Plasminogen Activator Inhibitor Type 1 Deficiency Research Articles

Fibrinolysis-Related Bleeding Troubles

An intricate enzymatic process called fibrinolysis is used to eliminate blood clots and stop vascular clogging. Numerous cofactors that make up the fibrinolytic system regulate fibrin breakdown and uphold the hemostatic balance. Different disease events that promote prothrombotic or hemorrhagic states based on the form of aberration are linked to the dysregulation of fibrinolysis. This review is centered on fibrinolysis ailments that can be either heritable or acquired and affect both adults and children. A shortage of one of the fibrinolysis inhibitors deficiencies, such as Plasminogen Activator Inhibitor Type 1 (PAI-1) or 2-plasmin inhibitor, or an overabundance of one of the activators, such as tissue-type plasminogen activator or urokinase-type plasminogen activator, can lead in hyperfibrinolytic bleeding. Delayed bleeding following trauma, surgery and dental interventions is a hallmark of fibrinolytic illnesses with a bleeding phenotype. Bleeding in states like menorrhagia and epistaxis, which has significant fibrinolytic activity, is also frequent. The most extreme bleeding episodes are experienced by patients with 2-plasmin inhibitor deficiency. Interestingly, it was found that, particularly in patients with PAI-1 deficiency, hyperfibrinolytic diseases are linked to a high prevalence of obstetric problems such as miscarriage and preterm delivery. Due to ignorance and a lack of reliable diagnostic tools, hyperfibrinolytic diseases are likely to be underdiagnosed. The vast majority of individuals whose bleeding was deemed to be “of unknown origin” may have a hyperfibrinolytic disease. Because these conditions may typically be successfully treated with antifibrinolytic drugs, early detection is crucial.

Fibrinolysis in patients with a mild-to-moderate bleeding tendency of unknown cause

In more than 50% of patients with a mild-to-moderate bleeding tendency, no underlying cause can be identified (bleeding of unknown cause, BUC). Data on parameters of fibrinolysis in BUC are scarce in the literature and reveal discrepant results. It was the aim of this study to investigate increased fibrinolysis as a possible mechanism of BUC. We included 270 patients (227 females, median age 44 years, 25–75th percentile 32–58) with BUC and 98 healthy controls (65 females, median age 47 years, 25–75thpercentile 39–55). Tissue plasminogen activator (tPA-) antigen and activity, plasminogen activator inhibitor type-1 (PAI-1), tPA-PAI-1 complexes, thrombin activatable fibrinolysis inhibitor (TAFI), α2-antiplasmin, and D-dimer were determined. While PAI-1 deficiency was equally frequent in patients with BUC and controls (91/270, 34%, and 33/98, 34%, p = 0.996), tPA activity levels were more often above the detection limit in patients than in controls (103/213, 48%, and 23/98, 23%, p < 0.0001). We found lower levels of tPA-PAI-1 complexes (6.86 (3.99–10.00) and 9.11 (7.17–13.12), p < 0.001) and higher activity of TAFI (18.61 (15.80–22.58) and 17.03 (14.02–20.02), p < 0.001) and α2-antiplasmin (102 (94–109) and 98 (90–106], p = 0.003) in patients compared to controls. Detectable tPA activity (OR 3.02, 95%CI 1.75–5.23, p < 0.0001), higher levels of TAFI (OR 2.57, 95%CI 1.48–4.46, p = 0.0008) and α2-antiplasmin (OR 1.03, 95%CI 1.01–1.05, p = 0.011), and lower levels of tPA-PAI-1 complexes (OR 0.90, 95%CI 0.86–0.95, p < 0.0001) were independently associated with BUC in sex-adjusted logistic regression analyses. We conclude that the fibrinolytic system can play an etiological role for bleeding in patients with BUC.

The Role of Plasminogen Activator Inhibitor Type-1 in Fibrosis.

Extracellular matrix (ECM) deposition during wound healing is a physiological response to an insult. Wound healing becomes deregulated in the setting of chronic injury or long-standing metabolic disease, leading to the accumulation of ECM components and fibrosis. Matrix protein turnover is determined by the rate of synthesis as well as the rate of proteolytic degradation and clearance by matrix metalloproteinases (MMPs). The persistent activation of interstitial myofibroblasts, coupled with defects in matrix proteolysis, ultimately disrupts tissue architecture and leads to biochemical and mechanical organ dysfunction with eventual organ failure. Plasminogen activator inhibitor type-1 (PAI-1) regulates tissue homeostasis and wound healing by inhibiting plasmin-mediated MMP activation. Multiple reports using models of liver, lung, and kidney fibrosis suggest that PAI-1 deficiency or inhibition of PAI-1 activity attenuates fibrosis. The disinhibition of plasmin-mediated MMP activation leads to collagen degradation and its diminished accumulation, resulting in the reduction of fibrotic matrix deposition in these organs. Paradoxically, homozygous deficiency of PAI-1 promotes age-dependent spontaneous cardiac fibrosis, suggesting a protective role for PAI-1 in the heart. It remains unclear whether PAI-1-deficient cardiac fibroblasts have increased proliferative, migratory, or differentiation capabilities, that allow them to overcome increased plasmin and MMP activity and matrix clearance. In this review, we examine the specific roles of PAI-1 in fibrosis of different organs including the lung, liver, kidney, and cardiovascular system.

