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PubMed1983

[Prothrombin--substrate for blood plasma kallikrein and factor XIIa].

A study was made of the interaction between prothrombin and enzymes: blood plasma kallikrein and factors alpha-XIIa and beta-XIIa immobilized on enzacryl-AH. Kallikrein-induced prothrombin proteolysis was accompanied by a decrease in prothrombin activity, appearance of BAME-esterase and poor clotting activity. As a result of fractionation of products on the column with DEAE-Sephadex A-50, some fractions that have thrombin amidase activity (splitting of the substrate S-2238) and high antithrombin activity were obtained. Antithrombin activity manifested in the inhibition of fibrinmonomer aggregation during fibrin formation. During incubation with prothrombin, factors alpha-XIIa and beta-XIIa also stimulated the appearance of BAME-esterase activity. None of the immobilized enzymes activated factor X.

Acrylic Resins↗
PubMed1983

Activation of human prothrombin by stoichiometric levels of staphylocoagulase.

The activation of human prothrombin by the bacterial protein staphylocoagulase proceeds via the formation of a very stable equimolar complex. Unmasking of the active center in the prothrombin moiety of the complex is not caused by limited proteolysis. The kinetics of activation of human prothrombin by staphylocoagulase has been studied. The second order rate constant at pH 7.5, 37 degrees C, is 3.3 X 10(6) M-1 S-1. This reaction rate is close to reported diffusion-controlled rates of protein-protein interaction. The dissociation constant of the complex was too low to be measurable. From the kinetic data it is assumed that the first order rate constant for dissociation is orders of magnitude less than 10(-5) S-1. However, dissociation of the complex did occur in the presence of sodium dodecyl sulfate. Equimolar amounts of staphylocoagulase protect human thrombin, but not human factor Xa and bovine thrombin, against inactivation by antithrombin III. From these findings we postulate that tertiary structural changes in the thrombin region of prothrombin caused by a highly specific interaction between staphylocoagulase and that region unmask the active site.

Amino Acids↗
PubMed1982

The role of augmented Hageman factor (factor XII) titers in the cold-promoted activation of factor VII and spontaneous shortening of the prothrombin time in women using oral contraceptives.

Spontaneous shortening of the Thrombotest time and an increase in factor VII activity after cold storage of plasma in plastic vials have been described in the plasma of women using OCAs. The phenomenon if CPA is dependent on the presence of HF (factor XII) and is associated with increased plasma kallikrein activity and the disappearance of C1-INH activity. In the present study, spontaneous CPA and shortening of the prothrombin time were observed in 66% of women using OCAs. The prothrombin time was inversely related to factor VII activity and HF coagulant titer, whereas factor VII activity was strongly associated with the titer of HF. The addition of purified HF to normal plasma to a degree observed in OCA users promoted CPA generation and shortening of the prothrombin time. In contrast, reduction of the HF coagulant titer in the plasma of OCA users to a normal level prevented the rise in factor VII activity and shortening of the prothrombin time after cold storage. These data indicate that a high titer of HF, such as that observed in OCA users, is needed for the spontaneous CPA and add support to evidence linking the extrinsic and intrinsic pathways of clotting. The relationship of this in vitro phenomenon to the pathogenesis of thrombosis in women using OCAs is presently unclear. (J Lab Clin Med 99:363, 1982.)

Adult↗
PubMed1982

Studies of the prothrombin activation pathway utilizing radioimmunoassays for the F2/F1 + 2 fragment and thrombin--antithrombin complex.

We have evaluated the efficacy of utilizing radioimmunoassays (RIAs) for prothrombin activation fragments (F2/F1 + 2) and for thrombin--antithrombin complex (TAT) in purified systems and in whole blood. During venipuncture, appropriate anticoagulants were employed in order to prevent the generation of thrombin and factor Xa. The RIAs were shown to be specific for F2/F1 + 2 as well as TAT and did not interact with other plasma components. Initially, thrombin generation was studied in a purified human system of prothrombin, antithrombin, factor Xa, and factor V as well as phospholipid and Ca++. Under these conditions, the kinetics of F2/F1 + 2 and TAT generation were virtually superimposable. However, when factor V was omitted from the reaction mixture, a significantly greater amount of F2/F1 + 2 as compared to TAT was observable. Subsequently, prothrombin activation was monitored during the spontaneous coagulation of freshly drawn blood. Throughout the entire course of thrombin generation, the observable rate of formation of F2/F1 + 2 was considerably greater than that of TAT. We have examined the levels of F2/F1 + 2 and TAT in normal individuals. Our studies indicate that the concentrations of F1 + 2 and TAT average 1.97 nM and 2.32 nM, respectively. We have also quantitated the concentrations of F2/F1 + 2 and TAT in patients with disseminated intravascular coagulation. In these individuals, the levels of both components are elevated. However, the ratio of F1 + 2 to TAT ranges from 2.37 to 5.55. Thus, we conclude that under in vivo conditions, prothrombin activation is characterized by the accumulation of a stable precursor, such as prethrombin-2, and that this phenomenon may be related to an alteration of factor V function.

