Thrombin-thrombomodulin Complex Research Articles

Thrombin activatable fibrinolysis inhibitor plasma levels and TAFI Thr325Ile genetic polymorphism in a cohort of Egyptian sickle cell disease patients and impact on disease severity.

Thrombin is a critical protease modulating thrombosis as well as inflammation, which are one of the main pathophysiological mechanisms in sickle vasculopathy, and its levels were reported to be high in sickle cell disease (SCD). The thrombin-thrombomodulin complex activates the TAFI inhibitor of fibrinolysis, which acts by reducing plasmin affinity for its substrate thus hindering fibrinolysis. We aimed to determine the influence of the Thr325Ile single nucleotide polymorphism (SNP) on TAFI antigen levels and potential effects on the severity of SCD in a cohort of Egyptian patients. Genotyping of Thr325lle polymorphism using Taq-Man SNP genotyping assay and TAFI level measurement using an enzyme-linked immunosorbent assay were performed for 80 SCD patients (45 homozygous HbSS, 16 S/β0 and 19 Sβ+) as well as 80 age- and gender-matched healthy control subjects. Plasma TAFI levels were higher in SCD patients with Thr325Ile polymorphism, yet the difference was not statistically significant (p=.204). SCD patients with polymorphic genotypes had a greater number of hospital admissions (p=.03). Ten patients with acute chest syndrome had the homozygous polymorphic genotype (GG), and all patients with pulmonary hypertension had the polymorphic genotype (six were homozygous [GG] and five were heterozygous [GA]). Patients with SCD complicated with pulmonary hypertension showed significantly higher plasma TAFI levels (p=.044). The analysis of Thr325Ile polymorphisms combined with plasma TAFI levels suggests that the analyzed SNP could influence plasma TAFL levels and SCD disease severity and hospitalization rates, which could be predictors for complex disease.

LPL rs264, PROCR rs867186 and PDGF rs974819 Gene Polymorphisms in Patients with Unstable Angina.

Coronary artery disease is caused by changes in the coronary arteries due to the atherosclerotic process and thrombotic changes. A very important role in the development of the atherosclerotic process in the coronary vessels is played by the inflammatory process and the immune response. Due to the important role of lipids and the coagulation process in the atherosclerotic process, research has also focused on genes affecting lipid metabolism and the coagulation system. Lipoprotein lipase (LPL) is an enzyme that metabolises lipids, hydrolysing triglycerides to produce free fatty acids and glycerol. Protein C (PC) is an essential component of coagulation and fibrinolysis. It is activated on the endothelial surface by the membrane-bound thrombin-thrombomodulin complex. Platelet-derived growth factor (PDGF) has a number of important functions in processes related to fibroblast and smooth muscle cell function. Due to their influence on lipid metabolism and coagulation processes, LPL, PROCR (endothelial cell protein C receptor) and PDGF may affect the atherosclerotic process and, thus, the risk of coronary heart disease. The aim of the study was to examine the associations between the LPL rs264, PROCR rs867186 and PDGF rs974819 gene polymorphisms and the risk of unstable angina and selected clinical parameters. The study included 232 patients with unstable angina and 144 healthy subjects as the control group. Genotyping was performed using real-time PCR. There were no statistically significant differences in the distribution of the polymorphisms tested between the patients with unstable angina and the control subjects. The results showed associations between the PROCR rs867186 and PDGF rs974819 polymorphisms and some clinical parameters in patients with unstable angina. In patients with the PDGF rs974819 CC genotype, there were increased values for cholesterol and LDL serum levels in comparison with patients with the PDGF rs974819 CT and TT genotypes. In patients with the PROCR rs867186 AA genotype, HDL serum levels were lower than in patients with the GA genotype. The results of our study did not show that the LPL rs264, PROCR rs867186 and PDGF rs974819 gene polymorphisms were significant risk factors for unstable angina in our population. The results of the study suggest that PDGF rs974819 and PROCR rs867186 may be associated with some parameters of lipid metabolism.

Ser252Asn Mutation Introduces a New N-Linked Glycosylation Site and Causes Type IIb Protein C Deficiency.

Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC). We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation. We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (252NTT254) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC. The new N-glycosylation site (252NTT254) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Dynamic allostery in thrombin-a review.