The obstetric, gynaecological and fertility implications of homozygous PAI‐1 deficiency: single‐centre experience

Complete plasminogen activator inhibitor type 1 (PAI-1) deficiency is an exceedingly rare autosomal recessive bleeding disorder previously identified and reported in a large Old Order Amish (OOA) kindred in Indiana [Fay etal. Blood 1997; 90: 204]. Mouse models suggest that proteolysis via the plasminogen activator/plasmin system plays a crucial role in reproduction including degradation of the follicular wall during ovulation, fertilization, embryo implantation and embryogenesis [Leonardsson etal., Proc Natl Acad Sci USA 1995; 92: 12446]. We report the obstetric, gynaecological and fertility histories of OOA individuals with homozygous PAI-1 deficiency. In this family, there are 10 affected members identified to date ranging in age between 10 and 32years, including seven female patients and three male patients. To date, two women have achieved pregnancies without difficulty; however, they experienced antenatal bleeding and preterm labour. The early initiation and continuation of antifibrinolytic agents, Epsilon-aminocaproic acid or tranexamic acid, during the pregnancy and in the postpartum period, was believed to be successful in preventing major bleeding complications in our patients with complete PAI-1 deficiency.

Plasminogen activator inhibitor type I may contribute to transient, non-specific changes in immunity in the subacute phase of murine tuberculosis

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1−/−) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2, 5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1−/− mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1−/− splenocytes released increased levels of IFN-γ compared to WT. No clear differences were found between PAI-1−/− and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis.

Remarkable extension of PAI-1 half-life surprisingly brings no changes to its structure

Plasminogen activator inhibitor type 1 (PAI-1) is a serpin protein, a natural inhibitor of urokinase (uPA) and tissue plasminogen activators (tPA). By inhibiting uPA it can block growth of the cancer tumors by suppressing angiogenesis, while when acting on tPA in the blood it can avert conversion of plasminogen to plasmin preventing lysis of the clot. Furthermore, blocking PAI-1 activity can protect against thrombosis. Thus PAI-1 makes great impact on human homeostasis and is desirable for clinical application. Wild-type PAI-1 (wt-PAI-1) has a short span of activity with a t1/2 of ~2 h, being spontaneously converted into a latent form. An enormous effort has been made to create a more stable molecule with >600 PAI-1 variants constructed to study its structure-function relationship. In the present study, we evaluate the structure of the active recombinant VLHL-PAI-1 (very long half life, active >700 h) which is glycosylated similarly to wt-PAI-1 at N232 and N288, with the extended reactive center loop, intact engineered -S-S-bridge (Q174C, G323C) that precludes latency without affecting structure, and can be controlled by a reducing agent to terminate activity at will. We have already proven its usefulness to control cancer in human cancer cells, as well as preventing clot lysis in human whole blood and plasma and in a mouse model. Our results demonstrate the potential therapeutic applications (topical or systemic) of this protein in the treatment of cancer, for the trauma patients to ward off an excessive blood loss, or for people with the PAI-1 deficiency, especially during surgery.

Plasminogen activator inhibitor type-1 deficiency exaggerates LPS-induced acute lung injury through enhancing Toll-like receptor 4 signaling pathway

Mice lacking plasminogen activator inhibitor-1 (PAI-1) did not affect lung injury induced by gram-positive bacteria pneumococcal pneumonia but worsened lung injury induced by gram-negative bacteria Klebsiella. The exact mechanisms have not been completely elucidated. In this study, we examined the signaling pathway of Toll-like receptor 4 (TLR4) with/without PAI-1 in acute lung injury (ALI) induced by lipopolysaccharides (LPS) in mice. PAI-1 knockout mice (n=60) and wild-type mice (n=60) were exposed to LPS intratracheal instillation. Different groups of mice were then sacrificed at 0 and 8 h after LPS instillation. PAI-1-/- mice showed increased excess lung water and elevated cytokines production and release. In addition, expression of TLR4 was up-regulated and the phosphorylation activation of extracellular regulating kinase (ERK) and c-Jun N-terminal kinase (JNK) were also increased in PAI-1 knockout mice compared to wild-type mice. Inversely, interleukin (IL)-1 receptor-associated kinase-M (IRAK-M) and suppressor of cytokine signaling 1 (SOCS1) were both significantly reduced in PAI-1-/-mice after LPS challenge. PAI-1 deletion increased lung injury induced by LPS through up-regulation of TLR4, ERK and C-JNK and down-regulation of TLR4 negative regulators.

