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.     

Coagulation and inflammation

The protein C anticoagulant pathway is critical for controlling microvascular thrombosis and is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. Protein C activation is augmented by an endothelial cell protein C receptor (EPCR). EPCR is shed from the vasculature by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravasation. EPCR can undergo translocation from the plasma membrane to the nucleus where it re-directs gene expression. During translocation, EPCR can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF-alpha and other inflammatory mediators can down-regulate EPCR and TM. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravasation in response to endotoxin and infusion of APC reverses these processes. In vitro, APC has been reported to inhibit TNF-alpha elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.     

Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation

Activated protein C (APC) is inhibited by two major plasma inhibitors (PCIs). To find evidence for in vivo complexation of APC, immunoblotting studies were performed on plasmas of 85 patients with suspected disseminated intravascular coagulation (DIC). Samples from 62 of these patients contained 5% to 35% of protein C antigen in APC:inhibitor complexes, indicating that protein C activation and inhibition had occurred. In 24 normal plasmas, no detectable APC:PCI complexes were observed (less than 5%). Patients with higher levels of complexes had more abnormal coagulation test data for DIC. The major band of APC complexes detected by anti-protein C antibodies did not react with antibodies to the heparin-dependent protein C inhibitor (PCI- 1) previously described. Rather, APC was complexed with another recently described plasma protein C inhibitor, PCI-2. Immunoblotting studies for protein S, the cofactor for APC, revealed that the majority of the DIC patient plasmas contained a higher than normal proportion of protein S in cleaved form, suggesting that protein S may have been proteolytically inactivated. Protein S total antigen levels were also found to be low in DIC patients, excluding those with malignancy. These studies support the hypothesis that the protein C pathway is activated during DIC.     

Effect of hyperhomocysteinemia on protein C activation and activity

Hyperhomocysteinemia has been proposed to inhibit the protein C anticoagulant system through 2 mechanisms: decreased generation of activated protein C (APC) by thrombin, and resistance to APC caused by decreased inactivation of factor Va (FVa). We tested the hypotheses that generation of APC by thrombin is impaired in hyperhomocysteinemia in monkeys and that hyperhomocysteinemia produces resistance to APC in monkeys, mice, and humans. In a randomized crossover study, cynomolgus monkeys were fed either a control diet or a hyperhomocysteinemic diet for 4 weeks. Plasma total homocysteine (tHcy) was approximately 2-fold higher when monkeys were on the hyperhomocysteinemic diet than when they were on the control diet (9.8 +/- 2.0 microM versus 5.6 +/- 1.0 microM; P <.05). After infusion of human thrombin (25 microg/kg of body weight), the peak level of plasma APC was 136 +/- 16 U/mL in monkeys fed the control diet and 127 +/- 13 U/mL in monkeys fed the hyperhomocysteinemic diet (P >.05). The activated partial thromboplastin time was prolonged to a similar extent by infusion of thrombin in monkeys fed the control diet and in those fed the hyperhomocysteinemic diet. The sensitivity of plasma FV to human APC was identical in monkeys on control diet and those on hyperhomocysteinemic diet. We also did not detect resistance of plasma FV to APC in hyperhomocysteinemic mice deficient in cystathionine beta-synthase (plasma tHcy, 93 +/- 16 microM) or in human volunteers with acute hyperhomocysteinemia (plasma tHcy, 45 +/- 6 microM). Our findings indicate that activation of protein C by thrombin and inactivation of plasma FVa by APC are not impaired during moderate hyperhomocysteinemia in vivo.     

Patients with severe sepsis vary markedly in their ability to generate activated protein C

Activated protein C (APC) supplementation significantly reduces mortality in patients with severe sepsis, presumably by down-regulating coagulation, inflammation, and apoptosis. In vivo, endogenous APC is generated from protein C (PC) "on demand" in response to elevated thrombin levels. Thrombomodulin and endothelial cell protein C receptor are endothelial receptors required to generate APC endogenously. Since these receptors may be down-regulated in sepsis, we measured plasma markers of APC generation in 32 patients with severe sepsis to determine whether APC generation is impaired and whether markers of APC generation correlate with 28-day mortality. Relative to normals, all patients had elevated F1 + 2 and thrombin-antithrombin complex (TAT) levels (markers of thrombin generation and inhibition, respectively), and 28 of 32 patients had reduced PC levels. In 20 patients, APC levels paralleled elevated F1 + 2 levels, whereas 12 patients had low APC levels despite elevated F1 + 2 levels, suggesting that APC generation is impaired in the latter. No significant differences exist between survivors and nonsurvivors with respect to baseline PC levels, F1 + 2 levels, and APACHE II (acute physiology and chronic health evaluation) scores. Baseline APC levels were higher in survivors (P = .024), and baseline F1 + 2/APC ratios were lower in survivors (P = .047). Larger studies are warranted to establish whether APC generation profiles aid in managing sepsis.     