Antithrombins↗
PubMed1981

Effect of thromboplastin and instrumentation on the prothrombin time test.

Prothrombin time was measured in three different plasma samples by 2580 laboratories in the 1977 CAP Proficiency Testing Program. Analysis of variance was used to show that instrument, as well as thromboplastin, has a significant effect upon observed prothrombin time. In addition, the instrument and thromboplastin effects were estimated, and all were shown to be linearly related to the prothrombin time of the plasma sample. This linear relationship was used to develop a formula for adjusting/correcting an observed prothrombin time for both the thromboplastin and instrument effect. This adjustment/correction method seems promising on the basis of its use in four different sets of data.

Hematologic Tests↗
PubMed1994

Homozygosity for a novel missense mutation in the prothrombin gene causing a severe bleeding disorder.

A patient with a severe bleeding tendency and hypoprothrombinemia (Factor II activity 2%, Factor II antigen 5%) was screened for the presence of alterations in his prothrombin gene. Direct sequencing of PCR fragments derived from the coding and flanking regions of the prothrombin gene, revealed that the patient was homozygous for an A-->G substitution in exon 3. This substitution predicts the replacement of Tyr44 (TAC) by Cys (TGC) in the prothrombin molecule. Both parents were found to be heterozygous for the same mutation. Further family studies revealed complete cosegregation of the mutation with the prothrombin deficiency. Only the five homozygous brothers and sisters of the propositus were clinically affected (severe hemorrhages including epistaxis, soft tissue, muscle and joint bleedings in all, and severe hemorrhages in the two women). The bleeding tendency therefore seems to inherit as an autosomal recessive trait.

Adenine↗
PubMed1994

A membrane-mediated catalytic event in prothrombin activation.

Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.

Amino Acid Sequence↗
PubMed1994

The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis.

The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.

Animals↗
PubMed1994

Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development.

The protease thrombin is a potent agonist for platelet aggregation, mesenchymal cell proliferation, and endothelial production of growth factors and adhesion molecules. Thrombin also modulates neurite outgrowth in neuronal cultures. These apparently disparate responses to thrombin appear to be largely mediated by the recently cloned thrombin receptor. In the adult, thrombin is generated from its zymogen prothrombin at sites of vascular injury when circulating coagulation factors meet extravascular tissue factor. In this context thrombin's varied actions may mediate responses to wounding. Whether thrombin's actions on cells may also play a role in development is unknown. We examined the expression of thrombin receptor, prothrombin, and tissue factor by in situ hybridization in mouse development. Thrombin receptor mRNA was expressed widely in mesenchymal cell populations during early organogenesis (E9.5) and was particularly abundant in developing heart and blood vessels. Robust receptor expression was also noted in the germinal epithelium of the hindbrain. Thrombin receptor expression became more restricted with time and by the fetal growth stage (E16.5) was most readily detected in certain neurons, endocardial and endothelial cells, and within lung and liver. In contrast to the thrombin receptor, prothrombin mRNA was limited to the embryonic liver and was not detected until E12.5, well after the onset of receptor expression. mRNA for tissue factor, one important trigger for thrombin generation in the adult, was detected in embryonic epithelia from E9.5-12.5. In several instances, tissue factor-expressing epithelia were surrounded by thrombin receptor-expressing mesenchyme. These data suggest a possible role for the thrombin receptor in development. The finding of robust thrombin receptor expression before prothrombin mRNA was detected raises the question of whether other proteases or peptide ligands can activate the thrombin receptor.

Animals↗
PubMed1993

Searching for inaccuracy in clinical laboratory testing using Medicare data. Evidence for prothrombin time.

