Statins Effects on Blood Clotting: A Review.

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.

IL16 and factor V gene variations are associated with asparaginase-related thrombosis in childhood acute lymphoblastic leukemia patients.

Aim: We previously conducted exome-wide association study in acute lymphoblastic leukemia patients and identified association of five SNPs with asparaginase-related thrombosis. Here we aimed to replicate these findings in an independent patient cohort and through analyses in vitro. Patients & methods: SNPs located in IL16, MYBBP1A, PKD2L1, RIN3 and MPEG1 genes were analyzed in patients receiving Dana-Farber Cancer Institute acute lymphoblastic leukemia treatment protocols 05-001 and 11-001. Thrombophilia-related variations were also analysed. Results: IL16 rs11556218 conferred higher risk of thrombosis and higher in vitro sensitivity to asparaginase. The association was modulated by the treatment protocol, risk group and immunophenotype. A crosstalk between factor V Leiden, non-O blood groups and higher risk of thrombosis was also seen. Conclusion: IL16 and factor V Leiden variations are implicated in asparaginase-related thrombosis.

This study looked at how certain genetic variations are related to a higher risk of blood clots in children with a type of cancer called acute lymphoblastic leukemia who are receiving a certain treatment (asparaginase). The study found that one specific genetic variation (IL16 rs11556218) was linked to a higher risk of blood clots (thrombosis), and that this risk was influenced by disease and treatment features. The study also found that a certain genetic variation (factor V Leiden), which makes blood more likely to clot, and blood type (non-O) were linked to a higher risk of thrombosis. The conclusion of this study is that genetic variations may play a role in blood clots in children with acute lymphoblastic leukemia receiving asparaginase, and if further confirmed, these variations can serve to advance personalized treatment strategies.

R399E, A Mutated Form of Growth and Differentiation Factor 5, for Disease Modification of Osteoarthritis.

OBJECTIVE: To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug. METHODS: Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. RESULTS: Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1ß (IL-1ß), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 µg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-µg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. CONCLUSION: R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

Genetic deficiency of nuclear factor of activated T cells 5 attenuates the development of osteoarthritis in mice.

OBJECTIVES: This study is aimed to investigate the role of nuclear factor of activated T cells 5 (NFAT5), originally known as the osmosensitive mammalian transcription factor, in the pathogenesis of osteoarthritis (OA) in mice. METHODS: OA was induced in male C57BL/6 (wild-type) and NFAT5 haplo-insufficient (NFAT5+/-) mice via destabilization of the medial meniscus (DMM) surgery. OA severity and synovial inflammation were histologically assessed. Expression of CCL2, inflammatory cytokines, cartilage degrading enzymes was determined in the knee joints and cultured chondrocytes from wild-type and NFAT5+/- mice. RESULTS: NFAT5 expression was significantly upregulated in the knee joint of a mouse after DMM surgery. NFAT5 deficiency decreased the severity of synovial inflammation and osteoarthritic changes in cartilage and subchondral bone. Moreover, NFAT5 deficiency also decreased the expression of CCL2, IL-1ß, MMP-13, ADMATS-5, and macrophage infiltration in the joint. In cultured chondrocytes, hyperosmolar or IL-1ß stimulation significantly enhanced the expression of NFAT5, CCL2, IL-1ß, IL-6, and MMP-13, and this effect was abolished in chondrocytes from NFAT5+/- mice. Hyperosmolarity or IL-1ß-induced NFAT5 and CCL2 downregulated by inhibiting p38 MAPK, JNK, and ERK pathways. CONCLUSIONS: Our results indicate that NFAT5 is a crucial regulator of OA pathogenesis by upregulating CCL2 expression and macrophage recruitment. In chondrocyte, NFAT5 plays an important role in the response to hyperosmolar or IL-1ß stimulation. Thus, NFAT5 could be an attractive therapeutic target for OA treatment.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice.

Acute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

"In vitro" correction of the severe factor V deficiency-related coagulopathy by a novel plasma-derived factor V concentrate.

INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.

