Which coagulation factors require calcium

Desmopressin D arginine vasopressin administered in the preoperative period is expected to raise the concentration of factor VIII in patients with quantitative disorders. Total or partial is an extremely rare inherited bleeding disorder. Afibrinogenemia is rather well tolerated and may manifest as subcutaneous haematoma or umbilical haematoma at birth.

The clinical findings are variable in childhood and adults. It often presents as diffuse bleeding associated with consumption of coagulation factors and thrombocytopenia secondary to widespread small vessel thrombosis.

Majority of clotting factors are synthesized in liver therefore severe liver disease is associated with coagulopathy. Since liver is also involved in the clearance of activated clotting factors and fibrinolytic products, it may predispose to DIC. Management of bleeding secondary to liver disease is based on the laboratory values of various coagulation tests. Hypothermia is also associated with anticoagulatory effects, which are more pronounced in the presence of acidosis.

All these features give newer oral anticoagulants a major pharmacological benefits over vitamin K antagonists. There are number of factors that are associated with the hypercoaguable states. In addition to the genetic and hereditary disorders that predispose to thrombosis, several risk factors such as smoking, obesity, pregnancy, immobility, malignancy, surgery, females on oral contraceptives may also contribute to its development.

Acquired Protein C and Protien S deficiency may be observed in vitamin K deficiency, warfarin therapy, pregnancy, liver cirrhosis and sepsis.

The risk of thromboembolism in the perioperative period is well recognized. Therefore, patients with herditary thrombophillia should be given thromboprophylaxis. During pregnancy stasis due to obstruction of inferior vene cava by gravid uterus along with increase in the majority of clotting factors, fibrinogen and vWF is observed. Activity of Protein S decreases with simultaneous resistance of protein C.

In addition, fibrinolytic system is also impaired thus contributing to a hypercoaguable state that makes the parturient more prone to thromboembolism. During surgery and trauma, prolonged immobility promotes stasis which results in local hypoxia.

Physical disruption leads to exposure of TF thus triggering thrombosis. Even a venepuncture cause vascular wall injury thus, predisposing to thrombus formation. Since lower limb is associated with stasis and immobilization during surgery, venepuncture preferably should be avoided in the lower limb.

Fibrinolytic system is a parallel system which is activated along with activation of coagulation cascade and serves to limit the size of clot. Fibrinolysis is an enzymatic process that dissolves the fibrin clot into fibrin degradation products FDPs by plasmin originating from fibrin bound plasminogen in liver.

This reaction is catalysed by tPA or urokinase plasminogen activator u-PA released from vascular endothelium. The release of t-PA is stimulated by tissue occlusion, thrombin, epinephrine, vasopressin and strenuous exercise.

In vivo activity of the fibrinolytic system is assessed clinically by measuring the FDP's. D dimers are produced by digestion of cross linked fibrin and are specific indicators of fibrinolysis used in the assessment and diagnosis of pulmonary embolism, DIC or deep vein thrombosis. Since plasmin has the potential to degrade fibrinogen leading to deleterious consequences, the fibrinolytic activity is limited by following factors:.

Plasminogen activator inhibitor - It is the main physiological inhibitor of fibriolysis and acts by inhibiting t-PA and u-PA irreversibly. TAFI - It is a plasma proenzyme synthesized by liver and activated by thrombin.

It decreases the affinity of plasminogen to fibrin and augments the action of anti-trypsin in inhibiting plasmin. Congenital disorders pertaining to fibrinolytic system are rare.

Although the hyperfibrinolytic state is associated with increased tendency to bleed, deficiency of the same predisposes to thromboembolism. Acquired hyperfibrinolysis may be encountered in trauma, liver cirrhosis, amniotic fluid embolism, multiple myeloma, snake bite and conditions associated with massive activation of t-PA, which can lead to DIC and haemorrhage. Haemostasis is a complex physiological process, maintaining the fluidity of blood and is regulated by delicate balance existing between thrombogenic and anti thrombogenic mechanisms present in the body.

Imbalance between the two components predisposes a patient to either bleed or present with thrombosis. The physiology of the same therefore, needs to be understood to predict the pathological and clinical consequences of the same before implementing any pharmacological interventions.