OBJECTIVE: To determine if the occurrence of health outcomes following clinical laboratory testing can be used to identify types of laboratories that may be having higher than expected error rates. DESIGN: Retrospective analysis of Medicare Part B outpatient claims, Part A hospitalization bills, and death records using a case-control study. SETTING: Medicare records from six carrier territories were sampled during the period 1985 through 1987. PATIENTS: A total of 14,755 Medicare patients receiving a prothrombin time test in either a physician office laboratory or a commercial laboratory. OUTCOME MEASURES: Occurrence of a hospitalization for stroke or acute myocardial infarction, death, or no adverse outcome within 6 days of a prothrombin time. RESULTS: In physician office laboratories where prothrombin time test volume is below 40 per month, the odds that a tested patient will experience a stroke or an acute myocardial infarction are up to 1.96 and 3.43 times greater, respectively, than for a similar patient tested in a commercial laboratory. Switching from one laboratory to another between successive prothrombin time tests increased the odds of a stroke or an acute myocardial infarction by 1.57 and 1.32, respectively. Patients in two states with strong laboratory regulatory programs had fewer adverse outcomes. CONCLUSION: Examining patient outcomes subsequent to clinical laboratory testing may be a useful tool for clinical laboratory quality assurance.

Case-Control Studies↗
PubMed1993

Decentralized testing for prothrombin time and activated partial thromboplastin time using a dry chemistry portable analyzer.

Previous work has established the precision and accuracy of a portable blood coagulation analysis system using paramagnetic particles contained in a dry reagent on a disposable test card. We examined the deployment of this technology in decentralized hospital settings and compared test results obtained in the surgical intensive care unit, coronary care unit, and outpatient cardiology clinic with those obtained in the central laboratory. Nursing personnel were instructed in the use of the system, and quality control testing was performed daily by the laboratory staff. In the intensive care units, patient subjects included those on whom tests of prothrombin time and activated partial thromboplastin time had been ordered. Immediate determinations were performed by the intensive care unit nursing staff on the same citrated, whole-blood samples that were subsequently sent to the central laboratory. In the outpatient cardiology clinic, fingerstick blood samples were obtained for prothrombin time determinations with the dry chemistry system. Paired prothrombin time samples obtained by venipuncture were run in the hospital laboratory. The study involved multiple users, multiple locations, two lots of activated partial thromboplastin time cards, and several different instruments, over an extended period. Correlation coefficients between the dry chemistry system and the hospital laboratory under these conditions were in an acceptable range in all sites studied. We concluded that, with appropriate training and quality assurance, the dry chemistry system provides an acceptable alternative to the hospital laboratory for prothrombin time and activated partial thromboplastin time determinations.

Chemistry Techniques, Analytical↗
PubMed1977

The prediction of prothrombin time system performance using secondary standards.

A method for the comparison of prothrombin time systems using single large lots of lyophilized plasma in the College of American Pathologists Surveys is presented. Systematic biases for prothrombin time methods as well as for thromboplastins have been measured, and their use allows a very accurate (within 2.25%) prediction of the prothrombin time for the great number of prothrombin time systems presently being used in the United States.

Blood↗
PubMed1997

Compound heterozygosity for two novel missense mutations in the prothrombin gene in a patient with a severe bleeding tendency.

The abnormal prothrombin gene of an Italian patient with a severe bleeding tendency and hypoprothrombinemia was selected for study and compared with the prothrombin genes of healthy controls. All the coding and their flanking regions and the 5'- and 3'-UT regions of the prothrombin gene were screened by analyzing the nucleotide sequence of the corresponding PCR products. The patient was found to be heterozygous for two novel point mutations: one at nucleotide 4251 in exon 6, which changes the codon for cysteine-138 (TGC) in the kringle 1 domain to that for tyrosine (TAC), and one at nucleotide 8812 in exon 10, which results in the replacement of tryptophan-357 (TGG) by cysteine (TGT) in the catalytic domain. Her mother was heterozygous for the Cys-138 Tyr mutation and her father heterozygous for the Trp-357 Cys mutation. Several other sequence variations were identified in the prothrombin genes from control individuals. Only the variations at nucleotide 4203 and 10253 could be established as polymorphisms.

Blood Coagulation Factors↗
PubMed1997

The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis.

