Prothrombin mRNA Research Articles

The novel FII c.*64_*66del prothrombin gene variant in women with pregnancy loss

Normal pregnancy associated with complex changes of hemostasis, leading to hypercoagulability states. The presence of acquired or genetic prothrombotic risk factors might affect the proper maternal-fetal circulation and result in pregnancy loss. Hence, the screening for the novel prothrombotic variants associated with pregnancy loss would be beneficial. Our aim was to investigate the potential association of recently reported c.*64_*66del variant in prothrombin gene with the etiology of pregnancy loss. Study included 105 women with pregnancy loss and 155 controls. Analyses in patients? plasma samples, as well as in vitro analyses on transfected Cos-7 cell line were performed in order to investigate the mechanism by which this variant could perturb the coagulation and lead to pregnancy loss. Analyses in patients' DNA and plasma samples involved: DNA sequencing and PCR-RFLP assay for detection of FII c.*64_*66del variant, routine thrombophilia screening, thrombin generation assay and Western blot analysis of prothrombin plasma level. In vitro analyses included transient transfections of Cos-7 cell line with wild-type and c.*64_*66del mutated constructs of pCIneo?SV40 expression vector. Real-Time PCR and Western blot analysis were used to determine the effect of FII c.*64_*66del variant on mRNA and protein level in constructs. Three women in patients group (2.9%) were detected as heterozygous carriers of FII c.*64_*66del, while none was found among controls. The carriers routine thrombophilia parameters were in reference range and similar prothrombin plasma level in FII c.*64_*66del carriers and non-carriers were detected. The endogenous thrombin potential was slightly increased in FII c.*64_*66del carriers compared to control plasma, but this difference was not statistically significant. Results of in vitro analyses showed significantly decreased prothrombin mRNA and protein level for c.*64_*66del variant compared to wild-type. Results of our pilot study have shown a trend of higher prevalence of FII c.*64_*66del variant in women with pregnancy loss. However, further studies are needed to completely elucidate whether FII c.*64_*66del variant affects prothrombin expression during pregnancy and to account its potential role in etiology of pregnancy loss.

The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

Thrombin and the Protease-Activated Receptor-1 in Organophosphate-Induced Status Epilepticus.

Organophosphates (OP) are a major threat to the health of soldiers and civilians due to their use as chemical weapons in war and in terror attacks. Among the acute manifestations of OP poisoning, status epilepticus (SE) is bearing the highest potential for long-term damages. Current therapies do not prevent brain damage and seizure-related brain injuries in OP-exposed humans. Thrombin is a serine protease known to have a fundamental function in the clotting cascade. It is highly expressed in the brain where we have previously found that it regulates synaptic transmission and plasticity. In addition, we have found that an excess of thrombin in the brain leads to hyperexcitability and therefore seizures through a glutamate-dependent mechanism. In the current study, we carried out in vitro, ex vivo, and in vivo experiments in order to determine the role of thrombin and its receptor PAR-1 in paraoxon-induced SE. Elevated thrombin activity was found in the brain slices from mice that were treated (in vitro and in vivo) with paraoxon. Increased levels of PAR-1 and pERK proteins and decreased prothrombin mRNA were found in the brains of paraoxon-treated mice. Furthermore, ex vivo and in vivo electrophysiological experiments showed that exposure to paraoxon causes elevated electrical activity in CA1 and CA3 regions of the hippocampus. Moreover, a specific PAR-1 antagonist (SCH79797) reduced this activity. Altogether, these results reveal the importance of thrombin and PAR-1 in paraoxon poisoning. In addition, the results indicate that thrombin and PAR-1 may be a possible target for the treatment of paraoxon-induced status epilepticus.

VKOR paralog VKORC1L1 supports vitamin K-dependent protein carboxylation in vivo.

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K-dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1-/- mice survived longer (1 week) than Ggcx-/- mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1-/-;Vkorc1l1-/- mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1-/- mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1-/- mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

Background and Aim: Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Methods: Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. Results: The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Conclusions: Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.

