Recombinant fibrinogen reveals the differential roles of α- and γ-chain cross-linking and molecular heterogeneity in fibrin clot strain-stiffening.

Essentials Fibrinogen circulates in human plasma as a complex mixture of heterogeneous molecular variants. We measured strain-stiffening of recombinantly produced fibrinogen upon clotting. Factor XIII and molecular heterogeneity alter clot elasticity at the protofibril and fiber level. This highlights the hitherto unknown role of molecular composition in fibrin clot mechanics. SUMMARY: Background Fibrin plays a crucial role in haemostasis and wound healing by forming strain-stiffening fibrous networks that reinforce blood clots. The molecular origin of fibrin's strain-stiffening behavior remains poorly understood, primarily because plasma fibrinogen is a complex mixture of heterogeneous molecular variants and is often contaminated by plasma factors that affect clot properties. Objectives and methods To facilitate mechanistic dissection of fibrin nonlinear elasticity, we produced a homogeneous recombinant fibrinogen corresponding to the main variant in human plasma, termed rFib610. We characterized the structure of rFib610 clots using turbidimetry, microscopy and X-ray scattering. We used rheology to measure the strain-stiffening behavior of the clots and determined the fiber properties by modeling the clots as semi-flexible polymer networks. Results We show that addition of FXIII to rFib610 clots causes a dose-dependent stiffness increase at small deformations and renders the strain-stiffening response reversible. We find that γ-chain cross-linking contributes to clot elasticity by changing the force-extension behavior of the protofibrils, whereas α-chain cross-linking stiffens the fibers, as a consequence of tighter coupling between the constituent protofibrils. Interestingly, rFib610 protofibrils have a 25% larger bending rigidity than plasma-purified fibrin protofibrils and a delayed strain-stiffening, indicating that molecular heterogeneity influences clot mechanics at the protofibril scale. Conclusions Fibrinogen molecular heterogeneity and FXIII affect the mechanical function of fibrin clots by altering the nonlinear viscoelastic properties at the protofibril and fiber scale. This work provides a starting point to investigate the role of molecular heterogeneity of plasma fibrinogen in fibrin clot mechanics and haemostasis.

Factor XIII stiffens fibrin clots by causing fiber compaction.

BACKGROUND: Factor XIII-induced cross-linking has long been associated with the ability of fibrin blood clots to resist mechanical deformation, but how FXIII can directly modulate clot stiffness is unknown. OBJECTIVES AND METHODS: We hypothesized that FXIII affects the self-assembly of fibrin fibers by altering the lateral association between protofibrils. To test this hypothesis, we studied the cross-linking kinetics and the structural evolution of the fibers and clots during the formation of plasma-derived and recombinant fibrins by using light scattering, and the response of the clots to mechanical stresses by using rheology. RESULTS: We show that the lateral aggregation of fibrin protofibrils initially results in the formation of floppy fibril bundles, which then compact to form tight and more rigid fibers. The first stage is reflected in a fast (10 min) increase in clot stiffness, whereas the compaction phase is characterized by a slow (hours) development of clot stiffness. Inhibition of FXIII completely abrogates the slow compaction. FXIII strongly increases the linear elastic modulus of the clots, but does not affect the non-linear response at large deformations. CONCLUSIONS: We propose a multiscale structural model whereby FXIII-mediated cross-linking tightens the coupling between the protofibrils within a fibrin fiber, thus making the fiber stiffer and less porous. At small strains, fiber stiffening enhances clot stiffness, because the clot response is governed by the entropic elasticity of the fibers, but once the clot is sufficiently stressed, the modulus is independent of protofibril coupling, because clot stiffness is governed by individual protofibril stretching.

Neonatal onset autosomal dominant polycystic kidney disease (ADPKD) in a patient homozygous for a PKD2 missense mutation due to uniparental disomy.

