Fibrinogen Fragments Research Articles

Expression and characterization of recombinant mast cell tryptase.

Tryptase, a serine protease, is the major protein component in mast cells. In an animal model of asthma, tryptase has been established as an important mediator of inflammation and late airway responses induced by antigen challenge. Human tryptase is notable for its tetrameric structure, requirement of heparin for stability, and resistance to endogenous inhibitors. Human protryptase was expressed as a recombinant protein in Pichia pastoris. The recombinant protein consisted of two forms of protryptase, one containing the entire propeptide and the other containing only the Val-Gly dipeptide at its amino terminus. Isolation of active recombinant tryptase required a two column purification protocol and included a heparin- and dipeptidyl peptidase I-dependent activation step. Purified recombinant tryptase migrated as a tetramer on a gel filtration column and displayed kinetic parameters identical to those of a native tryptase obtained from HMC-1 cells, a human mast cell line. Recombinant and HMC-1 tryptase exhibited comparable sensitivities to an array of protein and low-molecular-weight inhibitors, including one that is highly specific for tryptase (APC-1167). Similarly, the recombinant enzyme cleaved both alpha- and beta-chains of fibrinogen to generate fibrinogen fragments indistinguishable from those generated by HMC-1-derived tryptase. Thus, recombinant tryptase expressed in P. pastoris displays physical and enzymatic properties essentially identical to the native enzyme. This system provides a cost-effective and easy to manipulate expression system that will enable the functional characterization of this unique enzyme.

The receptor for the globular "heads" of C1q, gC1q-R, binds to fibrinogen/fibrin and impairs its polymerization.

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin gamma chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.

Envelope skeletonization as a means to determine monomer masks and non-crystallographic symmetry relationships: application in the solution of the structure of fibrinogen fragment D.

An algorithm is described which utilizes the solvent mask generated by the solvent-flattening technique to calculate a monomer molecular envelope. In the case where non-crystallographic symmetry (NCS) is present in the crystal and self-rotation angles are known from a self-rotation function, the resultant monomer envelopes can be used to search for the translation component of the NCS element by a three-dimensional search in real space. In the absence of self-rotation angles, the monomer envelope may be used to derive the NCS operators by reciprocal-space techniques. Thus, an automatic procedure for averaging directly from the solvent-flattening stage can be implemented. The procedure was instrumental in the structure solution of fibrinogen fragment D, which is presented as an example.

Structural Studies of Fibrinolysis by Electron and Light Microscopy

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

Presence of vitronectin and activated complement factor C9 on ventriculoperitoneal shunts and temporary ventricular drainage catheters.

The pathogenesis of cerebrospinal fluid (CSF) shunt infection is characterized by staphylococcal adhesion to the polymeric surface of the shunt catheter. Proteins from the CSF--fibronectin, vitronectin, and fibrinogen--are adsorbed to the surface of the catheter immediately after insertion. These proteins can interfere with the biological systems of the host and mediate staphylococcal adhesion to the surface of the catheter. In the present study, the presence of fibronectin, vitronectin, and fibrinogen on CSF shunts and temporary ventricular drainage catheters is shown. The presence of fragments of fibrinogen is also examined. The authors used the following methods: binding radiolabeled antibodies to the catheter surface, immunoblotting of catheter eluates, and scanning force microscopy of immunogold bound to the catheter surface. The immunoblot showed that vitronectin was adsorbed in its native form and that fibronectin was degraded into small fragments. Furthermore, the study demonstrated that the level of vitronectin in CSF increased in patients with an impaired CSF-blood barrier. To study complement activation, an antibody that recognizes the neoepitope of activated complement factor C9 was used. The presence of activated complement factor C9 was shown on both temporary catheters and shunts. Activation of complement close to the surface of an inserted catheter could contribute to the pathogenesis of CSF shunt infection.

Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis

Fibrinogen St. Gallen I was detected in an asymptomatic Swiss woman. Routine coagulation tests revealed a prolonged thrombin and reptilase time. Functionally measured fibrinogen levels were considerably lower than those determined immunologically. Polymerization of fibrin monomers derived from purified fibrinogen was delayed in the presence of either calcium or EDTA. Normal fibrinopeptide A and B release by thrombin was established. An abnormal degradation of fibrinogen St. Gallen I by plasmin was observed. Fragment D1 of normal fibrinogen was fully protected against further proteolysis in the presence of 10 mM calcium, whereas fibrinogen St. Gallen I was partially further degraded to fragments D2 and D3. In the presence of 10 mM EDTA, the conversion of variant fragment D1 to D2 was accelerated whereas the degradation of fragment D2 to D3 was delayed in comparison to degradation of fragments D1 and D2 of normal fibrinogen. Three high-affinity calcium binding sites were found in both normal and variant fibrinogen. Mutation screening with SSCP analysis suggested a mutation in exon VIII of the gamma-chain gene. Cycle sequencing of this gene portion revealed a single base substitution from G to T of the base 7527, leading to replacement of gamma 292 glycine by valine. The same mutation has already been described for the fibrinogen variant Baltimore I. Molecular modeling was performed of a part of the gamma-chain containing the mutation site, based on recently published X-ray crystal structures of human fibrinogen fragment D and of a 30 kD C-terminal part of the gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 297 Asp that is involved in interactions of fragment D with the Gly-Pro-Arg-Pro-peptide was noted by molecular modeling. The latter observation is compatible with delayed polymerization of fibrin monomers.

Three-dimensional structural studies on fragments of fibrinogen and fibrin

Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

Facile Purification of Fibrinogen Fragments Using a Computer-Based Model with General Applicability to the Generation of Salt Gradients

We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS–PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.

Identification of a Novel Recognition Sequence for Integrin αMβ2 within the γ-chain of Fibrinogen

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Measurement of plasma fibrinogen concentration by the prothrombin-time-derived method: applicability and limitations.

A prothrombin-time-derived method was used to measure plasma fibrinogen concentration (PFC) in 286 samples from 242 normal and 44 orally anticoagulated subjects. Absorbance changes at 405 nm (deltaOD) during the clotting process were obtained by an automatic coagulometer and their relationship with plasma fibrinogen concentration (range 90-1090 mg/dl), measured by the Clauss method, was investigated. A weighted linear regression between the deltaOD and the Clauss-derived PFC values provided the best fit of the experimental data. The fitting equation was found to be reliable and accurate for PFC determination in normal subjects, whereas a systematic overestimate of fibrinogen level was demonstrated in plasma with high fibrinogen concentrations (> 400 mg/dl) and in plasma from anticoagulated patients. The systematic overestimate in the latter samples could be a result of an increased fibrin gel turbidity, as shown by in-vitro experiments using purified fibrinogen clotted by different thrombin concentrations. The PFC overestimate by the prothrombin-time-derived method could also be experimentally reproduced by competitively inhibiting thrombin-fibrinogen interaction by hirudin 54-65 peptide and the fibrinogen fragment E. A similar qualitative result was also found for the prothrombin-time-derived method in the presence of the Gly-Pro-Arg-Pro peptide, which competitively inhibits the end-to-end fibrin aggregation process. Notably, under both the above experimental conditions, the Clauss method underestimated the PFC. On the other hand, the 'clot recovery' method was minimally affected by the above inhibitors. These results indicate that the prothrombin-time-derived method is accurate and precise for most routine purposes. Its precision seems inadequate, however, under those conditions where the prothrombin time is prolonged (such as anticoagulant therapy) and in the presence of high fibrinogen levels.

Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the ‘capsule eluate’) to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not β-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the begining of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of ‘free-floating’ capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, α 1-antitrypsin, fibronectin, albumin, α 2-macroglobulin, vitronectin, β 2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.

Prothrombin fragment 1+2 is a risk factor for myocardial infarction in treated hypertensive men.

Haemostatic factors may play a part in the development of acute coronary heart disease. To evaluate as predictors of major coronary events (fatal and non-fatal myocardial infarctions and sudden death) levels of fibrinogen, von Willebrand factor, prothrombin fragment 1+2, thrombin-antithrombin complex, plasminogen activator inhibitor activity and C-reactive protein. We studied 131 men, aged 56-77 years, with treated hypertension and at least one additional cardiovascular risk factor (hypercholesterolaemia, diabetes mellitus or smoking). These patients were recruited from a continuing risk factor intervention study. The mean observation time was 3.0 years. Fourteen patients died and 16 had a major coronary event during the follow-up period. After adjustments for other risk factors, levels of prothrombin fragment 1+2 and C-reactive protein were independent predictors of major coronary events. The other measured haemostatic variables were not significantly associated with major coronary events during follow-up. Fibrinogen and prothrombin fragment 1+2 levels were independent predictors for mortality. Among treated hypertensive men, levels of prothrombin fragment 1+2 and C-reactive protein were independent predictors of major coronary events.

