scholarly journals Complement Regulator Factor H is a Cofactor for Thrombin in both Pro- and Anticoagulant Roles

2021 ◽  
Author(s):  
Genevieve McCluskey ◽  
Gemma E Davies ◽  
Rebekah L Velounias ◽  
Timothy R Hughes ◽  
B Paul Morgan ◽  
...  
Keyword(s):  
Human Plasma ◽  
Protein C ◽  
Bleeding Time ◽  
Activated Protein C ◽  
Fibrin Clot ◽  
Factor H ◽  
Complement Regulator ◽  
Recombinant Thrombomodulin ◽  
Clot Structure ◽  
Maximum Turbidity

Background: Complement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin. Methods: Tail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR). Results: Tail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed. Conclusion: Complement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis.

Comparison of Enoxaparin to the Thrombin Variant Protein C Activator, W215A/E217A, in a Primate Model of Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1868-1868
Author(s):  
Ulla M. Marzec ◽  
Leslie Bush ◽  
Andras Gruber ◽  
Stephen R. Hanson ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Bleeding Time ◽  
Activated Protein C ◽  
Expansion Chamber ◽  
Primate Model ◽  
Endothelial Surface ◽  
Endogenous Protein ◽  
Platelet Deposition ◽  
Type Flow

Abstract Antithrombotic therapy by thrombomodulin-dependent activation of endogenous protein C might be safer than currently used therapies, since endogenous activated protein C (APC) is generated and probably concentrated on the endothelial surface and not in blood that escapes the blood vessels during injury. We compared the antithrombotic and antihemostatic activities of a protein C selective recombinant double mutant human thrombin (W215A/E217A; WE) that activates endogenous protein C on the endothelial surface, in vivo, with a low molecular weight heparin, enoxaparin in awake non anticoagulated baboons. Thrombosis was initiated by interposing a two part device consisting of a 2cm, 4mm ID, polyethylene terephthalate vascular graft (DVG) followed by a 2cm silicone tubing chamber, 9mm ID, into a chronic femoral arteriovenous shunt in baboons. Upon initiation of blood flow at 100mL/min, wall shear rate of 265sec−1 in the 4mm ID segment (arterial type flow) and 29sec−1 in the expansion chamber (venous type flow), platelet thrombus growth was monitored by gamma camera imaging of autologous 111In-labeled platelets for 1 hour. Fibrin accumulation was quantified by homologous 125I-labeled fibrinogen. Bleeding time and aPTT were monitored. WE was administered as a loading IV bolus (1/3 dose) 10 minutes before thrombus initiation followed by maintenance infusion (2/3 dose) for the duration of the study at 3 dose levels: 1.8 μg/kg (dose-1), 3.75 μg/kg (dose-2) and 7.5 μg/kg (dose-3). Results were compared to controls given normal saline and to 3 doses of IV enoxaparin (1/2 dose loading + 1/2 dose maintenance): 0.3 mg/kg (dose-1), 0.6 mg/kg (dose-2) and 1.2mg/kg (dose-3) (3–5 animals in each study group). WE inhibition of platelet deposition in the chamber was dose dependent and reduced by 57%, 76% and 95% for dose 1, 2 and 3 respectively, compared to controls (p < 0.05 each). Enoxaparin likewise reduced thrombosis in the chamber by 49%, 83% and 91% (p< 0.05 each) respectively at the 3 doses tested. Fibrin accumulation paralleled the platelet deposition data in the chamber. Thrombus growth on DVG was only reduced at dose level 3 for both WE and enoxaparin by 18% and 47%, respectively as determined by platelet deposition. Fibrin accumulation was reduced by 35% by WE and 51% for enoxaparin at dose 3 on the arterial thrombosis segment. Bleeding times did not increase significantly with any of the treatments except for dose-3 of enoxaparin which prolonged the bleeding time from 3.5±1.3 minutes to 5.3 ± 1.4 minutes. Prolongation of APTT was 1.14, 1.33, and 2.04 fold for WE doses 1, 2 and 3, respectively and enoxaparin prolonged the APTT by 1.25, 1.6 and 1.9 fold at dose 1, 2 and 3, respectively. We conclude that low dose WE infusion is safe and about two orders of magnitude more antithrombotic than enoxaparin infusion in the baboon model of thrombosis.