Autosomal dominant polycystic kidney disease (ADPKD), due to a heterozygous mutation in PKD1 or PKD2, is usually an adult onset disease. Renal cystic disease is generally milder in PKD2 patients than in PKD1 patients. Recently, several PKD1 patients with a severe renal cystic phenotype due to a second modifying PKD1 allele, or carrying two incomplete penetrant PKD1 alleles, have been described. This study reports for the first time a patient with neonatal onset of PKD homozygous for an incomplete penetrant PKD2 missense variant due to uniparental disomy.

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Anti-MCP-1 gene therapy inhibits vascular smooth muscle cells proliferation and attenuates vein graft thickening both in vitro and in vivo.

OBJECTIVE: Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease. METHODS AND RESULTS: MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures. CONCLUSIONS: These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.

Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

Inhibition of accelerated atherosclerosis in vein grafts by placement of external stent in apoE*3-Leiden transgenic mice.

OBJECTIVE: Vein grafts fail because of the development of intimal hyperplasia and accelerated atherosclerosis. Placement of an external stent around vein grafts resulted in an inhibition of intimal hyperplasia in several animal studies. Here, we assess the effects of external stenting on accelerated atherosclerosis in early vein grafts in carotid arteries in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice. METHODS AND RESULTS: Venous interposition grafting was performed in apolipoprotein E*3-Leiden mice fed standard chow or a highly cholesterol-rich diet for 4 weeks. After engraftment, external stents with different inner diameters (0.4 or 0.8 mm) were placed. In unstented vein grafts in hypercholesterolemic mice, thickening up to 50 times the original thickness, with foam cell-rich lesions, calcification, and necrosis, was observed within 28 days. The atherosclerotic lesions observed show high morphological resemblance to atherosclerotic lesions observed in human vein grafts. In stented vein grafts in hypercholesterolemic mice, no foam cell accumulation or accelerated atherosclerosis was observed. Compared with unstented vein grafts, stenting of vein grafts in a hypercholesterolemic environment resulted in a 94% reduction of vessel wall thickening. These effects were independent of stent size. CONCLUSIONS: Extravascular stent placement results in strong inhibition of accelerated vein graft atherosclerosis in hypercholesterolemic transgenic mice and thereby provides a perspective for therapeutic intervention in vein graft diseases.

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity.

BACKGROUND: The mesothelium has an important role in maintaining an adequate fibrinolytic capacity in the peritoneal cavity and thus in preventing the formation of fibrinous peritoneal adhesions by secreting the fibrinolytic enzyme tissue-type plasminogen activator (t-PA). The fibrinolytic activity of human mesothelial cells (HMCs) is counteracted by rapid uptake of t-PA via the low-density lipoprotein receptor-related protein (LRP). The 39 kD receptor-associated protein (RAP) is an inhibitor of binding of t-PA to LRP, but RAP itself is also rapidly degraded via LRP. METHODS: Adenovirus-mediated RAP gene transfer technology was used to evaluate the effect of prolonged overexpression of RAP on t-PA accumulation in conditioned medium of HMCs under basal and inflammatory conditions. RESULTS: Infection of HMCs with a recombinant adenovirus carrying the RAP cDNA resulted within one day in t-PA levels that were maximally twofold to threefold increased as compared with noninfected or adenovirus-beta-galactosidase-infected cells. Whereas upon prolonged incubation, t-PA levels in the conditioned medium of uninfected cells leveled off because of rapid uptake and degradation via LRP, t-PA concentrations in the medium of adenovirus-RAP-infected cells continued to increase, reaching fivefold control levels after 72 hours. The increased t-PA accumulation persisted for seven days and then slowly returned to control values over the next few weeks. In contrast, the production of a specific inhibitor of t-PA, plasminogen activator inhibitor-1 (PAI-1), was not affected by adenoviral RAP gene transfer. Northern blotting analysis showed that t-PA, PAI-1, and LRP mRNA concentrations were not changed after adenoviral infection, underlining that the elevated t-PA levels are the result of RAP-blocked uptake and degradation of t-PA rather than increased t-PA synthesis. RAP gene transfer also restored diminished fibrinolytic activity of cytokine-treated mesothelial cells. CONCLUSIONS: Adenovirus-mediated transfer of the RAP gene provides an efficient way of transiently increasing the fibrinolytic capacity of mesothelial cells.

