Activated Protein C Generation Research Articles

Antithrombin-resistant prothrombin Yukuhashi mutation also causes thrombomodulin resistance in fibrinogen clotting but not in protein C activation

IntroductionProthrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)–protein C (PC) anticoagulant system have not been previously elucidated. Materials and MethodsWe prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. Result and ConclusionsrTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.

Tamoxifen induces resistance to activated protein C

The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet. Blood samples were collected prospectively from women with breast cancer before (n=25) and monthly after start of adjuvant TAM treatment (n=75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally. APC sensitivity decreased by 41% (p=0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p=0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed. This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors.

Activation-Resistant Homozygous Protein C R229W Mutation Causing Familial Perinatal Intracranial Hemorrhage

Two first-degree cousins presented with similar phenotype characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis at the age of four months. One girl, now 9 yr old, was a full term baby presenting at 13 days of age with seizures and brain CT showing intracranial hemorrhage. At 4 months of age, she presented with multiple thrombotic lesions over both hands and feet that required extensive debridement and improved with anticoagulation and plasma replacement. She has been on enoxaparin with no new lesions afterward. At 9 years of age, protein C amidolytic activity (STACHROM® protein C) was 61% (nl 70-140%) and protein C antigen (ELISA) was 57% (nl 70-140%). Thrombophilia work up was otherwise normal. The second child is a 1 yr old boy who was delivered by cesarean section because of fetal distress. The mother gave history of decreased fetal movement for few weeks prior to his birth and was noted to be hypotonic. Brain MRI in the first week of life showed massive subacute hemorrhage in the brain parenchyma and upper spinal canal. Bleeding stopped with appropriate supportive care. He did not have purpura fulminans or any evidence of thrombosis during the neonatal period. At 4 months of age, a ventriculoperitoneal shunt was placed because of hydrocephalus and post operatively he developed thrombotic lesion in his left foot that resolved after starting enoxaparin and plasma replacement. He remains on enoxaparin with no new significant lesions. At 10 months of age, protein C amidolytic activity was 59% and protein C antigen was 73%. Both patients suffer from severe permanent neurological deficit and blindness. Dx of protein C homozygous deficiency was established after exome sequencing of the older child revealed a homozygous variant in exon 9 of PROC gene c.811 C>T (R271W equivalent to R229W in mature protein C numbering). Sanger sequencing validated this mutation. Both patients were homozygous (T/T) while tested parents and two siblings were heterozygous (C/T), and 55 controls were normal (C/C). No family history of thrombosis among family members who are heterozygous for this mutation was found. Complete exome sequence analysis also showed no mutations for ATIII, protein S, factors V or VIII, prothrombin, plasminogen, protein Z, or any ADAMTS genes. The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin (TM) interactions. To define functional abnormalities caused by the patients' mutation, recombinant protein C mutants R229W, R229Q and R229A were studied. Each was strikingly defective in rate of activation by thrombin:TM, showing an activation rate that was only 1%, 2% and 4%, respectively, compared to wildtype protein C. Once activated, each activated protein C (APC) mutant was moderately affected, showing a 50% reduction in anticoagulant activity and in rate of cleavage of purified factor Va at Arg506. The amidolytic activities were less affected and close to wildtype APC. The plasma half-lives were similar to wildtype APC. These properties of recombinant R229W protein C strongly suggest a severe in vivo deficit in these children for generation of APC, implying a severe deficiency of APC in spite of half-normal levels of protein C zymogen. As routine coag assays utilize snake venom protease, they fail to detect TM-activation-resistant protein C mutants. In summary, homozygous protein C R229W mutation in two related cousins was associated with significant leaky brain blood vessels in the perinatal period and significant neurological insult even prior to the first evidence of clinical thrombosis. This highlights the importance of protein C and APC in maintaining the integrity of the brain vascular endothelium in humans. Disclosures:No relevant conflicts of interest to declare.

Quantitative analysis of thrombomodulin-mediated conversion of protein C to APC: Translation from in vitro to in vivo

Thrombomodulin-bound thrombin cleaves protein C (PC) zymogen in blood plasma producing activated protein C (APC), which exerts anti-coagulant, anti-inflammatory, anti-apoptotic and CNS-protective effects. Recombinant APC and thrombomodulin (TM) are both in clinical studies for management of acute conditions including sepsis. Methods that permit accurate measurement of APC in plasma are needed for clinical monitoring and mechanistic studies in animal models. However, the two existing methods require either long incubation periods with substrate, resulting in high background or they also recognize protein C inhibitor (PCI) complexed with APC (APC:PCI), which convolutes analysis of the amount of APC generated. Here we describe a robust quantitative in vivo assay that measures APC generation at both low levels of human protein C seen in chronic inflammatory disease and at physiological levels that shows a >99% fit with in vitro data.