Mechanisms of fibrinogen domains : biomaterial interactions

Spontaneous adsorption of fibrinogen is critical to the pathogenesis of biomaterial-mediated inflammatory responses. However, the mechanism by which adsorbed fibrinogen affects phagocyte responses is still not clear. To investigate the molecular interaction between fibrinogen and biomaterials, fibrinogen fragments (D 100 and E50) were generated and used in the present study. The results indicate that biomaterial : D100 interaction is essential to fibrinogen-mediated inflammatory responses, because biomaterials precoated with D100, but not E50, prompt strong inflammatory responses. Furthermore, the results from in vitro studies show that whole molecule fibrinogen and D100 exhibit very similar protein:surface interactions. Specifically: (1) both D100 and fibrinogen have high affinity for biomaterial surfaces; and (2) the retention rates of adsorbed D 100 in both in vivo and in vitro environments are as high as that for adsorbed fibrinogen. On the other hand, E50 does bind to biomaterials but with low affinity because, once bound, it is not tightly adherent to the biomaterial surfaces. Taken together, the results suggest that the mechanism of fibrinogen-mediated inflammatory responses may involve the following three consecutive events: (1) after contact with blood or tissue fluid, the D domain tends to interact with biomaterial surfaces and is important in the tight binding of fibrinogen to implant surfaces; (2) the biomaterial surface then promotes conformational changes within the D domain, exposing P1 epitope (y 190-202, which interacts with phagocyte Mac-1 integrin); and (3) the engagement of Mac-1 integrin with P1 epitope then triggers subsequent phagocyte adherence and reactions.

Antibodies against Fibrinogen in Pregnant Women, in Post Delivery Women and in the Newborns

Pregnancy and delivery have measurable effects on haemostatic and immunological changes. Degradation of fibrinogen induces significant structural and conformational modulation and leads to the progressive loss of antigenic sites present on the parent molecule but also exposes some new sites. These neoantigens may be recognized by the immune system and may be elicited by the autologous host manifested by the production of autoantibodies. Therefore in the present study, in pregnant and post delivery women and in the newborns, levels of antibodies against fibrinogen, fibrin(ogen) degradation products (FDP) and fibrin degradation products (D-dimer) were examined. Enzyme immunoassay (ELISA) was used to detect and quantitate autoantibodies against fibrinogen (class G immunoglobulin) in human sera. In all sera there were found varying concentrations of autoantibodies and their levels were significantly higher in all pregnant women in comparison with non-pregnant ones. Significantly higher levels were found in Rh immunized and clinically complicated pregnancies. The level of autoantibodies, coagulation and fibrinolytic system components were higher in post delivery women than in normal pregnant women. Also antibodies to fibrinogen were studied in cord serum of newborns in different terms of delivery. The low levels of antibodies in all newborns raise questions of possible foetal-maternal immunologic interactions. Positive correlation between mothers and newborns was demonstrated after delivery at gestational age from 34th to 41st week, and negative in 42nd and more week. There were no significant differences in antibody level among the newborns delivered by the same mothers. It was found that autoantibodies bind selectively to the fibrinogen and fibrinogen fragments X, Y and D. These autoantibodies may represent a new interface between the coagulation and the immune systems which may be significant in controlling the pathologic activities of the cleavage fragments.

Enhancement of fibrin binding and activation of plasminogen by staplabin through induction of a conformational change in plasminogen

Staplabin (0.3–0.6 mM), a fungal triprenyl phenol, enhanced 3–6-fold the plasminogen activator-catalyzed activation of Glu-plasminogen and Lys-plasminogen as well as their binding to fibrin. Staplabin was not stimulatory to the amidolytic activity of plasmin and plasminogen activators. Even in the presence of ϵ-aminocaproic acid (EACA) and fibrinogen fragments, allosteric effectors for Glu-plasminogen, staplabin increased the activation of both forms of plasminogen. In size-exclusion chromatography of Glu-plasminogen and Lys-plasminogen, the molecular elution time, which varies as the conformation of a protein changes, was shortened by staplabin. These results suggest that staplabin causes plasminogens to be more susceptible to activation and fibrin binding by inducing a conformational change that is, at least in part, different from that induced by EACA and fibrinogen fragments.

Role of hemostatic risk factors for restenosis in peripheral arterial occlusive disease after transluminal angioplasty.