Thrombin is a serine protease that catalyzes a large number of different reactions including proteolytic cleave of fibrinogen to make the fibrin clot (procoagulant activity), of the protease activated receptors (for cell signaling) and of protein C generating activated protein C (anticoagulant activity). Thrombin has an effector binding site called the anion binding exosite 1 that is allosterically coupled to the active site. In this review, we survey results from thermodynamic characterization of the allosteric coupling as well as hydrogen-deuterium exchange mass spectrometry to reveal which parts of the thrombin structure are changed upon effector binding and/or mutagenesis, and finally NMR spectroscopy to characterize the different timescales of motions elicited by the effectors. We also relate the experimental work to computational network analysis of the thrombin-thrombomodulin complex.

An evaluation of circulating activated TAFI in septic DIC: a case series and review of the literature

BackgroundAdministration of recombinant human soluble thrombomodulin (rTM) is often used in Japan to treat septic disseminated intravascular coagulation (DIC). Thrombin-activatable fibrinolysis inhibitor (TAFI) is a fibrinolysis inhibitor activated by the thrombin-thrombomodulin complex, however, it is unknown whether circulating activated TAFI is increased after rTM administration in patients with DIC. Furthermore, the relationship between TAFI activation and the prognosis of septic DIC is not defined yet.Case presentationWe report a series of 8 patient’s TAFI activation with septic DIC treated by rTM. We sought to investigate the effect of rTM on TAFI activation and the association of plasma activated TAFI (TAFIa/ai) levels with the prognosis of septic DIC.Using plasma samples from clinical studies conducted from May 2016–March 2017 on eight patients with septic DIC at Kagoshima University Hospital, we measured plasma levels of total TAFI, TAFIa/ai, thrombin-antithrombin complex (TAT), prothrombin fragment 1 + 2 (F1 + 2), soluble fibrin (SF), antithrombin (AT), protein C (PC), protein S (PS), and plasminogen activator inhibitor-1 (PAI-1) before and after intravenous rTM administration. Then, we evaluated the relationship of these marker levels to prognosis. The thrombin-rTM complex activated TAFI in vitro in plasma from a healthy volunteer. However, TAFIa/ai levels did not significantly increase over baseline in the septic DIC patients after intravenous rTM administration. Baseline TAFIa/ai levels in non-survivors were significantly higher than those in survivors.ConclusionsPlasma TAFIa/ai did not increase with rTM administration. Elevated baseline TAFIa/ai concentration may be a negative prognostic indicator in septic DIC. Larger studies are needed to confirm the in vivo effect of rTM on TAFI activation.

Mechanisms for the allodynia caused by complementanaphylatoxin C5a in mice

The thrombin-thrombomodulin (TM) complex generates activated forms of protein C(APC) and thrombin-activatable fibrinolysis inhibitor (TAFIa), known ascarboxypeptidase B (CPB), and degrades high mobility group box 1 (HMGB1), apronociceptive molecule. We have shown that soluble TM (TMα) preventschemotherapy-induced peripheral neuropathy (CIPN) in rodents, and that APC andTAFIa/CPB, as well as HMGB1 degradation, contribute to the anti-CIPN effect ofTMα. Given our recent evidence for the involvement of complement C5a, degradableby TAFIa/CPB, in CIPN, we analyzed the mechanisms for peripheral C5a-mediatedpain in mice. Intraplantar (i.pl.) injection of C5a at 30-300 ng induceddose-dependent mechanical allodynia, as assessed by von Frey test, an effectabolished by i.p. administration of DF2593A, a mouse C5a receptor (C5aR)antagonist, TAFIa/CPB, TMα or an anti-HMGB1-neutralizing antibody. Systemic(i.p.), but not local (i.pl.), administration of liposomal clodronate, amacrophage depletor, partially reduced the C5a-induced allodynia. Inmacrophage-like RAW 264.7 cells, C5a caused HMGB1 release in the presence, butnot absence, of a sub-effective concentration of lipopolysaccharide. Together,C5a-induced allodynia is considered to involve C5aR-dependent HMGB1 release frommonocyte-derived macrophages and non-macrophage cells.

PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy.

Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.