The clinical value of activated protein C in pediatric leukemia patients with acute lung injury

Objective To investigate the changes of plasma activated protein C(APC)in the pediatric leukemia patients with acute lung injury(ALI),and the relationship with the outcome.Methods During the study period(from Jan to Dec 2009),17 pediatric leukemia patients with ALI were selected.They were divided into neutropenic(n =10)and non-neutropenic(n =7)group.We collected the basic data including the age,gender,stage of chemotherapy,pediatric critical illness score,mechanical ventilation time,ICU time.The blood gas and plasma APC levels were detected on day 1 and day 4 of ALI onset.Results Compared with the non-neutropenic group,the neutropenic group had a significantly longer mechanical ventilation time [(16.60 ± 1 0.83)d vs(3.79 ± 4.08)d,P =0.009 6],lower PaO2 level on day l[(54.90 ± 17.05)mm Hg vs (92.70 ± 27.53)mm Hg,P =0.009 7],and lower APC level on day 4[(193.06 ± 63.19)pg/mlvs(286.28 ±25.12)pg/ml,P =0.007 7].Conclusion The APC generation is severely impaired in the neutropenic group,especially in those who died.The lower APC level is,the longer is the mechanical ventilation time,the worse is oxygenation,the poorer is the prognosis.APC replacement therapy may be promising in these patients. Key words: Acute lung injury; Activated protein C; Leukemia; Childrer

Liver myofibroblasts activate protein C and respond to activated protein C

To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells. Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin. These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.

Modulation of Substrate Specificity of the Thrombin-Thrombomodulin Complex for Protein C Versus TAFI Activation by Platelet Factor 4 and Non-Anticoagulant Heparin-Derivatives.

Abstract 3181 Poster Board III-108 The endothelial cell receptor thrombomodulin (TM) is an essential cofactor for thrombin-mediated activation of protein C and the generation of activated protein C (APC), an enzyme with anticoagulant and multiple cytoprotective activities. TM also stimulates thrombin-mediated activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI), resulting in anti-fibrinolytic and anti-inflammatory effects. Based on these properties of TM, several potential strategies for therapeutic intervention have been proposed. In hemophilia patients with inhibitory antibodies, stimulation of TM-dependent TAFI activation might diminish bleeding complications by protection of clots against premature lysis. Alternatively, stimulation of endogenous APC generation in patients with severe sepsis might prove to augment the beneficial effects of APC, as pharmacologic applications of APC in these patients increased survival. Platelet Factor 4 (PF4), a platelet alpha-granule protein, acts as a soluble cofactor for protein C activation by the thrombin-TM complex and stimulated endogenous generation of APC in vivo. Here we show that in contrast to stimulation of protein C activation, PF4 dose-dependently inhibited activation of TAFI by thrombin-TM in a purified system, in normal pooled plasma and on TM-expressing cells. Functional consequences of inhibition of TAFI activation by PF4 were determined in hemophilia A plasma by analysis of clot resistance to tPA-induced fibrinolysis. In hemophilia A plasma, activation of TAFI was dependent on TM due to the ineffective amplification of thrombin generation via the tenase complex. PF4 diminished TM-dependent prolongation of clot lysis times but did not affect clot lysis in the absence of TM. Inhibition of TM-dependent prolongation of clot lysis time by PF4 was dependent on both the TM and tPA concentration used, consistent with the proposed threshold mechanism for inhibition of fibrinolysis by TAFIa. To determine the effect of PF4 on TM-dependent TAFI activation in normal plasma, the inactivation of bradykinin (BK) in plasma by TAFIa during tissue factor induced coagulation was analyzed. Addition of TM to plasma accelerated BK cleavage, i.e., generation of des-Arg9-BK, consistent with TM-mediated stimulation of TAFI activation and subsequent BK cleavage by TAFIa. PF4 neutralized the TM-enhanced BK inactivation and reversed BK and des-Arg9-BK levels to those seen in the absence of TM. Thus, PF4 alters the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, such that PF4 prevents generation of TAFIa's anti-fibrinolytic and anti-inflammatory activities. To find an antidote for PF4's inhibitory effects on TAFI activation, heparin-derivatives were screened for high affinity PF4 binding but greatly reduced anticoagulant activity. N-acetylated heparin (NAc-Hep), but not N-desulfated without N-acetylation or N-acetylated and O-desulfated heparins, retained high affinity binding to PF4 and effectively blocked PF4 binding to immobilized TM. Consistent with high affinity binding of NAc-Hep to PF4, sulfylamine group oxygens were relatively minor determinants of the interactions between heparin and PF4 based on the available structural model. NAc-Hep did not show detectable anticoagulant activity in APTT clotting assays but restored BK conversion to des-Arg9-BK by TAFIa in the presence of PF4, similar to that seen in the absence of PF4. In addition, NAc-Hep reversed PF4-mediated inhibition of TAFI activation and TAFIa's antifibrinolytic functions in clot lysis assays on TM-expressing cells using hemophilia A plasma. Thus, NAc-Hep reversed PF4-mediated inhibition of TAFI activation. In summary, PF4 modulates the substrate-specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation. NAc-Hep, a non-anticoagulant heparin derivative with high affinity for PF4, effectively reversed PF4-mediated inhibition of TM-dependent TAFI activation and restored TAFIa's antifibrinolytic and anti-inflammatory functions. Accordingly, NAc-Hep or similar non-anticoagulant heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis. Disclosures No relevant conflicts of interest to declare.

