The Prothrombin-Prothrombinase Interaction.

The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with exposure of tissue factor from the damaged endothelium and culminates with conversion of prothrombin to thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca2+, and phospholipids. This cofactor-dependent activation is paradigmatic of analogous reactions of the blood coagulation and complement cascades, which makes elucidation of its molecular mechanism of broad significance to the large class of trypsin-like zymogens to which prothrombin belongs. Because of its relevance as the most important reaction in the physiological response to vascular injury, as well as the main trigger of pathological thrombotic complications, the mechanism of prothrombin activation has been studied extensively. However, a molecular interpretation of this mechanism has become available only recently from important developments in structural biology. Here we review current knowledge on the prothrombin-prothrombinase interaction and outline future directions for the study of this key reaction of the coagulation cascade.

Effect of Xe/O2 Inhalation on Hemostasis in Experimental Thromboplastin Pneumonitis.

We studied the effectiveness of Xe/O2 mixture inhalation (30% Xe and 70% O2, 20 min for 5 days) in a model of experimental thromboplastin pneumonitis. Inhalation of the studied mixture decreased the intensity of the inflammatory process in the lung tissue assessed by the temperature response of animals, changed lung weight and lung weight coefficient. At acute stage of pneumonitis, an increase in xenon consumption was recorded due to its retention in the gas exchange zone and a natural decrease in oxygen consumption due to partial alveolar/capillary block. The formation of pneumonitis was accompanied by a pronounced procoagulant shift in the regulation system of the aggregate state of blood. The Xe/O2 inhalations ensured physiologically optimal levels of prothrombin and activated partial thromboplastin time against the background of a moderate decrease in fibrinogen level throughout the experiment. At the same time, the activity of the natural anticoagulant antithrombin III increased from day 5 to day 14.

The Etiology of the Thrombotic Phenomena Involved in the Process of Coronary Artery Disease-What Is the Role of Thrombophilic Genes in the Development of This Pathology?

Cardiovascular diseases, among which includes coronary artery disease, represent one of the most important causes of mortality and morbidity worldwide. Research aimed at determining the risk factors involved recognizes a group of "traditional" risk factors, but also more recent studies identified over 100 "novel" ones which may have a role in the disease. Among the latter is the thrombophilia profile of a patient, a pathology well-established for its involvement in venous thromboembolism, but with less studied implications in arterial thrombosis. This paper reviews the literature, explaining the pathophysiology of the thrombophilia causes associated most with coronary thrombosis events. Results of several studies on the subject, including a meta-analysis with over 60,000 subjects, determined the significant involvement of factor V Leiden, prothrombin G20210A mutation, plasminogen activator inhibitor-1 and antiphospholipid syndrome in the development of coronary artery disease. The mechanisms involved are currently at different stages of research, with some already established and used as therapeutic targets.

The Usefulness of Vitamin K-Dependent Proteins in the Diagnosis of Colorectal Carcinoma.

Colorectal cancer (CRC) is one of the most common neoplasms in developed countries, with increasing incidence and mortality, even in young people. A variety of serum markers have been associated with CRC (CEA, CA 19-9), but neither should be used as a screening tool for the diagnosis or evolution staging of CRC. The sensitivity and specificity of these markers are not as good as is required, so new ones need to be found. Matrix Gla protein and PIVKA II are involved in carcinogenesis, but few studies have evaluated their usefulness in predicting the presence and severity of CRC. Two hundred patients were divided into three groups: 80 patients were included in the control group; 80 with CRC and without hepatic metastasis were included in Group 1; 40 patients with CRC and hepatic metastasis were included in Group 2. Vitamin K-dependent proteins (VKDPs) levels in plasma were determined. Patients with CRC without methastasis (Group 1) and CRC patients with methastasis (Group 2) presented significantly higher values of CEA, CA 19-9, PIVKA II (310.05 ± 38.22 vs. 430.13 ± 122.13 vs. 20.23 ± 10.90), and ucMGP (14,300.00 ± 2387.02 vs. 13,410.52 ± 2243.16 vs. 1780.31 ± 864.70) compared to control group (Group 0). Interestingly, Group 1 presented the greatest PIVKA II values. Out of all the markers, significant differences between the histological subgroups were found only for ucMGP, but only in non-metastatic CRC. Studying the discrimination capacity between the patients with CRC vs. those without, no significant differences were found between the classical tumor markers and the VKDP AUROC curves (PIVKA II and ucMGP AUROCs = 1). For the metastatic stage, the sensitivity and specificity of the VKDPs were lower in comparison with those of CA 19-9 and CEA, respectively (PIVKA II AUROC = 0.789, ucMGP AUROC = 0.608). The serum levels of these VKDPs are significantly altered in patients with colorectal carcinoma; it is possible to find additional value of these in the early stages of the disease.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Cryo-EM structure and functional basis of prothrombin recognition by a type I antiprothrombin antiphospholipid antibody.