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

Platelet factor 4 (PF4), an abundant platelet alpha-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 microg 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 microg/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 controls from 10 through 120 minutes (P < or =.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.     

Activated protein C prevents development of phosphatidylserine-induced intrauterine growth restriction in mice

We examined the effect of activated protein C (APC) on the development of intrauterine growth restriction (IUGR) in an experimental animal model we established. The IUGR in mice was induced by artificial phosphatidylserine (PS)/phosphatidylcholine (PC) microvesicles that represent procoagulant phospholipids derived from activated platelets. This model represents the placental insufficiency associated with the phospholipid-induced hypercoagulable state in placental circulation. APC prolonged the activated partial thromboplastin time (aPTT) using mouse plasma and dose dependently inhibited thrombin generation in a chromogenic assay in defibrinated plasma of mice. Administration of exogenous APC at concentrations that maximally inhibited thrombin generation in defibrinated plasma prevented a significant reduction in fetal body weight and induced marked histological changes including congestion and fibrin depositions in IUGR mouse placentas. These results suggest that the inhibition of thrombin generation in the placental circulation by APC prevents the development of IUGR that is dependent on coagulation associated with PS/PC from activated platelets.     

Measuring thrombin generation based sensitivity to activated protein C using an automated coagulometer (ACL 9000)

Increased thrombin generation in the presence of activated protein C (APC) is known to be an independent risk factor for thrombosis. Although commercially available methods for measuring thrombin generation are available, none measure sensitivity to APC. We have developed an automated thrombin generation based APC sensitivity test, for use with defibrinated plasma using the automated coagulometer (ACL9000). A chromogenic substrate that is cleaved by thrombin at a slow rate, 7 pm tissue factor and 20 mum phospholipid (DOPC : DOPE : DOPS, 60 : 20 : 20) with and without 5 nm APC. Thrombin generation with [endogenous thrombin potential (ETP)(+APC)] and without APC (ETP), was expressed as a ratio relative to pooled normal plasma. Normal reference ranges were established in 30 normal subjects: ETP 0.73-1.06; ETP(+APC) 0.42-1.07. ETP(+APC) was found to be sensitive to factor V Leiden, oral contraceptive use, low levels of protein S and tissue factor pathway inhibitor and increased plasma factor VIII.     

Inhibition of endothelial cell-mediated generation of activated protein C by direct and antithrombin-dependent thrombin inhibitors.

The present study investigated the effect of the thrombin inhibitors antithrombin (AT) (with and without unfractionated heparin or low molecular weight heparin), hirudin, inogatran and melagatran on thrombin-thrombomodulin-mediated generation of activated protein C (APC), in solution and on endothelial cells. Sequential incubation with thrombin, thrombin inhibitors and protein C was followed by measurement of APC by an amidolytic assay. The approximate concentrations resulting in 50% inhibition of endothelial cell-mediated APC generation for AT, AT-unfractionated heparin, AT-low molecular weight heparin, hirudin, melagatran and inogatran were 200, 4, 9, 1, 8 and 60 nmol/l, respectively. The normal plasma level of AT is 2800 nmol/l and relevant therapeutic concentrations from clinical trials are 200 nmol/l for hirudin, 500 nmol/l for melagatran and 1000 nmol/l for inogatran. The present study indicates that clinically relevant concentrations of the tested thrombin inhibitors interfere with endothelial-mediated APC generation, which may offer an explanation for the lack of a dose-response effect in clinical trials with thrombin inhibitors.     

Anticoagulant factor concentrates in disseminated intravascular coagulation: rationale for use and clinical experience.

Natural inhibitors of coagulation, in other words, antithrombin (AT), the protein C system, and tissue factor pathway inhibitor (TFPI), play an important role in controlling the activation of coagulation during disseminated intravascular coagulation (DIC). Furthermore, they may not only influence coagulation but also attenuate inflammatory responses during sepsis. Low circulating levels of AT and protein C have been associated with poor outcome. Replacement therapy with AT, activated protein C (APC), and TFPI has been shown to attenuate thrombin generation and to reduce mortality in experimental sepsis models. Experience with AT and APC in patients is promising. Data from large phase III trials of AT and APC as treatment of patients with severe sepsis will soon be available. Recombinant TFPI is currently in phase II clinical trials for severe sepsis.     