Thrombomodulin Improves Early Outcomes After Intraportal Islet Transplantation

Primary islet nonfunction due to an instant blood mediated inflammatory reaction (IBMIR) leads to an increase in donor islet mass required to achieve euglycemia. In the presence of thrombin, thrombomodulin generates activated protein C (APC), which limits procoagulant and proinflammatory responses. In this study, we postulated that liposomal formulations of thrombomodulin (lipo-TM), due to its propensity for preferential uptake in the liver, would enhance intraportal engraftment of allogeneic islets by inhibiting the IBMIR. Diabetic C57BL/6J mice underwent intraportal transplantation with B10.BR murine islets. In the absence of treatment, conversion to euglycemia was observed among 29% of mice receiving 250 allo-islets. In contrast, a single infusion of lipo-TM led to euglycemia in 83% of recipients (p = 0.0019). Fibrin deposition (p < 0.0001), neutrophil infiltration (p < 0.0001), as well as expression TNF-alpha and IL-beta (p < 0.03) were significantly reduced. Significantly, thrombotic responses mediated by human islets in contact with human blood were also reduced by this approach. Lipo-TM improves the engraftment of allogeneic islets through a reduction in local thrombotic and inflammatory processes. As an enzyme-based pharmacotherapeutic, this strategy offers the potential for local generation of APC at the site of islet infusion, during the initial period of elevated thrombin production.

Platelet Factor 4 Impairs the Anticoagulant Activity of Activated Protein C

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Structural and Functional Studies to Define the Molecular Basis by Which Platelet Factor 4 (PF4) Increases Survival of Mice in Lipopolysaccharide (LPS)-Induced Endotoxicity