scholarly journals A Systematic Summary of Systematic Reviews on Anticoagulant Therapy in Sepsis

2019 ◽  
Vol 8 (11) ◽  
pp. 1869 ◽  
Author(s):  
Shuhei Murao ◽  
Kazuma Yamakawa
Keyword(s):  
Beneficial Effect ◽  
Anticoagulant Therapy ◽  
Systematic Reviews ◽  
Protein C ◽  
Activated Protein C ◽  
Specific Patient ◽  
Beneficial Effects ◽  
Increased Risk ◽  
Patient Groups ◽  
Recombinant Thrombomodulin

Many systematic reviews have been published regarding anticoagulant therapy in sepsis, among which there is substantial heterogeneity. This study aimed to provide an overview of existing systematic reviews of randomized controlled trials by using a comprehensive search method. We searched MEDLINE, EMBASE, and Cochrane Database of Systematic Reviews. Of 895 records screened, 19 systematic reviews were included. The target agent was as follows: antithrombin (n = 4), recombinant thrombomodulin (n = 3), heparin (n = 3), recombinant activated protein C (n = 8), and all anticoagulants (n = 1). Antithrombin did not improve mortality in critically ill patients but indicated a beneficial effect in sepsis-induced disseminated intravascular coagulation (DIC), although the certainty of evidence was judged as low. Recombinant thrombomodulin was associated with a trend in reduced mortality in sepsis with coagulopathy with no increased risk of bleeding, although the difference was not statistically significant and the required information size for any declarative judgement insufficient. Although three systematic reviews showed potential survival benefits of unfractionated heparin and low-molecular-weight heparin in patients with sepsis, trials with low risk of bias were lacking, and the overall impact remains unclear. None of the meta-analyses of recombinant activated protein C showed beneficial effects in sepsis. In summary, a beneficial effect was not observed in overall sepsis in poorly characterized patient groups but was observed in sepsis-induced DIC or sepsis with coagulopathy in more specific patient groups. This umbrella review of anticoagulant therapy suggests that characteristics of the target populations resulted in heterogeneity among the systematic reviews.

Effect of Human Plasma-Derived Protein C and Murine Recombinant Protein C on Severe Neonatal Sepsis in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 689-689
Author(s):  
Eva-Maria Muchitsch ◽  
Hans Peter Schwarz ◽  
Katalin Varadi ◽  
Charles Esmon ◽  
Giuseppe Mancuso ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Mortality Rate ◽  
Human Plasma ◽  
Neonatal Sepsis ◽  
Protein C ◽  
Activated Protein C ◽  
Risk Of Death ◽  
Neonatal Mice ◽  
Endogenous Protein ◽  
Septic Condition

Abstract Neonatal sepsis is a leading cause of infant morbidity and mortality frequently associated with activation of the coagulation system. Reduced levels of protein C are found in the majority of patients with sepsis and are associated with an increased risk of death. Although activated protein C is indicated for the treatment of severe sepsis in adults, the risk of severe bleeding may limit its use in neonates. Because the likelihood of inducing bleeding with the zymogen form of protein C is reduced we assessed both human and murine protein C zymogen in a murine neonatal sepsis model. In this model neonatal mice were challenged with viable group B streptococci (GBS). The effect of this septic condition on endogenous protein C levels was evaluated and the effect of treatment with either recombinant murine protein C or human plasma-derived (non-activated) protein C (Ceprotin) investigated. During severe GBS sepsis murine endogenous protein C levels decreased over time in neonatal mice, resulting in a maximum decrease of −30 % at 16 hours after GBS challenge and returned towards baseline at 30 hours. Concomitantly, there was an increase in endogenous protein C activation up to 59 % at 6 hours after GBS challenge, returning to baseline levels at 16 hours. Blocking endogenous murine protein C with an anti-mouse monoclonal antibody increased the mortality rate significantly from 62 to 91 %. Treatment of neonatal septic mice (n=36) with 300 U/kg murine protein C subcutaneously 4 hours before GBS challenge decreased the mortality rate significantly in severe sepsis (LD90) to 64 % (p=0.002). Similarly pretreatment with human plasma-derived protein C (200 IU/kg) 4 hours before GBS challenge increased survival rate significantly in severe septic mice. Treatment with 200 IU/kg (Ceprotin) was even effective given 18 hours after GBS challenge, leading to a decrease in the mortality rate in severe sepsis from 87.5 to 48 %. Despite this species difference and the septic condition, human protein C zymogen was activated to activated protein C and detectable in the circulation of mice. This is the first preclinical study were a beneficial effect of a non-activated protein C could be shown in an animal model of severe neonatal sepsis.

