Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase, which are largely used in patients with or at risk of cardiovascular disease. Available data on thromboembolic disease include primary and secondary prevention as well as bleeding and mortality rates in statin users during anticoagulation for VTE. Experimental studies indicate that statins alter blood clotting at various levels. Statins produce anticoagulant effects via downregulation of tissue factor expression and enhanced endothelial thrombomodulin expression resulting in reduced thrombin generation. Statins impair fibrinogen cleavage and reduce thrombin generation. A reduction of factor V and factor XIII activation has been observed in patients treated with statins. It is postulated that the mechanisms involved are downregulation of factor V and activated factor V, modulation of the protein C pathway and alteration of the tissue factor pathway inhibitor. Clinical and experimental studies have shown that statins exert antiplatelet effects through early and delayed inhibition of platelet activation, adhesion and aggregation. It has been postulated that statin-induced anticoagulant effects can explain, at least partially, a reduction in primary and secondary VTE and death. Evidence supporting the use of statins for prevention of arterial thrombosis-related cardiovascular events is robust, but their role in VTE remains to be further elucidated. In this review, we present biological evidence and experimental data supporting the ability of statins to directly interfere with the clotting system.

OBJECTIVE: Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V-abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA-based screening assay to distinguish PC pathway abnormality-related thrombophilia.
METHODS: Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated.
RESULTS: The peak ratio in healthy plasmas (n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high-FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC-deficient plasma or PS-deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose-dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV-deficient plasma spiked with 40 IU/dl rFV-R506Q (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios.
CONCLUSIONS: rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.

Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM.
To clone and express pEPCR, and determine its function in the human protein C pathway in vitro.
Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity.
The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18 ± 0.29 U) was 8-fold higher than vector-transfected cells (P = .004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01 ± 1.12 U, P = .004) or pEPCR (3.73 ± 0.65 U, P = .003), respectively.
Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting.

Babesiosis is a tick-borne zoonotic disease caused by haemoprotozoan parasites. The aim of this study were to assess markers of coagulation pathways in 25 dogs with naturally occurring babesiosis caused by B. canis, compared to 10 healthy controls. Protein C (PC) and antithrombin III (AT III) activity were assessed using a chromogenic substrate test, while levels of thrombin-antithrombin (TAT) complexes, activated protein C (APC) and endothelial protein C receptor were assessed using canine-specific ELISA. AT III activity was decreased as a result of a negative acute phase response, degradation by elastase, reduced availability of glycosaminoglycans, and, most importantly, consumption as a consequence of thrombin formation. Procoagulant state and haemostatic shift towards thrombin formation are also demonstrated by elevated TAT levels. Regarding PC pathway only significant difference was found for APC. Taken together, haemostatic alterations in uncomplicated babesiosis represent a procoagulant state that is mostly reversed during treatment.

Livedoid vasculopathy (LV) is a chronic disease with recurrent reticularis and ulcers, mainly affecting the feet and lower legs. The pathogenesis of LV has not been yet thoroughly understood, but thrombosis is thought to play a major role because fibrin deposition within both the wall and lumen of affected vessels is pathologically detected. A 68-year-old woman first presented to our hospital in 2004 with a 6-year history of a reticular rash and ulceration on the lower legs. Screening tests for vasculitis and collagen disease were mostly normal, leading to diagnosis of LV. After failed treatment with steroid and aspirin, she was started on warfarin, to which she had a favorable response. However, she had to be admitted to the hospital because complication of swelling and infection in her left lower leg in 2004 + 10. Contrast-enhanced computed tomography showed thrombosis in the left popliteal artery. Screening tests for thrombotic tendency revealed that protein S activity was low (27%) although total protein S antigen was within normal range (73%). Analysis of protein S-alpha gene revealed 155 Lys>Glu mutation in exon VI, which was reported in 1994 and named as protein S Tokushima. Thus, we conclude that protein S deficiency could contribute to LV.

