Summary

Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Seca) residing on αIIbβ3. The location of Seca on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G1818T) in exon 11 of the β3 gene (ITGB3), changing Lys580 (wild-type) to Asn580 (Seca). Two additional members of the family Sec were typed Seca positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn580, but not Lys580 αIIbβ3, bound anti-Seca, which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn580 variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Seca positive platelets, which, however, are heterozygous for the Lys580Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys580Asn mutation responsible for the formation of the Seca antigenic determinant affects αIIbβ3 receptor function.

SUMMARY

Bleeding and thrombotic disorders are major complications affecting patients with chronic kidney disease (CKD). Exposure of circulating platelets to uremic toxins and contact with artificial surfaces during dialysis induce platelet abnormalities and alter the platelet proteome. We hypothesized that these changes may be subsequent to changes in the composition and/or regulation of the platelet transcriptome. In this study, we investigated the circulating platelets of 10 CKD patients (i.e. 5 chronic hemodialysis patients and 5 stage 4 CKD uremic patients) and 5 age- and sex-matched healthy subjects. We observed an alteration of the platelet messenger RNA (mRNA) and microRNA transcriptome in CKD patients. Impaired in uremic platelets, the levels of some mRNAs and of most microRNAs appeared to be corrected by dialysis, which is consistent with a beneficial effect of dialysis and a mRNA regulatory role of platelet microRNAs. Reduced in platelets of uremic patients, phosphatidylcholine transfer protein (PCTP) and WD repeat-containing protein 1 (WDR1) were found to be regulated by microRNAs, the latter of which involving hsa-miR-19b, a microRNA increased in platelets of uremic patients and involved in platelet reactivity. These results suggest that an alteration of microRNA-based mRNA regulatory mechanisms may underlie the platelet response to uremia and entail the development of platelet-related complications in CKD.

Thrombin stimulates proliferation, invasion and metastasis by cleaving PAR1 on a human prostate cancer cells. Current direct thrombin inhibitors pose risks for bleeding in the cancer patients. We have developed an oral reversible direct thrombin inhibitor called FM19. FM19 inhibits thrombin-induced calcium mobilization of PC3 cells with an IC50 of 15 μM with a 95% confidence interval of 7.3–31.6 μM. Thrombin stimulation increases PC3 cell invasion 3-fold from 27.1 ± 11.4 to 66 ± 11.6. FM19 or bivalirudin reduces cell invasion at ≥ 0.1 μM (p≤0.02). After inoculation with PC3 cells, nude mice were treated with oral FM19 at 3 mg/ml in the drinking water. The treated mice do not have long bleeding times and only a 1.4-fold increase in their thrombin clotting time. However, with treatment, the mice have a reduced rate of tumor growth 0.26 ± 0.17 fold change/day versus 0.55 ± 0.35 for untreated (p = 0.038), reduced fold change in tumor size 5.3 ± 0.47 to 8.9 ± 1.8 (untreated) (p=0.048), and reduced overall tumor weight 0.5 ± 0.31 g versus 0.82 ± 0.32 g (untreated) (p=0.04). On microscopic examination, FM19 treatment reduces the number of large vessels in the tumors from 4.6±2.1 per high-powered field in untreated samples to 1.4±1.4 in treated samples (p≤0.04). These studies show FM19 reduces prostate tumor growth in vivo at a concentration below that needed for anticoagulation. These data suggest novel opportunities for oral direct thrombin inhibitors in cancer therapy.

A novel family of RGD-containing molecule (Tablysin-15) has been molecularly characterized from the salivary gland of the hematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity for platelet αIIbβ3 and endothelial cell αvβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~ 1 nM) and marginally to fibronectin (IC50 ~ 1 µM), but not to collagen. It also inhibits FGF-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilized fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilized Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow, without affecting platelet adhesion to collagen fibrils. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Summary

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membrane may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.

Rheumatic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), are associated with antibodies to “self” antigens. Persons with autoimmune diseases, most notably SLE, are at increased risk for developing accelerated cardiovascular disease. The link between immune and inflammatory responses in the pathogenesis of cardiovascular disease has been firmly established yet, despite our increasing knowledge, accelerated atherosclerosis continues to be a significant co-morbidity and cause of mortality in SLE. Recent animal models have been generated in order to identify mechanism(s) behind SLE-accelerated atherosclerosis. In addition, clinical studies have been designed to examine potential treatments options. This review will highlight data from recent studies of immunity in SLE and atherosclerosis and discuss the potential implications of these investigations.

Summary

Asymptomatic antiphospholipid antibody (aPL) carriers with high risk for thrombosis may benefit from preventive anticoagulation.