Аспекты применения транексамовой кислоты у пациентов с политравмой

Актуальность. В статье проанализированы особенности применения транексамовой кислоты у пациентов с политравмой. Цель исследования: изучить данные литературы об эффективности применения транексамовой кислоты у пациентов с политравмой и проанализировать частоту переливания компонентов крови у пациентов с политравмой в зависимости от времени назначения транексамовой кислоты после получения травмы. Материалы и методы. Проведено ретроспективное когортное исследование, в которое были включены 40 пациентов в возрасте от 22 до 56 лет с политравмой. Всех пациентов разделили на 2 группы: I группа — пациенты, у которых применяли транексамовую кислоту до 3 часов после возникновения политравмы, и II группа — больные, которые принимали транексамовую кислоту во временном промежутке 3–8 часов после возникновения политравмы. Результаты. Основным многофакторным условием для возникновения ранней коагулопатии у пациентов с политравмой является сочетание шока, вызванного кровотечением, и повреждения тканей, что приводит к генерации тромбин-тромбомодулинового комплекса, активации антикоагулянтной и фибринолитической систем. У пациентов, включенных в анализ данных в нашем исследовании, основными причинами политравмы были: дорожно-транспортное происшествие — у 34 пациентов (85 %), падение с высоты — у 6 пациентов (15 %). Средний возраст пациентов I группы составил 39,2 ± 17,3 года, во II группе — 41,9 ± 12,8 года. В исследование вошли 29 мужчин и 11 женщин. Не выявлено достоверных различий в тяжести состояния, структуре компонентов полиорганной недостаточности, антропометрических характеристиках пациентов обеих групп. Мы установили, что в I группе пациентов переливание эритроцитарной массы и свежезамороженной плазмы проводили в 14 случаях из 26, что составило 53 % от всех случаев политравмы в данной группе. Во II группе пациентов переливание компонентов крови проводили в 9 случаях из 14, то есть в 64 % от всех случаев из данной группы. Соответственно, частота переливания компонентов крови у пациентов II группы на 11 % выше по сравнению с пациентами I группы (p ≥ 0,05). Выводы. Полученные нами предварительные результаты свидетельствуют, что введение транексамовой кислоты до 3 часов после возникновения политравмы может снижать частоту гемотрансфузий.

Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Zymogen and activated protein C have similar structural architecture

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule FRET and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid epidermal growth factor domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing epidermal growth factor domains, such as factors VII, IX, and X.

Protein C Deficiency as a Risk Factor for Stroke in Young Adults: A Review.

Protein C (PC) is a 62-kD vitamin K dependent glycoprotein produced by the liver as a zymogen and is activated by binding to the thrombin-thrombomodulin complex, with protein S (PS) acting as a cofactor. Among its various functions, PC acts as a naturally occurring anticoagulant and its deficiency, either homozygous or heterozygous, predisposes the individual to a state of thrombosis, particularly venous thromboembolism, and mainfests as myocardial infarction (MI), deep venous thrombosis, pulmonary embolism, or stroke. This review discusses the pathophysiology of the anticoagulatory effect of PC, mode of inheritance of its deficiency, the arterial and venous involvement in patients with stroke, and its risk factors. A detailed analysis of published case reports on PC deficiency as a causative agent of stroke in young adults has also been included along with the management of such patients.

Use of Viscoelastography in Malignancy-Associated Coagulopathy and Thrombosis: A Review.

The relationship between malignancy and coagulopathy is one that is well documented yet incompletely understood. Clinicians have attempted to quantify the hypercoagulable state produced in various malignancies using common coagulation tests such as prothrombin time, activated partial thromboplastin time, and platelet count; however, due to these tests' focus on individual aspects of coagulation during one specific time point, they have failed to provide clinicians the complete picture of malignancy-associated coagulopathy (MAC). Viscoelastic tests (VETs), such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM), are whole blood analyses that have the advantage of providing information related to the cumulative effects of plasma clotting factors, platelets, leukocytes, and red cells during all stages of the coagulation and fibrinolytic processes. VETs have gained popularity in the care of trauma patients to objectively measure trauma-induced coagulopathy (TIC), but the utility of VETs remains yet unrealized in many other medical specialties. The authors discuss the similarities and differences between TIC and MAC, and propose a mechanism for the hypercoagulable state of MAC that revolves around the thrombomodulin-thrombin complex as it switches between activating the protein C anticoagulation pathway or the thrombin activatable fibrinolysis inhibitor coagulation pathway. Additionally, they review the current literature on the use of TEG and ROTEM in patients with various malignancies. Although limited research is currently available, early results demonstrate the utility of both TEG and ROTEM in the prediction of hypercoagulable states and thromboembolic complications in oncologic patients.

