Plasmin Proteolysis Research Articles

Influence of processing temperature on plasmin activity and proteolysis in process streams from cold microfiltration of skim milk

Plasmin in process streams derived from microfiltration of skim milk can result in casein hydrolysis, potentially affecting the quality and functionality of ingredients produced. To determine whether partitioning of plasmin into permeates was impacted by processing temperature, cold microfiltration of skim milk was investigated at 4, 8 and 12 °C. Permeate generated at 4 °C had the highest plasmin activity (0.0185 AMC units mL−1). When plasmin activity was expressed relative to β-casein content, differences in the extent of dissociation of β-casein, at the different processing temperatures, did not influence plasmin activity in the resulting permeate streams. Throughout storage at 37 °C, all retentates exhibited extensive plasmin-mediated hydrolysis of β- and αS2-casein; αS1-casein to a lesser extent. Particle size measurements before and after plasmin-mediated hydrolysis of permeates indicated that the proteolysis products of β-casein, γ-caseins, retained the ability to self-associate on heating, with particle size increasing with increasing temperature.

Influence of Processing Temperature on Plasmin Activity and Proteolysis in Process Streams from Cold Microfiltration of Skim Milk

Influence of Processing Temperature on Plasmin Activity and Proteolysis in Process Streams from Cold Microfiltration of Skim Milk

Plasmin‐mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer

BackgroundFactor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. ObjectiveInvestigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. MethodsNH2‐terminal sequencing, mass spectrometry analysis, and functional assays. ResultsPlasmin in the presence of Ca2+/phospholipid (PL) rapidly cleaved FIXaβ at Lys316↓Gly317 to yield FIXaγ followed by a slow cleavage at Lys413↓Leu414 to yield FIXaδ. FIXaγ/FIXaδ migrated indistinguishably from FIXaβ in nondenaturing gel system indicating that C‐terminal residues 317‐415/317‐413 of heavy chain remain noncovalently associated with FIXaγ/FIXaδ. However, as compared with FIXaβ, FIXaγ or FIXaγ/FIXaδ (25‐75 mixture, 8‐hour/24‐hour incubation analysis by mass spectrometry) was impaired ~ 10‐fold in hydrolyzing synthetic substrate CBS 31.39 (CH3‐SO2‐D‐Leu‐Gly‐Arg‐pNA), ~ 30‐fold (~ 5‐fold higher Km, ~ 6‐fold lower kcat) in activating FX in a system containing Ca2+/PL, and ~ 650‐fold in a system containing Ca2+/PL and FVIIIa. Further, FIXaγ or FIXaγ/FIXaδ bound FVIIIa with ~ 60‐fold reduced affinity compared with FIXaβ. Additionally, in ligand blots, plasminogen or diisopropylfluorophosphate‐inhibited plasmin (DIP‐plasmin) bound FIXaγ and FIXaδ but not FIXaβ. This interaction was prevented by ε‐aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP‐plasmin binds to FIXaγ/FIXaδ through newly generated C‐terminal Lys316 and Lys413. Importantly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tissue plasminogen activator (tPA)‐mediated plasminogen activation in a concentration dependent manner. Similarly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tPA‐induced clot lysis in FIX‐depleted plasma. ConclusionPlasmin cleavage at Lys316↓Gly317 abrogates FIXaβ coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer.

Impact of protease and lipase activities on quality of Swedish raw milk

An increasing demand for dairy products with long shelf-life is foreseen. A limiting factor for maintaining high quality of these products is the activity of spoilage enzymes during storage. Lipase and protease activities in Swedish raw milk at farm and dairy level were investigated, analysing milk samples from three geographical regions and two seasons. Lipase activity in milk at farm level was affected by regional and seasonal variations, whereas at dairy level only season had an effect. Lipase activity was positively correlated with ionic calcium. For protease activity, no effect of either region or season was seen. Degradation products, e.g., free fatty acids, peptides and plasmin proteolysis products, varied differently between season and geographical origin at both farm and dairy level. The results indicate that lipase and protease activities are important for raw milk quality, while ionic calcium might be a future indicator for milk fat stability.

Increased Mosquito Midgut Infection by Dengue Virus Recruitment of Plasmin Is Blocked by an Endogenous Kazal-type Inhibitor.