ABSTRACT: Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell's viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.

Protein kinase C delta enhances the diagnostic performance of hepatocellular carcinoma.

BACKGROUND: The conventional markers for hepatocellular carcinoma (HCC), α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), have several limitations; both have low sensitivity in patients with early-stage HCC; low sensitivity for AFP with HCC after eliminating hepatitis C virus (HCV); low specificity for DCP in patients with non-viral HCC, which is increasing worldwide; low specificity for AFP in patients with liver injury; and low specificity for DCP in patients treated with warfarin. To overcome these issues, the identification of novel biomarkers is an unmet need. OBJECTIVE: This study aimed to assess the usefulness of serum protein kinase C delta (PKCδ) for detecting these HCCs. METHODS: PKCδ levels were measured using a sandwich enzyme-linked immunosorbent assay in 363 chronic liver disease (CLD) patients with and without HCC. RESULTS: In both viral and non-viral CLD, PKCδ can detect HCCs with high sensitivity and specificity, particularly in the very early stages. Notably, the value and sensitivity of PKCδ were not modified by HCV elimination status. Liver injury and warfarin administration, which are known to cause false-positive results for conventional markers, did not modify PKCδ levels. CONCLUSIONS: PKCδ is an enhanced biomarker for the diagnosis of HCC that compensates for the drawbacks of conventional markers.

Vitamin K insufficiency and the prophylaxis strategy in term healthy infants: A multicentre study.

BACKGROUND/AIM: Late vitamin K deficiency bleeding (VKDB) during early infancy is a serious problem worldwide. Vitamin K (VK) deficiency commonly occurs in newborns who are exclusively breastfed. Protein Induced by VK Absence (PIVKA-II) has been identified as an early indicator of subclinical VK deficiency in neonates, surpassing prothrombin time. To assess PIVKA-II levels at 48 h, 1 and 3 months of age in full-term newborns who were exclusively breastfed and received varying VKDB prophylaxis regimens. METHODS: A prospective observational study was conducted in four hospitals, enrolling 105 newborns. PIVKA-II levels were measured using a sandwich-type enzyme-linked immunosorbent assay. RESULTS: At 48 h of age, there was no significant difference in PIVKA-II concentrations between newborns who received intramuscular administration of 1 mg of phylloquinone (VK1) and those who received oral administration of 2 mg of VK1 at birth. At 1 and 3 months of life, infants who received any supplementation regimen between 2 and 14 weeks exhibited significantly lower PIVKA-II concentrations compared to infants who received only 1 mg of intramuscular VK1 at birth. The prophylaxis involving a dose of 1 mg of intramuscular VK1 at birth followed by oral administration of 150 µg/day of VK1 from the 2nd to the 14th week of life showed the lowest PIVKA-II blood concentrations. CONCLUSIONS: Oral supplementation of VK1 after discharge significantly reduced PIVKA-II concentrations in exclusively breastfed term infants. These findings suggest the importance of oral VK1 supplementation in exclusively breastfed infants during their first 3 months of life to avoid the risk of VK insufficiency.

The use of shotgun label-free quantitative proteomic mass spectrometry to evaluate the inflammatory response in aqueous humor from horses with uveitis compared to ophthalmologically healthy horses.

OBJECTIVE: The objective of this study was to use shotgun label-free tandem mass spectrometry (LF-MS/MS) to evaluate aqueous humor (AH) from horses with uveitis (UH) compared to ophthalmologically healthy horses (HH). ANIMALS STUDIED: Twelve horses diagnosed with uveitis based on ophthalmic examination and six ophthalmologically healthy horses (postmortem) purchased for teaching purposes. PROCEDURES: All horses received a complete ophthalmic examination and physical exam. Aqueous paracentesis was performed on all horses and AH total protein concentrations were measured with nanodrop (TPn) and refractometry (TPr). AH samples were analyzed with shotgun LF-MS/MS and proteomic data were compared between groups using Wilcoxon rank-sum test. RESULTS: A total of 147 proteins were detected, 11 proteins had higher abundance in UH, and 38 proteins had lower abundance in UH. Proteins with higher abundance included apolipoprotein E, alpha-2-macroglobulin (A2M), alpha-2-HS-glycoprotein, prothrombin, fibrinogen, complement component 4 (C4), joining chain for IgA and IgM, afamin, and amine oxidase. There were positive correlations between TPn (p = .003) and TPr (p = .0001) compared to flare scores. CONCLUSION: Differential abundance of A2M, prothrombin, fibrinogen, and C4 indicate upregulation of the complement and coagulation cascade in equine uveitis. Proinflammatory cytokines and the complement cascade have potential as therapeutic targets for equine uveitis.

Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

INTRODUCTION: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. METHODS: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. RESULTS: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. CONCLUSIONS: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

The Effect of Vitamin K2 Supplementation on PIVKA-II Levels in Patients with Severe Motor and Intellectual Disabilities Undergoing Long-Term Tube Feeding.

Nutritional support is essential for patients with severe motor and intellectual disabilities (SMID) to ensure the smooth provision of medical care. These patients often require long-term tube feeding with enteral formulas, potentially leading to deficiencies in vitamins and trace elements. Additionally, frequent antibiotic use for infections often disrupts gut microbiota, inhibiting vitamin K2 production by intestinal bacteria. We assessed the serum protein induced by vitamin K absence or antagonists-II (PIVKA-II) and undercarboxylated osteocalcin (ucOC) levels to assess the vitamin K status in 20 patients with SMID (median age: 44.1 years, 11 men and 9 women) undergoing long-term tube feeding for durations ranging from 3 to 31 years. Thirteen (65%) and nine (45%) patients had elevated PIVKA-II (<40 mAU/mL) and serum ucOC levels (reference value < 4.50 ng/mL), respectively. Dietary vitamin K1 intake did not differ between patients with and without elevated PIVKA-II levels. Vitamin K2 supplementation for 3 months decreased serum PIVKA-II levels near those within the reference range. Approximately half of the patients with SMID on tube feeding had subclinical vitamin K deficiency. Further studies are needed to ascertain if long-term vitamin K2 supplementation effectively prevents vitamin K deficiency-induced hypercoagulation, osteoporosis, and vascular calcification in patients with SMID.

Protein induced by vitamin K absence or antagonist II: Experience to date and future directions.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with high mortality. The realization of precision medicine in HCC relies upon efficient biomarkers. Protein induced by vitamin K absence or antagonist II (PIVKA-II) is an immature prothrombin with insufficient coagulation activity, overexpressing in HCC cells. Previous evidence confirmed the role of PIVKA-II in screening and diagnosing HCC. However, the increased PIVKA-II was observed not only in HCC, but also in non-HCC individuals such as vitamin K deficiency. The joint detection of PIVKA-II and other biomarkers could significantly improve diagnostic accuracy in HCC. Furthermore, PIVKA-II serves as a valuable prognostic predictor, transplantation eligibility, resectability, tumor recurrence, therapeutic efficacy, and malignant tumor behaviors. Additionally, PIVKA-II represents a potential target for agent development to establish new therapeutic strategies. Besides HCC, PIVKA-II also serves as a biomarker of vitamin K status. In this review, we assess the role of PIVKA-II in diagnosis, prediction, and treatment. Over the past decades, substantial progress has been achieved in the application of PIVKA-II. Exploration and innovation are required for further advances in the field of PIVKA-II investigation.

Decrease of prothrombin level during thrombolysis in acute myocardium infarction.

Previously, the direct interactions of Bß26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or DDE-fragments causes non-enzymatic prothrombin activation. The direct measuring of the prothrombin level in the blood plasma of patients with acute myocardial infarction (AMI) allowed us to find a situation where such an activation can occur in vivo. Blood coagulation parameters in the blood plasma of patients with AMI were measured at 2 hours, three days, and seven days after the thrombolysis by streptokinase accompanied with intravenous administration of anticoagulants: unfractionated high molecular weight heparin (HMWH) and low-molecular-weight heparin (LMWH). The prothrombin level in the blood plasma of patients with AMI was normal before thrombolytic therapy and substantially decreased after streptokinase administration. This effect was prominent in the case of concomitant anticoagulant therapy with LMWH and was not observed when HMWH was applied. It can be explained by the fact that LMWH preferentially inhibits factor Xa, while the HMWH is an effective inhibitor of both factor Xa and thrombin. This observation suggested that the prothrombin level decrease was caused by the thrombin-like activity and possible autolysis of prothrombin by thrombin. Also, thrombolytic therapy with streptokinase caused the accumulation of fibrin degradation products (FDPs), some of which were able to bind prothrombin. The dramatic decrease of prothrombin level in the blood plasma of patients with AMI during thrombolysis allowed us to conclude the non-enzymatic prothrombin activation with the following autolysis of prothrombin that contributes to the pathology.

Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma.

Gamma (γ) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully γ-carboxylated clotting factors increase plasma des-γ-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of γ-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different γ-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different γ-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify γ-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 µL plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.

The C2H2 zinc finger transcription factor CF2-II regulates multi-insecticide resistance-related gut-predominant ABC transporters in Aphis gossypii Glover.

Clarifying the molecular mechanisms of cotton aphid resistance to various insecticides is crucial for the long-term safe application of insecticides in chemical control. ATP-binding cassette (ABC) transporters mediate the membrane transport of various substrates (including exogenous substances). Experiments confirmed that ABCB5, ABCF2, and MRP12 contributed to high levels of resistance to spirotetramat, cyantraniliprole, thiamethoxam or imidacloprid. Binding sites of the C2H2 zinc finger transcription factor CF2-II was predicted to be located in the promoters of ABCB5, ABCF2, and MRP12. The expression levels of ABCB5, ABCF2, and MRP12 were significantly upregulated after silencing CF2-II. The results of dual-luciferase reporter assays demonstrated a negative regulatory relationship between CF2-II and ABC transporter promoters. Furthermore, yeast one-hybrid (Y1H) and electrophoresis mobility shift assays (EMSAs) revealed that CF2-II inhibited the expression of ABC transporter genes through interaction with binding sites [ABCF2.p (-1149/-1140) or MRP12.p (-1189/-1181)]. The above results indicated that ABCB5, ABCF2, and MRP12 were negatively regulated by the transcription factor CF2-II, which will help us further understand the mechanism of transcriptional adaption of multi-insecticides resistant related ABC transporters in response to xenobiotics.

Irregularities of Coagulation in Hypertension.

PURPOSE OF REVIEW: This review article summarizes the role of coagulation in the pathogenesis of hypertension. It specifically focuses on significant factors and markers associated with coagulation, including D-dimer, fibrinogen and fibrin, prothrombin, P-selectin, soluble urokinase plasminogen activator receptor, thrombomodulin, tissue factor, tissue plasminogen activator, von Willebrand factor, ß-thromboglobulin, and Stuart-Prower factor. RECENT FINDINGS: D-dimer levels were elevated in hypertensive individuals compared to healthy controls, and the levels increased with the severity of hypertension. These findings indicate that increased coagulation activity of fibrin plays a role in the development of thromboembolic complications in hypertensive patients. Additionally, both fibrinogen levels and D-dimer levels displayed a positive correlation with the duration of hypertension, suggesting that these biomarkers were positively associated with the length of time an individual had been hypertensive. Increased systolic and diastolic blood pressures have been linked to higher levels of prothrombin time and activated partial thromboplastin time in individuals with hypertension as well as those with normal blood pressure. Also, the presence of P-selectin, produced by activated platelets and endothelial cells during angiotensin II stimulation, played a role in the development of cardiac inflammation and fibrosis associated with hypertension. Moreover, the change in systolic blood pressure was associated with baseline soluble urokinase plasminogen activator receptor (suPAR) in hypertensive participants, and the change in suPAR levels was associated with the development of hypertension. Moreover, it was observed a decrease in thrombomodulin expression in the placenta of preeclamptic patients, suggesting its potential involvement in placental dysfunction, possibly driven by an imbalance in angiogenic factors. Tissue factors and autophagy might have significant implications in the pathogenesis of chronic thromboembolic pulmonary hypertension, particularly in the context of vascular remodelling. Likewise, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) might be a promising biomarker for the early detection of pulmonary arterial hypertension and the von Willebrand factor is a candidate prognostic biomarker. The arterial ß-thromboglobulin levels were significantly lower than venous levels. This article concludes that D-dimer, fibrinogen and fibrin, prothrombin, P-selectin, soluble urokinase plasminogen activator receptor, thrombomodulin, tissue factor, tissue plasminogen activator, von Willebrand factor, and ß-thromboglobulin are important factors involved in the pathogenesis of hypertension.

PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.