Targeted Inhibition of Activated Protein C Anticoagulant Activity By Monoclonal Antibody HAPC1573 for Treatment of Hemophilia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 80-80 ◽  
Author(s):  
Xiao-Yan Zhao ◽  
Subramanian Yegneswaran ◽  
Maxine Bauzon ◽  
Derek Sim ◽  
Chandra Patel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protein C ◽  
Thrombin Generation ◽  
Bleeding Time ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Dependent Manner ◽  
Monkey Model ◽  
Binding Epitope ◽  
Dose Dependent

Abstract In patients with hemophilia, hereditary defects in coagulation factors result in unstable clots and recurrent bleeding. Although the current standard of care focuses on replacement factor therapy, we propose that inhibition of the anticoagulant pathways may offer novel therapeutic opportunities. Activated protein C (APC) is one of the major anticoagulants, which works by degrading factors Va and VIIIa to maintain hemostatic balance. APC also exhibits cytoprotective effects, which include antiapoptotic effects, endothelial barrier protection, and anti-inflammatory effects. Antibodies are ideally suited to inhibit the anticoagulant activity of APC while preserving its cytoprotective activity. We have developed an anti-APC monoclonal antibody (mAb), HAPC1573 (murine immunoglubulin G1/kappa), using hybridoma technology, that specifically binds to human APC (hAPC) at 3 to 7 nM binding affinity (Kd) but not to its zymogen, protein C (PC) as determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA). To investigate the binding epitope of mAb HAPC1573, APC was inhibited with Phe-Pro-Arg-chloromethylketone (PPACK). PPACK is an irreversible inhibitor of APC and forms a covalent bond with the catalytic triad Ser195 (chymotrypsin numbering). HAPC1573 bound comparably to PPACK-hAPC and untreated hAPC coated on an enzyme-linked immunosorbent assay (ELISA) plate, suggesting that the binding epitope of HAPC1573 is located outside the active site of APC. These ELISA results were also confirmed by SPR analyses. HAPC1573 inhibited the cleavage of a small peptide substrate Spectrozyme PCa (Sekisui Diagnostics, Lexington, MA) by hAPC up to 40%. The antibody protected factors Va and VIIIa from APC-mediated inactivation in a dose-dependent manner. HAPC1573 significantly reduced activated partial thromboplastin time of hemophilic plasma and enhanced thrombin generation (assessed by thrombin generation assay) in the presence of thrombomodulin. HAPC1573 inhibited the anticoagulant activity of APC without affecting its cytoprotective functions, as measured by histone-mediated cytotoxicity assays on human umbilical vein endothelial cells (up to 300 nM HAPC1573). Given its cross-reactivity with monkey APC, the antibody was evaluated in Cynomolgus monkeys for therapeutic efficacy and safety. Intravenous administration of the antibody at 3 and 10 mg/kg significantly shortened bleeding time after injury and restored hemostasis in a dose-dependent manner in an anti-FVIII antibody-induced hemophilia monkey model (Figure). Administration of a sheep-anti-FVIII antibody (Haematologic Technologies, Inc., Essex Junction, VT) reduced plasma FVIIIa activity to below the lower limit of quantification (LLOQ; Figure [left panel]) and led to a significantly longer bleeding time in normal monkeys (Figure; right panel), recapitulating the hemophilia A phenotype. This prolonged bleeding time was partially reduced by 270 µg/kg of recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Plainsboro, NJ) used as a positive control for these studies. There was a statistically significant dose-dependent reduction in bleeding time by HAPC1573 with the 10-mg/kg dose restoring the bleeding time back to normal. This study provides in vivo proof of concept of using anti-APC antibody for hemophilia. HAPC1573 represents an anti-APC antibody with therapeutic utility for patients with hemophilia with inhibitors. Figure The efficacy of HAPC1573 evaluated in an anti-FVIII antibody-induced hemophilia monkey model. Figure. The efficacy of HAPC1573 evaluated in an anti-FVIII antibody-induced hemophilia monkey model. Disclosures Zhao: Bayer Pharmaceuticals: Employment. Yegneswaran:Bayer Healthcare Pharmaceuticals: Employment. Sim:Bayer: Employment. Patel:Bayer Pharmaceuticals: Employment. Schneider:Bayer HealthCare LLC: Employment, Patents & Royalties. McLean:Bayer: Employment, Equity Ownership. Zhu:Bayer Healthcare: Employment. Jiang:Bayer Pharmaceuticals: Employment. Gu:Bayer Pharmaceuticals: Employment. Ivens:Bayer Pharmaceuticals: Employment. Xu:Shanghai RAAS Blood Products Co.Lt: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patent. Bringmann:Bayer Corporation, Parmaceuticals Division: Employment. Kauser:Bayer: Employment.