The influence of endothelial cell protein C receptor gene 6936A/G, 1651C/G, 4678G/C polymorphisms and soluble endothelial protein C receptor levels on in vitro fertilization outcomes

Hypercoagulability could be intrinsic or caused by the hormone treatment preceding the IVF procedure. Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C by the thrombin–thrombomodulin complex. Soluble EPCR plays a role in the maintenance of pregnancy evidenced by the findings that EPCR expression is critical for embryo development. Specific gene variants linked with altered soluble EPCR levels were associated with poor pregnancy outcome. The aim of this study was to assess the predictive value of EPCR gene polymorphisms (6936A/G, 1651C/G, and 4678C/G) and sEPCR level on the IVF outcome in Egyptian women with repeated IVF failure. They were compared to healthy control patients eligible for IVF. The present study was conducted on 45 women with repeated IVF failure, three or more previous IVF-embryo transfer cycles, and 45 healthy age-matched women eligible for IVF. PCR-RFLP for the EPCR polymorphisms (6936A/G, 1651C/G, 4678G/C) was done for cases and control groups. Plasma-soluble EPCR levels were measured with ELISA. As regards the mutant, EPCR (6936A/G) genotypes (AG, GG) were higher than the wild type (AA) (P < 0.001, OR 4.125, 95% CI 2.198–7.740). The homozygous mutant genotype (GG) was higher in comparison to the wild type (AA). The mutant allele (G) was higher than the wild allele (A) (P < 0.001, or 2.549, 95% CI 1.601–4.061). Higher frequencies of the (1651C/G) genotype and lower soluble EPCR levels were noted both in (C/C) (P = 0.004; Z = −0.2867) and (C/G) (P = 0.006; Z = −0.2767) genotype carriers. Regarding, EPCR polymorphism (4678G/C), the homozygous mutant genotype (CC) was significantly lower than the homozygous wild type (GG), (P = 0.014, OR 0.289, 95% CI 0.108–0.776). Our data suggest that the 6936A/G and 1651C/G EPCR gene variants coupled with procoagulant diminished levels of sEPCR may be associated with a higher tendency for repeated implantation failure.

Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin-Thrombomodulin Complex.

Thrombin activity, inhibition, and localization are regulated by two exosites that flank the active site. Substrates, cofactors, and inhibitors bind to exosite 1 to promote active site access, whereas exosite 2 interactions hold thrombin on cells, platelets, and proteins. The exosites also serve allosteric roles, whereby ligand binding alters thrombin activity. Previously, we showed that ligands that bind exosite 2 attenuate the exosite 1-mediated interaction of thrombin with fibrin, demonstrating allosteric connection between the exosites. To determine the functional consequences of these inter-exosite interactions, we examined the effect of exosite 2 ligands on thrombin's interaction with thrombomodulin, a key cofactor that binds exosite 1 and redirects thrombin activity to the anticoagulant protein C pathway. Exosite 2-directed ligands, which included the HD22 aptamer, glycoprotein 1bα-derived peptide, and fibrinogen γ'-chain peptide, reduced the level of exosite 1-mediated thrombin binding to the thrombomodulin peptide consisting of the fourth, fifth, and sixth epidermal-like growth factor-like domains, decreasing affinity by >10-fold, and attenuated thrombomodulin-dependent activation of protein C by 60-80%. The ligands had similar effects on thrombin-mediated protein C activation with intact soluble thrombomodulin and with thrombomodulin on the surface of cultured endothelial cells. Their activity was exosite 2-specific because it was attenuated when RA-thrombin, a variant lacking exosite 2, was used in place of thrombin. These results indicate that additional reactions mediated by exosite 1 are amenable to regulation by exosite 2 ligation, providing further evidence of inter-exosite allosteric regulation of thrombin activity.

Abstract 348: Lys 42, 43, 44 and Arg 12 of Thrombin Activable Fibrinolysis Inhibitor Comprise Thrombomodulin Binding Exosite Essential for Exerting Its Antifibrinolytic Activity

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin or plasmin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K 1/2 ) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues eliminated TM binding, decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K 1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay. When thrombin or plasmin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1- and 4.0-fold compared with wild-type TAFI, respectively, suggesting that the mutation only impacted activation kinetics by thrombin-TM. Surface plasmon resonance data show that mutation of the four residues results in complete loss of binding, either in the presence or absence of thrombin. Together, our findings suggest that these four residues are critical for the interaction of TAFI with the thrombin-TM complex that modulates its antifibrinolytic activity.