Factor X Mutant Zebrafish Tolerate a Severe Hemostatic Defect in Early Development Yet Develop Lethal Hemorrhage in Adulthood

Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis.We generated a 17 base pair deletion in the zebrafish f10 locus by genome editing with TALENs. Although indistinguishable morphologically from f10+/+ and f10+/- siblings at early stages, f10-/- mutants demonstrated progressive lethality between 1 and 5 months of age. Extensive hemorrhage was identified in multiple tissues starting at 3-4 weeks of age, particularly the brain. Notably, intracranial hemorrhage is a common feature in multiple zebrafish mutants with various vascular defects, including anomalies of endothelial differentiation and specification, apoptosis, and vessel integrity/permeability. Gross inspection of f10 mutant embryos and larvae in the first week of life revealed no apparent defects in circulation or vascular development. Expression of arterial and venous endothelial markers were examined by in situ hybridization at 24 and 72 hours post fertilization, the time period during which axial and intersegmental vessels form, along with early establishment of the vascular network. Markers included ephb2a, cdh5, flk1, flt4, and ephb4, and expression patterns were indistinguishable between mutants and wild type siblings. Acridine orange staining at 5 days post fertilization (dpf) did not detect any dysregulation of apoptosis. Hemoglobin staining found no specific hemorrhage in vehicle or warfarin treated mutants. However, 3 dpf f10-/- mutants did not develop occlusive thrombi in response to laser-mediated venous endothelial injury, indicating that F10 is required for hemostasis. We used quantitative PCR to measure transcription of f10 and downstream coagulation factors in 3 dpf larvae. f10 mRNA was undetectable in homozygous mutants, presumably due to nonsense-mediated decay as a consequence of the TALEN-induced frameshift mutation. fga (fibrinogen alpha) and at3 (antithrombin III) mRNAs were increased by 1.8 and 2.3-fold, respectively (p<0.05), when compared to wild type siblings. Prothrombin (f2) mRNA transcription was slightly decreased, although the effect was not statistically significant.In summary, we have produced a zebrafish model of human F10 deficiency that exhibits a spontaneous adult lethal bleeding phenotype, although early embryonic/larval survival is unaffected despite an underlying severe hemostatic defect. Further study of this mutant may identify species specific factors enabling this early survival. qPCR analysis in mutant larvae suggests that the level of F10 mRNA or protein indirectly regulates other coagulation factors during development. We have also used this model to evaluate the longstanding hypothesis that coagulation cascade mediated thrombin signaling is required for embryonic vasculogenesis. Thus far we have not found evidence for defects in vessel development or integrity, apoptosis, or endothelial differentiation/specification. Taken together, these data suggest that the effects of F10 loss are restricted to hemostasis, and thus therapies should continue to be focused on this in deficient patients. Further study as to why mutant embryos and larvae are able to tolerate what would likely cause lethal hemorrhage in mammals, as well as small molecule screens using this model, could potentially lead to innovative therapeutic modalities for patients with bleeding disorders. DisclosuresJoung:Horizon Discovery: Consultancy; Editas Medicine: Other: Financial interest; Poseida Therapeutics: Other: Financial interest; Hera Testing Laboratories: Other: Financial interest; Transposagen Biopharmaceuticals: Other: Financial interest. Shavit:CSL Behring: Consultancy; Octapharma: Consultancy; Bayer: Consultancy; Baxter: Consultancy.

Reversing Antisense Oligonucleotide Activity with a Sense Oligonucleotide Antidote: Proof of Concept Targeting Prothrombin.