Source of Support: Nil. Conflict of Interest: None declared. National Center for Biotechnology Information , U. Journal List Indian J Anaesth v. Indian J Anaesth. Author information Copyright and License information Disclaimer. Address for correspondence: Dr. E-mail: moc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.

This article has been cited by other articles in PMC. Abstract Coagulation is a dynamic process and the understanding of the blood coagulation system has evolved over the recent years in anaesthetic practice. Keywords: Anaesthesia, Coagulation system, haemostasis. Table 1 Thrombogenic and antithrombogenic components in the body. Open in a separate window. Platelet secretion After adhesion, degranulation from both types of granules takes place with the release of various factors.

Platelet aggregation Thromboxane A2 produced by activated platelets provide stimulus for further platelet aggregation. Table 2 Disorders of primary haemostasis. Table 4 Classification of coagulation factors.

Tissue factor plasminogen inhibitor It is a polypeptide produced by endothelial cells. Protein C pathway The propagation phase of the coagulation is inhibited by the Protein C pathway that primarily consist of four key elements: Protein C is a serine protease with potent anticoagulant, profibrinolytic and anti-inflammatory properties. It is activated by thrombin to form activated protein C APC and acts by inhibiting activated factors V and VIII with Protein S and phospholipids acting as cofactors Thrombomodulin - A transmembrane receptor on the endothelial cells, it prevents the formation of the clot in the undamaged endothelium by binding to the thrombin Endothelial protein C receptor is another transmembrane receptor that helps in the activation of Protein C Protein S is a vitamin K-dependent glycoprotein, synthesised by endothelial cells and hepatocytes.

Extrinsic pathway It is considered as the first step in plasma mediated haemostasis. Intrinsic pathway It is a parallel pathway for thrombin activation by factor XII. Common pathway Activated factor X along with its cofactor factor V , tissue phospholipids, platelet phospholipids and calcium forms the prothrombinase complex which converts prothrombin to thrombin.

Figure 1. Figure 2. Amplification Since the amount of thrombin generated is not sufficient, therefore numerous positive feedback loops are present that bind thrombin with platelets. Propagation The accumulated enzyme complexes tenase complex and prothrombinase complex on platelet surface support robust amounts of thrombin generation and platelet activation.

Figure 3. Stabilization Thrombin generation leads to activation of factor XIII fibrin stabilizing factor which covalently links fibrin polymers and provides strength and stability to fibrin incorporated in platelet plug. Table 5 Classification of disorders of coagulation. Fibrinogen deficiency Total or partial is an extremely rare inherited bleeding disorder.

Liver disease Majority of clotting factors are synthesized in liver therefore severe liver disease is associated with coagulopathy. Figure 4. SUMMARY Haemostasis is a complex physiological process, maintaining the fluidity of blood and is regulated by delicate balance existing between thrombogenic and anti thrombogenic mechanisms present in the body.

Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface. Thornton P, Douglas J.

Coagulation in pregnancy. Risk factors for venous and arterial thrombosis. Blood Transfus. Bombeli T, Spahn DR. Updates in perioperative coagulation: Physiology and management of thromboembolism and haemorrhage. Br J Anaesth. Point-of-care coagulation management in intensive care medicine. Crit Care. Endothelial cells in physiology and in the pathophysiology of vascular disorders. From normal to pathological hemostasis.

Can J Anesth. Regardless of whether the Extrinsic or Intrinsic pathway starts coagulation, completion of the process follows a common pathway. Both pathways are required for normal hemostasis and there are positive feedback loops between the two pathways that amplify reactions to produce enough fibrin to form a lifesaving plug. Deficiencies or abnormalities in any one factor can slow the overall process, increasing the risk of hemorrhage.

The coagulation factors are numbered in the order of their discovery. There are 13 numerals but only 12 factors. However, multiple alignments of a fairly large cohort of non-redundant sequences across the transglutaminase enzyme family indicate that at least in eukaryotic transglutaminases, three of these are of almost universal occurrence Unavailability of detailed structural information and the evolution of a compensatory regulatory motif might be the reason why some of these sites are not yet reported or not present in a few members of transglutaminase TGase family Amongst the structurally best-characterized transglutaminases, i.