Transfusion management of factor V deficiency: three case reports and review of the literature.

BACKGROUND: Factor V (FV) deficiency may be inherited as an autosomal recessive disease or acquired as a result of autoantibody formation, either spontaneously or secondary to exposure to bovine thrombin or medications. Congenital FV deficiency has traditionally been treated with plasma transfusions. However, recent evidence has suggested that platelet (PLT) transfusions may be a better alternative as FV stored within PLT alpha granules has greater procoagulant potential and is released locally at sites of vascular injury. We report three cases of FV deficiency, one congenital and two acquired, and emphasize the different management approaches. CASE REPORTS: Patient 1 was a 30-year-old man with congenital FV deficiency who presented with a trauma-induced hematoma of his lower extremity. He was treated with 5 PLT units over 48 hours. Patient 2 was a 64-year-old woman who presented with an upper-extremity thrombus and was discovered to have a FV inhibitor, likely secondary to antibiotics. Patient 3 was a 75-year-old woman with hepatitis C virus (HCV) who presented with minor ecchymosis and was found to have a FV inhibitor secondary to either HCV or antibiotic exposure. Corticosteroids alone were able to eradicate the inhibitors in both patients with acquired inhibitors. CONCLUSIONS: FV deficiency can present with a diverse range of symptoms. For bleeding patients, PLT transfusions should be the initial therapy. In patients with thrombosis, the risks and benefits of anticoagulation must be carefully assessed before treatment. For patients with minor bleeds, transfusions may be withheld, and elimination of the inhibitor should be the primary objective.

Inhibition of thrombin-mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ'.

BACKGROUND: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 µmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.

The anticoagulant function of coagulation factor V.

Almost two decades ago an anticoagulant function of factor V (FV) was discovered, as an anticoagulant cofactor for activated protein C (APC). A natural mutant of FV in which the R506 inactivation site was mutated to Gln (FV(Leiden)) was inactivated slower by APC, but also could not function as anticoagulant cofactor for APC in the inactivation of activated factor VIII (FVIIIa). This mutation is prevalent in populations of Caucasian descent, and increases the chance of thrombotic events in carriers. Characterisation of the FV anticoagulant effect has elucidated multiple properties of the anticoagulant function of FV: 1) Cleavage of FV at position 506 by APC is required for anticoagulant function. 2) The C-terminal part of the FV B domain is required and the B domain must have an intact connection with the A3 domain of FV. 3) FV must be bound to a negatively charged phospholipid membrane. 4) Protein S also needs to be present. 5) FV acts as a cofactor for inactivation of both FVa and FVIIIa. 6) The prothrombotic function of FV(Leiden) is a function of both reduced APC cofactor activity and resistance of FVa to APC inactivation. However, detailed structural and mechanistic properties remain to be further explored.

Low dose secondary prophylaxis reduces joint bleeding in severe and moderate haemophilic children: a pilot study in China.

The most common bleeding in haemophilic patients is in joints, and joint disability is the most common complications in these patients receiving inadequate treatment. Limited by economy and inadequate treatment, developing countries face huge challenge to reduce disability and improve quality of life (QoL) of haemophilic children. The aim of this study was to investigate the effect of low dose secondary prophylaxis in China. Children with moderate and severe haemophilia from the Beijing Children Hospital, Beijing, China, and with established joint disease, were followed for a 12-week observation period followed by a 12-week low dose secondary prophylaxis-study period (for haemophilia A, factor VIII concentrate 10 IU kg(-1) twice weekly; for haemophilia B, factor IX concentrate 20 IU kg(-1) weekly). The reduction of joint bleeding, improvement of joint function and QoL during prophylaxis were analysed. In total 34 children (median age 7.8 years) were analyzable. The number of joint bleeds decreased from a total of 337 (individual range 3-24, mean 9.9) during the observation period to 57 (range 0-6, mean 1.7) during the study period with an overall of reduction 83%. Joint function improved in 66.7% of disease joints, with 23.2% of which were considered good to moderate. School attendance improved in all subjects, sports participation and daily activity improved moderately. Low dose secondary prophylaxis significantly reduces frequency of joint bleeding; with moderate improvement in joint function, school attendance, sport participation and daily activities. Low dose secondary prophylaxis is therefore, cost-effective as applied to developing countries such as China, although there are still unresolved issues.