Rational Design of Protein C Activators

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

Plasma carboxypeptidase U (CPU, CPB2, TAFIa) generation during in vitro clot lysis and its interplay between coagulation and fibrinolysis.

Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.

Lys 42/43/44 and Arg 12 of thrombin-activable fibrinolysis inhibitor comprise a thrombomodulin exosite essential for its antifibrinolytic potential.

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K1/2) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay at physiologic levels of TAFI. Similar trends in the antifibrinolytic activity of the TAFI variants were observed when plasma clots were formed using HUVECs as the source of TM. When thrombin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1-fold compared with wild-type TAFI, suggesting that these mutations only impacted activation kinetics in the presence of TM. Surface plasmon resonance data suggest that mutation of the four residues abrogates TM binding with or without thrombin. Therefore, Lys 42, Lys 43, Lys 44 and Arg 12 are critical for the interaction of TAFI with the thrombin-TM complex, which modulates its antifibrinolytic potential.

Rivaroxaban Prophylaxis in Severe Congenital Protein C Deficiency

INTRODUCTIONProtein C is a vitamin K-dependent coagulation inhibitor produced by the hepatocytes. It is activated by the Thrombin-Thrombomodulin Complex, and in association with its cofactor, Protein S, induces proteolysis and inactivates Factor Va and Factor VIIIa thus limiting thrombin production.Congenital protein C deficiency may be inherited as an autosomal dominant disorder. Asymptomatic Protein C deficiency has been reported to be prevalent in 1 in 200 to 500 of healthy individuals, whereas the incidence of symptomatic deficiency is 1 in 20000. Autosomal recessive inheritance has been estimated to be around 1 in 4 million.CASE REPORTWe report a case of a 19 year old male, ex 33 week-premature first twin baby from Thailand, diagnosed with severe Protein C Deficiency after presenting to a local hospital with an acute manifestation of bilateral leg swelling and purpura fulminans when he was 2 years old. At that time, his Protein C activity was 2%. He was initially treated with quarantined FFP replacement 10-15ml/kg weekly and LMWH and later warfarin. MRI done at that time showed evidence of prior ischemic events.Upon arrival to the US at 16 years of age his treatment with warfarin was continued. During the following year, he presented several times with complications including deep venous thrombosis, warfarin skin necrosis, bleeding and transient neurological deficits. He was started on rivaroxaban - a direct thrombin inhibitor- 20mg orally daily at 17 years of age. For the following two years he did well and had no other acute thrombotic, bleeding events or medication side effects. Follow up brain MRI showed no new areas of infarct.DISCUSSIONNo well-defined general guidelines are available for the treatment of symptomatic patients with severe congenital protein C deficiency. Current treatment for this condition focuses on to Protein C replacement with Fresh Frozen Plasma or an infusion of a human plasma-derived, viral inactivated protein C concentrate - such as ceprotin.It has been shown that patients with severe congenital Protein C deficiency were more effectively treated with Protein C concentrates than those treated with FFP or conventional anticoagulants. Nevertheless, Protein C concentrates requires supervision of an experienced physician and long-term treatments require the need of a central venous access. Side effects include higher risk of infections due to frequent transfusions, fluid overload, allergic reactions, high sodium intake, heparin induced thrombocytopenia and high cost. New anticoagulation medications open a window of possibilities for management of this medical condition.There is no literature on the use of rivaroxaban in children. Few studies have been done and most of them are case reports. In 2013, Martinelli et al published a case report of a 6 year old girl with severe protein S deficiency. Initially started on warfarin but had recurrent episodes of warfarin-induced skin necrosis. Her hypercoagulable state was due to warfarin-induced reduction of protein C so the switch of anticoagulant therapy to rivaroxaban resulted in the disappearance of skin necrosis at 1 year of follow-up.Our case demonstrates an autosomal recessive Protein C deficiency successfully managed with the use of direct Xa inhibitor- rivaroxaban- given orally on a daily basis. This treatment approach appears to be safe and effective and could be studied in prospective large cohorts of patients with Protein C deficiency. DisclosuresNo relevant conflicts of interest to declare.

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin‐activatable fibrinolysis inhibitor activation and activated thrombin‐activatable fibrinolysis inhibitor activity

Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.