The tissue half-life of second-generation antisense oligonucleotide drugs (ASOs) is generally longer than traditional small molecule therapeutics. Thus, a strategy to reverse the activity of antisense drugs is warranted in certain settings. In this study, we describe a strategy employing the administration of a complementary sense oligonucleotide antidote (SOA). As a model system we have chosen to target the coagulation factor and antithrombotic drug target, prothrombin, to assess the feasibility of this approach. ASO targeting mouse prothrombin specifically suppressed >90% hepatic prothrombin mRNA levels and circulating prothrombin protein in mice. These effects were dose- and time-dependent, and as expected produced predictable increases in anticoagulation activity [prothrombin time/activated partial thromboplastin time (PT/aPTT)]. Treatment with prothrombin SOAs resulted in a dose-dependent reversal of ASO activity, as measured by a return in prothrombin mRNA levels and thrombin activity, and normalization of aPTT and PT. The antithrombotic activity of prothrombin ASOs was demonstrated in a FeCl3-induced thrombosis mouse model, and as predicted for this target, the doses required for antithrombotic activity were also associated with increased bleeding. Treatment with SOA was able to prevent prothrombin ASO-induced bleeding in a dose-dependent manner. These studies demonstrate for the first time the utility of SOAs to selectively and specifically reverse the intracellular effects of an antisense therapy.

Lack of association between the prothrombin rs1799963 polymorphism and juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure.

Thrombin induces ischemic LTP (iLTP): implications for synaptic plasticity in the acute phase of ischemic stroke.

Acute brain ischemia modifies synaptic plasticity by inducing ischemic long-term potentiation (iLTP) of synaptic transmission through the activation of N-Methyl-D-aspartate receptors (NMDAR). Thrombin, a blood coagulation factor, affects synaptic plasticity in an NMDAR dependent manner. Since its activity and concentration is increased in brain tissue upon acute stroke, we sought to clarify whether thrombin could mediate iLTP through the activation of its receptor Protease-Activated receptor 1 (PAR1). Extracellular recordings were obtained in CA1 region of hippocampal slices from C57BL/6 mice. In vitro ischemia was induced by acute (3 minutes) oxygen and glucose deprivation (OGD). A specific ex vivo enzymatic assay was employed to assess thrombin activity in hippocampal slices, while OGD-induced changes in prothrombin mRNA levels were assessed by (RT)qPCR. Upon OGD, thrombin activity increased in hippocampal slices. A robust potentiation of excitatory synaptic strength was detected, which occluded the ability to induce further LTP. Inhibition of either thrombin or its receptor PAR1 blocked iLTP and restored the physiological, stimulus induced LTP. Our study provides important insights on the early changes occurring at excitatory synapses after ischemia and indicates the thrombin/PAR1 pathway as a novel target for developing therapeutic strategies to restore synaptic function in the acute phase of ischemic stroke.

Placental prothrombin mRNA levels in APC resistance (APCR) women with increased placental fibrin deposition

We investigated the link between the mRNA of the procoagulant prothrombin in the placental tissue with the increased placental fibrin deposition associated with activated protein C resistance (APCR). Women with APCR were not found to produce higher levels of prothrombin transcript compared to women with a normal APC ratio. This indicates that accumulated fibrin in the placenta is not the consequence of too much production of the procoagulant prothrombin transcript, but may be associated with altered function of other haemostatic factors interacting with APC in the placenta.

