We expect that the versatility of the device concept, the simple operation principle, and the compatibility with cost-efficient mass production will make the concept a highly attractive option for applications in biophotonics and point-of-care diagnostics.

Influence of different concentrations of fibrinogen on the properties of a fibrin matrix for vascular tissue engineering

Aim. To evaluate the potential utility of fibrin matrices containing 10, 20, and 25 mg/ml of fibrinogen (fibrin-10, fibrin-20, and fibrin-30, respectively) in vascular tissue engineering (VTE).
 Materials and Methods. Fibrinogen was isolated using the method of ethanol cryoprecipitation and polymerized using a solution of thrombin and CaCl2. The fibrin structure was studied in a scanning electron microscope, and the physical and mechanical properties of the material were tested on a Zwick/Roell test machine. The metabolic activity of endothelial cells (EC) on the fibrin surface was evaluated by the MTT assay, and the viability of fibroblasts in the thickness of fibrin and possibility for migration by in fluorescent and light microscopy. Percent of fibrin shrinkage was determined from the difference in the sample volumes before and after removal of moisture.
 Results. The fiber diameter did not differ among all fibrin samples, but the pore diameter in fibrin-30 was smaller than those in fibrin-10 and fibrin-20. A possibility for migration of fibroblasts into the depth of the fibrin matrix and preservation of 97-100% viability of cells at a depth 5 mm was confirmed. The metabolic activity of EC on the surface of fibrin-20 and fibrin-30 exceeded that on collagen, fibronectin, and fibrin-10. All fibrin samples shrank in volume to 95.5-99.5%, and the highest shrinkage was seen in fibrin-10. The physical and mechanical properties of fibrin were inferior to those of human A. mammaria by a factor of 10.
 Conclusion. Fibrin with fibrinogen concentrations of 20 and 30 mg/ml maintains a high metabolic and proliferative activity of EC on the surface and also a high viability of fibroblasts in the matrix. Its availability, ease of preparation, and a number of other favorable properties make fibrin a promising material for VTE. However, the problem of insufficient strength requires further investigations.

Enhanced therapeutic index of liposomal doxorubicin Myocet locally delivered by fibrin gels in immunodeficient mice bearing human neuroblastoma

Caelyx and Myocet are clinically used liposomal forms of doxorubicin (Dox). To explore ways to improve their therapeutic index, we have studied their activity in vitro and in vivo when locally delivered by fibrin gels (FBGs). In vivo local toxic and anti-tumour activities of loaded FBGs were assessed in two immunodeficient mouse orthotopic human neuroblastoma (NB) models after application in the visceral space above the adrenal gland, either still tumour-bearing or after tumour removal. In parallel, in vitro assays were used to mimic the in vivo overlaying of FBGs on the tumour surface.FBGs were prepared with different concentrations of fibrinogen (FG) and clotted in the presence of Ca2+ and thrombin. The in vitro assays showed that FBGs loaded with Myocet possess a cytotoxic activity against NB cell lines generally greater than those loaded with free Dox or Caelyx.In vivo FBGs loaded with Myocet showed lower general and local toxicities as compared to gels loaded with Caelyx or free Dox, and also to free Dox administered i.v. (all treatments with Dox at 2.5 mg/Kg). The anti-tumour activity, evaluated in the two mouse orthotopic NB models of adjuvant and neo-adjuvant therapy, resulted in a better performance of FBGs loaded with Myocet compared to the other local (FBGs loaded with Caelyx or free Dox) or systemic (free Dox) treatments (administered at 2.5 and 5 mg/Kg Dox). Specifically, the application of FBGs at 40 mg/mL in the adjuvant model caused 92 % tumour volume reduction, while by the neo-adjuvant application of FBGs at 22 mg/mL a re-growing tumour volume reduction of 89 % was obtained.Taken together, our in vitro and in vivo results indicate a significantly higher activity for the FBGs loaded with Myocet. In particular, the lower toicity coupled with the higher anti-tumour activity on both the local treatment modalities strongly suggest a better therapeutic index when Myocet is administered through FBGs. Therefore, FBGs loaded with Myocet may be considered as a possible new tool for the loco-regional treatment of NB or even other tumour histotypes treatable by loco-regional chemotherapy.