Thrombin and activated protein C (aPC) bound to the endothelial protein C receptor (EPCR) both activate protease-activated receptor 1 (PAR1) generating either harmful or protective signaling respectively. In the present study we examined the localization of PAR-1 and EPCR and thrombin activity in Schwann glial cells of normal and crushed peripheral nerve and in Schwannoma cell lines. In the sciatic crush model nerves were excised 1h, 1, 4, and 7days after the injury. Schwannoma cell lines produced high levels of prothrombin which is converted to active thrombin and expressed both EPCR and PAR-1 which co-localized. In the injured sciatic nerve thrombin levels were elevated as early as 1h after injury, reached their peak 1day after injury which was significantly higher (24.4±4.1mU/ml) compared to contralateral uninjured nerves (2.6±7mU/ml, t-test p<0.001) and declined linearly reaching baseline levels by day 7. EPCR was found to be located at the microvilli of Schwann cells at the node of Ranvier and in cytoplasm surrounding the nucleus. Four days after sciatic injury, EPCR levels increased significantly (57,785±16602AU versus 4790±1294AU in the contralateral uninjured nerves, p<0.001 by t-test) mainly distal to the site of injury, where axon degeneration is followed by proliferation of Schwann cells which are diffusely stained for EPCR. EPCR seems to be located to cytoplasmic component of Schwann cells and not to compact myelin component, and is highly increased following injury.

Deficiency of antithrombin (AT), protein C (PC) or protein S (PS) constitutes a major risk factor for venous thromboembolism (VTE). Individuals at high risk for recurrence who benefit from screening need to be identified. The primary study objective was to determine the individual recurrence risk among children with a first non-central-venous-catheter-associated VTE with respect to their thrombophilia status and to evaluate if the clinical presentation at first VTE onset differs between children with AT, PC or PS deficiency versus no thrombophilia. We calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia, age, sex and positive family VTE history in 161 consecutively enrolled paediatric VTE patients. The presence of a deficiency relative to no thrombophilia was evaluated as a potential predictor of recurrence. Predictors for recurrence were AT deficiency (hazard ratio/95% CI: 6·5/2·46-17·2) and female gender (2·6/1·1-6·35). The annual recurrence rates (95% CIs) were 5·4% (2·6-10) in AT-deficient children, 1·3% (0·3-3·8) in patients with PC deficiency, 0·7% (0·08-2·4) in the PS-deficient cohort and 0·9% (0·4-1·8) in patients with no thrombophilia. Positive family VTE history or combined thrombophilias did not predict recurrence. Given the overall annual incidence rate of recurrence of 1·5% we suggest screening for AT deficiency in children with VTE.

BACKGROUND: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells.
METHODS: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells.
RESULTS: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C-treated ovalbumin-pulsed dendritic cells had significantly less airway hyperresponsiveness, significantly decreased lung concentrations of Th1 and Th2 cytokines, and less plasma concentration of immunoglobulin E when compared to control mice.
CONCLUSIONS: These results suggest that dendritic cells mediate the immunosuppressive effect of activated protein C during allergic inflammation.

In mice, trophoblasts are equipped with a potent anticoagulant mechanism, the protein C pathway. In human placenta, no functional studies of the protein C pathway are available. Human first-trimester trophoblasts (CK(++) HLA-G(+/-) Vim(-)) were isolated and kept in culture for a maximum of 48 hours. Activation of protein C on trophoblasts was at least as efficient as in endothelial cells (4.43 × 10 (-) (7) nmol/L/min/cell). Endothelial protein C receptor (EPCR) was expressed in syncytiotrophoblasts and extravillous trophoblasts. Downregulation of the messenger RNA of trophoblast EPCR occurred when trophoblasts were challenged with tumor necrosis factor α, and it could be prevented by unfractionated heparin but not by low-molecular-weight heparin at therapeutic doses. In conclusion, there is a functional protein C pathway on human first-trimester trophoblasts which can be modulated by inflammation. This finding has implications for the pathogenesis and prevention of placenta-mediated obstetric complications.

Eosinophils and mast cells play critical roles in the pathogenesis of bronchial asthma. Activation of both cells leads to the release of pro-inflammatory mediators in the airway of asthmatic patients. Recently, we have shown that inhaled thrombomodulin inhibits allergic bronchial asthma in a mouse model. In the present study, we hypothesize that thrombomodulin can inhibit the activation of eosinophils and mast cells. The effect of thrombomodulin on the activation and release of inflammatory mediators from eosinophils and mast cells was evaluated. Thrombomodulin inhibited the eotaxin-induced chemotaxis, upregulation of CD11b and degranulation of eosinophils. Treatment with thrombomodulin also significantly suppressed the degranulation and synthesis of inflammatory cytokines and chemokines in eosinophils and mast cells. Mice treated with a low-dose of inhaled thrombomodulin have decreased number of eosinophils and activated mast cells and Th2 cytokines in the lungs compared to untreated mice. The results of this study suggest that thrombomodulin may modulate allergic responses by inhibiting the activation of both eosinophils and mast cells.