It was our objective to test whether the risk of thrombosis increases with: 1) increasing titres of anticardiolipin antibodies (aCL) after adjustment for other cardiovascular risk factors and 2) the number of aPL detected.

In a cross-sectional study, blood was collected from clinics in two teaching hospitals. The study included 208 individuals suspected of having an aPL and 208 age- and sex-matched controls having blood drawn for a complete blood count.

Clinical variables included history of previous arterial (ATE) or venous (VTE) thrombotic events, traditional risk factors for cardiovascular disease, and systemic lupus erythematosus (SLE). Laboratory variables included IgG/IgM aCL, lupus anticoagulant, and IgG/IgM anti-β2-glycoprotein I.

Mean age was 46.5 years and 83% were female. Seventy-five of the 416 participants had ≥ 1 aPL, and 69 had confirmed ≥ 1 ATE or VTE. Family history was positive in 48% of participants, smoking in 28%, hypertension in 16%, diabetes in 6%, and SLE in 20%. A 10-unit increase in aCL IgG titre was associated with an odds ratio (OR) [95% CI] of 1.07 [1.01–1.13] for ATE and 1.06 [1.02 – 1.11] for VTE. The odds of a previous thrombosis increased with each additional aPL detected: 1.5 [0.93–2.3] for ATE and 1.7 [1.1–2.5] for VTE.

These results indicate that increased titres of aCL and multiple aPL were associated with an increased risk of a previous thrombotic event.

Summary

Although antiphospholipid antibodies (aPL) are associated with thrombosis, it is not known who with aPL is at higher risk for thrombosis. It was the aim of this cross-sectional study to investigate how thrombophilic factors contribute to venous or arterial thrombosis in aPL-positive individuals. In outpatient test centres at two tertiary care hospitals, two hundred and eight (208) persons requiring aPL testing were matched by age, gender and centre to 208 persons requiring a complete blood count. Persons were classified as aPL-positive (having anticardiolipin, lupus anticoagulant and/or anti-β2-glycoprotein I antibodies) or aPL-negative. Several thrombophilic factors were studied using logistic regression modelling. Results showed that the aPL-positive group had three-fold more events (37%) than the aPL-negative group (12%). In unadjusted analyses, clinically important associations were observed between factor V Leiden and venous thrombosis, hyperhomocysteinemia and arterial thrombosis, and activated protein C resistance (APCR) and venous thrombosis (OR, 95% CI = 4.00, 1.35–11.91; 4.79, 2.03–11.33; and 2.03, 1.03–3.97, respectively). After adjusting for recruitment group, persons with both APCR and aPL had a three-fold greater risk (OR, 95% CI = 3.31, 1.30–8.41) for venous thrombosis than those with neither APCR nor aPL. Similarly, after adjusting for hypertension, family history of cardiovascular disease, gender and recruitment group, persons with both hyperhomocysteinemia and aPL had a five-fold increased risk (OR, 95% CI = 4.90, 1.37–17.37) for arterial thrombosis compared to those with neither risk factor. In conclusion, APCR phenotype and hyperhomocysteinemia are associated with a higher risk of venous and arterial thrombosis, respectively, in the presence of aPL.

Although intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualized by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalization. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalization within 1 hour after treatment. Skin hemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1−/−) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.

Summary

We performed a randomized pilot trial of PerMIT, a novel decision support tool for genotype-based warfarin initiation and maintenance dosing, to assess its efficacy for improving warfarin management. We prospectively studied 26 subjects to compare PerMIT-guided management with routine anticoagulation service management. CYP2C9 and VKORC1 genotype results for 13 subjects randomly assigned to the PerMIT arm were recorded within 24 h of enrollment. To aid in INR interpretation, PerMIT calculates estimated loading and maintenance doses based on a patient’s genetic and clinical characteristics and displays calculated S-warfarin plasma concentrations based on planned or administered dosages. In comparison to control subjects, patients in the PerMIT study arm demonstrated a 3.6-day decrease in the time to reach a stabilized INR within the target therapeutic range (4.7 vs. 8.3 days, p = 0.015); a 12.8% increase in time spent within the therapeutic interval over the first 25 days of therapy (64.3% vs. 55.3%, p = 0.180); and a 32.9% decrease in the frequency of warfarin dose adjustments per INR measurement (38.3% vs. 57.1%, p = 0.007). Serial measurements of plasma S-warfarin concentrations were also obtained to prospectively evaluate the accuracy of the pharmacokinetic model during induction therapy. The PerMIT S-warfarin plasma concentration model estimated 62.8% of concentrations within 0.15 mg/L. These pilot data suggest that the PerMIT method and its incorporation of genotype/phenotype information may help practitioners increase the safety, efficacy, and efficiency of warfarin therapeutic management.