SummaryDengue symptoms include alteration of blood coagulation and fibrinolysis, causing severe hemorrhage and death. Here, we demonstrate that higher concentration of plasmin, the human fibrinolytic factor, in blood meal enhances dengue virus (DENV) infection in mosquito midgut and dissemination in mosquitoes. We also show that mosquitoes express a plasmin-selective Kazal-type inhibitor (AaTI) in the midgut to inhibit plasmin proteolysis and revert the enhanced infection. Using bio-layer interferometry, we show that DENV, plasmin, and AaTI interact to form a tripartite complex. Eventually, plasmin increases midgut internalization of dextran molecules and this is reverted by AaTI. Our study demonstrates that (1) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glycocalyx and enhancing DENV internalization and (2) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.

Dengue Virus Recruitment of Plasmin Proteolysis Increases Mosquito Midgut Internalization, Enhancing Infection Onset, and this Can Be Blocked by an Endogenous Kazal-Type Inhibitor

Deciphering the impact of dengue pathogenesis on vector transmission is critical for understanding virulence evolution, epidemiology and developing transmission-blocking tools. Dengue infection can alter blood coagulation and fibrinolysis pathways, resulting in severe hemorrhage and death. Here, we demonstrate that when plasmin, the human fibrinolytic factor, is added to blood meal, it enhances DENV infection in mosquito midgut, resulting in higher dissemination in mosquitoes. In addition, the mosquito midgut-expressed AaTI, a plasmin-selective mosquito Kazal-type inhibitor, reverted this enhanced infection by inhibiting plasmin proteolysis in the midgut lumen. Using bio-layer interferometry, we showed that DENV, plasmin and AaTI interact to form a tripartite complex. Eventually, plasmin supplementation in infectious blood meal increased midgut internalization of dextran molecules and this was reverted by AaTI. Our study demonstrates that: (a) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glyocalyx and enhancing DENV infection onset, and (b) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.

2124-P: The Tissue Plasminogen Activator (tPA)/Plasmin System Reduces Amyloid Formation by Cleaving Human Islet Amyloid Polypeptide (hIAPP)

Type 2 diabetes is characterized by islet amyloid deposition, which is associated with β-cell loss. hIAPP is the unique peptide component of these deposits. We previously found that expression of tPA, the initiator of fibrinolysis, is upregulated in islets by amyloid formation in vitro. As plasmin, the proteolytic product of tPA activity, cleaves Aβ and attenuates amyloid deposition in Alzheimer’s disease, we hypothesized that the increase in islet tPA by amyloid deposition serves a protective function by generating plasmin to limit further accumulation of hIAPP. We therefore sought to determine whether the tPA/plasmin system reduces amyloid formation by hIAPP and, if so, what islet cell type produces tPA. Using a thioflavin T assay, we found 4 µM plasmin abrogated 20 µM hIAPP fibril formation by 91.6±0.9% (n=5; p<0.01). To determine whether this could be by plasmin proteolysis of hIAPP, 20 µM hIAPP was incubated with 4 µM plasmin or buffer and then mass spectrometry was performed. After 10 minutes of incubation with plasmin, intact hIAPP was absent and expected cleavage products were present. As tPA is produced by macrophages and endothelial cells, we next determined whether tPA expression is increased by hIAPP in either or both of these cell types. In rat bone-marrow-derived macrophages treated for 24 h with 10 µM hIAPP, non-amyloidogenic 10 µM rat IAPP (rIAPP) or vehicle, tPA expression was increased only by hIAPP (4.2±1.0 vs. 1.5±0.6 vs. 1.0±0.5 fold, respectively; n=4; p<0.05 for hIAPP vs. rIAPP and vehicle). In contrast, in primary rat islet endothelial cells, tPA expression was not increased by 20 µM hIAPP (0.7±0.3 vs. 1.0±0.4 fold; n=3; p=0.2 for hIAPP vs. vehicle). In summary, plasmin rapidly degrades hIAPP, curbing its aggregation into amyloid. As tPA is expressed in macrophages, enhancing its activity or its production by islet macrophages may represent a novel strategy to generate plasmin, cleave hIAPP and thus reduce islet amyloid formation and β-cell loss. Disclosure N. Esser: None. M.F. Hogan: None. A. Aplin: None. A.T. Templin: None. S. El-Osta: None. D. Raleigh: None. J.S. Edgar: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. Consultant; Self; Neurimmune. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. Funding U.S. Department of Veterans Affairs (BX001060); National Institutes of Health (GM078114); Belgian American Educational Foundation; Société Francophone du Diabète

