Factor V Leiden and Prothrombin G20210A in Portuguese Women with Recurrent Miscarriage: Is it Worthwhile to Investigate?

OBJECTIVE: To compare the prevalence of factor V Leiden (FVL) and prothrombin (PT) G20210A mutations in Portuguese women with unexplained recurrent miscarriage (RM) and a control group of parous women. MATERIALS AND METHODS: FVL and PT G20210A analysis were carried out in 100 women with three or more consecutive miscarriages and 100 controls with no history of pregnancy losses. Secondary analysis was made regarding gestational age at miscarriage (embryonic and fetal losses). RESULTS: Overall, the prevalence of FVL and PT G20210A was similar in women with RM (5 and 3%) compared with controls (5 and 1%) OR 1.36 (CI 95% 0.45-4.08). In RM embryonic subgroup, PT G20210A was observed in 1.3% of women and FVL prevalence (2.6%) was inclusively lesser than that of controls. Both polymorphisms were more prevalent in women with fetal losses than in controls, although statistical significance was not reached due to the small size of the >10 weeks' subgroup. CONCLUSION: These data indicate that neither FVL nor PT G20210A is associated with RM prior to 10 weeks of gestation. Therefore, its screening is not indicated as an initial approach in Portuguese women with embryonic RM and negative personal thromboembolic history.

Evaluation of the marker of hypercoagulability prothrombin fragment F 1+2 in patients with mechanical or biological heart valve prostheses

OBJECTIVE: To investigate whether patients with heart valve prostheses and similar International Normalized Ratios (INR) have the same level of protection against thromboembolic events, that is, whether the anticoagulation intensity is related to the intensity of hypercoagulability supression. METHODS: INR and plasma levels of prothrombin fragment 1+2 (F1+2) were assessed in blood samples of 27 patients (7 with mechanical heart valves and 20 with biological heart valves) and 27 blood samples from healthy donors that were not taking any medication. RESULTS: Increased levels of F1+2 were observed in blood samples of 5 patients with heart valve prostheses taking warfarin. These findings reinforce the idea that even though patients may have INRs, within the therapeutic spectrum, they are not free from new thromboembolic events. CONCLUSION: Determination of the hypercoagulability marker F1+2 might result in greater efficacy and safety for the use of oral anticoagulants, resulting in improved quality of life for patients.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

The prothrombin time/international normalized ratio (PT/INR) Line: derivation of local INR with commercial thromboplastins and coagulometers--two independent studies

The WHO scheme for prothrombin time (PT) standardization has been limited in application, because of its difficulties in implementation, particularly the need for mandatory manual PT testing and for local provision of thromboplastin international reference preparations (IRP).

Point-of-care prothrombin time testing in paediatric intensive care: an observational study of the ease of use of two devices

Two major difficulties arise when taking blood samples in children: the challenge of venous access and the comparatively large amount of blood required.

Does point of care prothrombin time measurement reduce the transfusion of fresh frozen plasma in patients undergoing major surgery? The POC-OP randomized-controlled trial

BACKGROUND: Bleeding is a frequent complication during surgery. The intraoperative administration of blood products, including packed red blood cells, platelets and fresh frozen plasma (FFP), is often live saving. Complications of blood transfusions contribute considerably to perioperative costs and blood product resources are limited. Consequently, strategies to optimize the decision to transfuse are needed. Bleeding during surgery is a dynamic process and may result in major blood loss and coagulopathy due to dilution and consumption. The indication for transfusion should be based on reliable coagulation studies. While hemoglobin levels and platelet counts are available within 15 minutes, standard coagulation studies require one hour. Therefore, the decision to administer FFP has to be made in the absence of any data. Point of care testing of prothrombin time ensures that one major parameter of coagulation is available in the operation theatre within minutes. It is fast, easy to perform, inexpensive and may enable physicians to rationally determine the need for FFP. METHODS/DESIGN: The objective of the POC-OP trial is to determine the effectiveness of point of care prothrombin time testing to reduce the administration of FFP. It is a patient and assessor blind, single center randomized controlled parallel group trial in 220 patients aged between 18 and 90 years undergoing major surgery (any type, except cardiac surgery and liver transplantation) with an estimated blood loss during surgery exceeding 20% of the calculated total blood volume or a requirement of FFP according to the judgment of the physicians in charge. Patients are randomized to usual care plus point of care prothrombin time testing or usual care alone without point of care testing. The primary outcome is the relative risk to receive any FFP perioperatively. The inclusion of 110 patients per group will yield more than 80% power to detect a clinically relevant relative risk of 0.60 to receive FFP of the experimental as compared with the control group. DISCUSSION: Point of care prothrombin time testing in the operation theatre may reduce the administration of FFP considerably, which in turn may decrease costs and complications usually associated with the administration of blood products. TRIAL REGISTRATION: NCT00656396.