In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI.

Injury-induced neointimal development results from migration and proliferation of vascular smooth muscle cells (SMC). Cell migration requires controlled proteolytic degradation of extracellular matrix surrounding the cell. Plasmin is a major contributor to this process by degrading various matrix proteins directly, or indirectly by activating matrix metalloproteinases. This makes it an attractive target for inhibition by gene transfer. An adenoviral vector, Ad.ATF.BPTI, was constructed encoding a hybrid protein, which consists of the aminoterminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) linked to bovine pancreas trypsin inhibitor (BPTI), a potent inhibitor of plasmin. This hybrid protein binds to the u-PA receptor, thereby inhibiting plasmin activity at the cell surface, and was found to be a potent inhibitor of cell migration in vitro. Local infection with Ad.ATF.BPTI of balloon-injured rat carotid artery resulted in detectable expression of ATF.BPTI mRNA and protein in the vessel wall. Morphometric analysis of arterial cross-sections revealed that delivery of Ad.ATF.BPTI to the carotid artery wall at the time of balloon injury inhibited neointima formation by 53% (P < 0.01) at 14 days and 19% (P = NS) at 28 days after injury when compared with control vector-infected arteries. Intima/media ratios were decreased by 60% (P < 0.01) and 35% (P < 0.05) at 14 and 28 days, respectively, when compared with control vector-infected arteries. Furthermore, a small but significant increase in medial area was found in the Ad.ATF.BPTI-treated arteries at 28 days (P < 0.05). These results show that local infection of the vessel wall with Ad.ATF.BPTI reduces neointima formation, presumably by inhibiting SMC migration, thereby offering a novel therapeutic approach to inhibiting neointima development.

Improved adenovirus vectors for infection of cardiovascular tissues.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.

Adenoviral expression of a urokinase receptor-targeted protease inhibitor inhibits neointima formation in murine and human blood vessels.

BACKGROUND: Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha(2)-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. METHODS AND RESULTS: The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x10(9) pfu/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9+/-2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. CONCLUSIONS: These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.

Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation.

A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.

Adenoviral gene transfer of a u-PA receptor-binding plasmin inhibitor and green fluorescent protein: inhibition of migration and visualization of expression.

Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Telemetric monitoring of blood pressure in freely moving mice: a preliminary study.

This paper describes for the first time the possibility for recording the systolic pressure (SP), diastolic pressure (DP), and the mean arterial pressure (MAP) as well as the heart rate (HR) and locomotor activity (LA) in freely moving mice, using a commercially available telemetry and data acquisition system. The system comprises a new, small radio-telemetry transmitter implanted in the peritoneal cavity, a receiver board placed underneath the home cage, a multiplexer and a computer-based data acquisition system. The signals from the receiver were consolidated by the multiplexer and were stored and analysed by the computer. The telemetered pressure signals (absolute pressure) were corrected automatically for changes in atmospheric pressure measured by an ambient pressure monitor. The effects of implantation on animal behaviour, and, after the animals had recovered, the effects of handling on the SP, DP, MAP and HR were examined. The radio-telemetry system for recording the SP, DP, MAP and HR provides an accurate and reliable method for monitoring the direct effects of handling on SP, DP, MAP and HR. In addition, by using this new blood pressure (BP) transmitter, we maintain that BP measurements in freely moving mice are more efficient, reliable, and less labour-intensive than the measurement techniques described in the literature thus far.

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells.

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.

Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser-->Cys substitution associated with thrombosis.

A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor.

OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.