Sequential therapy with activated prothrombin complex concentrates and recombinant FVIIa in patients with severe haemophilia and inhibitors: update of our previous experience.

Haemophilia patients with inhibitors can develop bleeding episodes, which are refractory to monotherapy with either recombinant factor VIIa (rFVIIa) or activated prothrombin complex concentrates (APCC). Management of such bleeds is often difficult. We previously reported the safety of using a combination of rFVIIa and APCC given in sequential fashion. In this report, we update our experience with sequential therapy. A retrospective review of medical records was conducted including all reports of sequential therapy defined as receiving both rFVIIa and APCC within 6 h. Data extracted included demographic data, treatment prior to and following hospital admission, clinical data including type and location of bleed, response to therapy, physical examination and laboratory data. In addition, for some patients, thromboelastography was performed to document the effect of sequential therapy on clot formation characteristics. Four patients comprising 35 admissions, 209 hospital days and 115 days of sequential therapy were included in the updated dataset. No patient developed thrombosis or overt disseminated intravascular coagulation (DIC) although elevations in the D-dimer above 5 microg mL(-1) were noted in 42% of the courses that lasted >3 days. Efficacy is suggested by the fact that patients had resolution of their bleeds after a median of 3 days of sequential therapy after failing to respond to a median of 3 days of monotherapy. Thromboelastography demonstrated an additive effect. Sequential therapy is a safe, potentially efficacious approach in the management of refractory bleeding episodes in patients with haemophilia and inhibitors.

Clinical evaluation of a new functional test for detection of activated protein C resistance (Pefakit APC-R Factor V Leiden) at two centers in Europe and the USA.

A new clotting assay, Pefakit APC-R Factor V Leiden (Pentapharm Ltd., Switzerland), for the detection of an increased resistance of coagulation factor V against degradation by activated protein C, caused mainly by the factor V Leiden mutation, was evaluated in clinical studies at two University Centers in Europe and the US. The performance was compared with the performance of the routinely used predicate device COATEST APC Resistance V (Chromogenix IL, USA). Both tests were run in parallel on a STA-R analyzer (Diagnostica Stago, France). Samples from subjects undergoing routine laboratory thrombophilia screening were examined, 187 at the Institute of Medical and Chemical Laboratory Diagnostics (IMCLD), University of Vienna, Austria, and 236 at the Duke University Medical Center (DUMC), Durham/Raleigh NC, USA. All samples were analyzed for factor V Leiden mutation and prothrombin 20210 G/A mutation using standard PCR methods. The data show that the Pefakit APC-R Factor V Leiden assay discriminates very well between healthy controls and carriers of the factor V Leiden mutation, even in patients with lupus anticoagulant or with deficiency in Protein C or Protein S. Furthermore, this new test is able to discriminate well between heterozygous and homozygous carriers of the factor V Leiden mutation. In both studies the Pefakit assay showed 100% sensitivity and 100% specificity for detection of the factor V Leiden mutation, compared to 93.1% sensitivity and 93.0% specificity for the COATEST APC Resistance V in the IMCLD study and 93.9% sensitivity and 95.6% specificity in the DUMC study. The new test has PCR-like discrimination power which will help to decrease costs by reducing the need for PCR verification of borderline cases.

Platelet factor V supports hemostasis in a patient with an acquired factor V inhibitor, as shown by prothrombinase and tenase assays.

