Plasmin Prevents Dystrophic Calcification After Muscle Injury.

Extensive or persistent calcium phosphate deposition within soft tissues after severe traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood; consequently, current treatments are suboptimal. Here, we show that plasmin protease activity prevents dystrophic calcification within injured skeletal muscle independent of its canonical fibrinolytic function. After muscle injury, dystrophic calcifications either can be resorbed during the process of tissue healing, persist, or become organized into mature bone (HO). Without sufficient plasmin activity, dystrophic calcifications persist after muscle injury and are sufficient to induce HO. Downregulating the primary inhibitor of plasmin (α2-antiplasmin) or treating with pyrophosphate analogues prevents dystrophic calcification and subsequent HO in vivo. Because plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration. © 2016 American Society for Bone and Mineral Research.

Plasmin deficiency leads to fibrin accumulation and a compromised inflammatory response in the mouse brain.

BACKGROUND: Excess fibrin in blood vessels is cleared by plasmin, the key proteolytic enzyme in fibrinolysis. Neurological disorders and head trauma can result in the disruption of the neurovasculature and the entry of fibrin and other blood components into the brain, which may contribute to further neurological dysfunction. OBJECTIVES: While chronic fibrin deposition is often implicated in neurological disorders, the pathological contributions attributable specifically to fibrin have been difficult to ascertain. An animal model that spontaneously acquires fibrin deposits could allow researchers to better understand the impact of fibrin in neurological disorders. METHODS: Brains of plasminogen (plg)- and tissue plasminogen activator (tPA)-deficient mice were examined and characterized with regard to fibrin accumulation, vascular and neuronal health, and inflammation. Furthermore, the inflammatory response following intrahippocampal lipopolysaccharide (LPS) injection was compared between plg(-/-) and wild type (WT) mice. RESULTS AND CONCLUSIONS: Both plg(-/-) and tPA(-/-) mice exhibited brain parenchymal fibrin deposits that appear to result from reduced neurovascular integrity. Markers of neuronal health and inflammation were not significantly affected by proximity to the vascular lesions. A compromised neuroinflammatory response was also observed in plg(-/-) compared to WT mice following intrahippocampal LPS injection. These results demonstrate that fibrin does not affect neuronal health in the absence of inflammation and suggest that plasmin may be necessary for a normal neuroinflammatory response in the mouse CNS.

Role of copper in human neurological disorders.

Copper is a trace element present in all tissues and is required for cellular respiration, peptide amidation, neurotransmitter biosynthesis, pigment formation, and connective tissue strength. Copper is a cofactor for numerous enzymes and plays an important role in central nervous system development; low concentrations of copper may result in incomplete development, whereas excess copper maybe injurious. Copper may be involved in free radical production, via the Haber-Weiss reaction, that results in mitochondrial damage, DNA breakage, and neuronal injury. Evidence of abnormal copper transport and aberrant copper-protein interactions in numerous human neurological disorders supports the critical importance of this trace metal for proper neurodevelopment and neurological function. The biochemical phenotypes of human disorders that involve copper homeostasis suggest possible biomarkers of copper status that may be applicable to general populations.

Functions for proteinases in the ovulatory process.

The ovary is a unique and dynamic organ in respect to rapid and extensive degrees of tissue development and remodeling that are periodically repeated in the female reproductive activity. Ovulation is a directed and sequential process accompanied by broad-spectrum proteolysis and culminates in the follicular rupture to release the matured oocyte. This review will focus on the potential roles of six representative proteinases that are involved in various aspects of ovulatory processes: matrix metalloproteinases (MMPs), plasminogen activator (PA)/plasmin, a disintegrin and metalloproteinase domain with thrombospondin motif (ADAMTS), cathepsin-L, pregnancy-associated plasma protein-A (PAPP-A), and bone morphogenetic protein 1/mammalian Tolloid (BMP-1/mTld). Based on the studies of expression and function, these selected proteinases provide and share diverse functions ranging from cleaving components of the extracellular matrix (ECM) to modulating non-ECM molecules, such as various growth factors and their binding proteins. Consistently, the genetic deletion of each individual gene in mice shows their functional overlap in the reproductive activity.

Plasmin deficiency does not alter endogenous murine amyloid beta levels in mice.

Deposition of amyloid beta (A beta) into extracellular plaques is a pathologic characteristic of Alzheimer's disease. Plasmin, neprilysin, endothelin-converting enzyme and insulin-degrading enzyme (IDE) have each been implicated in A beta degradation; data supporting the role of the latter three enzymes have included increased levels of endogenous murine A beta in mice genetically deficient for the respective enzyme. In this study, we sought to determine if plasminogen deficiency increases endogenous A beta. We report that plasminogen deficiency did not result in an A beta increase in the brain or in the plasma of adult mice. Hence, although plasmin is potentially important in the degradation of A beta aggregates, we interpret these data as suggesting that plasmin does not regulate steady-state A beta levels in non-pathologic conditions.

Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer.

During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the beta-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system.

BACKGROUND/AIMS: The regeneration after liver injury is regulated by the release and activation of several growth factors. The role of the plasmin/alpha(2)-antiplasmin (alpha(2)-AP) system in liver regeneration was investigated. METHODS: CCl(4) was injected intraperitoneally into the mice deficient (-/-) in fibrinolytic factors: alpha(2)-AP-/-, plasminogen (Plg) -/-, and Plg-/-.alpha(2)-AP-/-, and wild-type (WT) mice. The liver tissue was examined for its microscopic appearance, fibrinolytic activity, and fibronectin levels. RESULTS: In the gene deficient and WT mice, the livers exhibited the same extent of necrosis 2 days after the CCl(4) injection. The livers of the WT mice normalized after 7 days, and the alpha(2)-AP-/- mice normalized after 5 days. In contrast, the livers of the Plg-/- and Plg-/-.alpha(2)-AP-/- mice remained in the damaged state until 14 days after the liver injury. The injection of anti-alpha(2)-AP antibody in the WT mice improved the regeneration after the liver injury, and the injection of tranexamic acid in the alpha(2)-AP-/- mice reduced. CONCLUSIONS: These results suggest that the plasmin/alpha(2)-AP system played an important role in hepatic repair via clearance from the injury area.