Regulation Of Hematopoietic Stem Cell Trafficking By The Coagulation Pathway

Hematopoeitic stem and progenitor cells (HSPC) dynamically switch between a quiescent, non-motile mode in the bone marrow (BM), to an active state, in which they proliferate, differentiate and egress to the circulation. Injection of the coagulation protease thrombin induced rapid HSPC mobilization to the blood via activation of its major receptor, protease activated receptor 1 (PAR1) on BM hematopoietic and stromal cells. We hypothesized that coagulation factors control stem cells fate in the BM. We examined if thrombin is generated in the murine BM and found by immunohistochemistry prothrombin associated with bone lining osteoblasts in the endosteum region. These cells also highly express osteopontin which induces stem cell quiescence and retention. Cleavage of osteopontin by thrombin or by osteoclast derived cathepsin K induces stem cell mobilization. In addition, a unique structure of multinucleated CD45+ cell clusters in the trabecular-rich area of the murine femoral metaphysis express the cell surface receptor Tissue Factor (TF), a potent initiator of the coagulation cascade leading to thrombin generation. These clusters were found adjacent to multinucleated TRAP (tatrate resistant acid phosphate) positive active osteoclasts. In vitro, we found that immature osteoclasts expressed TF in cell fusion areas, suggesting that osteoclast maturation also activates the coagulation thrombin/PAR1 axis, thus mediating HSPC recruitment to the circulation. Supporting this notion, bleeding which prompts a hemostatic response and thrombin production, is a strong inducer of osteoclasts activation and HSPC mobilization. In addition, injection of bacterial lipopolysaccharides (LPS) is known to activate osteoclasts and induce HSPC mobilization (Kollet et al Nat Med 06). We found that LPS injection upregulated TF expression by CD45+ myeloid cells in the murine BM. LPS treatment provoked massive HSPC mobilization, which was attenuated by PAR1 inhibition. To further address the role of thrombin in stem cell maintenance, we targeted prothrombin in vivo by applying Antisense Oligonucleotides (ASO) knockdown technology, previously shown to induce a dose- and time-dependent up to 90% reduction of prothrombin mRNA levels in the murine liver (Monia et al Blood 2010). Prothrombin depletion altered the BM niche microenvironment by expanding the mesenchymal stem and progenitor (MSPC) population and the long-term repopulating CD34-/ROSlow/LSK HSPC population in the BM. In untreated mice, TF was also expressed by a small MSPC population, suggesting that the bone stomal compartment may also contribute to the regulation of HSPC mobilization upon demand. To further asses the role of thrombin generation in HSPC development, we examined the involvement of the endothelial cell receptor Thrombomodulin (TM) that is pivotal for the anticoagulant pathway which mediates activation of protein C. TM protein is expressed by BM small blood vessels resembling sinusoids and by neighboring MSPC. By immunohistochemistry, we also detected activated protein C on the same blood vessels. A mouse model with a mutation in the TM gene (TMPro/Pro) is characterized by reduced capacity for activated protein C generation which in turn increases thrombin levels in these mice. We found increased circulating hematopoietic stem cells in TMPro/Pro mice, suggesting that chronically increased basal levels of thrombin generation can promote HSC egress. Conversely, short term (5 day) intermittent treatment of mice with low dose thrombin that mainly causes activated protein C formation in vivo, display higher levels of CD34-/ROSlow/LSK and EPCR+LSK stem cells in the BM, indicating additional roles for the anticoagulant pathway in BM stem cell pool maintenance. In summary, our results provide evidence that the activator of the coagulation cascade, TF, and coagulation factors Thrombin and activated protein C are present in the BM and regulate and integrate functions of hematopoietic stem and progenitor cells and BM stromal progenitor cells. Disclosures: Crosby: Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other. Monia:Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other.

Mice with Genetic Modifications in Prothrombin Limiting Activation Cleavage Events to Meizothrombin Survive to Adulthood, but Exhibit Alterations in Hemostasis and Thrombus Formation