Evaluation of cell viability of human dental pulp stem cells in two dimensional and three dimensional fibrin glue scaffold

Context:Regenerative endodontics uses the concept of tissue engineering to restore the diseased immature tooth, allowing for continued development of the root to a healthy state. For regenerative endodontics, the use of human dental pulp stem cells (HDPSCs) with appropriate scaffolds and growth factors is imperative.Aims:The aim of the study was to evaluate the human dental pulp cell viability in two-dimensional (2D) and 3D fibrin glue scaffold to be used in regenerative endodontics.Subjects and Methods:Regenerative potential of HDPSCs was comparatively assessed usings 2D and 3D fibrin glue scaffold. 3D scaffold was made with different concentrations of fibrinogen. Cell morphology was studied under inverted phase-contrast microscopy, and cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at various time intervals.Statistical Analysis Used:Collected data underwent a two-way ANOVA test. The P value of the study was kept 0.05 according to the sample size.Results:Study revealed a significant increase in the proliferation of HDPSCs in fibrin glue precoated wells of 2D fibrin glue compared to preseeded cells on day 1 and day 3. The concentration of fibrinogen has a major role in cell viability in 3D fibrin glue scaffold. Homing of HDPSCs in the 3D scaffold improves with time.Conclusions:This study concludes that the concentration of fibrin glue has a significant role in HDPSC Viability in 3D scaffold.

Fibrinogen and thrombin concentrations are critical for fibrin glue adherence in rat high-risk colon anastomoses

OBJECTIVE:Fibrin glues have not been consistently successful in preventing the dehiscence of high-risk colonic anastomoses. Fibrinogen and thrombin concentrations in glues determine their ability to function as sealants, healers, and/or adhesives. The objective of the current study was to compare the effects of different concentrations of fibrinogen and thrombin on bursting pressure, leaks, dehiscence, and morphology of high-risk ischemic colonic anastomoses using fibrin glue in rats.METHODS:Colonic anastomoses in adult female Sprague-Dawley rats (weight, 250-350 g) treated with fibrin glue containing different concentrations of fibrinogen and thrombin were evaluated at post-operative day 5. The interventions were low-risk (normal) or high-risk (ischemic) end-to-end colonic anastomoses using polypropylene sutures and topical application of fibrinogen at high (120 mg/mL) or low (40 mg/mL) concentrations and thrombin at high (1000 IU/mL) or low (500 IU/mL) concentrations.RESULTS:Ischemia alone, anastomosis alone, or both together reduced the bursting pressure. Glues containing a low fibrinogen concentration improved this parameter in all cases. High thrombin in combination with low fibrinogen also improved adherence exclusively in low-risk anastomoses. No differences were detected with respect to macroscopic parameters, histopathology, or hydroxyproline content at 5 days post-anastomosis.CONCLUSIONS:Fibrin glue with a low fibrinogen content normalizes the bursting pressure of high-risk ischemic left-colon anastomoses in rats at day 5 after surgery.

Injectable tissue‐engineered cartilage using commercially available fibrin glue

To achieve injectable tissue-engineered cartilage using a commercially available fibrin sealant, and to determine the most suitable fibrin glue concentration, cartilage source, and cultured chondrocyte concentration. Animal research. A total of 28 immunocompetent New Zealand white rabbits were divided into four groups. The cultured chondrocytes from different anatomical sources carried in fibrin glue with and without aprotinin in different concentrations of fibrinogen and thrombin (Tisseell), were injected into forehead and interocular regions of the rabbits. The new tissue formation was harvested at 8 weeks and analyzed through gross and histological analysis. The new tissue formations were found in round, elliptical, and flat forms. The mean value of Tisseell and cell suspension was 0.8 cc in all of the rabbits' injection regions, but the mean volume of the samples in which immature cartilage matrix and mature cartilage was 0.1 cc. In the 20 of the 55 injection regions of rabbits (36, 36%), mature and/or immature cartilage formation were observed. We observed inflammatory reactions, abscess formation, and foreign body reactions around the new cartilage tissue of tissue-engineered cartilage. The comparison of results using different cartilage sources, chondrocyte concentrations, or different fibrin glue concentrations did not show any significant difference. We observed that changing the concentrations of ingredients of commercially available fibrin glue, the source of the cartilage, or the cultured chondrocyte concentration did not have significant effect on neocartilage formation.