A Second Plasma Inhibitor of Activated Protein C: α1-Antitrypsin

1989 ◽  
Vol 62 (02) ◽  
pp. 756-762 ◽  
Author(s):  
Felix J M van der Meer ◽  
Nico H van Tilburg ◽  
Aat van Wijngaarden ◽  
Irma K van der Linden ◽  
Ernest Briët ◽  
...  
Keyword(s):  
Human Plasma ◽  
Protein C ◽  
Activated Protein C ◽  
Order Rate Constant ◽  
High Plasma ◽  
Normal Human Plasma ◽  
Glycoprotein I ◽  
First Order Kinetics ◽  
Neutralizing Capacity ◽  
Normal Human

SummaryInactivation of activated protein C (APC) in normal human plasma was studied in the absence and presence of heparin. In the absence of heparin APC inactivation followed pseudo-first order kinetics. In the presence of heparin the neutralization of APC was found to be biphasic. Up to 500 nM APC could be readily inactivated in normal plasma, indicating that the concentration of the APC inhibitor must be higher than previously assumed. Plasma deficient in the protein C inhibitor (PCI-I, as described by Suzuki and coworkers) and deficient in p2-glycoprotein I still possessed APC neutralizing capacity, presumably through the formation of complexes of APC with another plasma protein as was demonstrated by immunoblotting with anti-protein C antibodies. Together these data made us to conclude that a second inhibitor of APC (PCI-II) must be present in normal human plasma. This second inhibitor should be heparin independent, have a relatively high plasma concentration and form complexes with APC.Subsequently, we purified this PCI-II by isolating APC-PCI-II complexes from plasma deficient of vitamin K dependent proteins, PCI-I and β2-glycoprotein-I, to which purified human APC had been added. Purified PCI-II has a molecular weight of 50,000 daltons and aminoacid analysis revealed that PCI-II is identical with α1-antitrypsin (α1-AT). The second order rate constant for the reaction between purified α1-AT and APC was found to be 269 M−1 min−1 in the absence of calcium and 602 M−1 min−1 in the presence of calcium.Finally, the analysis of the kinetics of APC inactivation in the plasma of a patient congenitally deficient in α1-AT (3% α1-AT antigen) revealed that there probably is a third inhibitor of APC in plasma, different from PCI-I and from α1-AT.

scholarly journals The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2093-2100 ◽  
Author(s):  
L Bajzar ◽  
ME Nesheim ◽  
PB Tracy
Keyword(s):  
Human Plasma ◽  
Polyclonal Antibody ◽  
Protein C ◽  
Activated Protein C ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Normal Human Plasma ◽  
Concentration Dependent Manner ◽  
Lysis Time ◽  
Normal Human