Our previous studies have demonstrated that platelet-released PF4 (chemokine CXCL4) promotes survival in a murine LPS-induced endotoxicity model, although the molecular basis for PF4's protective effects was not fully defined. We hypothesized that enhanced generation of cytoprotective activated protein C (APC) by PF4 might contribute to the molecular mechanism of PF4's beneficial effects in vivo, based on the observation that PF4 stimulates protein C (PC) activation by the thrombin/thrombomodulin complex both in vitro and in vivo. Here we show that PF4 in vitro affects human (h) PC activation in the presence of human thrombomodulin (hTM) in a bell-shaped concentration curve, i.e. stimulation at low, but inhibition at high PF4 concentrations with a peak around 3 μM. This curve is similar to that seen with PF4 for surface-bound heparin-induced thrombocytopenia (HIT) antigenicity, suggesting that similar complexes of PF4 with glycosaminoglycans (GAGs) that occur in HIT (termed ultralarge complexes (ULC)) are relevant to PF4's interaction with the hPC/hTM. Addition of heparin blocks PF4 increase of APC generation in a similar fashion as it does for surface-bound ULC. A PF4 variant PF4K50E that poorly forms PF4 tetramers requires 8-fold higher concentrations to enhance APC generation, supporting that PF4 tetramers are central for APC generation as they are for formation of ULC. Neither PF4 nor PF4K50E accelerated in vitro generation of APC in the presence of hTM that was depleted of its chondroitin sulfate chain, suggesting that PF4 binds to this domain on hTM. In vivo studies involving simultaneous infusions of PF4 and thrombin into PF4 knock out (mPF4−/−) mice showed that PF4 leads to enhanced mouse (m) APC generation not seen with infused PF4K50E, consistent with our in vitro studies. We then asked if surface heparan sulfate on the endothelial lining was necessary for the observed PF4 effect on in vivo mAPC formation. We studied mice with a Tie2-Cre conditional knock out of N-deacetylase-N-sulfotransferase-1 activity (NDST-1−/−) that have only 15% of normal endothelial cell surface heparan sulfate content using a similar thrombin/PF4 infusion model. We found that mAPC generation was accelerated by PF4 to the same extent both in NDST1−/−/mPF4−/− and mPF4−/− mice, suggesting that surface GAGs are not involved in the PF4 effect. We have also tested the in vivo effect of PF4 on mAPC formation in TM mutant (TMpro/pro) mice that have impaired capacity for APC formation to further demonstrate that PF4's positive effect in LPS endotoxic shock survival involves enhanced mAPC generation. Upon injection of high doses of thrombin (40 U/kg), mAPC levels are increased to the same extent in WT and TMpro/pro mice. After injection of low amounts of thrombin (8 U/kg), generation of mAPC was impaired in TMpro/pro as compared to WT mice. Concurrent infusion of PF4 increased mAPC formation in TMpro/pro mice after injection of low doses of thrombin, approximately equal to that seen in WT mice with no PF4 injected. As previously described, TMpro/pro mice had increased mortality after injection of LPS as compared to WT mice; however, with concurrent platelet PF4 overexpression, mortality decreased to that seen in WT mice, suggesting that the biological value of PF4 in LPS endotoxicity is related to its effect on the generation of APC. Thus, these studies support enhanced APC generation as the basis for the positive effect of PF4 on LPS endotoxicity and further define the molecular basis for increased APC generation by PF4 by forming ULC with the chondroitin sulfate domain of TM, but not with heparan sulfate on the vascular surface.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

Treatment of endothelium with the chemotherapy agent vincristine affects activated protein C generation to a greater degree in newborn plasma than in adult plasma

Introduction Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and α 2macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. Materials and methods APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-α 1AT were assayed by ELISA. APC-α 2M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. Results Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-α 1 AT and APC-α 2 M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. Conclusions The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.

Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge

AbstractPharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4−/−) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C–deficient (PC+/−) mice. Transgenic mice overexpressing human PF4 (hPF4+) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC+/− mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4+ and PC+/−/hPF4+ mice had improved survival. Further, infusion of hPF4+ platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting.

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase-PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways.

Polymorphisms in the endothelial protein C receptor gene and thrombophilia

The protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. Once formed, activated protein C (APC) down-regulates thrombin formation by inactivating factors (F)Va and FVIIIa. Endothelial protein C receptor (EPCR) is able to bind protein C and increase the rate of protein C activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of thrombin, thrombomodulin, protein C and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced/increased APC generation and modify the risk of thrombosis. This review highlights the different mutations/polymorphisms reported in the EPCR gene and their association with the risk of thrombosis.

Release of High Levels of Platelet Factor 4 (PF4) from Platelets Improves Survival after a Lethal Lipopolysaccharide (LPS) Challenge.