Enhanced Outside-In Signaling Through the Prothrombotic Pro33 Variant of Integrin αIIbβ3 in Adherent Platelets Under Static and Flow Dynamic Conditions

Abstract 2199 Objectives:Platelet integrin αIIbβ3 is polymorphic at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). In patients with coronary artery disease, the Pro33 isoform is associated with premature manifestation of myocardial infarction (Zotz et al., JTH 2005). Based on this finding, it has been postulated that the Pro33 variant of αIIbβ3 has a prothrombotic phenotype. Moreover, several studies have shown that elevated shear stress can activate platelets leading to shear-induced platelet aggregation contributing to acute myocardial infarction. Thus, it has been reported that pathological shear stress directly regulates αIIbβ3 (Feng et al., Am J Physiol Cell Physiol 2006). We have shown that adherent Pro33 platelets have a higher resistance than Leu33 platelets upon exposure to high shear rates (Loncar et al, Thromb J 2007). We now studied the impact of the Leu33/Pro33 polymorphism on αIIbβ3-mediated outside-in signaling under static and flow dynamic conditions in the presence or absence of Mn2+, analyzing the Src pY418 and FAK pY397 activities. Methods:Adhesion assays were performed with 10 or 100 μg/ml of immobilized fibrinogen for different incubation times with washed human platelets in the presence or absence of Mn2+ (0.5 mM). Mn2+ induces the active conformation of αIIbβ3. Src pY418 and FAK pY397 activities were determined by Western blotting and quantified densitometrically. Control experiments were performed with 1% BSA. Adhesion experiments under flow conditions were carried out with a cone-plate viscometer. Results:Under static conditions, Pro33 platelets adherent onto fibrinogen exhibited a 2.5-fold higher Src pY418 activity than Leu33 platelets after incubation for 20 min (p<0.01). Presence of Mn2+ (0.5 mM) in suspended Leu33 platelets stimulated their Src pY418 activity in an extent comparable to that of platelets adherent onto fibrinogen, while addition of Mn2+ (0.5 mM) to platelets adherent onto fibrinogen yielded a 3.5-fold increase in Src pY418 activity (p<0.05). Increase of the Mn2+ concentration (2 mM) raised the Src pY418 activity 1.5-fold, as compared to 0.5 mM Mn2+ (p<0.05). In parallel experiments with both HPA-1 isoforms of αIIbβ3, Pro33 platelets adherent onto fibrinogen in the presence of 0.5 mM Mn2+ revealed a 2.5-fold higher Src pY418 activity than Leu33 platelets after 5 min (p<0.05). This difference further increased upon prolonged adhesion for 10 or 20 min with a 5-fold increase after 40 min (p<0.01). The concentration of immobilized fibrinogen (10 or 100 μg/ml) had no influence on this effect. Addition of abciximab completely abolished the Src pY418 activation. Upon exposure to abnormal (5000 s−1) but not physiological (500 s−1) shear rates for 2 or 5 min, Pro33 platelets adherent onto 100 μg/ml fibrinogen exhibited a 2- or 2.5-fold higher Src pY418 activity than under static conditions (p<0.05 or 0.02, respectively). Under the same conditions, the Src pY418 activity of Pro33 platelets was 2-fold higher than that of Leu33 platelets (p<0.05). Again, different concentrations of fibrinogen (10 μg/ml and 100 μg/ml) did not affect these results. In comparison to Src (pY418), phosphorylation of FAK (pY397) increased slower both in Leu33 and Pro33 platelets adherent onto fibrinogen, when exposed to a shear rate of 5000 s−1 for 10min. Both isoforms (Leu33 or Pro33) exhibited a significantly higher FAK pY397 activity than under static conditions (3-fold increase, p<0.05). The Pro33 variant of αIIbβ3 showed a higher FAK pY397 activation than the Leu33 isoform (p<0.05). Again, abciximab completely blocked both the Src and FAK activation not only under static conditions but also upon exposure to shear. Conclusion:Our observations indicate that the HPA-1 polymorphism of αIIbβ3 has a considerable impact on the integrin-mediated outside-in signaling. The significantly higher Src (pY418) and FAK (pY397) activities of Pro33 platelets adherent onto fibrinogen under static and flow conditions are in agreement with the contention that the Pro33 (HPA-1b) isoform of αIIbβ3 is indeed a prothrombotic integrin variant. Disclosures:No relevant conflicts of interest to declare.