Thrombin-activatable fibrinolysis inhibitor (TAFI) is the precursor of an exopeptidase that is identical to plasma procarboxypeptidase B. Upon activation by thrombin, activated TAFI (TAFIa) attenuates fibrinolysis, presumably by catalyzing the removal of C-terminal lysines from partially degraded fibrin. Activated protein C (APC) proteolytically inactivates the essential cofactor in prothrombinase, factor Va, and limits both the formation of thrombin and subsequent activation of TAFI, thereby appearing profibrinolytic. TAFI is able to reconstitute an APC-dependent shortening of lysis time in a purified system; however, it remained to be determined the extent to which TAFI is involved in the profibrinolytic effect of APC in a plasma-based system. To aid in addressing this question, two monoclonal antibodies (MoAbTAFI#16 and #13) and a polyclonal antibody were produced against purified TAFI. MoAbTAFI#16 was shown to inhibit TAFI activation and thereby appears to stimulate fibrinolysis. Furthermore, an enzyme- linked immunosorbent assay was developed using MoAbTAFI#13 and the polyclonal antibody. Through its use, the plasma concentration of TAFI was determined to be 73 nmol/L. In addition, a turbidity assay was used to determine the effect of APC on tissue plasminogen activator-induced fibrinolysis of clots produced from normal human plasma (NHP), plasma immunodepleted of TAFI (TdP), and TdP reconstituted with purified TAFI. APC shortened lysis time of clots produced from NHP in a saturable and concentration-dependent manner. However, APC had no effect on lysis time of clots formed from either TdP or NHP in the presence of 80 nmol/L MoAbTAFI#16. The APC effect could be reconstituted in TdP by the addition of purified TAFI. The lysis time in TdP was increased from 50 to 180 minutes in a TAFI concentration-dependent manner. The EC50 was 15 nmol/L and saturation was approached at physiologically relevant concentrations (60 nmol/L). The profibrinolytic effect of APC was also compared with that of MoAbTAFI#16 and two competitive inhibitors, an inhibitor of the carboxypeptidase A and B family purified from potato tubers and 2-Guanidinoethylmercaptosuccinic acid (GEMSA). All were able to reduce lysis time of clots formed from normal human plasma by 90 minutes, yielding respective EC50 values of 5 nmol/L, 15 nmol/L, 50 nmol/L, and 90 mumol/L. Therefore, the majority of the profibrinolytic effect of APC, in an in vitro plasma system, is dependent on TAFI. Because TAFIa dramatically influences lysis time, inhibitors of TAFIa or TAFI activation may prove to be important adjuvants for thrombolytic therapy.

Protein C Inhibitor from Human Plasma: Characterization of Native and Cleaved Inhibitor and Demonstration of Inhibitor Complexes with Plasma Kallikrein

1989 ◽  
Vol 62 (03) ◽  
pp. 885-891 ◽  
Author(s):  
Martin Laurell ◽  
Johan Stenflo
Keyword(s):  
Sequence Analysis ◽  
Human Plasma ◽  
Protein C ◽  
Activated Protein C ◽  
Immunoaffinity Chromatography ◽  
Plasma Kallikrein ◽  
Cleavage Sites ◽  
Protein C Inhibitor ◽  
Secondary Cleavage

SummaryProtein C inhibitor (PCI) was purified from human plasma using immunoaffinity chromatography and heparin Sepharose chromatography, a method that allowed the purification of active and inactive inhibitor. PCI purified from outdated plasma was inactive and either in complex with plasma kallikrein or proteo-lytically degraded. Sequence analysis of cleaved PCI and of complexes between PCI and activated protein C or urokinase identified the previously recognized inhibitor cleavage site Arg354-Ser355. Two additional cleavage sites were observed in the modified inhibitor i.e. Arg357-Leu358 and Arg362-Leu363 which probably represent secondary cleavage of the inhibitor. Furthermore the sequence analysis of the inhibitor, whether purified from fresh or outdated plasma, revealed that it was microheterogeneous in the NH2-terminus as a result of cleavage by a trypsin like enzyme(s).

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