Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates

2001 ◽  
Vol 354 (2) ◽  
pp. 309-313 ◽  
Author(s):  
Kazuya HOSOKAWA ◽  
Tomoko OHNISHI ◽  
Midori SHIMA ◽  
Masanori NAGATA ◽  
Takehiko KOIDE
Keyword(s):  
Factor Viii ◽  
Factor Xiii ◽  
Protein C ◽  
Factor Viia ◽  
Activated Protein C ◽  
Factor Xa ◽  
Serine Proteinase ◽  
Coagulation Factors ◽  
Kd Values ◽  
Alkaline Conditions

Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin–agarose in a reversible manner. The Kd values for factor VIII, fibrinogen, factor XIII and protein C were 1.2×10-8, 4.4×10-8, 2.8×10-7 and 8.1×10-5M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (kass) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

scholarly journals The coagulopathy in sepsis: significance and implications for treatment

2011 ◽  
Vol 3 (4) ◽  
pp. 30 ◽  
Author(s):  
Paola Saracco ◽  
Pasquale Vitale ◽  
Carlo Scolfaro ◽  
Berardino Pollio ◽  
Mauro Pagliarino ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Tissue Factor ◽  
Protein C ◽  
Factor Viia ◽  
Activated Protein C ◽  
Microvascular Dysfunction ◽  
Clinical Effectiveness ◽  
Coagulation Factors ◽  
Anticoagulant System ◽  
Adults And Children

Sepsis related coagulopathy ranges from mild laboratory alterations up to severe disseminated intravascular coagulation (DIC). There is evidence that DIC is involved in the pathogenesis of microvascular dysfunction contributing to organ failure. Additionally, the systemic activation of coagulation, by consuming platelets and coagulation factors, may cause bleeding. Thrombin generation via the tissue factor/factor VIIa route, contemporary depression of antithrombin and protein C anticoagulant system, as well as impaired fibrin degradation, due to high circulating levels of PAI-1, contribute to enhanced intravascular fibrin deposition. This deranged coagulopathy is an independent predictor of clinical outcome in patients with severe sepsis. Innovative supportive strategies aiming at the inhibition of coagulation activation comprise inhibition of tissue factor-mediated activation or restoration of physiological anticoagulant pathways, as the administration of recombinant human activated protein C or concentrate. In spite of some promising initial studies, additional trials are needed to define their clinical effectiveness in adults and children with severe sepsis.

Evidence of Abnormal Coagulation: Production of Factor VIII α-Fragment In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1020-1020
Author(s):  
Pierre F. Neuenschwander
Keyword(s):  
Factor Viii ◽  
Factor Viia ◽  
Activated Protein C ◽  
Factor Xa ◽  
Major Product ◽  
Western Blots ◽  
Coagulation Inhibitors ◽  
Normal Hemostasis

Abstract Treatment of thrombosis typically involves the administration of coagulation inhibitors that must be carefully monitored and balanced so as to reduce unwanted coagulation (thrombosis) while maintaining normal or near-normal hemostasis. This balancing is necessary since the anticoagulants used alter enzymatic activities that are involved in both processes. This therapeutic strategy is based entirely on the view that thrombosis occurs by the same general pathways as normal hemostasis. While the enzymatic cascade of blood coagulation is well described and well accepted, numerous other minor reactions have been shown to occur in vitro but have not been examined in great detail due to the belief that they do not occur significantly during normal coagulation in vivo. We postulate that in certain pathological environments some of these minor procoagulant reactions may in fact become significant and lead to thrombogenic situations. If true, this could potentially allow novel targets for anticoagulation to be identified. In addition, the inhibition of these abnormal reactions could attenuate pathological coagulation whilst having limited or no effect on normal hemostatic reactions. One candidate reaction is the proteolysis of factor VIII (fVIII) by the factor VIIa-tissue factor (fVIIa-TF) complex, which results in a mixture of active and inactive fVIII molecules. We have previously shown that this reaction occurs in vitro using purified plasma components and in situ in a plasma-based system. Both of these systems produce a low level of fVIII activation with sustained (albeit low) fVIIIa activity. While it remains possible that this reaction is important in early hemostasis the elevated levels of TF in many pathological situations raises the possibility that this reaction may be more pronounced under certain circumstances in disease states. Examination of the importance of this reaction in vivo is an extremely important issue, but very difficult to address due to the inability to ascertain if fVIII activity or fragments found in vivo derive from fVIIa-TF proteolysis or proteolysis by other enzymes such as thrombin, factor Xa, or activated protein C. With this in mind we have developed an antibody reagent that can specifically detect a fVIII fragment that is a unique product of fVIII proteolysis by the fVIIa-TF complex. This antibody detects only fVIIa-TF proteolyzed fVIII (fVIII cleaved at Arg336) and its major product (α-fragment) on Western blots but not intact (unactivated) fVIII or thrombin-activated fVIII. Using this antibody we screened samples of pulmonary lavage and pleural fluid from normal patients as well as patients with acute respiratory distress syndrome, interstitial lung disease, pneumonia and lung cancer—all of which have associated procoagulant pathologies. Sandwich ELISAs of patient samples showed variably elevated levels of α-fragment (from 100 – 2000 pM) compared to normal controls (~5 pM). Western blots of lavage samples confirmed the presence of α-fragment in samples as well as the elevated levels compared to normals. These data strongly support the notion that alternative “abnormal” coagulation products can be and are generated in vivo in certain pathological settings. The data are also strongly suggestive that the fVIIa-TF complex is the most likely source of fVIII α-fragment. Although it remains unclear if fVIII α-fragment is one of the causative agents in the procoagulant pathologies of these disorders or merely an indicator of the abnormal procoagulant state, its presence in vivo indicates that the role of abnormal coagulation reactions should be further investigated.

