Circulating Activated Protein C Levels Research Articles

Genetic Upregulation of Activated Protein C Mitigates Delayed Effects of Acute Radiation Exposure in the Mouse Plasma.

Exposure to ionizing radiation, accidental or intentional, may lead to delayed effects of acute radiation exposure (DEARE) that manifest as injury to organ systems, including the kidney, heart, and brain. This study examines the role of activated protein C (APC), a known mitigator of radiation-induced early toxicity, in long-term plasma metabolite and lipid panels that may be associated with DEARE in APCHi mice. The APCHi mouse model used in the study was developed in a C57BL/6N background, expressing the D168F/N173K mouse analog of the hyper-activatable human D167F/D172K protein C variant. This modification enables increased circulating APC levels throughout the mouse's lifetime. Male and female cohorts of C57BL/6N wild-type and APCHi transgenic mice were exposed to 9.5 Gy γ-rays with their hind legs shielded to allow long-term survival that is necessary to monitor DEARE, and plasma was collected at 6 months for LC-MS-based metabolomics and lipidomics. We observed significant dyslipidemia, indicative of inflammatory phenotype, upon radiation exposure. Additionally, observance of several other metabolic dysregulations was suggestive of gut damage, perturbations in TriCarboxylic Acid (TCA) and urea cycles, and arginine metabolism. We also observed gender- and genotype-modulated metabolic perturbations post radiation exposure. The APCHi mice showed near-normal abundance for several lipids. Moreover, restoration of plasma levels of some metabolites, including amino acids, citric acid, and hypoxanthine, in APCHi mice is indicative of APC-mediated protection from radiation injuries. With the help of these findings, the role of APC in plasma molecular events after acute γ-radiation exposure in a gender-specific manner can be established for the first time.

Circulating activated protein C levels are not increased in septic patients treated with recombinant human soluble thrombomodulin

BackgroundRecombinant human soluble thrombomodulin (rTM) has been used for the treatment of disseminated intravascular coagulation in Japan, and an international phase III clinical trial for rTM is currently in progress. rTM mainly exerts its anticoagulant effects through an activated protein C (APC)-dependent mechanism, but the circulating APC levels after rTM treatment have not been clarified. This prospective observational study investigated plasma APC levels after rTM treatment.MethodsPlasma levels of soluble thrombomodulin, thrombin-antithrombin complex (TAT), protein C, and APC were measured in eight septic patients treated with rTM. APC generation in vitro was assessed in the presence or absence of rTM.ResultsrTM significantly increased thrombin-mediated APC generation in vitro. In septic patients, soluble thrombomodulin levels were significantly increased during a 30–60-min period of rTM treatment and TAT levels were decreased. However, APC activity was not increased during the treatment period.ConclusionsPlasma APC activity is not increased in septic patients treated with rTM. It is possible that APC acts locally and does not circulate systemically.

Activated Protein C Levels Found In Trauma Patients Are Insufficient To Inactivate Platelet Factor Va and Produce Coagulopathy In An In Vitro Model