A woman with gross hematuria was shown to have a severe isolated factor V deficiency due to a factor V inhibitor of 200 U/ml titer. Hematuria persisted despite multiple infusions of plasma but, after one transfusion with 1 U platelets, urine red blood cells decreased by more than 98%. To evaluate the patient's platelet function we performed prothrombinase and tenase assays with platelets from the patient and from normal donors. By prothrombinase assay, ionophore-activated patient platelets showed 42% of the activity of normal platelets in their ability to support prothrombin activation by activated factor X; whereas in a 'tenase' assay, which measures the platelets' ability to support factor X activation by activated factor IX + activated factor VIII, their activity was 117% of normal. The addition of excess bovine activated factor V to the prothrombinase assay fully corrected the defect. The results demonstrate the benefit of platelet transfusion and indicate that in this case the platelets are the primary source of factor V for hemostasis.

A Bayesian approach to the assessment of inhibitor risk in studies of factor VIII concentrates.

The assessment of inhibitor risk is a crucial component in the clinical development of new and modified factor VIII (FVIII) preparations. There has been a recent discussion about the design of studies and the assessment of inhibitors and inhibitor risk in such studies at a recent FDA-sponsored FVIII Inhibitor Workshop, and new requirements for the success of these trials have been proposed to evaluate inhibitor data based on the use of an upper 95% confidence bound. We review the consequences of these requirements and demonstrate that for any product to succeed, it must have an extremely low underlying risk of inhibitor development. Furthermore, several existing commercially available FVIII products with an excellent safety record would not necessarily pass these endpoints. As a result, we propose an alternative set of acceptance criteria based on a Bayesian statistical paradigm. This approach is based on the determination of a probability that the product in question actually has an inhibitor risk below some pre-set limit, a concept that we believe is more intuitive than the traditional confidence interval method. We show that all existing products would pass this approach, but a product (Bisinact) with known inhibitor risk would not.

Sequential therapy with activated prothrombin complex concentrate and recombinant factor VIIa in patients with severe haemophilia and inhibitors.

Patients with haemophilia and inhibitors have bleeding episodes that can be refractory to home therapy with either activated prothrombin complex concentrate (APCC) or recombinant factor VIIa (rFVIIa). Sequential therapy with these products has not been widely used because of concern regarding the possibility of thrombosis. This report describes the results of a retrospective chart review of five hospitalized children with severe haemophilia and inhibitors who have been treated with sequential doses of APCC and rFVIIa for refractory bleeding. These patients all had failed home therapy with APCC and rFVIIa alone. A total of 20 admissions were documented covering 170 hospital days, including 91 days of combination therapy. While being closely monitored in the hospital, they received alternating doses of APCC and rFVIIa every 6 h. Anywhere from one to three doses of rFVIIa were given every 2 h between APCC doses. Doses of APCC ranged from 35 to 80 U kg(-1) dose(-1), and doses of rFVIIa ranged from 80 to 225 mcg kg(-1) dose(-1). There was no clinical or laboratory evidence of thrombosis, thrombocytopenia, or disseminated intravascular coagulation (DIC). We found the combination of these factors to be safe and effective for patients with refractory bleeds. However, we recommend this aggressive therapy only in the inpatient setting with careful monitoring of the physical examination and frequent laboratory screening to assess for thrombosis and DIC, and without the concurrent use of antifibrinolytic medications.

Prednisone and low-dose activated prothrombin complex concentrates for FVIII inhibitor in nonhaemophilic patients.

Eight nonhaemophilic patients with factor VIII (FVIII) inhibitors were reported. There was no difference in sex distribution. Median age at diagnosis was 62 years (ranging from 14 to 73 years). No associated disorders were revealed and all the patients were presented with severe muscular or arthral bleeding. Inhibitor titre was measured by the Bethesda method, which were 6.4, 126.0, 155.0, 4.8, 56.0, 13.5, 35.0 and 150.0 BU mL-1, respectively, at diagnosis. FVIII:C levels were less than 1 U dL-1 in seven patients and less than 2 U dL-1 in one patient. The median vWF:Ag level was 210% (ranging from 80% to 340%). All the patients had good response to activated prothrombin complex concentrates for acute bleeding episodes and prednisone for inhibitor elimination. Inhibitors completely eliminated in seven patients within a follow-up duration over 1 year, and one patient died of intracranial haemorrhage when her inhibitor titre decreased to 4.5 BU mL-1 and FVIII:C increased to 21 U dL-1.