PAI-1: An Integrator of Cell Signaling and Migration

Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.

Systemic or topical application of plasminogen activator inhibitor with extended half-life (VLHL PAI-1) reduces bleeding time and total blood loss

Civilian and military trauma patients consist of a disproportional number of young people, causing a considerable burden to society in terms of disability and premature death. Hemorrhage is a leading cause of mortality in this group of patients and the novel methods to reduce bleeding would be welcomed. Management of bleeding following major trauma includes hemostatic agents that offer effective clotting. However a very limited number of agents control secondary bleeding triggered by lysis of the clot. Fibrinolysis depends on the balance between tissue plasminogen activator (tPA), activating plasminogen to plasmin initiating fibrinolysis, and plasminogen activator inhibitor type 1 (PAI-1) inhibiting tPA and preventing lysis. The drugs available on the market that prevent the activation of plasminogen have been used successfully, but have some side effects and limited efficacy for the control of localized bleeding in the surgical setting. Inhibitors of tPA, initiator of clot fibrinolysis, have not yet found their way into the clinical arena. Plasminogen activator inhibitor-1, the major specific inhibitor of tPA, can be used to limit fibrinolysis. Unfortunately, PAI-1 has a short half-life of approximately 2 h and is rapidly converted to the latent form. A recombinant PAI-1 with very long half-life developed in our laboratory (a two-point mutant, VLHL PAI-1, half-life over 700 h) has clinical potential as an agent to promote hemostasis in several scenarios including surgical injury, trauma, and PAI-1 deficiency. Here we report testing of VLHL PAI-1 as a potent inactivator of fibrinolysis reducing total blood loss while applied systemically or topically in experimental animals. The very long half-life of VLHL PAI-1 may provide an advantage in the important physiological mechanism to protect clots from premature dissolution, when applied topically or systemically to prevent excessive bleeding in the surgical and trauma setting and possibly in PAI-1 deficient patients.

Plasminogen activator inhibitor type 1 deficiency

Plasminogen activator inhibitor type 1 (PAI-1) is an important component of the coagulation system that down-regulates fibrinolysis in the circulation. Reduced PAI-1 levels may result in increased fibrinolysis and an associated bleeding diathesis. Clear documentation of PAI-1 deficiency as a cause of a bleeding disorder has been rare. PAI-1 was initially identified in the 1980s, and the first reported case of PAI-1 deficiency appeared in 1989. Several reports followed, although only two identified an underlying genetic defect. These reports of PAI-1 deficiency suggest that affected individuals exhibit mild to moderate bleeding symptoms, including epistaxis, menorrhagia, and delayed bleeding after trauma or surgical procedures. Affected individuals rarely exhibit spontaneous bleeding events commonly seen in other procoagulant deficiencies. The majority of bleeding events are controlled with antifibrinolytic agents, such as tranexamic acid and epsilon-aminocaproic acid. A major issue that contributes to difficulty in establishing an accurate diagnosis of PAI-1 deficiency is that the activity assay is accurate in detection of elevated levels but not at the lowest range. Reported normal ranges begin at zero, thereby making a deficiency state because of a dysproteinaemia difficult to distinguish from that of a normal unaffected individual. Although the antigen assay may be helpful in some circumstances, it assists only with complete quantitative disorders. Because of lack of standardized commercially available PAI-1 activity assay sensitive in the lowest range, the true prevalence of this rare condition has not been established.

Bleeding Complications and Pregnancies in an Established Plasminogen Activator Inhibitor, Type-1 (PAI-1) Deficient Population.