Is perioperative point-of-care prothrombin time testing accurate compared to the standard laboratory test?

There is no accepted way of measuring prothrombin time without time loss for patients undergoing major surgery who are at risk of intraoperative dilution and consumption coagulopathy due to bleeding and volume replacement with crystalloids or colloids. Decisions to transfuse fresh frozen plasma and procoagulatory drugs have to rely on clinical judgment in these situations. Point-of-care devices are considerably faster than the standard laboratory methods. In this study we assessed the accuracy of a Point-of-care (PoC) device measuring prothrombin time compared to the standard laboratory method. Patients undergoing major surgery and intensive care unit patients were included. PoC prothrombin time was measured by CoaguChek XS Plus (Roche Diagnostics, Switzerland). PoC and reference tests were performed independently and interpreted under blinded conditions. Using a cut-off prothrombin time of 50%, we calculated diagnostic accuracy measures, plotted a receiver operating characteristic (ROC) curve and tested for equivalence between the two methods. PoC sensitivity and specificity were 95% (95% CI 77%, 100%) and 95% (95% CI 91%, 98%) respectively. The negative likelihood ratio was 0.05 (95% CI 0.01, 0.32). The positive likelihood ratio was 19.57 (95% CI 10.62, 36.06). The area under the ROC curve was 0.988. Equivalence between the two methods was confirmed. CoaguChek XS Plus is a rapid and highly accurate test compared with the reference test. These findings suggest that PoC testing will be useful for monitoring intraoperative prothrombin time when coagulopathy is suspected. It could lead to a more rational use of expensive and limited blood bank resources.

The risk of early mortality of polytrauma patients associated to ISS, NISS, APACHE II values and prothrombin time

BACKGROUND: The early hemodynamic normalization of polytrauma patients may lead to better survival outcomes. The aim of this study was to assess the diagnostic quality of trauma and physiological scores from widely used scoring systems in polytrauma patients. METHODS: In total, 770 patients with ISS > 16 who were admitted to a trauma center within the first 24 hours after injury were included in this retrospective study. The patients were subdivided into three groups: those who died on the day of admission, those who died within the first three days, and those who survived for longer than three days. ISS, NISS, APACHE II score, and prothrombin time were recorded at admission. RESULTS: The descriptive statistics for early death in polytrauma patients who died on the day of admission, 1--3 days after admission, and > 3 days after admission were: ISS of 41.0, 34.0, and 29.0, respectively; NISS of 50.0, 50.0, and 41.0, respectively; APACHE II score of 30.0, 25.0, and 15.0, respectively; and prothrombin time of 37.0%, 56.0%, and 84%, respectively. These data indicate that prothrombin time (AUC: 0.89) and APACHE II (AUC: 0.88) have the greatest prognostic utility for early death. CONCLUSION: The estimated densities of the scores may suggest a direction for resuscitative procedures in polytrauma patients.Trial registration: "Retrospektive Analysen in der Chirurgischen Intensivmedizin" StV01-2008.http://www.kek.zh.ch/internet/gesundheitsdirektion/kek/de/home.html.

MASP-1 of the complement system promotes clotting via prothrombin activation.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

MASP-1 Induced Clotting--The First Model of Prothrombin Activation by MASP-1.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.

Prothrombin deficiency results in embryonic and neonatal lethality in mice

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII−/−) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII−/− embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII−/− embryos within this gestational time frame. However, at least one-quarter of the FII−/− mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice

Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2−/− embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2−/− embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2−/− neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.

Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FIIRHH and FIIHRR, FIIRRH and FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.