scholarly journals Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 642-647 ◽  
Author(s):  
K Murakami ◽  
K Okajima ◽  
M Uchiba ◽  
M Johno ◽  
T Nakagaki ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Vascular Injury ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

Recombinant Human Soluble Thrombomodulin Reduces Endotoxin-Induced Pulmonary Vascular Injury Via Protein C Activation in Rats

1995 ◽  
Vol 74 (05) ◽  
pp. 1265-1270 ◽  
Author(s):  
Mitsuhiro Uchiba ◽  
Kenji Okajima ◽  
Kazunori Murakami ◽  
Katsuhico Nawa ◽  
Hiroaki Okabe ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Soluble Thrombomodulin ◽  
Intravascular Coagulation ◽  
At Iii

SummaryAdult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dan- syl Glu Gly Arg chloromcthyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase.Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.

Attenuation of endotoxin-induced pulmonary vascular injury by antithrombin III

1996 ◽  
Vol 270 (6) ◽  
pp. L921-L930 ◽  
Author(s):  
M. Uchiba ◽  
K. Okajima ◽  
K. Murakami ◽  
H. Okabe ◽  
K. Takatsuki
Keyword(s):  
Endothelial Cells ◽  
Vascular Permeability ◽  
Vascular Injury ◽  
Antithrombin Iii ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor ◽  
At Iii

We evaluated the effects of antithrombin III (AT III) on the pulmonary vascular injury induced by injecting rats with lipopolysaccharide (LPS) to investigate the possible usefulness of AT III as a treatment for acute respiratory distress syndrome. The intravenous administration of AT III prevented the pulmonary accumulation of leukocytes (as evaluated by myeloperoxidase activity) and the increase in pulmonary vascular permeability to 125I-bovine serum albumin induced by LPS. The increase in pulmonary vascular permeability induced by LPS administration was unaffected by various anticoagulants but was inhibited by the leukocytopenia induced by nitrogen mustard or by the administration of a granulocyte elastase inhibitor, ONO-5046. AT III given alone, but not heparin plus AT III or Trp49-modified AT III, which lacks affinity for heparin, significantly increased the plasma concentration of 6-keto-prostaglandin F1alpha, suggesting that the interaction of AT III with heparin-like substances at the endothelial cell surface promotes the release of prostacyclin from endothelial cells in vivo. Trp49-modified AT III failed to prevent the LPS-induced accumulation of leukocytes and vascular injury. The pulmonary accumulation of leukocytes and vascular injury induced by LPS were not prevented by administering AT III to rats that were pretreated with indomethacin. The continuous intravenous infusion of prostacyclin prevented the LPS-induced pulmonary accumulation of leukocytes and vascular injury. Findings suggest that AT III depends on its ability to promote the release of prostacyclin, a potent inhibitor of leukocyte activation, from endothelial cells to prevent pulmonary vascular injury induced by LPS.

Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production

1997 ◽  
Vol 272 (2) ◽  
pp. L197-L202 ◽  
Author(s):  
K. Murakami ◽  
K. Okajima ◽  
M. Uchiba ◽  
M. Johno ◽  
T. Nakagaki ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Protein C ◽  
Cytokine Production ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Weight Ratio ◽  
Dry Weight ◽  
Endothelial Damage ◽  
Factor X

We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.

rhs-TM prevents ET-induced increase in pulmonary vascular permeability through protein C activation

1997 ◽  
Vol 273 (4) ◽  
pp. L889-L894 ◽  
Author(s):  
Mitsuhiro Uchiba ◽  
Kenji Okajima ◽  
Kazunori Murakami ◽  
Masayoshi Johno ◽  
Mitsunobu Mohri ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vascular Permeability ◽  
Histological Examination ◽  
Vascular Injury ◽  
Protein C ◽  
Neutrophil Infiltration ◽  
Leukocyte Activation ◽  
Pulmonary Vascular Permeability ◽  
Intravascular Coagulation

We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation. In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C. rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12). Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM. ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM. However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C. These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.