A dimorphism in the 3'-untranslated region of the prothrombin gene (G to A transition at position 20210) has recently been reported to be associated with increases in plasma prothrombin levels and in the risk of venous thrombosis. We have examined the prothrombin dimorphism among 99 unselected outpatients with phlebography verified deep venous thrombosis, and in 282 healthy controls. The prevalence of the 20210 A allele was 7.1% (7/99) in the patient group, and 1.8% (5/282) in the healthy control group (p = 0.0095). The relative risk of venous thrombosis was calculated to be 4.2 (95% CI, 1.3 to 13.6), and was still significant when adjustment was made for age, sex and the factor V:R506Q mutation causing APC resistance [odds ratio 3.8 (95% CI, 1.1 to 13.2)]. As previously reported, 28% of the patients were carriers of the factor V:R506Q mutation. Thus, 34% (one patient carried both traits) of unselected patients with deep venous thrombosis were carriers of an inherited prothrombotic disorder. To sum up, our results confirm the 20210 A allele of the prothrombin gene to be an important risk factor for venous thrombosis.

Adult↗
PubMedDate not supplied

[Effect of Glycine max lectin on the interaction of prothrombin and erythrocytes].

Native porcine erythrocytes do not initiate blood coagulation, though even the weak association (Kd = 4,25 +/- 9,35 microM) of prothrombin with their surface which is limited to the projection of two phospholipid polar head groups onto the external cell membrane, exerts a slight but authentic influence on the acceleration of blood clotting. There are 11.9 x 10(7) sites for 125I-prothrombin on one erythrocytes. At physiological concentration of prothrombin the surface of erythrocytes is far from saturation. The binding of 125I-labelled prothrombin to erythrocytes after their surface was covered by lectin Glycine max by means of the accessible residues of N-acetyl-D-galactosamine and D-galactose, glycoproteins and dlycolipids can never become saturated with any used concentration of the ligand. In the presence of the suspension of erythrocytes treated lectin platelet free plasma coagulates at the same speed as the control plasma.

Animals↗
PubMed1998

Geographic distribution of the 20210 G to A prothrombin variant.

A variant in prothrombin (clotting factor II), a G to A transition at nucleotide position 20210, has recently been shown to be associated with the prothrombin plasma levels and the risk of both venous and arterial thrombosis. The purpose of this study was to investigate the prevalence of carriership of this mutation in various populations. We combined data from 11 centres in nine countries, where tests for this mutation had been performed in groups representing the general population. We calculated an overall prevalence estimate, by a precision-weighted method, and, since the distribution of the prevalences did not appear homogeneous, by an unweighted average of the prevalences. We examined differences in the prevalences by geographical location and ethnic background as a possible explanation for the heterogeneity. Among a total of 5527 individuals who had been tested, 111 heterozygous carriers of the 20210A mutation were found. The prevalence estimates varied from 0.7 to 4.0 between the centres. The overall prevalence estimate was 2.0 percent (CI95 1.4-2.6%). The variation around the summary estimate appeared more than was expected by chance alone, and this heterogeneity could be explained by geographic differences. In southern Europe, the prevalence was 3.0 percent (CI95 2.3 to 3.7%), nearly twice as high as the prevalence in northern Europe (1.7%, CI95 1.3 to 2.2%). The prothrombin variant appeared very rare in individuals from Asian and African descent. The 20210A prothrombin variant is a common abnormality, with a prevalence of carriership between one and four percent. It is more common in southern than in northern Europe. Since this distribution within Europe is very different to that of another prothrombotic mutation (factor V Leiden or factor V R506Q), founder effects are the most likely explanation for the geographical distribution of both mutations.

Brazil↗
PubMed1998

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia.

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.

Factor V↗
PubMed1998

Inhibition of lupus anticoagulant activity by hexagonal phase phosphatidylethanolamine in the presence of prothrombin.

We have previously demonstrated that lupus anticoagulant antibodies from patients with systemic lupus erythematosus (SLE) specifically recognize hexagonal (II) phase phosphatidylethanolamine (PE), but not bilayer PE (Thromb Haemost 1989; 62: 892). In those studies, the involvement of proteins in this recognition was not evaluated. To address this issue, we have isolated IgG lupus anticoagulant antibodies from the plasma of SLE patients and evaluated the inhibition of lupus anticoagulant activity by hexagonal (II) phase PE in the presence and absence of purified plasma proteins. All six of the IgG lupus anticoagulant antibodies tested were inhibited by hexagonal (II) phase PE in the presence, but not the absence, of human prothrombin. In contrast, little or no inhibition was observed with prothrombin alone or with PE in combination with either beta2-glycoprotein I or annexin V. These data indicate that, for certain lupus anticoagulant antibodies, inhibition by hexagonal (II) phase PE is dependent on prothrombin, suggesting that these antibodies recognize a complex of PE and prothrombin.

Adult↗