Regulation of Cell Adhesion by the Physical Properties of Fibrin(ogen) Substrates

The physical properties of substrates are crucial to the regulation of cell adhesion through integrin‐mediated signaling. The matrices formed of fibrin(ogen) represent the natural substrates whose variable rigidity may control adhesion of blood cells. Binding of fibrinogen to the surface of fibrin gel prevents firm adhesion of leukocytes and platelets creating the anti‐adhesive layer. Also, fibrinogen immobilized on surfaces at high density supports weak adhesion whereas at low density it is highly adhesive. To understand the mechanism underlying differential cell adhesion, we have characterized the structural and physical properties of surfaces prepared by deposition of different concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposited at high density results in the multilayered material characterized by low rupture forces. In contrast, immobilization of fibrinogen at low density produces a single layer in which molecules are directly attached to the solid surface resulting in higher rupture forces. Consistent with their distinct physical properties, low, but not high density fibrinogen induces strong intracellular signaling and cell spreading. The data suggest that multilayered fibrinogen prevents cell adhesion by modifying the physical properties of surfaces which results in reduced force generation and insufficient signaling.HL63199

Scaffold permeability as a means to determine fiber diameter and pore size of electrospun fibrinogen

The purpose of this study was to construct a flowmeter that could accurately measure the hydraulic permeability of electrospun fibrinogen scaffolds, providing insight into the transport properties of electrospun scaffolds while making the measurement of their topographical features (fiber diameter and pore size) more accurate. Three different concentrations of fibrinogen were used (100, 120, and 150 mg/mL) to create scaffolds with three different fiber diameters and pore sizes. The fiber diameters and pore sizes of the electrospun scaffolds were first analyzed with scanning electron microscopy and image analysis software. The permeability of each scaffold was measured with the flowmeter and used to calculate permeability-based fiber diameters and pore sizes, which were compared to values obtained through image analysis. Permeability measurement revealed scaffold permeability to increase with fibrinogen concentration, much like average fiber diameter and pore size. Comparison between the two measurement methods demonstrated the efficacy of the flowmeter as a way to measure scaffold features.

Microplate reader analysis of triatomine saliva effect on erythrocyte aggregation

Our hypothesis is that the action of aggregating and disaggregating substances in the blood can be detected and quantified by the Microplate Reader. To ascertain the validity of this hypothesis, we selected two types of blood: one that naturally presents erythrocyte aggregation (pig blood) and the other that does not present aggregation (bovine blood). One important reason for the choice of pig blood is that its erythrocyte aggregation resembles that of human blood. T. infestans saliva was added to the pig blood as a disaggregating substance, while bovine fibrinogen was added to the bovine blood as a substance that promotes erythrocyte aggregation. We investigated the dynamic viscosity (h) of these mammals' blood, of T. infestans saliva and of the absorption (A) by Microplate Reader, carrying out UV-Vis spectrophotometric assays of pig plasma with different concentrations of triatominae saliva and of bovine blood with different concentrations of fibrinogen. Our findings indicate that spectroscopic techniques such as the Microplate Reader complement and expand the study of blood rheology, erythrocyte sedimentation and aggregation.

The Effect of Fibrinogen Substitution on Reversal of Dilutional Coagulopathy: An In Vitro Model

Colloids and crystalloids are usually administered as treatment for hypovolemia in severely injured patients. However, dilution of clotting factors and platelets together with impaired fibrinogen polymerization are associated with fluid therapy and may aggravate hemorrhage, thus worsening final outcome of these patients. We investigated, in an in vitro model, whether the addition of fibrinogen to diluted blood samples can reverse dilutional coagulopathy. Blood from 5 healthy male volunteers was diluted by 60% using lactated Ringer's solution, 4% modified gelatin solution, or 6% hydroxyethyl starch 130/0.4, as well as the combination of lactated Ringer's solution with either of the 2 colloid solutions. Thereafter, aliquots of diluted blood samples were incubated with 3 different concentrations of fibrinogen (0.75, 1.5, and 3.0 mg/mL). Measurements were performed by modified thrombelastography (ROTEM; Pentapharm, Munich, Germany). After 60% dilution, clotting times increased, whereas clot firmness and fibrin polymerization decreased significantly. After administration of fibrinogen, clotting times decreased and clot firmness, as well as fibrin polymerization, increased in all diluted blood samples. The effect of in vitro fibrinogen substitution on ROTEM variables was dependent on the fibrinogen dosage and the type of solution used to dilute the blood samples.

Localized surface plasmon resonance based optical biosensor using surface modified nanoparticle layer for label-free monitoring of antigen–antibody reaction

In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV–Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biomolecular interactions in an arraybased format. In this research,the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmissionmeasurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen–antibody reaction. The anti-fibrinogen antibody wasimmobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen–antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml.