Mechanisms for Mortality Reduction by Activated Protein C in Severe Sepsis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1-1 ◽  
Author(s):  
Edward J. Kerschen ◽  
Brian C. Cooley ◽  
Francis J. Castellino ◽  
Shaun R. Coughlin ◽  
Jose A. Fernandez ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Severe Sepsis ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Mortality Reduction ◽  
Bacterial Sepsis ◽  
Cellular Effects ◽  
Anticoagulant Action

Abstract Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC’s beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over < 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50% to 0–10% at LD-50 LPS doses). APC treatment did not alter the extent of circulating inflammatory cytokine levels at 3 or 24 hr after endotoxin exposure. The survival benefit conferred by wt APC infusions was abolished in mice with genetically reduced levels of endothelial protein C receptor (EPCR) (< 10% of normal) or in mice genetically lacking protease-activated receptor-1 (PAR-1). Murine APC variants with either 3 or 5 Ala substitutions, 3K3A-APC (KKK192-194AAA) or 5A-APC (RR230/231AA + KKK192-194AAA) that had reduced anticoagulant activity (25 % and < 10 % of wt APC, respectively), but normal cytoprotective activities, were as effective as wt APC in reducing mortality after LPS challenge. A murine APC variant lacking proteolytic activity (active site S360A) did not enhance survival after LPS, showing a requirement for APC’s enzymatic activity. Thus, the survival-promoting efficacy of APC in this model requires the enzymatic active site of APC and the presence of two receptors, EPCR and PAR-1, that are known to mediate APC’s in vitro beneficial cytoprotective effects on cells. In a whole bacteria sepsis model, when APC was given to mice at the time of initiation of peritoneal Staphylococcus aureus-induced sepsis and again at 24 hr, wt APC surprisingly increased mortality (100% mortality vs. 50% at LD-50 bacteria dose). In contrast, when 3K3A-APC or 5A-APC variants with attenuated anticoagulant activity was given at 0 and 24 hr, they prevented mortality due to bacterial sepsis (0–10% vs. 50% mortality for saline control at LD-50 dose). This implies that APC’s anticoagulant action might impair beneficial coagulation-dependent host defense mechanisms in early stages of bacterial sepsis whereas the 5A-APC variant, with very low anticoagulant activity but normal cytoprotective activity, might provide beneficial cellular effects to help prevent death during bacterial sepsis. In summary, the full anticoagulant activity of APC is not required for protection against mortality in each of these models. These results highlight the importance of the cellular protein C pathway for APC therapy and suggest that APC variants with reduced anticoagulant action but normal potency for beneficial direct cellular effects merit further evaluation for sepsis therapy.

Antithrombotic Action of Endogenous Porcine Protein C Activated with a Latent Porcine Thrombin Preparation

1995 ◽  
Vol 74 (03) ◽  
pp. 879-885 ◽  
Author(s):  
Robert D McBane ◽  
Waldemar E Wysokinski ◽  
James H Chesebro ◽  
Whyte G Owen
Keyword(s):  
Active Site ◽  
Protein C ◽  
Carotid Arteries ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Mechanical Trauma ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
Endogenous Protein

SummaryEndogenously activated protein C is evaluated for antithrombotic activity in porcine carotid arteries subjected to mechanical trauma. Protein C is activated by intravenous administration of guanidinobenzoyl- thrombin, which binds to thrombomodulin and there deacylates to yield thrombin. The bound, transiently active thrombin yields a peak of anticoagulant activity between 5 and 10 min after infusion of the latent thrombin. Inhibition of thrombin binding in vivo by co-infusing an active-site-blocked thrombin preparation elicits acute and lethal systemic thrombosis. Nearly occlusive platelet thrombosis, which occurs within 30 min of crushing 1 cm segments of carotid arteries with a standard hemostat, is blocked by endogenous protein C activation initiated 2 min before the crush injury. It is concluded that activated protein C blocks thrombosis in deeply injured musculo-elastic arteries, and that activation of latent thrombin bound to thrombomodulin in vivo is a practical means for delivery of pharmacologically effective concentrations of activated protein C.