Increased activation of protein C, but lower plasma levels of free, activated protein C in uraemic patients: relationship with systemic inflammation and haemostatic activation

Chronic renal failure (CRF) courses with both systemic inflammatory reaction and haemostatic activation. We explored the relationship of these processes with plasma levels of free, activated protein C (APC) and complexes of APC with its inhibitors in patients with CRF under conservative treatment. Plasma concentrations of inflammatory cytokines [tumour necrosis factor alpha (TNFalpha) and interleukin 8], acute-phase proteins (C-reactive protein, fibrinogen, alpha1-anti-trypsin and von Willebrand factor), and markers of haemostatic activation (thrombin-anti-thrombin complexes, plasmin-anti-plasmin complexes, and fibrin and fibrinogen degradation products) were higher in patients than in controls. Inflammatory and haemostatic markers were significantly and positively correlated. Total plasma APC and APC:alpha1-anti-trypsin (alpha1AT) complexes were 44% and 75% higher in patients than in controls (P = 0.0001), whereas free APC was 20% lower (P < 0.015). No significant difference was observed in APC:protein C inhibitor (PCI) complexes between both groups. The free/total APC ratio was significantly lower in patients than in controls (P < 0.0001). Total plasma APC and APC:alpha1AT were positively correlated with activation markers of haemostasis and acute-phase proteins, whereas free APC was inversely correlated with plasma levels of creatinine, acute-phase proteins and fibrin degradation products (FnDP). Systemic inflammation and activation of haemostasis are interrelated processes in CRF. APC generation was increased in response to elevated thrombin production, but the inflammatory reaction, associated with increased synthesis of alpha1AT, reduced its anticoagulant effect. Lower free plasma APC in CRF may be pathogenically associated with atherothrombosis, a major cause of death in this disease.     

Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency.     

Thrombomodulin activity of fat-derived microvascular endothelial cells seeded on expanded polytetrafluorethylene

Lining the luminal surface of prosthetic vascular grafts with endothelial cells (cell seeding) will lower its thrombogenicity. Commonly used macrovascular human adult endothelial cells (HAEC) require in vitro cultivation before large enough numbers are obtained to cover grafts confluently. Fat-derived microvascular endothelial cells (MVEC) prove to be a good alternative as they can be harvested in much larger numbers while showing similar antithrombotic and fibrinolytic characteristics. An important anticoagulant function of macrovascular endothelial cells is due to the activity of thrombomodulin (TM) on their surface. In this study, the presence and functional activity of TM on fat-derived microvascular cells used in cell seeding was investigated. The expression and localization of TM on MVEC was studied using immunohistochemistry. Functional activity of TM on MVEC was measured by the generation of activated protein C (APC) and was compared to human umbilical vein endothelial cells (HUVEC). TM activity was studied in MVEC seeded on expanded polytetrafluorethylene (ePTFE) vascular prostheses and compared to blank prostheses. We found that TM was expressed on the surface of MVEC, both in vivo and vitro. TM-dependent generation of APC differed significantly between MVEC and HUVEC (3.98 +/- 1.2 vs. 3.0 +/- 0.7 nM, respectively). After seeding MVEC on vascular prostheses, TM activity did not change. APC generation was significantly higher on MVEC-seeded vascular grafts compared to blank grafts (4.0 +/- 0.7 vs. 1.7 +/- 0.5 nM, respectively). We conclude that TM is present and highly active on cultured MVEC. When seeded on ePTFE, MVEC retain the possibility to inhibit thrombin coagulant activity and to activate protein C. Therefore, since MVEC are readily available, the anticoagulant properties demonstrated here indicate that this cell type is suitable for cell seeding of vascular prostheses.     