A freeze-dried fibrin disc as a biodegradable drug release matrix

A fibrin clot loaded with soluble tetracycline (TET) was prepared and lyophilized to make discs of a size and shape to use as a drug delivery matrix. On subcutaneous implantation of these discs in mice, they were found to have degraded in 15 days as evidenced by gross and histological examination. The in vitro discharge kinetics of tetracycline from the disc into phosphate buffered saline (PBS) and human serum were compared. It was observed that the release rate of tetracycline from the matrix into serum remained steady from day 1 to day 12, maintaining sufficient concentration that may be required to control microbial growth in the medium. Two different concentrations of fibrinogen were used to fabricate discs denoted as FG200 and FG100, and in both cases the retention rate was comparable when the study medium was serum. In contrast, when suspended in PBS instead of serum, the delivery of the drug into the medium was found to be high for up to the 3rd day when a sharp decline in discharge was observed. The fibrinogen used is a factor that determines not only the longevity of discharge but also fibrinolysis. The degradation of the disc in vitro was visible when the discs were suspended in the buffer, and correspondingly fibrin degradation product (FDP) measured in the medium using an antibody-based assay system was high. Fibrin disc is haemostatic and biodegradable in vivo, and in vitro release of a small molecule at a controlled rate is demonstrated here. Hence, it may be a suitable candidate as a drug delivery implant for short-term use.

Locking the β3 Integrin I-like Domain into High and Low Affinity Conformations with Disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.

Involvement of Fibrinogen in Protamine-Induced Pulmonary Hypertension

Protamine reversal of heparin anticoagulation occasionally results in pulmonary hypertension as well as systemic hypotension. To examine the contribution of blood components to this induction of pulmonary hypertension, we developed an isolated rat lung perfusion model and perfused heparinized plasma, heparinized serum, and Hepes (4% bovine serum albumin, 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 5 mM glucose, in warm physiological saline) buffer solution with or without fibrinogen. Perfusion with heparinized plasma and Hepes buffer solution with fibrinogen caused pulmonary hypertension; perfusion with heparinized serum or Hepes buffer solution without fibrinogen did not, suggesting that fibrinogen is involved in the induction of pulmonary hypertension. We also labeled protamine with125I and compared the amounts of protamine accumulating in the lung with different concentrations of fibrinogen. The amount of protamine trapped in the lung increased according to the concentration of fibrinogen. Fibrinogen may accelerate the reaction between pulmonary endothelial cells and protamine or protamine–heparin complexes. In the mechanism of protamine-induced pulmonary hypertension, fibrinogen, as well as heparin and protamine, may be an essential component.

The Morphology of Thrombin-activated Platelets with Reference to Different Fibrinogen Concentrations as Revealed by Computer-aided Three-dimensional Reconstruction

Washed human platelets incubated in different concentrations of fibrinogen were activated by thrombin and aggregated in the presence of Ca(2+) or did not aggregate when EDTA was present. They were analyzed by transmission electron microscopy and computer-aided three-dimensional reconstruction. The volumes and surface areas of the reconstructed models were calculated. The quotients of the values calculated for the whole platelet and the surface-connected canalicular system were taken as measures of the degree of surface invagination. Increasing the concentrations of fibrinogen reduced the values of the quotients indicating enhanced internalization of surface membranes, and tended to smoothen the outer surfaces to obtain spherical shapes. The invaginations are much more pronounced in platelets that did not aggregate in the presence of EDTA suggesting that aggregation fixes some membrane areas that otherwise would be redistributed to the platelet's inner compartments.

Fibrinogen-containing membrane-associated structures arising at the surfaces of ADP-stimulated blood platelets

Ultrastructural studies of ADP-stimulated gel-filtered human platelets incubated with different concentrations of fibrinogen reveal unusual extracellular structures composed at least partly of the aggregated fibrinogen. Development of these structures depends on the exogenous fibrinogen concentration and duration of the incubation. Fibrinogen-containing extracellular material exists in two different structural forms. As was shown earlier, one of them is represented by dense, amorphous intercellular matrix and is localized mainly in platelet microaggregates (Belitser et al.,Thromb. Res., in press). Another one consists of huge sheet-like structures bearing individual or clumped platelets bound either to one or to both of their surfaces. After thrombin treatment, these structures could not be found anymore; sometimes they seem to be substituted by the fibrin-like fibers spatially connected to each other and/or to the platelet membranes. It has been suggested that soluble fibrinogen interacting with its specific membrane receptors undergoes conformational changes promoting intermolecular interactions and resulting in the fibrinogen aggregates formation at the surfaces of the activated platelets. A probable physiological significance of the structures described is discussed. It is supposed that under certain conditions in vivo they may play an important role, in particular as a highly concentrated substrate for the thrombin action, providing in such a way a possibility of rapid, effective consolidation of the initial platelet thrombi with the fibrin fibers.