Anti-Inflammatory and Anti-Apoptotic Activities of Activated Protein C Are Independent of Anticoagulant Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 65-65 ◽  
Author(s):  
Xia V. Yang ◽  
John H. Griffin ◽  
Laurent O. Mosnier
Keyword(s):  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Relative Importance ◽  
Endothelial Apoptosis ◽  
Maximum Inhibition ◽  
Anti Inflammatory ◽  
Apoptotic Activity

Abstract Activated protein C (APC), a well known anticoagulant enzyme, reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models, including neuroprotective activity in rodent ischemic stroke, attenuation of inflammatory lung injury, and survival benefits in rodent sepsis models. For these in vivo benefits, the relative importance of APC’s anticoagulant activity vs. APC’s direct cytoprotective effects on cells is unclear. APC anticoagulant activity involves inactivation of factors Va and VIIIa whereas cytoprotection by APC involves two receptors, Endothelial Protein C Receptor (EPCR) and Protease Activated Receptor-1. To distinguish the cytoprotective from the anticoagulant activities of APC, we made recombinant human wild type (wt) APC and a protease domain mutant, 5A-APC (RR229/230AA + KKK191−193AAA), a Gla domain mutant, PTGla-APC (APC with prothrombin residues 1–46)(Smirnov et al JBC 1998) and an active site mutant, S360A-APC. Active site titration and chromogenic assays showed that wtAPC, 5A-APC and PTGla-APC had full enzymatic activity while S360A-APC had none. 5A-APC had almost no anticoagulant activity (< 3 %) whereas PTGla-APC had 300% of wtAPC anticoagulant activity. APC binding to EPCR was assayed as binding to immobilized soluble EPCR and to K293 cells transfected with wtEPCR. We verified that wtAPC and 5A-APC each could bind to EPCR with similar affinity (Kd,app = 37 nM and 29 nM, respectively) whereas, in contrast, PTGla-APC bound very weakly to EPCR (Kd,app > 300 nM ). We then compared the anti-inflammatory and anti-apoptotic activities of the hypo-anticoagulant 5A-APC and the hyper-anticoagulant PTGla-APC to those of wtAPC. To assay APC anti-inflammatory activity, APC-mediated inhibition of LPS-induced TNFα secretion from U937 monocytic cells was determined. The ability of 5A-APC to inhibit LPS-induced TNFα secretion was indistinguishable from that of wtAPC with half-maximum inhibition at 5.4 nM and 6.5 nM, respectively. Neither the enzymatically inactive S360A-APC not the protein C zymogen inhibited LPS-induced TNFα secretion, indicating that a functional APC active site was required. Anti-EPCR antibodies blocking APC binding prevented the anti-inflammatory activity of wtAPC, indicating binding of APC to EPCR on U937 cells was required. The anti-apoptotic activity of each APC species was determined in staurosporine-induced endothelial cell apoptosis assays. Dose-dependent inhibition of apoptosis by 5A-APC was indistinguishable from that by wt-APC with half-maximum inhibition at 0.70 and 2.0 nM, respectively. In contrast to this potent anti-apoptotic activity of wtAPC and 5A-APC, the hyper-anticoagulant PTGla-APC required a 24-fold higher concentration for half-maximal inhibition of endothelial apoptosis. As expected, S360A-APC showed no significant inhibition of endothelial apoptosis. Hence, the 5A-APC variant with < 3% anticoagulant activity exhibited normal anti-inflammatory and anti-apoptotic activities in vitro on monocytic and endothelial cells. These APC variants may be useful for in vivo assessment of the relative importance of APC’s anticoagulant vs. cytoprotective activities. In summary, these data highlight important distinctions between structural requirements for APC’s anticoagulant functions compared to its anti-inflammatory and anti-apoptotic activities. These structural insights may lead to safer therapeutic APC variants that retain APC’s beneficial cytoprotective effects but that have reduced bleeding risk due to reduction in anticoagulant activity.

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.

scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

2012 ◽  
Vol 107 (03) ◽  
pp. 468-476 ◽  
Author(s):  
Ilze Dienava-Verdoold ◽  
Marina R. Marchetti ◽  
Liane C. J. te Boome ◽  
Laura Russo ◽  
Anna Falanga ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Values ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Intact Protein ◽  
The Impact ◽  
Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

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