Background A subset of severe trauma victims suffering from uncontrolled hemorrhage present to the emergency room with an acute coagulopathy of trauma (ACoT). This condition occurs within the first 30 min following injury, is not the result of resuscitation-associated dilutional effects, and is defined by an INR > 1.2. The mechanisms behind this coagulopathy have not been completely identified, but recent studies posit that the exuberant activation of protein C is a key contributor to ACoT. Protein C is activated through the interaction of thrombin and thrombomodulin on the endothelial surface. Activated protein C (APC) has anti-inflammatory and cytoprotective functions, in addition to regulating coagulation by inactivating factor Va (FVa). Evidence that APC is implicated in ACoT includes: 1) circulating APC levels are elevated 3-5 fold in patients with high injury severity scores (>15) and base deficit (>6); 2) high APC levels (>6 ng/ml, or 105 pM) in these patients are correlated with an increase in prothrombin time (PT) and partial thromboplastin time [Cohen et al. Ann Surg 2012; 255(2): 379-85]. FVa exists in a soluble plasma fraction and in a platelet-associated fraction (in the membrane or alpha-granules). The platelet pool of FVa is more resistant to APC degradation [Camire et al. J Biol Chem 1995; 270(35): 20794-800] and is sufficient to maintain thrombin generation even in patients with severe congenital FV-deficiencies [Duckers et al. Blood 2010; 115(4): 879-86]. We hypothesize that healthy platelets in sufficient quantity will serve as an effective promoter of hemostasis in patients with ACoT, overcoming the effects of APC. Methods and Results We studied the effects of APC on coagulation in commercially obtained FV-deficient plasma (FVdp) and platelets/plasma from healthy volunteers. Tests included prothrombin time, turbidimetric assessment of fibrin cross-linking, and thromboelastography (TEG). Prothrombin time in platelet-free plasma or FVdp subjected to exogenous APC display a dose-dependent response where clotting is only significantly delayed at APC >1 nM, much higher than the systemic concentrations found in the ACoT patients (105pM) or even in subjects treated with drotrecogin alfa, a recombinant APC product intended for the treatment of sepsis (steady-state concentration was found to be ∼790pM) [Macias et al. Clin Pharmacol Ther 2002; 72(4): 391-402]. Examining fibrin crosslinking in a static 96-well turbidimetric plate assay demonstrated that concentrations of APC of 1nM or below had no effect on the initiation time, rate, or strength of clotting in FVdp or PFP from healthy volunteers. Titration of plasma FV into FVdp illustrated that clotting time and rate (as measured by TEG) were not significantly affected at 10nM FV, less than 50% of the “normal” 23nM concentration. Clot strength was unaffected by FV depletion. To examine the contributions of platelet FVa, isolated and washed platelets were introduced in increasing amounts to FVdp and PFP; the rate of clotting and clot strength aligned between the two plasmas given equivalent numbers of platelets, emphasizing the importance of platelet FVa. At lower platelet concentrations (particularly 10,000 to 100,000 / µl), there were statistically significant differences in clotting time between FVdp and normal plasma, but these differences diminished with increasing platelet concentration. To evaluate the resistance of the platelet fraction to APC effects, 200,000 platelets / µl were suspended in FVdp and treated with a titration of APC from 1pM to 100nM. The samples were unaffected by APC up to a dose of at least 10nM as measured by TEG clotting parameters, well above the amounts found in the trauma patients or drotrecogin alfa-treated subjects. Conclusions FV-deficiency affects clotting only in cases of severe depletion (<50%). In a low shear in vitro closed system, APC does not affect coagulation except at very high levels (>1nM). Healthy platelets in sufficient quantity are capable of rescuing FV-deficiency and are resistant to APC effects. APC is unlikely to be the main driver of ACoT. Disclosures: No relevant conflicts of interest to declare.

New Direction for WE Thrombin

An appealing strategy for developing therapeutic agents is to modify the design of human proteins, thereby taking advantage of target-specificity, multifunctionality of many proteins, and human compatibility—that is, provided any undesirable functional effects of the parent molecule can be selectively engineered away. In 2000, Cantwell & Di Cera1 reported a mutant form of the human serine protease, α-thrombin, a multifunctional enzyme involved in both pro- and antithrombotic pathways. This mutant, termed WE thrombin, containing Trp215Ala and Glu217Ala substitutions, still promoted formation of activated protein C (APC) that inhibits coagulation factors Va and VIIIa (antithrombotic), but inefficiently converted fibrinogen to fibrin (prothrombotic), and was an effective antithrombotic agent in primate models of thrombosis.2,3 But a number of observations suggested this was not the full story. For instance, at low WE thrombin concentrations, there was a greater antithrombotic effect than expected based on circulating APC levels and minimal systemic anticoagulation, and in addition, labeled WE thrombin was incorporated into a developing thrombus, suggesting it might be interacting with platelets. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Berny and colleagues4 have provided further information (see Figure), revealing an unanticipated additional antithrombotic effect of WE thrombin, that is, binding to platelet glycoprotein (GP)Ibα (the major ligand-binding subunit of the GPIb-IX-V complex),5–7 and inhibiting platelet adhesion to von Willebrand factor (vWF) under hydrodynamic flow and thrombus formation on a collagen matrix. The interaction of both wild-type and WE thrombin with GPIbα involves the N-terminal ligand-binding region of GPIbα (residues 1 to 282), but with profoundly different functional consequences. Figure. An antithrombotic form of mutant thrombin, WE, is ineffective at converting fibrinogen to fibrin, but is able to generate APC, which inhibits activation of coagulation factors V and VIII (to FVa and FVIIIa, respectively). Berny and colleagues4 now …

Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates

AbstractThe anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity.

Circulating activated protein C is reduced in young survivors of myocardial infarction and inversely correlates with the severity of coronary lesions.

