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The Journal of Biological Chemistry Aug 2013The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B...
The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.
Topics: Crystallography, X-Ray; Energy Transfer; Models, Molecular; Protein Conformation; Prothrombin; Static Electricity
PubMed: 23775088
DOI: 10.1074/jbc.M113.466946 -
Bioscience Trends Apr 2008Des-gamma-carboxyprothrombin (DCP) is an abnormal prothrombin with a decreased number of gamma-carboxylated glutamic acid residues in the Gla domain. DCP is also known... (Review)
Review
Des-gamma-carboxyprothrombin (DCP) is an abnormal prothrombin with a decreased number of gamma-carboxylated glutamic acid residues in the Gla domain. DCP is also known to be an effective serological tumor marker for hepatocellular carcinoma (HCC), and highly sensitive methods of detecting serum DCP have enabled the detection of early and smallsized HCC in clinical settings. Several immunohistochemical studies have suggested that excessive production of DCP in HCC tissues may relate to worse tumor behavior such as the presence of vascular invasion and intrahepatic metastasis of HCC cells. Clinical availability of DCP, therefore, might be a more significant factor in the diagnosis of tumor behavior in HCC patients. Recently, some studies have suggested that DCP may play an important role in cancer progression via induction of cancer cell proliferation and angiogenesis around HCC tissues. Thus, DCP is expected to be effectively used not only as a tumor marker but also as a target of drug discovery.
Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Models, Biological; Protein Precursors; Prothrombin
PubMed: 20103901
DOI: No ID Found -
PLoS Pathogens Sep 2022The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57...
The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.
Topics: Adenoviridae; Adenoviruses, Human; Animals; Capsid Proteins; Cattle; Genetic Vectors; Humans; Mice; Prothrombin; Transduction, Genetic
PubMed: 36156097
DOI: 10.1371/journal.ppat.1010859 -
Animal Models and Experimental Medicine Feb 2023Knowing the variability of blood coagulation responses to liver damage of different origins can provide a key to curing liver tissues or to mitigating treatment side...
BACKGROUND
Knowing the variability of blood coagulation responses to liver damage of different origins can provide a key to curing liver tissues or to mitigating treatment side effects. The aim of the present work was to compare the changes in the main components of hemostasis under experimental drug-induced hepatosis and hepatitis in rats.
METHODS
We modeled diclofenac-induced hepatitis and tetracycline-induced hepatosis. Hemostasis response was gauged by measuring fibrinogen, factor X, protein C (PC), and prothrombin in plasma. The decarboxylated form of prothrombin was detected by measuring prothrombin index and ecamulin index. Platelet reactivity was studied using aggregometry.
RESULTS
Both hepatitis and hepatosis decreased the synthesis of fibrinogen, factor X, and prothrombin. However, protein carboxylation was not disrupted in hepatosis but was much impaired in hepatitis. PC decreased in both models as a consequence of its consumption possibly during inflammatory response. Platelet aggregation rate was lower in hepatosis but higher in hepatitis.
CONCLUSIONS
Our findings imply the need for a thorough monitoring of the hemostasis system in liver diseases to avoid possible thrombotic complications. Its state indicates the disorder's rate and character.
Topics: Rats; Animals; Prothrombin; Factor X; Blood Coagulation Factors; Fibrinogen; Liver Diseases; Hemostatics; Chemical and Drug Induced Liver Injury
PubMed: 36574273
DOI: 10.1002/ame2.12301 -
Plastic and Reconstructive Surgery Oct 2022Surgical and technological advances have resulted in the widespread adoption of microsurgical breast reconstruction. Many comorbidities that potentially might impair... (Review)
Review
BACKGROUND
Surgical and technological advances have resulted in the widespread adoption of microsurgical breast reconstruction. Many comorbidities that potentially might impair vasculature and wound healing are no longer considered contraindications for these procedures. However, some uncertainty still prevails regarding the perioperative management of patients with disorders of hemostasis.
METHODS
The authors combined a literature review with a retrospective chart review of patients with disorders of hemostasis who had undergone microsurgical breast reconstruction at the senior author's (J.F.) center between 2015 to 2020. Several disorders associated with thrombotic and/or hemorrhagic complications were identified, and a standardized risk assessment and management strategy was developed in cooperation with a hematologist.
RESULTS
Overall, 10 studies were identified comprising 29 patients who had a defined disorder of hemostasis and underwent microsurgical breast reconstruction. Seventeen microsurgical breast reconstructions were performed on 11 patients at the senior author's (J.F.) center. High factor VIII levels, heterozygous factor V Leiden, and heterozygous prothrombin mutation G20210A were the most common genetic or mixed genetic/acquired thrombophilic conditions. As expected, hereditary antithrombin, protein C, or protein S deficiencies were rare. Among hemorrhagic disorders, thrombocytopenia, platelet dysfunction, and von Willebrand disease or low von Willebrand factor levels were those factors most frequently associated with increased perioperative bleeding.
CONCLUSIONS
Patients should be screened for elevated risk of thrombosis or bleeding before undergoing microsurgical breast reconstruction, and positive screening should prompt a complete hematologic evaluation. Interdisciplinary management of these disorders with a hematologist is essential to minimize risks and to obtain optimal reconstructive results.
CLINICAL QUESTION/LEVEL OF EVIDENCE
Risk, IV.
