Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model.

BACKGROUND: Low level laser therapy (LLLT) has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK) is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration) and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A(1) (Control-saline), A(2) (Carrageenan-only), A(3) (laser 660 nm only) and A(4) (Carrageenan + laser 660 nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660 nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.

Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model / Inibição da expressão de RNA mensageiro de pré-calicreínas tecidual e plasmática pela laserterapia em modelo de edema de pata induzido pela carragenina em ratos

BACKGROND: Low level laser therapy (LLLT) has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK) is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration) and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A1 (Control-saline), A2 (Carrageenan-only), A3 (laser 660nm only) and A4 (Carrageenan + laser 660nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.

CONTEXTUALIZAÇÃO: A laserterapia de baixa potência tem sido usada para o tratamento de processos inflamatórios diversos em que a calicreína tecidual e a plasmática possuem participação ativa. A pré-calicreína plasmática (PPK) é sintetizada pelos hepatócitos e secretada na corrente sanguínea, onde participa da ativação da coagulação, fibrinólise, geração de cininas e inflamação. A pré-calicreína tecidual está associada com importantes doenças (incluindo câncer, inflamação e neurodegeneração) e tem sido utilizada ou sugerida clinicamente como importante biomarcador ou alvo terapêutico. OBJETIVO: Avaliar se a laserterapia altera a expressão gênica da pré-calicreína tecidual e da plasmática no modelo de inflamação aguda induzida pela carragenina. MÉTODOS: Quarenta ratos foram separados em quatro grupos experimentais: A1 (controle), A2 (carragenina, apenas), A3 (laser 660nm, apenas) e A4 (Carragenina + laser 660nm). O edema foi medido por um pletismômetro. Tecido subplantar foi coletado para a quantificação de RNA mensageiro (RNAm) de pré-calicreínas tecidual e plasmática por PCR em tempo real. RESULTADOS: Observou-se uma diminuição significativa no volume de edema após irradiação com laser 660nm. A expressão de RNAm de pré-calicreínas tecidual e plasmática aumentou após a inoculação de carragenina, entretanto a expressão gênica das pré-calicreínas diminuiu significantemente após laserterapia de baixa potência de 660nm. CONCLUSÃO: Os resultados sugerem que a expressão de RNAm das pré-calicreínas tecidual e plasmática é modulada pela laserterapia de baixa potência, podendo ser alvo terapêutico para tratamento de processos inflamatórios.

[Study of kini-kallikrein and renin--angiotensin systems in patients with primary open angle glaucoma]. / Studiul sistemelor biochimice kinina-kalikreina si renina--angiotensina în glaucomul primitiv cu unghi deschis.

PURPOSE: To evaluate the activity of Renin-Angiotensin (SRA) and Kinin-Kalikrein (SKK) systems in patients with primary open-angle glaucoma (POAG) in tears, blood and aqueous flow. METHODS: Components of SRA and SKK were analysed in the blood, tears and aqueous flow of 38 patients found in different stages of POAG. The samples of aqueous flow were harvested during glaucoma surgery The results were compared to those from a healthy group of patients (for tears and blood) and to a group of normal patiens that had ocular surgery for cataract and high ametropias (for samples af aqueous flow). RESULTS: In patients with high pressure primary open-angle glaucoma, when comparing them to those from the control group, the measurements showed: a high level of angiotensin converting enzyme (ACE) activity (in tears up to 108-125%, p < 0.001 and in aqueous flow up to 40-47% p < 0.001), a high level of kalikrein (in tears up to 21-29%, p < 0.001 and in aqueous flow up to 35-44% p < 0.001) and a high level of the summed activity of prekalikrein +kalikrein (up to 11-14, p < 0.001). A decrease in the prekalikrein/kalikrein ratio was found (up to 21-25% p < 0.01 in tears and aqueous flow) and this decrease was proved to be in direct correlation to the glaucoma stages of evolution. A decrease in prekalikrein activity was also found; up to 7% in the tears for each developing glaucoma stage. After the glaucoma surgery, the levels of ACE activity and kalikrein measured in tears decreased (up to 17% p < 0.001 and 17% p < 0.001 respectively) without reaching the levels in the normal group while the levels of prekalikrein and the prekalikrein/kalikrein ratio grew (up to 7% p < 0.01 and 16% < 0.001 respectively). CONCLUSIONS: The results show a high level of kalikrein and angiotensin converting enzyme (ACE) activity (measured in aqueous flow and tears) when comparing the group with POAG to the normal group. A decrease in prekalikrein activity was found, and the prekalikrein/kalikrein ratio was also low in the aqueous flow and tears. After the glaucoma surgery the levels of ACE activity and kalikrein decreased without reaching the levels in the normal group while the levels of prekalikrein and the prekalikrei/kalikrein ratio grew.

Active plasma kallikrein localizes to mast cells and regulates epithelial cell apoptosis, adipocyte differentiation, and stromal remodeling during mammary gland involution.

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development.

Expression of the plasma prekallikrein gene: utilization of multiple transcription start sites and alternative promoter regions.