Cardiovascular risk factors for myocardial infarction (MI) are less frequent in younger than in older MI survivors. Therefore, the thrombotic component of MI may play a more important role at a young age. As activated protein C (APC) provides systemic anticoagulant and anti-inflammatory protection, a low plasma APC level may be an arterial thrombotic risk factor. To determine whether there is an association between reduced APC levels and early MI and severe coronary lesions. APC was measured in 231 young MI survivors and 231 controls. Low APC levels were significantly associated with MI. Compared with the fourth quartile, the odds ratio (OR) for APC values in the first quartile was 3.7 [95% confidence interval (CI) = 2.1-6.4], and 3.2 (1.5-7.0) after adjustment for cardiovascular risk factors. Moreover, each decrease of 0.43 ng mL(-1) (1 SD) in APC increased the OR 1.7 times (1.4-2.2), and 1.5 times (1.2-1.9) after adjustment for cardiovascular risk factors. Low APC levels were also associated with the number of coronary arteries affected and with the severity of coronary lesions (P < 0.001). There is a significant association between low circulating APC levels and both early MI and the extent and severity of coronary atherosclerosis, which might be related to the anticoagulant and anti-inflammatory properties of APC.

Association of increased fibrinogen concentration with impaired activation of anticoagulant protein C.

Low levels of activated protein C (APC) are a risk factor for venous thrombosis. The mechanisms leading to interindividual differences in APC are not totally elucidated. Protein C is activated by the thrombin-thrombomodulin complex. As thrombin binds to fibrinogen and thrombomodulin through a common region, it is conceivable that fibrinogen influences the activation of protein C. This would help to explain the association between high levels of fibrinogen and an increased thrombotic risk. We analyzed the association between circulating APC levels and fibrinogen concentration in 382 healthy subjects. Subsequently, we studied the effect of increasing fibrinogen concentrations on the APC generation on cultured endothelial cells. An independent inverse association between circulating APC levels and fibrinogen was found [betacoefficient, -0.16; 95% confidence interval (95% CI) -0.26, -0.06; P = 0.001]. For each 100 mg dL(-1) increase in fibrinogen, the independent risk of having low APC levels (<0.7 ng mL(-1)) was almost three times higher (OR 2.8; 95% CI 1.1, 7.2; P = 0.04). Accordingly, a notable association between increasing fibrinogen concentrations and the reduction in the thrombin-thrombomodulin dependent activation of protein C on endothelial cells was found (r = -0.57; P = 0.002). We present evidence of an inverse association between circulating APC and fibrinogen levels. According to this finding together with the results of our in vitro experiments, we propose that the impairment in the generation of APC on endothelial cells constitutes a new prothrombotic mechanism of fibrinogen.

PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

The Multifunctional Protein C System

The protein C pathway is a major regulator of blood coagulation, since it controls the conversion of prothrombin to thrombin through a feedback inhibition mechanism. Protein C circulates in plasma as an inactive zymogen and is activated on the surface of endothelial cells by the thrombin-thrombomodulin complex, a process that can be further enhanced when protein C binds to its membrane receptor, the endothelial-cell protein C receptor. Activated protein C (APC) is then released from the complex, binds protein S and inhibits thrombin formation by inactivating coagulation factors Va and VIIIa. The importance of the protein C anticoagulant pathway is emphasized by the increased risk of venous thromboembolism (VTE) associated with protein C and protein S deficiencies, the factor V Leiden mutation, and reduced circulating APC levels. The protein C pathway also plays a significant role in inflammatory processes, since it prevents the lethal effects of E. coli-associated sepsis in animal models and improves the outcome of patients with severe sepsis. APC seems to display anti-apoptotic and neuroprotective activities. Thus, it reduces organ damage in animal models of sepsis, ischemic injury, endothelial cell injury, or stroke. Further research will hopefully widen the current therapeutic perspectives in all these illnesses, where these effects might play a crucial role in their treatment. This review will summarize the mechanisms that contribute to these biological activities of the protein C pathway.

Platelet factor 4 modulation of the thrombomodulin-protein C system.