Plasmin deficiency in Alzheimer's disease brains: causal or casual?

Substantial recent evidence suggests that defects in amyloid peptide degradation can be at the base of cases of sporadic Alzheimer's disease (AD). Among the discovered brain enzymes with the capacity to degrade amyloid peptide, the serine protease plasmin acquires special physiological relevance because of its low levels in areas of AD human brains with a high susceptibility to amyloid plaque accumulation. In this article we comment on a series of observations supporting the fact that plasmin paucity in the brain is not simply a secondary event in the disease but rather a primary defect in certain cases of sporadic AD. We also refer to recent data pointing to alterations in raft membrane domains and diminished membrane cholesterol as the underlying cause. Finally, we discuss the possibility that plasmin deficiency in the brain could lead to AD symptomatology because of amyloid aggregation and the triggering of cell death signaling cascades.

[The fibrinolysis system in recurrent myocardial infarction]. / Sistema fibrinoliza pri povtornykh infarktakh miokarda.

Studies of blood fibrinolytic activity in 112 patients with repeated acute myocardial infarction or injury to the myocardium have revealed reduced fibrinolytic activity on non-heated fibrin plates, decreased plasmin activity and euglobulin+ fraction lysis, lowered levels of plasminogen activator, total nonenzymic fibrinolysis, antithrombin III, ++FFDP, and elevated soluble complexes of fibrin-monomer level. Complications of myocardial infarction presenting as thromboembolism; ciliary arrhythmia, chronic aneurysm with thrombosis are associated with still more marked disorders of the fibrinolysis system.

Open-heart surgery in a patient with heterozygous alpha 2-antiplasmin deficiency. Perioperative strategies in the first reported case.

alpha 2-Antiplasmin deficiency is a serious coagulation disorder that results in unrestrained fibrinolytic activity. Clinically, it is manifested by instability of the fibrin hemostatic plug and prolonged or delayed bleeding, which is more serious in patients who are homozygous for this trait. A patient scheduled for aortic valve replacement and coronary bypass presented with a history of repeated episodes of postoperative bleeding. Hemostatic laboratory evaluation revealed that the patient had the heterozygous form of alpha 2-antiplasmin deficiency with a serum concentration of 52 percent (normal, greater than 65 percent of the activity of pooled plasma). He underwent preoperative plasmapheresis with administration of 3,000 ml of fresh frozen plasma, which resulted in an increase in the preoperative level of alpha 2-antiplasmin to 78 percent. Although postoperative blood loss was greater than normal, it was easily managed. Preoperative identification of this rare coagulation abnormality permitted appropriate treatment and probably prevented a postoperative death from hemorrhage.

Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI-Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor.

The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3' end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H-sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

[Distribution of the causes of congenital thrombophilia]. / A veleszületett thrombosis-hajlam (thrombophilia) okainak megoszlása.

In recent years there have been discovered more and more such connatal mostly hereditary coagulopathies, which can explain the thrombosis susceptibility of the given individual or/and the family. The International Thrombosis and Haemostasis Society made a survey to estimate the frequency of those defects causing thrombophilias. In this survey the authors analysed the cases of their patients according to the given points of view. In their work they discuss some theoretical and practical problems of the theme, which can have an importance in respect to the everyday medical practice.

Fibrinolysis in normal plasma and blood: evidence for significant mechanisms independent of the plasminogen-plasmin system.

Fibrinolytic activity of normal plasma and blood has been measured by 125l-fibrin solid phase assay. Activity of plasma is not affected by removal of plasminogenplasmin by affinity chromatography. Activities of euglobulin and pseudoglobulin fractions are approximately equal. epsilon-aminocaproic acid (EACA) (10 mM), tranexamic acid (10 mM), diisopropylfluorophosphate (DFP, 50 mM), and soybean and lima bean trypsin inhibitors (100 mug/ml) do not inhibit plasma activity at concentrations that inhibit pure plasmin and urokinase-activated plasma. Activity is not affected by glass contact and is not inhibited by inhibitors of contact or enzymatic activation of Hageman factor (hexadimethrine bromide, 100 mug/ml; cytochrome C, 250 mug/ml; spermidine, 2 mM; phenylmethylsulfonylfluoride, 1 mM). It is inhibited partially (30%-40%) by heating (56 degrees C, 30 min) and by zymosan (2.5 mg/ml; 40%-90% inhibition), and is increased by hydrazine (20 mM), salicylaldoxime (20 mM), DFP (50 mM), and tosyl-L-arginine methyl ester (TAMe, 10 mM)-the latter two at concentrations known to inhibit Cls of the classic, and factor D of the alternate complement pathways. Increase fibrinolytic activity with TAMe is associated with reciprocal decrease in classic and alternate complement pathway activity. It is concluded that normal plasma fibrinolytic activity is relatively independent of plasmin as the ultimate fibrinolytic enzyme, that Hageman factor-dependent pathways are of minor importance, and that significant heat-stable and heat-labile nonplasmin fibrinolytic activities are operative. These may include proteinases involved in complement activation, and in common control of classic and alternate complement pathways, as well as other nonplasmin proteinases.