The plasma prekallikrein gene is expressed in many different human tissues at distinctly different levels and therefore tissue-specific control of the gene transcription is likely. In this study we demonstrate that transcription of the plasma prekallikrein gene can be initiated at multiple sites, for which at least four different promoters are utilized. A comparison of the genomic and mRNA sequences of mouse plasma prekallikrein revealed that the sequence segment that was formerly regarded as the first exon of the mouse plasma prekallikrein gene consists of three exons, with the first exon localized 14.2 kbp upstream of the translation start. For the rat and human plasma prekallikrein genes, in silico analysis suggested an analogous exon-intron organization. Determination of the transcription start sites showed that in both mouse and human, the proximal and distal regions could be utilized for transcription initiation; however, the proximal region is preferred. A deletion mutation analysis of the proximal promoter region using a 1.7-kbp segment revealed a strong activating region immediately upstream of the known mRNA, followed by both a modest repressor and an enhancer region.

Contact system. New concepts on activation mechanisms and bioregulatory functions.

This review considers the data of recent years concerning the contact system initiating the activation of blood plasma proteolytic systems, such as hemocoagulation, fibrinolysis, kininogenesis, and also complement and angiotensinogenesis. The main proteins of the contact system are the factors XII and XI, prekallikrein, and high-molecular-weight kininogen. The data on the structure, functions, and biosynthesis of these proteins and on their genes are presented. Studies in detail on the protein-protein interactions during formation of the ensemble of the contact system components on the anionic surface resulted in the postulation of the mechanism of activation of this system associated with generation of the XIIa factor and of kallikrein. This mechanism is traditionally considered a trigger of processes for the internal pathway of the hemocoagulating cascade. However, the absence of direct confirmation of such activation in vivo and the absence of hemorrhagia in the deficiency of these components stimulated the studies designed to find another mechanism of their activation and physiological role outside of the hemostasis system. As a result, a new concept on the contact system activation on the endothelial cell membrane was proposed. This concept is based on the isolation of a complex of proteins, which in addition to the above-mentioned proteins includes cytokeratin 1 and the receptors of the urokinase-like plasminogen activator and of the complement q-component. The ideas on the role of this system in the biology of vessels are developed. Some of our findings on the effect of leukocytic elastase on the key components of the contact system are also presented.

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia.

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.

Extra-hepatic transcription of plasma prekallikrein gene in human and rat tissues.

The expression of plasma prekallikrein (PPK) mRNA has been investigated applying reverse transcription, followed by polymerase chain reaction, of mRNA (RT-PCR) in various human and rat tissues. PPK gene transcripts were detected in liver and kidney in both species and, in addition, in human adrenal gland and placenta. No PPK mRNA was identified in rat adrenal gland, heart, aorta, lung, brain cortex and medulla, hypothalamus, and uterus. These results show that PPK gene expression is not restricted to the liver.

[Polysorbates for hemoperfusion. The effect of polysorbate surface modification on morphology, coagulation, fibrinolysis, and kallikrein formation]. / Sorbenty polimerowe do hemoperfuzji. 2. Wplyw modyfikacji powierzchniowej sorbentów polimerowych na morfologie, krzepliwosc, fibrynolize i kalikreinogeneze.

In the work the influence of surface sulfonated polymer sorbents with styrene-divinylbenzene (Vinylsorb SS) modified by amino acids upon the morphological picture of blood and proteins of the coagulation system was tested. Those sorbents do not exert a remarkable influence upon erythrocytes and coagulation proteins but they adsorb blood platelets, leucocytes and they activate prekallikrein system. Vinylsorb SS has the best influence because it adsorbs few platelets and activates prekallikrein system to the least extent.

Thrombin generation during collection of blood from donors taking oral contraceptives.

Thrombin generation, as evidenced by plasma fibrinopeptide A (FPA) concentrations, was studied during blood collection from donors taking oral contraceptives (OC). 450 ml blood were drawn into Fenwal PVC bags from 26 OC users and 28 nonusers. Blood samples for determination of FPA, beta-thromboglobulin (BTG), thrombotest (TT), prekallikrein (PKK), antithrombin-III (AT-III) and factor VIII procoagulant activity (FVIII:C) were drawn from the bags immediately after ending blood donation and following storage for 24 h at 4 degrees C. The FPA concentrations following donation were significantly higher in the OC than in the control group (p less than 0.05). The levels of PKK were also higher in blood obtained from OC users (p less than 0.001), as was the FVIII:C level, the latter difference, however, was not significant (p = 0.06). No cold-promoted activation of factor VII, as evidenced from TT, was detected following storage at 4 degrees C, neither was any change observed in the FPA, PKK and AT-III levels. The BTG concentrations increased significantly during storage, most pronounced in the control group (p less than 0.05). The decay of FVIII:C was similar in the two groups, averaging 24.7%. No correlation was observed between the FPA levels and the other parameters determined. We conclude that thrombin generation is more pronounced during routine blood collection from donors taking OC.

Modulation of the proteolytic cascade systems by high dose corticosteroids.