Thrombin Generation Impairments in Platelet Function Disorders

Many platelet function disorders (PFD) present as uncharacterized disorders that impair aggregation responses to multiple agonists and/or cause non-syndromic dense granule deficiency (DGD). We postulated that some of these commonly encountered PFD might also impair the ability of platelets to support thrombin generation, given that an important subgroup are caused by mutations in transcription factors, such as RUNX1, that impair multiple aspects of platelet function. Accordingly, we initiated a study of thrombin generation (TG) in a prospective cohort of individuals presenting with an uncharacterized PFD. In addition, we reassessed how Quebec platelet disorder (QPD, previously called Factor V Quebec) affects coagulation as this disorder (which is caused by a duplication mutation of PLAU) triggers intraplatelet (but not systemic) plasmin generation and proteolysis of platelet but not plasma factor V (FV), and the normal plasma FV might potentially compensate for the loss of platelet FV in QPD.The study was conducted with written informed consent of participants and with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire Sainte Justine. Participants included: 1) five individuals with QPD; 2) eighteen individuals presenting with an uncharacterized PFD and confirmed reduced maximal aggregation responses to ≥2 agonists and/or confirmed DGD, including five that had a pathogenic RUNX1 mutation; and 3) eighteen similarly-aged general population controls. TG was assessed by the calibrated automated thrombogram (CAT) procedure on a Fluoroskan (Thermo Fisher Scientific AG, Reinach, Switzerland) using Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), manufacturer- and ISTH-recommended protocols and reagents for testing platelet poor plasma (PPP) and platelet rich plasma (PRP). Endpoints included: endogenous thrombin potential (ETP, nM·min), peak thrombin concentration (nM), time-to-peak (min) and lag time (min). Platelet lysate and plasma FV concentrations were determined by enzyme-linked immunoassay. Data were analyzed using Mann-Whitney tests with Bonferroni correction for multiple comparisons.All TGA endpoints for PPP were comparable for controls and participants with PFD, including QPD (p values ≥0.10). In TGA with PRP, most PFD participants had findings similar to controls, however, the subgroups with pathogenic RUNX1 mutations or QPD had significantly reduced ETP and peak thrombin concentration compared to controls (data as median [range]: ETP: controls: 1860 [1530-2630]; RUNX1 mutation subgroup: 1520 [805-1640], p=0.004; QPD: 1370 [981-2010], p=0.01; peak thrombin concentration: controls: 111 [65-152]; RUNX1 mutations: 66 [32-93], p=0.006; QPD: 59 [41-91], p=0.005). Plasma FV levels were similar in all subjects (µg/ml PPP, median [range]: controls: 7.7 [6.3-10.7]; QPD: 8.4 [7.0-10.2], p=0.33; other PFD: 7.5 [5.4-12], p=0.74) and showed no significant association to TG endpoints for PPP or PRP (p values ≥0.14). Only QPD subjects had platelet FV deficiency (µg FV/mg platelet protein, median [range]: controls: 0.89 [0.63-1.54]; QPD: 0.35 [0.18-0.46], p<0.001; other PFD: 0.83 [0.47-1.75], p=0.48; RUNX1 mutation subgroup: 0.73 [0.50-1.53], p=0.41). In QPD, but not other participants, platelet FV showed a significant association to ETP (R2=0.81, p=0.04) and peak TG endpoints (R2=0.88, p=0.01).Our study illustrates that platelet-dependent thrombin generation, evaluated by CAT, is abnormal in QPD but normal in many uncharacterized PFD with defective aggregation and/or DGD except for the important subgroup with pathogenic RUNX1 mutations, which impair TG but do not reduce platelet FV (unlike QPD). In QPD, the platelet-dependent TG defect shows a unique relationship to the platelet FV deficiency, which suggests that the normal levels of plasma FV are insufficient to compensate for the platelet procoagulant abnormalities in this PFD. DisclosuresNo relevant conflicts of interest to declare.

Human cathepsins K, L, and S: Related proteases, but unique fibrinolytic activity

BackgroundFibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described. MethodsFibrin gels were polymerized and incubated with recombinant human cathepsins (cat) K, L, or S, or plasmin, for dose-dependent and time-dependent studies. Dissolution of fibrin gels was imaged. SDS-PAGE was used to resolve cleaved fragments released from fibrin gels and remnant insoluble fibrin gel that was solubilized prior to electrophoresis to assess fibrin α, β, and γ polypeptide hydrolysis by cathepsins. Multiplex cathepsin zymography determined active amounts of cathepsins remaining. ResultsThere was significant loss of α and β fibrin polypeptides after incubation with cathepsins, with catS completely dissolving fibrin gel by 24 h. Binding to fibrin stabilized catL active time; it associated with cleaved fibrin fragments of multiple sizes. This was not observed for catK or S. CatS also remained active for longer times during fibrin incubation, but its association/binding did not withstand SDS-PAGE preparation. ConclusionsHuman cathepsins K, L, and S are fibrinolytic, and specifically can degrade the α and β fibrin polypeptide chains, generating fragments unique from plasmin. General significanceDemonstration of cathepsins K, L, and S fibrinolytic activity leads to further investigation of contributory roles in disrupting vascular hemostasis, or breakdown of fibrin-based engineered vascular constructs where non-plasmin mediated fibrinolysis must be considered.

Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.

Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin. We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain. Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.

Plasmin activity and proteolysis in milk protein ingredients

Despite the widespread use of milk protein ingredients and the well-known detrimental effects of plasmin-induced casein hydrolysis on product flavour and stability, little research has been carried out on the occurrence and activity of plasmin in milk protein ingredients. In this study, 19 sodium caseinate (NaCas), 13 calcium caseinate (CaCas), 2 micellar casein isolate (MCI) and 14 milk protein concentrate (MPC) samples were studied for plasmin and plasminogen-derived activity and proteolysis after reconstitution. Results indicated a higher occurrence and activity of plasmin in MPC and MCI than in NaCas and CaCas. During storage, activation of plasminogen to plasmin and autolysis of plasmin were observed. Furthermore, extensive plasmin-induced casein hydrolysis was observed. The specificity of casein hydrolysis was similar in all samples and proteolysis per unit plasmin activity was most extensive in MPC. These results indicate that residual plasmin activity should be a factor of consideration in milk protein ingredient selection.

Soluble urokinase receptor conjugated to carrier red blood cells binds latent pro-urokinase and alters its functional profile

Coupling plasminogen activators to carrier red blood cells (RBC) prolongs their life-time in the circulation and restricts extravascular side effects, thereby allowing their utility for short-term thromboprophylaxis. Unlike constitutively active plasminogen activators, single chain urokinase plasminogen activator (scuPA) is activated by plasmin proteolysis or binding to its receptor, uPAR. In this study we conjugated recombinant soluble uPAR (suPAR) to rat RBC, forming RBC/suPAR complex. RBC carrying suPAR circulated in rats similarly to naïve RBC and markedly prolonged the circulation time of suPAR. RBC/suPAR carrying ~ 3 × 10 4 suPAR molecules per RBC specifically bound up to 2 × 10 4 molecules of scuPA, retained ~ 75% of scuPA-binding capacity after circulation in rats and markedly altered the functional profile of bound scuPA. RBC carrying directly conjugated scuPA adhered to endothelial cells, while showing no appreciable fibrinolytic activity. In contrast, RBC/suPAR loaded with scuPA did not exhibit increased adhesion to endothelium, while effectively dissolving fibrin clots. This molecular design, capitalizing on unique biological features of the interaction of scuPA with its receptor, provides a promising modality to deliver a pro-drug for prevention of thrombosis.

D005 Proteolysis of angiogenic factors associated with intraplaque hemorrhages in human atherothrombotic plaques

Increased permeability of, and bleeding from, microvessels within the atheromatous arterial wall participate in atherothrombosis progression. The VEGF family and angiopoietin system components govern microvessel development and maturation. Serine protease activities are proportional to atherothrombosis progression and linked to intraplaque hemorrhage. Here we investigated the relationship between angiogenic factors and proteases in human hemorrhagic and non-hemorrhagic atheromatous carotid samples by immunohistochemistry, in situ hybridization and ELISA. Plaque activity of plasmin and leukocyte elastase and proteolysis of angiogenic factors by plaque extracts were determined. Smooth muscle cell migration induced by hemorrhagic and nonhemorrhagic plaques was assessed. The absence of á-actin-positive cells characterized microvessels in hemorrhagic areas in spite of similar expression of VEGF and angiopoietin system components in microvessels of hemorrhagic and non-hemorrhagic areas. However, VEGF, PlGF and angiopoietin-1 levels were signifi cantly decreased in hemorrhagic compared to non-hemorrhagic plaques, whereas angiopoietin-2 did not change and soluble Tie-2 levels increased. Consequently, smooth muscle cell migration stimulatory activity of hemorrhagic plaques was reduced. Recombinant PlGF, VEGF and angiopoietin-1 added to plaque extracts were only degraded by hemorrhagic lesions. This proteolysis was prevented by inhibitors of plasmin and elastase, whose activity was increased in hemorrhagic plaques. No degradation was observed for angiopoietin-2. Decreased angiogenic factor levels caused by proteolysis may destabilize plaque microvessels via impairment of mural cell recruitment, thus leading to a vicious circle of intraplaque hemorrhages and lesion progression.

Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease

Amyloid beta-peptide (Abeta) accumulation leads to neurodegeneration and Alzheimer disease; however, amyloid metabolism is a dynamic process and enzymic mechanisms exist for Abeta removal. Considerable controversy surrounds whether the intracellular domain of the amyloid precursor protein (AICD) regulates expression of the Abeta-degrading metalloprotease, neprilysin (NEP). By comparing two neuroblastoma cell lines differing substantially in NEP expression, we show by chromatin immunoprecipitation (ChIP) that AICD is bound directly to the NEP promoter in high NEP-expresser (NB7) cells but not in low-expresser (SH-SY5Y) cells. The methylation status of the NEP promoter does not regulate expression in these cells, whereas the histone deacetylase inhibitors trichostatin A and valproate partly restore NEP expression and activity in SH-SY5Y cells. ChIP analysis also reveals AICD binding to the NEP promoter in rat primary neurons but not in HUVEC cells. Chromatin remodelling of crucial Alzheimer disease-related genes by valproate could provide a new therapeutic strategy.

Enhanced clearance of Aβ in brain by sustaining the plasmin proteolysis cascade

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

Plasmin Digestion of Photooxidized Milk Proteins

Plasmin-mediated hydrolysis of 6 different milk protein preparations [αS-casein (αS1 + αS2), β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of αS-casein, consisting of a mixture of αS1- and αS2-casein and a preparation of β-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.

The effect of plasminogen activator inhibitor type 1 on apoptosis

Plasminogen activator inhibitor type-1 (PAI-1), an inhibitor of plasminogen activators, inhibits formation of plasmin and plasmin-mediated proteolysis. Apoptosis, or programmed cell death, is a potentially important phenomenon in mediating overall cell death. This review focuses on the influence of PAI-1 on apoptosis. Greater expression of PAI-1 has been associated with increased survival of cells and resistance to apoptosis. PAI-1 appears to influence apoptosis by decreasing cell adhesion (anoikis) as well as its effect on intracellular signaling. Mechanisms by which PAI-1 may render a cell resistant to apoptosis include its ability to inhibit generation of plasmin, its ability to inhibit caspase 3, and its ability to inhibit cell adhesion mediated by vitronectin. Inhibition of caspase 3 by PAI-1 may divert intracellular signalling from induction of apoptosis to induction of proliferation.

Changes in fibrinogen and fibrin induced by a peptide analog of fibrinogen gamma365-380.

The effects of synthetic peptides with sequences derived from the gamma-chain of fibrinogen on the functional properties of fibrinogen and fibrin were investigated. Methods included thrombelastography, clot turbidity measurement, clot elasticity measurement, platelet aggregation, and scanning transmission electron microscopy (STEM). Peptide gamma369-380 (NH(2)-WATWKTRWYSMK-COOH) showed the greatest impact on fibrin structure, compared with the 76 other overlapping dodecapeptides. Addition of this peptide, or peptide gamma365-380 (NH(2)-NGIIWATKTREWYSMK-COOH) to a mixture of fibrinogen and thrombin resulted a shorter clotting time, higher clot turbidity, lower clot elastic modulus, a higher degree of D-trimer and D-tetramer formation, and impaired plasmin proteolysis of the clot. In STEM, fibrin formed in the presence of peptide gamma369-380 consisted of a more extensive array of linear fibrils typically consisting of 20 or more molecules. Fibrils were better organized than those from non-peptide containing mixtures. Replacement of the tryptophan residue gamma376 massively reduced the effect of the peptide on fibrin structure. Binding of the peptide to fibrinogen induces conformational changes, which result in accelerated clotting and increased lateral association of fibrin protofibrils. The results imply a relevant functional role of sites interacting with peptide gamma369-380 region in the fibrinogen molecule.

Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site.

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.

Distribution of the Tissue Plasminogen Activator (t‐PA) Promotor to Perivascular Sympathetic Nerves and Other Neural Crest Derivatives: Evidence of a Dispersed Plasmin Proteolysis

Distribution of the Tissue Plasminogen Activator (t‐PA) Promotor to Perivascular Sympathetic Nerves and Other Neural Crest Derivatives: Evidence of a Dispersed Plasmin Proteolysis