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.

Phosphoinositides and Other Glycerophospholipids Interact with Protein C Inhibitor (PCI) and Modify Its Activity towards Activated Protein C (APC).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2122-2122
Author(s):  
Felix Wahlmüller ◽  
Margareta Furtmüller ◽  
Barbora Sokolikova ◽  
Bernd R Binder ◽  
Margarethe Geiger
Keyword(s):  
Molecular Weight ◽  
Phosphatidic Acid ◽  
Serine Protease ◽  
Protein C ◽  
Activated Protein C ◽  
Low Molecular Weight ◽  
Native Page ◽  
Stimulatory Activity ◽  
Protein C Inhibitor ◽  
Stimulation Of

Abstract Abstract 2122 Poster Board II-99 Protein C Inhibitor (PCI, SERPINA5, PAI3) is a non-specific, secreted serine protease inhibitor (serpin) which circulates at low levels (5μg/mL or 90nM) in blood plasma (review: Geiger 2007, Suzuki 2008). First described as an inhibitor of activated protein C (APC) an anticoagulant serine protease in human plasma, it has been shown that PCI inactivates a variety of other proteases and has a wide tissue distribution. Some compounds like glycosaminoglycans (e.g. heparin, heparan sulfate) and certain phospholipids can modify PCI activity. Recently it was shown that single-stranded DNA aptamers stimulates the inhibitory activity of PCI towards APC in a glycosaminoglycan-like fashion (Müller 2009). In 2007 Malleier et al. analyzed the interaction of PCI with phosphatidylserine (PS), oxidized PS (OxPS) and oxidized phosphatidylethanolamin (OxPE). PS, OxPS and OxPE bind to PCI and enhance the stimulation of APC-inhibition 130 to 190-fold. In addition, PE supports the internalization of PCI by cells, and internalized PCI promotes phagocytosis of bacteria (Baumgärtner et al. 2007). JFC1 (synaptotagmin-like protein 1) was identified by our group as a new intracellular interaction partner of PCI and colocalization of PCI and CSN6, a subunit of the COP9 signalosome could be observed in lymphocytes. Here, we analyzed the interaction of PCI with phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL), phosphoinositides and derived second messengers like inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). To identify lipid-regions which are important for PCI binding we were focusing on differences in fatty acid composition and headgroup phosphorylation. The binding was studied by native PAGE, protein overlay assays (dot blot analysis) or ELISA. The stimulation of PCI activity towards APC was analyzed in functional assays using a low molecular weight substrate (S-2366). IP3 and inositol-1,3,4,5-tetrakisphosphate (IP4) did neither interact with PCI nor stimulate its activity towards APC. PCI bound to saturated, unsaturated and oxidized PA. The oxidized form of 1-palmitoyl-2-arachidonoyl-phosphatidic acid (OxPAPA) exhibited lower binding to PCI, but higher stimulatory activity on APC inhibition, as compared to unoxidized PAPA. Saturated dipalmitoyl-PA did not modulate PCI activity. From all studied lipids 1-palmitoyl-2-arachidonoyl-PG (PAPG) had the strongest stimulatory effect on APC-inhibition similar to 0.1μM low molecular weight heparin. Oxidation of PAPG led to a slight decrease and saturation to a complete loss in stimulatory activity. Also tetra-oleoyl-CL bound to PCI with high affinity and had a similar effect as oxidized PAPG and OxPAPA. All mono- and diphosphorylated phosphoinositides as well as phosphatidylinositol-3,4,5-triphosphate (PI3,4,5P3) bound to PCI as judged from binding assays. A mobility shift of PCI antigen on native PAGE was observed when PCI was incubated with phosphatidylinositol-3,5-diphosphate and phosphatidylinositol-4,5-diphosphate (PI4,5P2). Therefore different phospholipids modulate the activity of PCI, but on the other hand PCI may as well effect lipid signaling. As PI4,5P2 plays an important role as substrate for PI3-kinase we will take a closer look at the effect of PCI on the PI3K/PTEN system and on the activation of AKT (PKB) by PDK1. We will also study the influence of PCI on the generation of IP3 and DAG and its possible role in calcium signaling and protein kinase activation. So far we conclude that phosphoinositides and other glycerophospholipids may function as additional intracellular interaction partners of PCI. Disclosures: No relevant conflicts of interest to declare.

Specificity of the Thrombin-Induced Release of Tissue Plasminogen Activator from Cultured Human Endothelial Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 115-119 ◽  
Author(s):  
Eugene G Levin ◽  
David M Stern ◽  
Peter P Nawroth ◽  
Richard A Marlar ◽  
Daryl S Fair ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Protein C ◽  
Primary Cultures ◽  
Activated Protein C ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Human Endothelial Cells ◽  
Tissue Plasminogen

SummaryThe addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of α-thrombin, promoted tPA release but was less effective than α-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of γ-thrombin 20 times greater than α-thrombin was required. The response to Factor Xa was similar to α-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active α-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

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.

Involvement of Lys 62[217] and Lys 63[218] of Human Anticoagulant Protein C in Heparin Stimulation of Inhibition by the Protein C Inhibitor

1999 ◽  
Vol 82 (07) ◽  
pp. 72-79 ◽  
Author(s):  
Lei Shen ◽  
Bruno Villoutreix ◽  
Björn Dahlbäck
Keyword(s):  
Rate Constants ◽  
Protein C ◽  
Double Mutant ◽  
Activated Protein C ◽  
Human Kidney ◽  
Peptide Bond ◽  
Dimensional Structure ◽  
Heparin Binding ◽  
Protein C Inhibitor ◽  
Stimulation Of

SummaryInhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated by heparin, whereas inhibition by α1-antitrypsin (AAT) is heparin-independent. Three lysine residues located in a positively charged cluster in the serine protease domain of protein C (PC) were mutated to probe their involvement in the heparin stimulation of inhibition by PCI. These mutations were selected after analysis of the three-dimensional structure of APC and of molecular models for PCI and the APC-PCI complex. A double mutant, K62[217]N/K63[218]D, a single mutant, K86[241]S, and wild-type PC were expressed in embryonic human kidney 293 cells. Heparin stimulated the rate of inhibition of wt-APC by PCI approximately 400-fold, with second order rate constants (k 2 ) in the absence and presence of heparin of 0.72 × 103 M–1s–1 and 2.87 × 105 M–1s–1, respectively. In contrast, heparin only yielded a 52-fold stimulation of the rate of inhibition of the double mutant APC by PCI as the rate constants in the absence and presence of heparin were k 2 = 2.44 × 103 M–1s–1 and k 2 = 1.26 × 105 M–1s–1, respectively. The double mutant K62N/K63D eluted at approximately 10% lower NaCl concentration from a heparin Sepharose column than the K86S mutant or wt-APC. These data suggest K62 and K63 in APC to be part of a heparin binding site which is important for heparin-mediated stimulation of inhibition of APC by PCI. Abbreviations: APC, activated protein C; PC, protein C; PCI, protein C inhibitor, AAT, α1-antitrypsin also called α1-proteinase inhibitor, AT, antithrombin; TM, thrombomodulin. The chymotrypsinogen nomenclature for APC (1) is used in the text while the PC numbering is indicated between brackets whenever appropriate. P1, P2... and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S1, S2.. and S1’, S2’.. the corresponding subsites of the protease (2). The antitrypsin numbering (3) for PCI is used along this article while the PCI numbering is mentioned between brackets whenever appropriate.

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.

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.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

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).

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

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