The effects of high-dose corticosteroids (HDC) on activities within the proteolytic cascade systems were studied in vitro and in vivo using chromogenic peptide substrate assays. In in vitro experiments 20 mg methylprednisolone sodium succinate (Solu-Medrol) per ml plasma significantly inhibited activation of plasma prekallikrein, prothrombin and plasminogen and reduced functional plasma kallikrein inhibition, antithrombin and antiplasmin activities. The effects of HDC on activities within these proteolytic cascade systems were further evaluated in experimental acute pancreatitis in pigs. Acute pancreatitis was induced by injection of Na-taurocholate into the pancreatic duct. Seven test animals received methylprednisolone sodium succinate 30 mg per kg intravenously for 30 minutes before the induction of pancreatitis as pretreatment. Eight animals remained untreated. Trypsin (TRY), plasma prekallikrein (PKK), plasma kallikrein (KK) and functional plasma kallikrein inhibition capacity (KKI) were studied in the peritoneal exudate. Cardiac output (CO) and mean arterial pressure (MAP) were monitored regularly before and during a 6 hour observation period. During untreated pancreatitis a reduction of PKK levels of about 40% were found, paralleled by an increased KK activity and a reduction of KKI capacity. Several of the animals experienced high TRY activities. The mortality rate was 63% (5 out of 8 animals). In the pretreated groups, all animals survived the observation period. CO and MAP were significantly less reduced than the untreated group at 6 hours. HDC was also found to reduce significantly plasma kallikrein activities in the peritoneal exudate compared with untreated animals. No changes in TRY activities were found in pretreated animals. Furthermore, plasma prekallikrein and functional plasma kallikrein inhibition values in the exudate were elevated significantly in HDC treated animals compared with untreated animals.

Acute effect of smoking on fibrinolysis: increase in the activity level of circulating extrinsic (tissue-type) plasminogen activator.

The acute effect of cigarette smoking on the fibrinolytic enzyme system in blood was studied. It was found imperative to have an initial 30 min rest period, after venipuncture, to obtain a stable baseline in the fibrinolytic studies. The average heart rate, in inhaling smokers, increased from 64 to a peak of 79 beats min-1, 5-10 min after commencement of smoking. A peak in fibrinolytic activity was found to occur later, at 22.5 min. Analysis of the increase in fibrinolytic activity revealed no demonstrable activation of intrinsic systems via factor XII, nor changes in plasminogen, prekallikrein and C1-inactivator. No plasmin-alpha 2-antiplasmin complexes were detectable. The increase (P less than 0.01) was found to be due to extrinsic (tissue-type) plasminogen activator, revealed as C1-inactivator-resistant plasminogen activator activity, and further identified by quenching with anti-tissue plasminogen activator IgG. Thus, smoking appears to elicit a significant increase in the level of activity of circulating extrinsic plasminogen activator.

The kinetics of blood coagulability: fibrinolytic and kallikrein-kinin system at the onset and during labor.

The relationship between the kallikrein-kinin system, the coagulation system, and the fibrinolytic system was evaluated on 58 patients during pregnancy, labor and puerperium. Also included in this study were 7 patients with premature placental separation. The most prominent changes were those in the kallikrein-kinin system. After the onset of labor, prekallikrein decreases rapidly which may trigger changes in the blood coagulation and fibrinolytic system. The three systems have a close interrelationship possibly affecting uterine contractility during labor and delivery.

A platelet derived inhibitor of plasma prekallikrein activation.

Platelet products released by ADP mediated platelet aggregation or by repeated freeze-thawing of platelet-rich-plasma caused inhibition of the dextran sulphate induced activation of prekallikrein, measured amidolytically, in subsequently prepared platelet-poor-plasma. Platelet products did not inhibit the amidolytic activity of contact activated-plasma. Addition of antiserum to platelet factor-4 (PF4) neutralized the inhibitory effect of platelet products towards prekallikrein activation. When platelet released products were fractionated by heparin affinity chromatography, 93% of the PF4 applied and 86% of the inhibitory activity of the starting material was recovered in the high-affinity fraction.

Reduced plasma kininogen concentration during sickle cell crisis.

Eight patients with sickle cell anemia (SS hemoglobin) were found to have decreased plasma levels of kininogen compared to normal control subjects. The kininogen level of the patients with sickle cell anemia was decreased further during sickle cell crises. The results suggest that components of the kinin system are profoundly affected in patients with sickle cell anemia, and during crises may play a role in the clinical presentation of patients.

Acute turpentine inflammation and kinin release in rat-paw thermic oedema.

Livers from rats at 2-3 days after s.c. injection of turpentine, when perfused, synthesized prekallikrein nearly 3 times faster than did livers from normal rats. On the other hand paw oedema, produced by heating to 46 degrees, in rats injured in this way was less marked. Likewise in such rats the amount of bradykinin release by 50 min. of coaxial perfusion of the paw was only 13.6 +/- 4.6 compared with 63.1 +/- 13.4 ng in normal rats. A possible explanation for the observed reduction in production of bradykinin may be inhibition of kallikrein due to an increased concentration of alpha 2-macroglobulin.