In a prospective study, the role of various hemostatic factors known to be associated with thrombotic risk was investigated in 71 patients with peripheral arterial occlusive disease (PAOD, stages II through IV, Fontaine; aged 68 +/- 13 years). Laboratory investigations were done before; 1, 24, and 48 hours after; and 3 and 6 months after percutaneous transluminal angioplasty (PTA). Thirty of 71 (42.3%) patients developed restenosis (> 50% reduction of the lumen diameter) at the site of PTA within 6 months, verified by color-coded duplex sonography. Significantly increased levels of thrombin-antithrombin III complexes (P < .01), prothrombin fragments 1 + 2 (P < .01), and D-dimers (P < .01) were found 1 hour, as well as 24 to 48 hours, after PTA. Fibrinogen (P < .01) and von Willebrand factor (P < .01) were significantly higher 48 hours after PTA. Restenotic patients as a whole had higher plasma fibrinogen (3.46 +/- 1.12 versus 2.95 +/- 0.62 g/L, P < .01) and C-reactive protein (25.4 +/- 46.7 versus 7.9 +/- 6.9 mg/L, P < .05) at baseline, as well as higher fibrinogen (P < .05) and prothrombin fragments 1 + 2 (P < .01) during months 3 to 6 after PTA. There was a nonsignificant tendency for higher values of von Willebrand factor (206 +/- 98% versus 184 +/- 100%, P = .2) at baseline in patients with restenosis, whereas tissue plasminogen activator, plasminogen activator inhibitor, coagulation screening tests, blood cell counts, and serum lipids showed no significant difference between the two groups. The relative risk for developing restenosis within 6 months while having high fibrinogen (> 2.8 g/L) or C-reactive protein at baseline was 2.80 (95% CI: 1.30-6.02, P < .01) and 1.96 (95% CI: 1.07-3.58, P < .05), respectively. Patients with critical limb ischemia (stage III/IV, Fontaine) had significantly higher fibrinogen and von Willebrand factor at repeated points of time, as well as significantly higher C-reactive protein and lower creatinine clearance at entry. In the logistic regression risk factor analysis, baseline plasma fibrinogen, C-reactive protein concentration, and the severity of the arterial disease were significantly predictive of restenosis. Our results indicate that high procoagulant factors and persistent thrombin generation of the hemostatic system might promote restenosis, particularly in patients with extended atherosclerosis. This finding suggests that new treatment strategies should be taken under consideration for patients with PAOD and PTA.

Dimeric αs2-casein, a novel matrix for tissue-type plasminogen activator catalyzed plasminogen activation

Abstract In previous studies, we demonstrated a differential distribution of plasminogen, tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA) between various milk fractions. Plasminogen and t-PA were mainly confined to the casein micelle fraction, whereas u-PA was restricted to the cell fraction. These studies also showed that the differential distribution of these components was caused by specific binding components in milk. The u-PA binding component in the cell fraction was identified as the u-PA receptor. The t-PA binding components of bovine casein micelles were identified as dimeric αs2-casein and multimeric forms of κ-casein. In order to further explore the action of the plasminogen activation system that influences endogenous degradation of proteins in bovine milk, we evaluated the effect of individual bovine caseins on the rate of plasminogen activation by t-PA. Using a coupled peptidyl anilide plasminogen activator assay, we found that dimeric αs2-casein induced a concentration-dependent enhancement of the t-PA-catalyzed plasminogen activation. On a weight basis, the stimulating effect of dimeric αs2-casein was comparable to that of cyanogen bromide cleaved fibrinogen fragments. In a direct catalytic assay with a peptidyl anilide substrate, dimeric αs2-casein had no effect on t-PA activity. Ligand blotting experiments showed binding of both plasminogen and t-PA to the dimeric αs2-casein. The combined results from the binding experiments and the enzymatic assays suggest that the observed enhancement in plasminogen activation is mediated via formation of a ternary complex between t-PA, plasminogen, and dimeric αs2-casein. We further investigated the binding specificity of t-PA towards dimeric αs2-casein using t-PA deletion mutants and monoclonal antibodies. The results were compatible with the binding being mediated by kringle 2 and the finger domain of t-PA. These findings suggest that casein, analogous to fibrin clots in the intravascular space, serve as a molecular matrix for t-PA-catalyzed plasminogen activation.

The Role of Putative Fibrinogen Aα-, Bβ-, and γA-chain Integrin Binding Sites in Endothelial Cell-mediated Clot Retraction

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking Aalpha-chain RGD residues at 572-574 or lacking the gamma-chain residues AGDV 408-411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin alphavbeta3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin alphaIIbbeta3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by alphavbeta3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.

Central obesity, insulin resistance, syndrome X, lipoprotein(a), and cardiovascular risk in Indians, Malays, and Chinese in Singapore.

STUDY OBJECTIVE: To examine the hypothesis that the higher rates of coronary heart disease (CHD) in Indians (South Asians) compared with Malays and Chinese is at least partly explained by...