Infusion of activated protein C (APC) improves survival in sepsis. PF4 is a CXC chemokine predominantly expressed during megakaryopoiesis and stored in platelet alpha-granules whose biological function(s) are not well understood. We have shown that recombinant PF4 enhances APC generation by thrombin/thrombomodulin complexes both in vitro and in vivo. Would endogenous PF4 released from platelets activated during an inflammatory state similarly affect APC production? We addressed this issue using mice that were either completely devoid of PF4 (mPF4−/−) or had a 6-fold excess of human PF4 (hPF4+). Using a lethal LPS challenge model (O111:B4, 40 mg/kg IP), we examined whether platelets become activated and release PF4 during endotoxemia. Two hours after LPS injection, the platelet count in mice decreased to ~70% of baseline levels (p=0.006). Serum PF4, as measured by ELISA, also dropped to ~60% of baseline from 47±5 kU/ml to 30±5 kU/ml (p=0.002). At the same time, plasma PF4 level was increased by 20%, consistent with LPS resulting in PF4 release. The smaller increase than expected suggests that much of the released PF4 binds immediately to the surface of vascular cells. Consistent with this, we have observed higher accumulation of PF4 in mouse lungs after LPS injection compared to uninjected mice (990±220 and 660±120 U/mg, respectively, p=0.017). APC generation was assessed 10 min after thrombin infusion (80 U/kg. IV) as a measure of endogenous platelet PF4's effect in an inflammatory/procoagulant state. In mPF4−/− mice APC levels were 72% of that in wild type (WT) mice (p=0.0006) while in hPF4+ mice APC formation increased to 178% (p=0.003). Survival of mice 24 hrs after LPS (25 mg/kg) challenge was then examined. hPF4+ mice had a mortality rate of 9% compared to ~40% in both WT and mPF4−/− (p< 0.001). To examine the role of APC in this improved survival, we performed similar experiments with mice heterozygous for protein C deficiency (PC+/−). More PC+/− mice died 16 hrs after injection of 40 mg/kg LPS than WT mice (61% vs. 29% mortality respectively, p=0.005), while mortality for hPF4+/PC+/− mice was significantly lower (14%, p< 0.001 compared to PC+/− mice), supporting the hypothesis that the protective effect of PF4 is at least in part due to increased APC generation. Next we asked if infusion of platelets with high PF4 is protective in the LPS model. We injected either vehicle buffer or mPF4−/− or hPF4+ platelets (3×108 per 20 g mouse) into WT mice prior to treatment with LPS. Mortality of mice at 24 hrs after LPS injection with mPF4−/−platelet infusion was not significantly different than mice with buffer infusion (86% vs. 96% respectively, p=0.4), but mortality was significantly lower when hPF4+ platelets were infused (58% vs. 96%, p=0.01). Our results suggest that PF4 is released from platelets after an inflammatory stimulus and that this may have a positive physiological role by enhancing APC generation. High endogenous platelet PF4 levels may have a survival advantage after exposure to endotoxins. Infusion of platelets containing high levels of PF4 in sepsis may be a novel therapeutic strategy that warrants further investigation.

Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.

The effect of aprotinin on activated protein C‐mediated downregulation of endogenous thrombin generation

Thrombin plays a central role in coagulation and haemostasis. Binding of thrombin to thrombomodulin generates activated protein C (APC), which exerts a negative feedback on thrombin formation. Aprotinin, a natural proteinase inhibitor is used extensively during cardiac surgery because this procedure is often associated with profound activation of coagulation and inflammatory pathways. Some in vitro evidences suggest that aprotinin inhibits APC, but the clinical relevance is unclear. The recombinant human soluble thrombomodulin (rhsTM)-modified thrombin generation (TG) assay was used to investigate the effects of aprotinin on APC in plasma samples obtained from healthy volunteers, aprotinin-treated cardiac surgical patients and in protein C (PC)-depleted plasma. Based on the results of in vitro TG assay, addition of rhsTM (0.75-3.0 microg/ml) to volunteer or patient platelet-poor plasma significantly reduced (70.8 +/- 21.9 and 95.3% +/- 4.6%, respectively) thrombin formation when compared with PC-depleted plasma (8.3% +/- 5.2%). Aprotinin (100-200 KIU) caused a small, statistically insignificant decrease in the peak thrombin formation in normal and PC-deficient plasma (12.0 +/- 6.1%). In cardiac surgical patients, levels of functional PC, factor II, antithrombin and platelet significantly decreased after cardiopulmonary bypass (CPB). Soluble thrombomodulin concentrations were increased after CPB (3.5 +/- 2.2 to 5.0 +/- 2.2 ng/ml), but they were still within the normal range for human plasma. Our results showed that, even though endogenous PC level is decreased after CPB, it retains its activity in the presence of thrombomodulin, and aprotinin has limited inhibitory effect on APC generation.

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.

Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates.

Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 microg kg(-1) i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 microg kg(-1) loading + 5 microg kg(-1) h(-1) infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL(-1) to 86 ng mL(-1) by 40 min, then declined, but remained elevated at 34 ng mL(-1) at 240 min. A 22-fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL(-1) at 40 min and decreased to 20 ng mL(-1) within 120 min despite continued activator infusion. Co-infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low-grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.