AbstractAbstract 496Thrombin-mediated proteolysis is the central event not only of hemostasis and thrombosis but also in distinct physiological and pathological contexts such as development, inflammation, cancer biology, and cardiovascular disease. The proteolytic conversion of prothrombin to α-thrombin (fIIa) by the prothrombinase complex occurs through two possible pathways: i) the inactive intermediate termed prethrombin 2, or ii) the proteolytically active two-chain intermediate termed meizothrombin (fIIaMZ). fIIaMZ, unlike fIIa, retains the γ-carboxyglutamic acid-rich gla domain and two kringle domains of prothrombin and has distinct catalytic properties relative to fIIa that could be biologically meaningful in vivo, including a diminished capacity to cleave fibrinogen and a significantly increased capacity to activate protein C in the presence of thrombomodulin. Here, the endogenous prothrombin gene was modified in mice to better explore the properties of fIIaMZin vivo and to test the hypotheses that mice carrying a mutant form of prothrombin with a terminal activation product of fIIaMZ will: 1) develop to term and survive to adulthood despite an altered hemostatic profile; and 2) exhibit significantly dampened inflammatory responses due to enhanced protein C activation. In order to limit cleavage events to the single site yielding fIIaMZ, alterations were introduced into the endogenous prothrombin gene resulting in three amino acid substitutions (R157A, R268A, and K281A) located at the P1 positions of potential fXa or autocatalytic cleavage sites. Unlike prothrombin-null mice, mice homozygous for these mutations (hereafter referred to as fIIMZ mice) were found to be present at weaning age and viable into adulthood. Furthermore, unlike mice with a major deficit in either clotting function (e.g., fibrinogen-null) or platelet function (e.g., Gαq-null), fIIMZ females were capable of successfully carrying a litter to term. However, analysis of over 700 progeny generated from crosses of heterozygous fIIMZ/WT mice revealed that only about half of the number homozygous fIIMZ mice expected, based on Mendelian transmission rates, were observed in weaning-age offspring. Complementary studies suggest that the fraction of fIIMZ offspring that fail are lost primarily in utero, but occasional post-partum failures were observed associated with hemorrhagic events. Successful adult fIIMZ mice were found to have similar prothrombin mRNA levels and equivalent fII protein expression to fIIWT mice. Furthermore, fIIMZ animals exhibited normal complete blood cell counts and predictably normal thrombin times, but prolonged PTs and aPTTs. Thrombin generation assays revealed a prolonged time to peak thrombin production, but no significant difference in peak thrombin levels. Consistent with these findings, tail bleeding times in fIIMZ mice were significantly prolonged. None of the fIIMZ mice assayed achieved hemostasis within the 10 minute observation window, whereas fIIWT mice all stopped within 2 minutes of challenge. More sophisticated comparative studies of thrombus formation in mesenteric arterioles following FeCl3 injury using a real-time intravital microscopy approach established that the time to first thrombus formation in fIIMZ mice was almost twice that of wildtype animals. The majority of the fIIMZ mice failed to occlude the injured vessel within a 25 min observation window, whereas all fIIWT mice successfully formed occlusive thrombi with a mean time of 12.5 minutes. In summary, site-directed alterations of the endogenous prothrombin gene in mice leading to a terminal prothrombin activation product of meizothrombin were found to be compatible with development, growth to adulthood and reproductive success, albeit with modified hemostatic function. Based on the catalytic properties of fIIaMZ favoring protein C activation, studies are underway to compare APC generation in control and fIIMZ mice and explore the theory that physiological and pathological inflammatory responses will be dampened in fIIMZ mice. Disclosures:No relevant conflicts of interest to declare.

P38 MAPK Controls Prothrombin Expression by Regulated RNA 3′ End Processing

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide invivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.

A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR

The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3′-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

A Sense Oligonucleotide Specifically Reverses the Prothrombin Depletion and Anticoagulation Caused by ISIS 401025, an Antisense Oligonucleotide Targeting Prothrombin RNA in Mice.

The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.

Multiplex PCR to Assay the Effect of Nucleic Acid‐Based Inhibitors on Prothrombin Transcript Level

We compared an antisense-oligodeoxynucleotide and four DNAzymes directed to the prothrombin mRNA for their efficiency to reduce prothrombin transcript level in HepG2 cells. The DNAzymes have different binding arm symmetry and cleavage sites, but are directed to the identical target site of the antisense-oligodeoxynucleotide. The nucleic acid-based inhibitors were transfected into HepG2 cells and prothrombin transcript level was quantified and normalized to the beta-actin transcript level by multiplex PCR. All nucleic acid-based inhibitors reduced prothrombin transcript level and the effect was in almost all cases, strongest 24 h after transfection, but still remarkable up to 68 h after transfection. The antisense-oligodeoxynucleotide was more effective than the DNAzymes tested.