Clinical Trials Registration

http://www.clinicaltrials.gov. Unique identifier: NCT00993200

Summary

Antiphospholipid antibodies (aPL) are associated with vascular events, but the magnitude of this risk, alone, or in combination with other atherogenic and thrombophilic risk factors, remains unclear. A prospective cohort of 415 persons was studied for arterial and venous events (AE and VE) over a median time of 7.4 years. aPL and coagulation abnormalities were measured upon beginning of the study and annually for the first four years. Within the cohort, a nested case-control study was conducted to investigate the role of endothelial and inflammatory markers in predicting new vascular events. Forty-five individuals had new vascular events: 18 occurred during the first year of follow-up. The proportion of event-free survivors at eight years was 90% (95%CI = 87%, 94%) for aPL-negative and 72% (60%, 85%) for aPL-positive individuals, respectively. Predictors for new AE were previous AE (HR=5.7 [2.7, 12.0]), diabetes (5.6 [2.4, 13.2]), aPL positivity (2.6 ([1.2, 5.9]), and age (1.04 [1.01, 1.07]). New VE were predicted by previous VE (6.1 [1.9, 19.9]), anti-β2-glyco-protein I (aβ2GPI) positivity (5.8 [1.4, 24.1]), activated protein C resistance (APCR) (4.1 [1.1, 15.1]), and gender (3.7 [1.1, 12.9]). In the nested case-control study, similar predictors were observed for AE, while abnormal APCR (OR=5.5 [1.1, 26.6]) and elevated von Willebrand factor (vWF) (OR=5.0 [1.2, 19.8]) best predicted VE. We demonstrate that aPL independently predict new vascular events and discriminate between individuals with and without events in the first two years of follow-up, indicating that aPL are associated with a short-term risk of developing new and recurrent vascular events.

BACKGROUND

Abnormal plasma fibrin architecture is a major determinant of both premature coronary artery disease (CAD) and hypofibrinolysis. The presence of the FXIIIVal34Leu genetic variant increases and accelerates fibrin stabilization. Its association with premature CAD remains controversial.

AIM

To evaluate fibrin clot structure/function in patients with premature CAD compared to healthy controls and whether the presence of the FXIII Val34Leu variant is an independent correlate of both impaired fibrinolysis and premature CAD.

METHODS

Fibrin phenotype and FXIII Val34Leu genetic variant were determined in a cohort of 242 young patients (<45 years) who survived an MI and compared to 242 healthy controls matched for age and gender. Fibrin clot stiffness (elastic modulus) and response to rt-PA mediated fibrinolysis (clot lysis time and fibrinolysis rate) were measured using the Hemodyne analyser and by confocal microscopy. The effect of FXIII Val34Leu on long term survival was also evaluated.

RESULTS

CAD patients produced stiffer fibrin clots as compared to healthy controls (24.7±16 vs. 13.6±6 kdynes/cm2; p<0.0001) and displayed a reduced response to fibrinolysis with longer clot lysis time (16.5±12 vs. 10±7 min; p<0.0001) and lower fibrinolysis rate (8.3±7. vs. 14.7±19 sec−1×10−4; p<0.0001). Factor XIII Val34Leu presence led to a stepwise decrease in fibrinolysis rate with a gene dose effect in both patients (9.4±8 vs. 6.9±7 vs. 5.5±4 sec−1×10−4, for wildtype, heterozygous and homozygous, respectively, p value for all =0.02) and healthy controls, suggesting an effect independent from CAD. A similar impact of the Factor XIII Val34Leu substitution was observed on clot lysis time. Increased clot stiffness and hypofibrinolysis were both independent correlates of premature CAD. Factor XIII Val34Leu presence was neither protective of premature CAD (adjOR 0.83 [0.49–1.4] nor did it impact long term clinical outcome during a median follow-up of 6.3 years (±2.4).

CONCLUSIONS

Stiff fibrin that is more resistant to fibrinolysis is a major determinant of premature CAD. Presence of the factor XIII Leu34 genetic variant provides a pharmacogenetic resistance to fibrinolysis ex-vivo but neither relates to premature CAD nor to recurrent acute coronary events.