In 1992, a young Amish girl with marked post-procedural bleeding was found to have a PAI-1 deficiency with a homozygous frame shift mutation in exon 4 of the PAI-1 gene that results in a non-functional protein. This mutation was tracked within the ancestors of this Amish community and two individuals were identified that likely introduced this gene into the community. In 1997, seven children (including the proband) from two families within the kindred were described with symptoms consistent with a mild bleeding disorder. Two further homozygous children, one from a third family within the kindred, have subsequently been identified. Presently, over 300 members of this kindred have been tested with nearly 100 identified heterozygotes, none of whom have experienced bleeding complications. The homozygotes all have had bleeding problems with injury a common cause. Five of the nine homozygotes have had head trauma, resulting in significant bleeding complications. ε-aminocaproic acid (EACA) initiated early controls bleeding. One child had a recurrence of his subdural hematoma when he discontinued the EACA upon discharge from the hospital. But the bleeding was subsequently controlled with reinstitution of EACA, and the child had a full recovery. Another common bleeding complication is menorrhagia with all homozygous females experiencing this symptom, and two even requiring red blood transfusions due to severe anemia. Currently, these women require concurrent EACA with onset of menses to control bleeding. Of interest PAI-1 and the plasminogen activation system have been shown to play an important role in embryo implantation, and it was unknown whether homozygous PAI-1 deficient women would be able to conceive and maintain a pregnancy. We now report 2 of these homozygous deficient females have become pregnant; one remains pregnant at 28 weeks gestation, and one delivered at 32 weeks. This child represents that first documented birth to a woman with complete PAI-1 deficiency. The mother experienced abnormal cervical bleeding beginning at 27 weeks requiring EACA therapy which was continued until delivery and for about 7 days post-partum without abnormal bleeding at delivery or in the post-partum period, and without ill effect on the fetus. This cohort is the largest group of PAI-1 deficient patients ever documented. The molecular defect is well defined so that it is simple to differentiate normals from heterozygotes and homozygotes. Continued follow-up will allow further understanding of the role of PAI-1 in human physiology.

Gender-dependent physiological implications of combined PAI-1 and TIMP-1 gene deficiency characterized in a mouse model

The endogenous proteinase inhibitors plasminogen activator inhibitor type 1 (PAI-1) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) are two distinct proteins with separate molecular pathways. However, a close relationship between PAI-1 and TIMP-1 has been proposed indicating some degree of functional overlap due to their involvement in ECM turnover, tissue remodeling, and cellular migration and signaling. To study the housekeeping physiological implications of PAI-1 and TIMP-1, we generated a combined PAI-1 and TIMP-1 gene-deficient mouse model. We present the results on generating this specific mouse model with particular emphasis on phenotypical characteristics, blood leukocyte counts, histology, and gene expression studies of PAI-1 and TIMP-1 in various organs. We observed a significant deviation in segregation of offspring only in male mice (P < 0.01) predominantly caused by PAI-1 deficiency. In addition, the body weight in 3- and 20-wk-old male and 20-wk-old female mice was significantly different between genotypes (P <or= 0.0008). Furthermore, blood leukocyte counts were significantly different between genotypes in 20-wk-old male mice (P <or= 0.0002), whereas no significant differences were observed between genotypes in 20-wk-old female mice (P >or= 0.13). Quantifying the relative expression of PAI-1 and TIMP-1 revealed upregulation of PAI-1 (P < 0.001) in male mice only. Our data highlight the complex roles of PAI-1 and TIMP-1 on physiological parameters such as segregation of offspring (embryonic development and survival), body weight (metabolism), blood leukocyte counts (immunity), and gene expression (regulatory redundancy). We conclude that PAI-1 and TIMP-1 seem to possess gender-dependent regulatory properties on various housekeeping physiological parameters and stress the potential implications in pathological conditions.

The role of plasminogen activator inhibitor type 1 in the inflammatory response to local tissue injury

The plasma levels of the plasminogen activator-inhibitor type 1 (PAI-1) are consistently elevated in patients with sterile tissue injury, often accompanied by a systemic acute phase protein response. It remains unknown, however, whether and to what extent PAI-1 affects the host response to trauma. By using the well-established murine model of turpentine-induced tissue injury we compared local and systemic inflammatory responses in PAI-1 gene-deficient (PAI-1-/-) and normal wild-type (Wt) mice. Subcutaneous turpentine injection elicited strong increases in PAI-1 protein concentration in plasma and at the site of injury, but not in liver. PAI-1 mRNA was locally increased and expressed mainly by macrophages and endothelial cells. PAI-1 deficiency greatly enhanced the early influx of neutrophils to the site of inflammation, which was associated with increased edema and necrosis at 8 h after injection. Furthermore, PAI-1-/- mice showed a reduced early interleukin (IL)-6 induction with subsequently lower acute phase protein levels and a much slower recovery of body weight loss. These findings suggest that PAI-1 is not merely a marker of tissue injury but plays a functional role in the local and systemic host response to trauma.

Recombinant plasminogen activator inhibitor type 1

Plasminogen activator inhibitor type 1 (PAI-1), a member of the serpin family of serine protease inhibitors, inhibits both tissue-type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). High PAI-1 levels are associated with an increased risk of thromboembolic disease while PAI-1 deficiency may represent an inherited autosomal recessive bleeding disorder. This review describes the biochemistry of PAI-1 including its purification, conversion between active and latent forms, and interaction with its target serine proteases and its protein cofactor, vitronectin. In addition, an overview of animal studies with PAI-1 is presented to examine its role in regulating fibrinolysis in vivo. For this review, particular emphasis is placed on studies with a recombinant form of bacterially expressed PAI-1 (rPAI-1), which shares many features in common with the active form of native PAI-1.