These additional regulatory mechanisms explain the need of six calcium binding sites in TG2 to induce the conformational pull needed for overall activation of the molecule which therefore would be higher than FXIII or TG3. Calcium coordination by Asp induces the movement of a loop region, enabling substrate access to the active site. The activation events initiated by thrombin-mediated cleavage of its N-terminal activation peptide expose the other two calcium binding sites to coordinate, resulting in a series of conformational changes involving the formation of substrate binding pockets and ultimately exposing the catalytic triad Therefore, while retaining these three highly conserved calcium binding sites, these enzymes have functionally evolved differently to use these sites in a way that is best suited to their physiological milieu.

The spatial position of all three calcium binding sites in all transglutaminase structures is similar Fig. However, the orientation of the binding site residue side chains varies along with their inter-atomic distance, between transglutaminases.

The difference in inter-atomic distances is reflected in the similarity of the coordination pattern, as mentioned earlier for these proteins. Based on a structural relationship tree, we find that TG1 is more similar to FXIII-A than any other transglutaminase, which also is true evolutionarily since these two proteins are closely related Fig.

Transglutaminases at a prokaryotic level have evolved from ancient cysteine proteases papain-like thiol proteases ; and several microbial transglutaminases, as well as cysteine proteases, show a dependency on calcium for the regulation of activity.

Therefore one might assume that the mammalian FXIII calcium-binding sites have evolved from ancestral calcium binding sites in microbial transglutaminases and cysteine proteases However, microbial transglutaminases themselves have evolved divergently to eukaryotic transglutaminases.

The only structurally fully characterized primitive transglutaminase from Streptoverticllium mobaraense PDB ID: 1iu4 does not even show calcium dependence. All the above observations lead us to conclude that calcium binding sites in eukaryotic transglutaminases including FXIII have evolved divergently and have no evolutionary connection to the calcium binding sites in microbial transglutaminases or ancestral cysteine proteases.

The unique role of Cab1 is confirmed by the accelerated rate of activation of the molecule upon disrupting the calcium coordinating shells of Cab1 EK , disengaging calcium binding to Cab1, in the presence of an intact Cab2 Fig. The mutation EK which introduces an additional positive charge within Cab1 has the same effect as the coordination at Cab2 i. The endpoint assays, however, record lower activity of this mutant compared to the wild type Fig.

The strong differences between the generation assay parameters and the endpoint assays like photometric assay indicate that the isopeptidase activity on which the generation assay is based 26 and the transglutaminase crosslinking activity should not be considered synonymous especially when dealing with mutants. Our study is in close agreement with an earlier report that suggests that by mutating hydrophobic residues around the active site it is possible to have transglutaminase variants that are deficient in crosslinking activity but have normal or raised isopeptidase activity Certain calcium binding mutants like EK therefore influence cross-linking activity negatively by altering the substrate binding sites of the acceptor or donor cross-linking activity as also mentioned in the methods section is a two-step ping pong reaction.

These observations present an interesting pharmaceutical possibility of engineering enzymes with multiple but quantitatively varying enzymatic capacities. Since FXIII-A possesses a transglutaminase, isopeptidase and also protein disulfide isomerase activity, it can serve as a model protein to generate variants with higher enzymatic efficiency than normal in one aspect but neutralized concerning the other activities 27 , Therefore, the calcium binding site residues critically govern the structural integrity of the core domain with a possible distal allosteric effect on FXIII-AP.

Whether these variants alter the binding affinity of FXIII-A to thrombin or directly impede the cleavage reaction cannot be ascertained from our data. The different endpoint-based assays between themselves revealed consistency only to a limited extent, i. However, quantitatively, these assays also revealed relative differences from one another for the mutants.

For example, one mutation from Cab3 DK and two from Cab2 ND, QD , reported consistently low levels in all these assays, indicating that the mutations distorted the substrate binding site Fig. However, the extent of decrease in each assay was different, i.