Inhibition of thrombin generation by protein S at low procoagulant stimuli: implications for maintenance of the hemostatic balance

The activated protein C (APC)-independent anticoagulant activity of protein S on tissue factor-induced thrombin generation was quantified in plasma. In absence of APC, protein S significantly decreased the endogenous thrombin potential (ETP) in a concentration-dependent manner. The APC-independent anticoagulant activity of protein S in plasma was not affected by phospholipid concentrations but strongly depended on tissue factor concentrations: protein S inhibited the ETP from 6% at 140 pM tissue factor to 74% at 1.4 pM tissue factor. Plasma with both 60% protein S and 140% prothrombin showed an ETP of 240% compared to normal plasma, suggesting an APC-independent protective role of protein S in the development of thrombosis as a result of protein S deficiency and the prothrombin-G20210A mutation. At high tissue-factor concentrations, protein S hardly expressed APC-independent anticoagulant activity but exerted potent APC-cofactor activity when thrombomodulin or APC were added to plasma. Neutralization of protein S under these conditions resulted in a 20-fold reduction of the anticoagulant activity of APC. The present study shows that protein S effectively regulates coagulation at 2 levels: at low procoagulant stimuli, protein S maintains the hemostatic balance by directly inhibiting thrombin formation, and at high procoagulant stimuli, protein S restores the hemostatic balance via its APC-cofactor activity.     

ART‐123: Recombinant Human Soluble Thrombomodulin

ART‐123 is a soluble form of recombinant human thrombomodulin comprising all extracellular domains of thrombomodulin. Bound to thrombin, ART‐123 inhibits its procoagulant activity and promotes activation of protein C. ART‐123 inhibits thrombin generation by the activation of protein C and the subsequent inactivation of factor Va in the presence of protein S. ART‐123 attenuates the extension of the clot by inhibiting further thrombin generation on clots, while other anticoagulants inhibit the initiation of clot formation. A higher concentration of ART‐123 is needed to affect clotting time and platelet aggregation than thrombin generation. Intravenous administration of ART‐123 is effective in animal models of disseminated intravascular coagulation (DIC), AV‐shunt, and other models with a wider safety margin than conventional anticoagulants. The plasma half‐life of ART‐123 is long, especially on subcutaneous injection. Clinical studies have been performed or are planned to evaluate the efficacy of ART‐123 in DIC and deep venous thrombosis (DVT). In clinical trials against DIC ART‐123 was well tolerated and showed a good dose‐response effect. These findings suggested that ART‐123 is a promising drug for thrombotic diseases.     

Inverse correlation between activated protein C generation and carotid atherosclerosis in Type 2 diabetic patients.

AIMS: Activated protein C (APC) is a key regulator of the clotting system and immune responses. We studied the relationship between the degree of atherosclerosis as measured by the intima-media thickness (IMT) of carotid artery and APC generation in Type 2 diabetic patients. METHODS: Eighty-seven Type 2 diabetic patients and 35 control subjects participated. APC generation was assessed by the plasma APC-protein C inhibitor complex (APC-PCI) levels and the mean IMT of carotid artery was measured by ultrasonography. The plasma levels of the thrombin-anti-thromobin complex (TAT) and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunoassays. RESULTS: Plasma TAT levels were significantly higher in diabetic patients [2.03 (1.12, 2.56) ng/ml, median (25th, 75th percentile)] compared with control subjects [0.85 (0.55, 2.08) ng/ml, P < 0.01]. Plasma APC-PCI levels were significantly lower in diabetic patients [0.93 (0.74, 1.22) ng/ml], than in control subjects [1.66 (1.25, 2.36) ng/ml, P < 0.001]. The mean IMT was significantly increased in diabetic patients (0.881 +/- 0.242 mm; mean +/- sd) compared with control subjects (0.669 +/- 0.140 mm; P < 0.01). Univariate analysis showed a significant and inverse correlation between plasma APC-PCI levels and mean IMT (r = -0.32, P < 0.005), and multivariate regression analysis confirmed the independent correlation (P < 0.05). Moreover, plasma APC-PCI levels significantly and inversely correlated with plasma PDGF levels in diabetic patients (r = -0.30, P < 0.01). CONCLUSIONS: These results suggest that decreased APC generation is associated with vascular atherosclerotic changes in Type 2 diabetic patients.     

Poor outcome in disseminated intravascular coagulation or thrombotic thrombocytopenic purpura patients with severe vascular endothelial cell injuries