Topics: Antithrombins; Factor VIII; Hemostasis; Humans; Mammaplasty; Protein C; Prothrombin; Retrospective Studies; von Willebrand Factor
PubMed: 35943960
DOI: 10.1097/PRS.0000000000009499 -
The Journal of Biological Chemistry Aug 2016The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the...
The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr(93) in kringle-1 onto Trp(547) in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1 onto the active site in the closed form to prevent autoactivation. The open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase.
Topics: Amino Acid Substitution; Humans; Mutation, Missense; Protein Domains; Prothrombin; X-Ray Diffraction
PubMed: 27435675
DOI: 10.1074/jbc.M116.738310 -
The Journal of Biological Chemistry Dec 2013Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for...
Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation. The effect is abrogated by mutation of the catalytic Ser-525 and requires the presence of the Gla domain. Fluorescence titrations document direct binding of histone H4 to prothrombin with an affinity in the low nm range. Stopped flow data and luminescence resonance energy transfer measurements indicate that the binding mechanism obeys conformational selection. Among the two conformations of prothrombin, collapsed and fully extended, histone H4 binds selectively to the collapsed form and induces a transition toward a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases by 13 Å. These findings confirm the molecular plasticity of prothrombin emerged from recent structural studies and suggest that different conformations of the inter-kringle linker domain determine the functional behavior of prothrombin. The results also broaden our mechanistic understanding of the prothrombotic phenotype observed during cellular damage due to the release of histones in the blood stream. Prothrombin autoactivation induced by histone H4 emerges as a mechanism of pathophysiological relevance through which thrombin is generated independently of activation of the coagulation cascade.
Topics: Coenzymes; Enzyme Activation; Histones; Kinetics; Prothrombin
PubMed: 24178300
DOI: 10.1074/jbc.M113.509786 -
Journal of Thrombosis and Haemostasis :... Dec 2006Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G...
The association of prothrombin A19911G polymorphism with plasma prothrombin activity and venous thrombosis: results of the MEGA study, a large population-based case-control study.
BACKGROUND
Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G carriers of the prothrombin A19911G polymorphism has not been extensively investigated.
OBJECTIVES AND METHODS
We assessed prothrombin activity, FIIG20210A, and FIIA19911G polymorphisms in a large population-based case-control study, the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Four thousand three hundred and sixty-five consecutive patients with a first episode of deep vein thrombosis of the leg or pulmonary embolism were included. The control group (n = 4779) consisted of partners of patients or persons gathered using a random-digit dialing method. We studied the effect of FIIA19911G polymorphism on prothrombin activity and thrombosis risk, also in combination with factor V Leiden.
RESULTS
Among FII20210-GG control subjects, FII19911-GG carriers had 7.1% [95% confidence interval (CI): 5.7-8.5] higher mean prothrombin activity than FII19911-AA carriers and the risk for GG carriers was 1.43-fold increased compared to AA carriers [odds ratio (OR) 1.43; 95% CI: 1.27-1.61]. Among FII20210-GA control carriers, the mean prothrombin activity in both FII19911-AA and -AG carriers was nearly equivalent [131.7% and 133.4%; mean difference (95% CI) = 1.7% (-7.2-10.7)]. Because of genetic linkage, FII19911-GG carriers were very rare on a FII20210-GA background, as only one FII20210A carrier had FII19911-GG. In FII20210-GA carriers, the OR increased from 3.05 (95% CI: 2.17-4.27) in subjects with FII19911-AA to 3.33 (2.28-4.85) in subjects with FII19911-AG, compared to those with FII20210-GG and FII19911-AA.
CONCLUSIONS
The FIIA19911G polymorphism is associated with mildly elevated prothrombin activity and is a risk factor for venous thrombosis.
Topics: Adenine; Adult; Aged; Case-Control Studies; Factor V; Female; Gene Frequency; Genetic Predisposition to Disease; Guanine; Heterozygote; Humans; Male; Middle Aged; Netherlands; Odds Ratio; Polymorphism, Genetic; Population Surveillance; Prothrombin; Risk Factors; Venous Thrombosis
PubMed: 17059428
DOI: 10.1111/j.1538-7836.2006.02257.x -
The Journal of Biological Chemistry Mar 1975The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots...
The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).
Topics: Binding Sites; Calcium; Enzyme Activation; Factor V; Factor X; Kinetics; Peptide Fragments; Phospholipids; Protein Binding; Prothrombin; Time Factors
PubMed: 1117001
DOI: No ID Found -
Cellular and Molecular Life Sciences :... Dec 2006Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However,... (Review)
Review
Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However, although bites can be deadly, snake venom is a natural biological resource that contains several components of potential therapeutic value. Venom has been used in the treatment of a variety of pathophysiological conditions in Ayurveda, homeopathy and folk medicine. With the advent of biotechnology, the efficacy of such treatments has been substantiated by purifying components of venom and delineating their therapeutic properties. This review will focus on certain snake venom components and their applications in health and disease.
Topics: Animals; Cardiovascular System; Disintegrins; Fibrinogen; Hemostasis; Lectins, C-Type; Muscles; Neurotoxins; Phospholipases A; Platelet Aggregation Inhibitors; Protein Structure, Tertiary; Prothrombin; Signal Transduction; Snake Venoms; Thrombin
PubMed: 17103111
DOI: 10.1007/s00018-006-6315-0