Their coordination subsequently plays a major role in the correct orientation of the substrate binding sites as can be seen in the transition state intermediate models Since the three assays are related to three different substrates, the relative impact on substrate affinity would depend on the extent to which each mutation affects the substrate binding site.

In several transglutaminases cross-talk between calcium and other ions depending on the physiological milieu influences the activity status of the protein. In TGM3, calcium ion coordination induces the movement of a loop region that enables substrate access to the active site. Tighter coordination with magnesium ion instead of calcium keeps the loop in its inactive configuration.

Therefore, the relative concentrations of calcium and magnesium act as a regulatory switch for transglutaminase activity in TGM3 The principle behind the effect of sodium on the non-proteolytic calcium-induced activation of FXIII-A is very much different from that observed for TGM3 in the case of magnesium and calcium since unlike in TGM3, where binding of both magnesium and calcium is observed, in FXIII-A there is no actual coordination of sodium.

This event most likely disturbs the pKa ionization constant of internal buried residues, thereby affecting intra-domain residue interactions and changing the local secondary structure, which results in subtle conformational changes. The fact that ionic coordination alone does not influence protein conformation at the secondary structure level is further strengthened from our MD simulations in which we subjected only the core domain of the FXIII-A subunit to increasing concentrations of calcium Fig.

Again, as with high sodium concentrations, we observed a change in surface electrostatic potential with increasing calcium concentration even though in the period of simulation, no actual coordination was observed for calcium as well. Therefore, the activity status of FXIII-A is not only the result of the coordination of calcium ions to its three binding sites; it is the net impact of calcium coordination as well the response to ancillary solvent ion concentration surrounding the molecule.

Intracellularly FXIII-A is present as a dimer, and since no thrombin is accessible intracellularly, any enzymatic role, e.

Intracellular non-proteolytic FXIII activation could be achieved either through sudden ion fluxes like calcium release from endoplasmic reticulum that might alter the levels of calcium to supra-physiological levels fit for non -proteolytic activation The other option might involve an increase in sodium concentration, which, as mentioned earlier, will support non-proteolytic activation even at low concentrations of calcium.

If FXIII-A is considered as a therapeutic option for reparative processes related to these body fluids, knowledge of the subtle conformational changes brought about by the combination of ions in these fluids will help design easy to activate FXIII-A and with possibly a high specific activity. A cartoonist impression of the FXIII-A zymogenic state where no ions are influencing the structure followed by pre-activation state where the both the non-coordinating assumed to be sodium in purple as well as later coordinating ions assumed to be calcium in yellow influence the structure and conformation of FXIII-A molecule by altering its surface electrostatic properties.

The last part of the figure depicts the fully activated molecule which is the final outcome of the coordination of the coordinating ions to its binding sites in a FXIII-A molecule that has already been moved from its zymogenic to a pre-activation state. The FXIII complex assembly and its dissociation in plasma primarily involve the formation and disruption of non-covalent hydrophobic interactions Intrinsic ion binding to proteins involves hydration energies, which are entropically driven Also, calcium binding to transglutaminases is reported to induce conformational changes, as is observed in the crystal structures of activated forms of FXIII-A as well as TG2 15 , These conformational changes result in a change of hydrophobic surface area while exposing the core domain active site.

The high K 1 for Cab1 would ideally be enthalpically favorable since this strong ion binding would constrict the molecular motions by bringing about an order in the spatial secondary structure 41 , 42 , Therefore, the first event cannot be independently thought of as binding of calcium at Cab1 but rather the combined saturation of Cab1 and simultaneous coordination of Cab2 to the point of transient disruption of Cab1.

Once the Cab1 is transiently disrupted, the molecule overcomes the zymogenic constraint and moves conformationally towards the open, active structure as a result of a gain of entropy. At this point of time, the conformational changes taking place in FXIII-A have set into motion another event, which is the disruption of the zymogenic dimeric interface Major hydrophobic and non-covalent interactions between the opposing dimers are lost, resulting in a solvent-protected to a solvent-exposed state, and b exposure of Cab3.