To review published studies of the influence of platelet factor 4 (PF4) and other cationic proteins on the generation of activated protein C (APC) by the thrombomodulin-protein C system. Using the PubMed citation index, literature published from 1973 to 2003 regarding cationic proteins, PF4, and the thrombomodulin-protein C system was reviewed. All other cationic proteins studied to date either impair or do not affect APC generation via the thrombomodulin-protein C system; however, the platelet alpha-granule protein PF4 causes a 25-fold increase in the ability of thrombomodulin polypeptides to generate APC and a ten-fold increase in the ability of cultured endothelial cell-associated thrombomodulin to generate APC. The mechanism underlying this phenomenon depends on binding of the cationic PF4 to the anionic, vitamin K- dependent gamma-carboxyglutamic acid domain of protein C. The extent of PF4's stimulation of APC generation is further increased by its interaction with the anionic glycosaminoglycan moiety that is variably expressed through posttranslational, O-linked glycosylation of thrombomodulin. In an in vivo thrombin-infusion model of thrombomodulin activation in cynomolgus monkeys, previous intravenous infusion of pharmacologic amounts of PF4 resulted in circulating APC levels and APC-dependent prolongation of activated partial thromboplastin times that were two- to three-fold greater than those observed in saline-infused control animals. These findings raise the possibility that PF4 plays a hitherto unsuspected physiologic role in enhancing APC generation in vivo. They also provide a rationale for considering the infusion of PF4 or PF4-related peptides or peptidomimetics as a way of beneficially stimulating "endogenous" APC generation from circulating protein C in pathologic human disease states such as sepsis.

Platelet factor 4 enhances generation of activated protein C in vitro and in vivo

AbstractPlatelet factor 4 (PF4), an abundant platelet α-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 μg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 μg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 ± 5 ng/mL and 39 ± 2 ng/mL, respectively (P < .001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 ± 6 seconds in PF4-treated animals and by 9 ± 1 seconds in control animals at 30 minutes (P < .001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of control’s from 10 through 120 minutes (P ≤ .04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation. (Blood. 2003;102:146-151)

Brain-specific protein C activation during carotid artery occlusion in humans.

Activation of plasma protein C (PC) zymogen by thrombin-thrombomodulin at the endothelial surface is an important endogenous antithrombotic mechanism. It is unknown whether activated protein C (APC) is generated in vivo in the cerebrovasculature, because there is only limited thrombomodulin expression in human brain vascular endothelium. Therefore, we tested the hypothesis that carotid occlusion produces brain-specific PC activation. Blood samples were simultaneously collected from the ipsilateral internal jugular vein and radial artery before and during carotid cross-clamping and on "de-occlusion" in 8 awake patients undergoing routine carotid endarterectomy. Plasma PC zymogen and circulating APC levels were measured using enzyme immunocapture assay and expressed as percent of pooled plasma controls. Internal jugular vein APC levels increased 28% exclusively during carotid occlusion and then decreased 32% with de-occlusion (F=8.1, P<0.005). PC zymogen increased only 5.9% with occlusion (F=6.3, P<0.02), consistent with hemoconcentration. There were no changes in radial artery PC or APC levels. These findings demonstrate brain-specific protein C activation in humans during carotid occlusion and suggest a protective role for endogenous APC generation during cerebrovascular occlusion.

Impairments of the protein C system and fibrinolysis in infection-associated stroke.

Infection/inflammation appears to be an important predisposing risk factor for brain infarction, but little is known regarding underlying molecular mechanisms. We examined the hypothesis that patients with brain infarction preceded by infection/inflammation within 1 week could be identified by a distinctive procoagulant laboratory profile characterized by abnormalities in the protein C system and endogenous fibrinolysis. We performed a case-control study examining the relationship between preceding systemic infectious/inflammatory syndromes and selected immunohematologic variables in 36 patients with acute brain infarction and 81 control subjects (community control subjects [n = 47] and hospitalized nonstroke neurological patient controls [n = 34]). The stroke group had a lower mean level of the circulating antithrombotic enzyme activated protein C (APC) (4.33 +/- 0.34% [log-transformed percentage of control value, mean +/- SD]) than community control subjects (4.51 +/- 0.27%, P < .02) or hospitalized neurological patient controls (4.57 +/- 0.31%, P < .005). The lowest circulating APC levels were found in the stroke group with antecedent infection/inflammation within 1 week preceding index brain infarction (4.23 +/- 0.4%, n = 12). Within the stroke group, circulating APC levels were inversely related to IgG isotype anticardiolipin antibody titers (r = -.55, P < .001). Only the stroke group with infection/inflammation within 1 week had elevated plasma C4b binding protein compared with control subjects (141 +/- 61% versus 112 +/- 44%, P < .05). Stroke patients with antecedent infection/inflammation had a distinctively lower ratio of active tissue plasminogen activator to plasminogen activator inhibitor (0.11 +/- 0.04, n = 9) than other stroke patients (0.19 +/- 0.06, n = 9, P < .01) and control subjects (0.22 +/- 0.16, n = 17, P < .02). Impairments in the protein C pathway and endogenous fibrinolysis may contribute to the increased risk for brain infarction after recent (< or = 1 week) infection/inflammation. A decrease in the circulating anticoagulant APC may be related to elevated antiphospholipid antibody titers.