Development of Hyperfibrinogenemia in Spontaneously Hypertensive and Hyperlipidemic Rats: A Potentially Useful Animal Model as a Complication of Hypertension and Hyperlipidemia

According to current concepts, hypertension and hyperlipidemia cause vascular damage that leads to a hypercoagulative state. In this study, we investigated whether spontaneously hypertensive and hyperlipidemic rats (SHHR) can be a useful experimental model for complications in combined hypertension and hyperlipidemia, by comparing coagulative and fibrinolytic activities in SHHR with those in spontaneously hypertensive rats (SHR) and spontaneously hyperlipidemic rats (HLR). We measured coagulation and fibrinolysis markers in plasma and levels of fibrinogen and prothrombin mRNA in livers of eight-month-old male Wistar Kyoto rats (WKY), Sprague-Dawley rats (SD), SHR, HLR and SHHR. The plasma levels of fibrinogen in SHR, HLR and SHHR were significantly higher than those in WKY and SD, and were highest in SHHR. Higher plasma levels of antithrombin III and plasminogen were detected in increasing order in SHR, HLR and SHHR as compared to those in WKY and SD. Hepatic mRNA expressions of fibrinogen chains and prothrombin were enhanced in SHR, HLR and SHHR, resulting in increased plasma fibrinogen levels in SHHR. These results suggest that hypertension and hyperlipidemia can each cause hypercoagulation, with hyperlipidemia being a stronger factor than hypertension. Since a greater hypercoagulative state is a complication of combined hypertension and hyperlipidemia, the SHHR model is a good system for studying the early stage of atherosclerosis ensuing from hyperfibrinogenemia.

The G20210A Mutation Alters the Structure and Function of Prothrombin mRNA.

The activated form of prothrombin (F.II) plays a pivotal role in physiologically important thrombotic and fibrinolytic processes. Heterozygotes for a G->A transversion at position 20210 of the F.II 3′UTR display elevated prothrombin levels and exhibit a corresponding increase in the risk of cardiac and cerebral thromboses. Previous studies of endogenous F.IIG and F.IIA mRNAs in primary liver cells, and analyses of transiently expressed F.IIG and F.IIA reporter mRNAs expressed in cultured heterologous cells, have reached different conclusions on the molecular mechanism through which the G20210A mutation effects F.II overexpression. To resolve this issue, we designed a cell-homologous culture system for directly assessing functional attributes of F.II reporter mRNAs, including their stabilities as well as their translational efficiencies. An hepatocellular-phenotype HepG2 cell line was generated that stably expresses the tetracycline transactivator (tTA), a chimeric factor that mediates tetracycline-conditional silencing of genes linked to a tetracycline regulatory element (TRE). These cells, which permit the stabilities of mRNAs encoded by TRE-linked genes to be determined by transcriptional chase analysis, were validated in studies utilizing stable and unstable control mRNAs. TRE-linked β-globin reporter genes were subsequently constructed to contain the F.II 5′UTR as well as the full-length F.II 3′UTR and contiguous flanking region, with either the native G (βF.IIG) or the mutant A (βF.IIA) at position 20210. The βF.IIG and βF.IIA mRNAs displayed equal stabilities when transiently expressed in intact tTA-expressing HepG2 cells, an observation corroborated by formal studies in multiple cell lines containing stably-integrated TRE-linked βF.IIG and βF.IIA genes. This data suggests that the G20210A mutation is likely to effect clinical hyperprothrombinemia through inductive effects on the translational efficiency of the F.II mRNA rather than by altering its stability. The mechanistic basis for this property may reflect differences in the 3′-cleavage patterns exhibited by nascent F.IIG mRNA (occurring at nts 20210 and 20212) and F.IIA mRNA (occurring only at nt 20210). Consistent with this possibility, we observed that 20212-terminal F.IIG 3′UTRs fail to bind three cytoplasmic factors that exhibit a strong affinity for both 20210-terminal F.IIG and F.IIA 3′UTRs in vitro. A candidate binding site for one factor–at positions 20202-06–may be sterically inhibited by potential secondary structure that forms in 20212-, but not the 20210- terminal F.II mRNA 3′-cleavage isotypes. We propose that the differential affinity of this factor for 20210- and 20212-terminal F.II mRNAs may mediate a difference in the aggregate translational efficiencies of F.IIG and F.IIA mRNAs. To test this hypothesis, we established a sucrose-gradient fractionation method that estimates the translational efficiency of an individual mRNA by assessing the number of ribosomes that it carries. Initial studies indicate a difference in the ribosomal loading of βF.IIG and βF.IIA mRNAs that is consistent with their anticipated relative translational efficiencies. The results of these studies will provide a rational basis for the design of strategies to therapeutically manipulate the expression of F.II, and will afford potential insights into the regulation of other, evolutionarily related coagulation factors whose expression levels may define independent thrombotic risk.