The third event in our thermodynamic cycle is the final coordination and saturation of Cab3, which is occurring simultaneously to the monomerization of the activated FXIII molecule. This event is aided by a major influx of water molecules in FXIII-A regions which were previously inaccessible to water owing to the closed structural fold or the dimeric interface.

The rise in entropy is the result of the enthalpic contribution of the final Cab3 saturation and dimer dissociation. One must, of course, remember that these events can occur in two different species, i. In the latter, there are two dissociative processes in action, i. However, both dissociative events most likely run simultaneously each contributing to the success of the other.

Both are brought about by conformational changes upon calcium binding and influx of water molecule into previous hydrophobic cores formed by the respective folds of the protein and the pattern in which the zymogenic complex is assembled. The figure s represent in successive order the zymogenic crystal structure of FXIII-A monomer, the third and sixth transition state intermediate models reported earlier and the fully activated structure of FXIII-A monomer.

All structures are depicted in the ribbon format. The N-terminal beta sandwich domain has been hidden for the sake of clarity. The hinge region of almost 20 amino acids is coloured red.

The water solvation shell is depicted with blue circles. Since the final event in the thermodynamic cycle of FXIII-A activation is enthalpically driven instead of entropically i. Our thermodynamic analysis highlights the role of the influx of bulk solvent that in continuation of the conformational changes induced by calcium binding enables the global domain movements observed during FXIII-A activation.

At a structural level, we can observe these changes if we follow the conformation of the transition state models between the activated and zymogenic form of FXIII-A Fig.

With the binding of calcium, conformational changes occurring at the core domain disrupt the non- covalent association of core domain with the barrel domains. This primary disruption enables the entry of the solvent molecules to this conformationally shielded region. In other words, the activation of FXIII-A is majorly driven by conformational entropy brought about by calcium binding. Since all reactions follow a common energetic purpose, i.

Recent experimental evidence has also shown that the activated form of FXIII-A is, in fact, monomeric and is capable of reconversion to its zymogenic dimeric state upon removal of calcium from the medium Ever since FXIII-A has been recognized as a pharmaceutical candidate for inhibitor development, inhibitors have been designed primarily against the active site or the thrombin cleavage mechanism of this protein 44 , 45 , 46 , 47 , Our results suggest that two other areas of FXIII can potentially serve as regions against which inhibitors can be designed.

The regions in and around the calcium binding sites belong to one group of potential candidates since they not only dictate the rate of activation but also the proper orientation of substrate binding sites. Cab1 being spatially more accessible gets saturated faster and strongly than Cab2 yielding a low-energy, relatively stable state.

Subsequently, the Cab2 coordination transiently disrupts Cab1 giving rise to a high-energy, unstable, transient state event 1. The increasing saturation of Cab2 accompanied by the conformational changes directed by it result in the stabilization of unstable intermediate bringing down free energy. Meanwhile, slow disruption of the dimeric interface proceeds as the molecule proceeds towards full saturation Cab3 gets exposed with the dimeric interface coming apart event 2.

As the dimer loosens up, water molecules seep through and expose the dimeric interface further. Molecule solvation shell is represented by dotted circle. Calcium ion saturation at Cab1, Cab2 and Cab3 is depicted by red, blue and green solid dots respectively.

Black rod is the dimeric interface. Yellow star is the catalytic center. As a final commentary to this study, we would like to acknowledge some limitations of our study.

Our work focusses on calcium binding without discriminating between proteolytic and non-proteolytic modes of activation A significant part of the work is based on transient transfections and in silico analysis performed on modeled structures. Similarly, the thermodynamic analyses are based on custom fitting models which themselves are built around hints from in silico experiments.

We present this work to the readers as an exploratory characterization of individual calcium ion binding sites of FXIII-A. All experiments were done on sub-cultured cells in logarithmic phase below passage Site-directed mutagenesis was performed on the aforementioned construct, using GeneArt Site-directed mutagenesis system Life Technologies. Although a total of 13 different residues contribute to calcium binding in the three FXIII-A calcium binding sites, we were successful in cloning and expressing only 8 of them.