Rapid Activation of Protein C by Factor Xa and Thrombin in the Presence of Polyanionic Compounds

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4572-4580 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Chondroitin Sulfate ◽  
Protein C ◽  
Dextran Sulfate ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Activation Rate ◽  
Thrombin Activation

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (∼50 to 100 μmol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations (∼80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.

Rapid Activation of Protein C by Factor Xa and Thrombin in the Presence of Polyanionic Compounds

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4572-4580 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Chondroitin Sulfate ◽  
Protein C ◽  
Dextran Sulfate ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Activation Rate ◽  
Thrombin Activation

Abstract A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (∼50 to 100 μmol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations (∼80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.

Heparin Stimulation of the Inhibition of Activated Protein C and other Enzymes by Human Protein C Inhibitor - Influence of the Molecular Weight of Heparin and Ionic Strength

1996 ◽  
Vol 76 (06) ◽  
pp. 0983-0988 ◽  
Author(s):  
Justo Aznar ◽  
Francisco España ◽  
Amparo Estellés ◽  
Montserrat Royo
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Plasminogen Activator ◽  
Enzyme Inhibition ◽  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Protein C Inhibitor ◽  
Stimulation Of

SummaryThe ability of unfractionated (UF) heparin and low-molecular-weight heparin (LMWH) to potentiate the inhibition of fibrinolytic and coagulation factors by protein C inhibitor (PCI) was studied. Inhibition of activated protein C (APC), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), thrombin, factor Xa (Xa), factor XIa (XIa) and plasma kallikrein (KK) by PCI was found to be dependent on the size of the polysaccharide. In general, maximal stimulation was reached with UF heparin, except in the case of KK. Differences in heparin stimulation were more pronounced for thrombin, APC, uPA, tPA and XIa, whereas inactivation of Xa by PCI was less dependent on the presence of heparin, and kallikrein showed higher potentiation with LMWH than with UF heparin. The second-order rate constants for enzyme inhibition by PCI were strongly dependent on the ionic strength, and, in general, with an ionic strength higher than 0.15 the heparin stimulation of the inhibition reactions was drastically reduced. These results may explain the large discrepancies in the literature on the effect of heparin on the stimulation of enzyme inhibition by PCI. They also show that LMWH is less efficient in stimulating the PCI inhibition of APC, uPA and tPA, which could contribute to the antithrombotic effect of these enzymes.

APC Resistance and other Haemostatic Variables during Pregnancy and Puerperium

1999 ◽  
Vol 81 (04) ◽  
pp. 527-531 ◽  
Author(s):  
U. Kjellberg ◽  
N.-E. Andersson ◽  
S. Rosén ◽  
L. Tengborn ◽  
M. Hellgren
Keyword(s):  
Factor Viii ◽  
Plasminogen Activator ◽  
Protein C ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Protein S ◽  
Reference Level ◽  
Soluble Fibrin ◽  
Prothrombin Fragment ◽  
D Dimer

SummaryForty-eight healthy pregnant women were studied prospectively and longitudinally. Blood sampling was performed at 10-15, 23-25, 32-34 and 38-40 weeks of gestation, within one week and at eight weeks postpartum. Classic and modified activated protein C ratio decreased as pregnancy progressed. In the third trimester 92% of the ratios measured with the classic test were above the lower reference level whereas all modified test ratios were normal. Slight activation of blood coagulation was shown with increased levels of prothrombin fragment 1+2, soluble fibrin and D-dimer. Fibrinogen, factor VIII and plasminogen activator inhibitor type 1 and type 2 increased. Protein S and tissue plasminogen activator activity decreased. Protein C remained unchanged. No correlation was found between the decrease in classic APC ratio and changes in factor VIII, fibrinogen, protein S, prothrombin fragment 1+2 or soluble fibrin, nor between the increase in soluble fibrin and changes in prothrombin fragment 1+2, fibrinogen and D-dimer.

Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

1989 ◽  
Vol 61 (03) ◽  
pp. 386-391 ◽  
Author(s):  
Guido Tans ◽  
Truus Janssen-Claessen ◽  
Jan Rosing
Keyword(s):  
Gel Electrophoresis ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Product Formation ◽  
Chromogenic Substrate ◽  
Nitrocellulose Membrane ◽  
Activation Products ◽  
Factor Xia ◽  
Prothrombin Activation

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

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