Renal prothrombin mRNA is significantly decreased in a hyperoxaluric rat model of nephrolithiasis

Although urinary prothrombin fragment 1 (UPTF1) possesses several hallmarks expected of a regulatory protein in urolithiasis, its precise role remains unknown. To determine the relationship between renal prothrombin (PT), the parent molecule of UPTF1, and lithogenesis, this study quantified and compared levels of renal PT mRNA in healthy rats (n = 10) and rats rendered lithogenic (n = 10) by ingestion of 0.75% ethylene glycol for 8 weeks. Studies included morphological and histological examination of the kidneys with scanning electron microscopy of the urinary filtrates of control and experimental animals. Haematuria and calcium oxalate (CaOx) crystals occurred in the urine of all experimental rats, but not in those of controls. Histological examination showed birefringent nephroliths and associated damage in kidneys of lithogenic rats, which were not seen in the control group. The amounts of total RNA extracted from both groups of rats were similar, but the median ratio of PT to beta-actin transcript of 11.14 x 10(-4) (10.65 x 10(-4) +/- 2.24 x 10(-4)) in the control rats was significantly (p < or = 0.001) reduced to 6.47 x 10(-4) (6.57 x 10(-4) +/- 2.72 x 10(-4)) in the lithogenic group. These results demonstrate that renal PT mRNA is reduced by approximately 42% in lithogenic rats and confirm the existence of a direct association between renal PT synthesis and calculogenesis. Attempts to compare renal PT and urinary levels of PTF1 were unsuccessful because of interference from hepatic PT circulating in the blood, haematuria, and the presence of urinary CaOx crystals. This is the first report of a significant reduction in the renal expression of a urinary protein well documented to inhibit CaOx crystal growth and aggregation in undiluted human urine in vitro.

Cellular localization of thrombin in prostate: Implications for thrombin targeted therapy in prostate cancer

14527 Background: Tumor and coagulation interaction is complex and heterogeneous. Expression of an intact repertoire of coagulation proteins is not detected in all tumor types. Thrombin, a key coagulation protein, which has been observed to promote tumorigenesis in experimental models, is not expressed on breast, colon and squamous lung cancer cells, but has been detected on small cell lung, melanoma and renal cancer epithelium. In prostate cancer, expression of its receptor, protease activated receptor-1 (PAR-1) has been previously reported but thrombin expression on epithelium not determined. Therefore, we evaluated thrombin and prothrombin expression in prostate specimens. Methods: Quantitative real-time PCR was performed for (pro)thrombin mRNA expression in fresh prostate cancer tissue lysates obtained from localized disease (n = 9 prostatectomy specimens) and advanced disease (n = 7 channel trans-urethral prostate resection specimens). Prothrombin and thrombin protein immunohistochemistry was performed on anonymized prostate tissue micro arrays which included benign (n = 117), prostate intra-epithelial neoplasia (PIN; n = 26) and prostate cancer (n = 112) cores. Results: (Pro)thrombin mRNA expression (normalized to 18s mRNA) was detected in all cancer tissue lysates. Mean intensity of expression in advanced cancer specimens was 20.3 compared to 14.0 for local stage specimens (p = 0.09; Wilcoxon rank sum test). Spatially, expression of thrombin was detected in stroma, benign and malignant epithelium (see Table) and on vascular endothelium. Prothrombin localization was similar to thrombin expression. Conclusions: Thrombin’s tumorigenic effects via PAR-1 activation provide novel therapeutic targets in tumors expressing thrombin-PAR1 axis. The current study confirms thrombin expression on prostate epithelium and taken with previous results of PAR1 epithelial expression, provides a rationale for exploring specific thrombin inhibitors and/or PAR inhibitors in prostate cancer. [Table: see text] No significant financial relationships to disclose.