Various hemostatic and vascular endothelial cell markers were measured in patients with disseminated intravascular coagulation (DIC), non-DIC, or thrombotic thrombocytopenic purpura (TTP) and in healthy volunteers to examine the relationships between the hemostatic abnormalities or vascular endothelial cell injuries and the patients' outcomes. Although the plasma levels of soluble fibrin monomer, thrombin-antithrombin complex, plasmin-plasmin inhibitor complex, and D-dimer were significantly increased in the DIC patients, there were no significant differences in these markers between the DIC patients who survived and those who died, suggesting that these markers might not be directly related to the patient outcome. The plasma thrombomodulin (TM) levels in the DIC and TTP patients were significantly higher than those in the healthy volunteers, and the plasma TM levels in the patients who died were significantly higher than those in the patients who survived. These findings showed that the TM level reflected the outcome, and that the outcome of the diseases underlying DIC and TTP might depend on vascular endothelial cell injuries. The plasma protein C and antithrombin activities were markedly reduced in the DIC, non-DIC, and TTP patients who died compared to those who survived. These findings suggest that reduced plasma antithrombin and protein C activities are useful markers of systemic vascular endothelial injuries. Although the plasma tissue factor (TF) levels were significantly increased in the DIC patients, there was no significant difference in the plasma TF levels between the DIC patients who died and those who survived. In conclusion, we found that the outcome of the diseases underlying DIC and TTP is related to vascular endothelial cells, and that plasma TM, antithrombin, and protein C are useful markers for systemic vascular endothelial cell injury. Am. J. Hematol. 58:189–194, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of 125I-fibrinogen kinetics and fibrinopeptide A in patients with disseminated neoplasias

To provide more information on the pathways of fibrinogen catabolism in generalized cancer, the effect of heparin on fibrinopeptide A (fpA) and on 125I-fibrinogen kinetics was studied in 15 patients with disseminated neoplasia. Three patients had evidence of venous thrombosis and in 2 additional patients a low fibrinogen level together with increased amounts of FDP/fdp and a positive ethanol test indicated disseminated intravascular coagulation (DIC). The plasma levels of fpA were grossly elevated (4.6--20, mean 11.4 ng/ml, normal values 1.01 +/- 0.45 ng/ml) in patients with thrombosis or DIC, and normal to grossly elevated (0.4--10.4, mean 6.1 ng/ml) in the other patients. Intravenous heparin bolus lowered the fpA level in 11/11 patients, and continuous heparin treatment led to an impressive suppression or complete normalization of the plasma fpA in 5/6 patients. This finding is thought to reflect heparin suppression of thrombin activity on fibrinogen. In some cases, the fpA fall after heparin bolus was slow and/or incomplete, suggesting fpA generation at sites not easily accessible to heparin or insufficient heparin dosage. The 125I- fibrinogen kinetics were characterized by a significantly shorter half- life (t1/2: 2.5 days), increased catabolic rate constant (j: 0.44 days- 1), and increased absolute turnover (68.9 mg fibrinogen/kg/day) as compared to 4 normal subjects (t1/2: 4.2 days; j: 0.26 days-1; turnover 21.7 mg fibrinogen/kg/day). As estimated from the fpA generation rates, intravascular thrombin action on fibrinogen contributed only in minor part to increase the turnover of 125I-fibrinogen. In particular, the turnover was greatly accelerated in heparin-treated patients despite impressive suppression or normalization of the fpA levels in 5/6 cases.     

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

Pharmacologic 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.     

Activated protein C levels in Behçet's disease and risk of venous thrombosis

Behçet's disease is a multi‐systemic inflammatory disorder of unknown cause. Most abnormalities have been associated with endothelial injury caused by vasculitis. Thrombosis occurs in about 25% of patients, although the mechanism is unknown. The objective of this study was to evaluate the protein C activation system in Behçet's disease and its correlation with venous thromboembolism (VTE). Thirty‐nine patients (12 with VTE) and 78 age‐ and sex‐matched controls were included in the study, and levels of protein C, protein S, activated protein C (APC), protein C inhibitor (PCI), soluble thrombomodulin (TM), antithrombin (AT), α₁‐antitrypsin, fibrinogen, factor VIII, von Willebrand factor (VWF) and C‐reactive protein (CRP) were measured. APC and TM levels were significantly lower in patients than in controls, whereas protein S, AT, α₁‐antitrypsin, fibrinogen, factor VIII, VWF and CRP levels were significantly higher in patients than in controls. APC, PCI and TM levels were lower in patients with VTE (0·65 ± 0·19 ng/ml, 86% ± 22% and 15·5 ± 7·1 ng/ml respectively) than in those without VTE (0·78 ± 0·17 ng/ml, 100% ± 15% and 22·1 ± 15·3 ng/ml) (P < 0·05). In patients, APC levels below 0·75 ng/ml (10th percentile of the control group) increased the risk of VTE about fivefold (odds ratio = 5·1; 95% confidence interval = 1·1–23·4). These results show that reduced APC levels are associated with the high incidence of VTE in Behçet's disease.