All vector construct clones were completely sequenced and verified for the correct incorporation of mutations in-house. Briefly, 2. Each transfection lot was accompanied with a positive control, i. Results for all assays performed on the transfection lysates were normalized accordingly. Intracellular lysate from transfected cells was quantified by BCA estimation Pierce, Life technologies. Subsequently, after a wash with PBS-Tween 0. After washing thrice in PBS-Tween 0. Using known amounts of recombinant FXIII-A positive control , the percentage antigenicity and absolute quantity of antigenically active FXIII-A was calculated in transfected samples based on the chemiluminescent intensity of the signal.

The antigenic levels were used in combination with activity levels from photometric assay to calculate the specific activity of the individual samples. All the experiments were performed in duplicates, with three sets of transfections to ensure reproducibility.

Cross-linking of the 5- biotinamido pentylamine into the fibrin by recombinant FXIII was detected using streptavidin-alkaline phosphatase Life Technologies and p-Nitrophenyl phosphate Sigma. Optical density OD values at time 0 were subtracted from the minute readings for each lysate to remove background and a standard curve of known concentrations of FXIII were used to extrapolate activity of rFXIII in each lysate.

OD values at time 0 were subtracted from the minute readings for each lysate to remove background and a standard curve of known concentrations of FXIII were used to extrapolate activity of rFXIII in each lysate. Human standard plasma Siemens Healthcare, Erlangen, Germany was used as an internal assay control. The transglutaminase crosslinking reaction is a two-step reaction a ping-pong mechanism , in which two substrates sequentially accesses the active site to first form intermediates and then get crosslinked to each other 22 , Theoretically, both continuous-generation assay and end-point activity assay should show correlation, but that might not be the case for mutant variants, especially those that alter substrate binding sites.

The tlag is the time required for the activation of FXIII-A by thrombin in plasma background , in the generation assay. Note: An illustration of the generation assay curve along with a small commentary is provided in Supplementary Fig.

We used FXIII-A subunit activation transition state intermediate models we had earlier generated and reported, to study the changes in the substrate binding regions as the FXIII-A molecule unfolds from its closed zymogenic state to its open fully activated state during the process of activation Three major putative substrate binding regions i.

Finally these binding regions were mapped and highlighted on the FXIII-A subunit activation transition state intermediate models.

The final gap resolved structure was chosen from the output file based on scores that were a combination of all backbone atom anchor match RMSD corresponds to the base structure and all backbone atom loop match RMSD corresponds to the loop structure.

The file with the lowest energy in the simulation trajectory was chosen for conducting further simulations. The macro was modified for running simulations at different ionic concentrations. Hydrogen bond networks were optimized using the method of Hooft and co-workers The simulation cell was filled with water at a density of 0.

Most importantly, the simulation cell net charge was neutralized with different NaCl and CaCl 2 concentrations. The entire system was energy minimized by steepest descent to remove conformation stress within the structure, followed by simulated annealing minimization until convergence was achieved. The output is in the form of a colour coded point and connector network pattern.

The colour codes are explained in the inset diagram for all network pattern outputs. The degree to which an amino or nucleic acid position is evolutionarily conserved i. The resulting alignment output was viewed on the Jalview alignment viewer.

The structural conservation of the calcium binding sites was evaluated by structurally aligning all human transglutaminase structures, including FXIII-A subunit using the Multiseq tool embedded in VMD The highest scoring best C-score model among the output files was chosen for multiple alignments Supplementary Fig.

Post alignment, a structure-based phylogenetic tree was generated using delta QH values, which is a measure of structural homology. Subjects closer to each other on this tree are structurally similar. Isothermal titration calorimetry experiments were carried out on a MicroCal microcalorimeter Malvern Panalytical, UK. The reference cell was filled with Autoclaved MiliQ water. Hence, titrations were performed at a high c-value as 25,; and a standard c-value of 20 For each experiment, thrombin was added before the start of the reaction; hence rFXIII-A 2 was incubated with thrombin, in the absence of calcium for the period of pre-titration delay.

To account for the heat of dilution, we performed blank experiments under the same conditions without rFXIII-A 2 in the sample cell Thrombin in cell titrated against CaCl 2 in the syringe. Peak integration was done in the software Origin 7.