Salivary glands from hematophagous animals express a notable diversity of negative modulators of platelet function. Triplatin is an inhibitor of collagen-induced platelet aggregation which has been described as an antagonist of glycoprotein VI (GPVI). Because triplatin displays sequence homology to members of the lipocalin family of proteins, we investigated whether triplatin mechanism of action could be explained by interaction with pro-hemostatic prostaglandins. Our results demonstrate that triplatin inhibits platelet aggregation induced by low doses of collagen, thromboxane A2 (TXA2) mimetic (U46619), and arachidonic acid (AA). On the other hand, it does not inhibit platelet aggregation by convulxin, PMA, or low-dose ADP. Isothermal titration calorimetry (ITC) revealed that triplatin binds AA, cTXA2, TXB2, U46619 or PGH2 mimetic (U51605). Consistent with its ligand specificity, triplatin induces relaxation of rat aorta contracted with U46619. Triplatin also interacts with PGF2α and PGJ2, but not with leukotrienes, AA or biogenic amines. Surface plasmon resonance experiments failed to demonstrate interaction of triplatin with GPVI; it also did to inhibit platelet adhesion to fibrillar or soluble collagen. Because triplatin displays sequence similarity to apolipoprotein D (ApoD)—a lipocalin associated with HDL, it was tested as a putative TXA2-binding molecule. ITC failed to demonstrate binding of ApoD to all prostanoids described above, or to AA. Furthermore, ApoD was devoid of inhibitory properties towards platelets activation by AA, collagen, or U46619. In conclusion, Triplatin mechanism of action has been elucidate without ambiguity as a novel TXA2- and PGF2α- binding protein. It conceivably blocks platelet aggregation and vasoconstriction, thus contributing to successful blood feeding at the vector-host interface.

Background

Constitutional deficiency in Factor XI (FXI) is a rare bleeding disorder in the general population, with the exception of Ashkenazi Jews. During the last decade, the detection of FXI-deficient patients has shifted from clinical screening identifying mostly severe bleeders to biological screening combining findings of prolonged activated partial thromboplastin time and FXI coagulation activity (FXI:C) below 50 U/dL.

Objectives

The goal of this study was to determine the molecular basis of FXI deficiency in western Brittany, France.

Patients/Methods

Over the course of 4 years, we detected 98 FXI-deficient patients through biological screening, and 44 patients agreed to participate in this study corresponding to 25 index cases. We developed an efficient mutation detection strategy (combining direct sequencing and QFM-PCR to search for heterozygous rearrangements in a routine setting) that detected F11 mutations in 24 out of the 25 index cases.

Results

An unexpected allelic heterogeneity was found, with 14 different single point mutations being detected, among which 9 are new. Moreover, a large heterozygous deletion of the entire F11 gene was detected, then further defined using a CGH array as a 4q34.2 telomeric deletion of 7 Mb containing 77 genes.

Conclusion

We propose that the observed recurrent mutations may be considered as genetic tags of a population. This study highlights the importance of screening for large deletions in molecular studies of F11.

Summary

In July 1982, the occurrence of three cases of acquired immunodeficiency syndrome (AIDS) in men with haemophilia was an immediate signal to Oscar Ratnoff that AIDS was transmissible through blood products. Work that he led provided important and clear indication that the AIDS agent was transmissible through pooled plasma products and had rapidly infected many men who had haemophilia. Before the blood supply was protected, the risk for infection in haemophilia was related directly to the intensity of therapy with pooled anti-haemophilic factor concentrates. Studies performed among the small proportion of haemophiliacs who remained uninfected despite heavy exposure to these plasma products revealed that the rare protective genotype – homozygosity for the 32 base pair deletion in the CCR5 gene was heavily concentrated in this population. Among those who did not have this protective genotype, a state of diminished immune activation distinguished these high risk uninfected haemophiliacs from haemophiliacs who later acquired human immunodeficiency virus (HIV) infection and from healthy uninfected controls. Immune activation state may not only predict risk for HIV acquisition but also appears to be an important predictor and likely determinant of HIV disease progression. The potential drivers of immune activation in chronic HIV infection include HIV itself, other co-infecting pathogens, homeostatic responses to cytopenia as well as the recently recognised phenomenon of translocation of microbial products across a damaged gut mucosal surface. This latter process is particularly compelling as clinical studies have shown a good relationship between indices of microbial translocation and markers of both immune activation and T cell homeostasis in chronic HIV infection. More recently, we have also found evidence that these microbial products also may drive a heightened tendency to thrombus formation in HIV infection via induction of monocyte tissue factor expression. Thus systemic exposure to microbial elements that are translocated through a gut mucosa damaged in the first few weeks of HIV infection may contribute to the pathogenesis of both immune deficiency and the heightened risk for vascular events that have been noted in persons with HIV infection.

Several studies have reported that taller individuals are at greater risk of venous thromboembolism (VTE). We hypothesized that longer leg length would be positively associated with incident VTE, and would explain the height association. LITE ascertained VTE in a prospective population-based sample of 21,860 individuals aged 45 and older. Leg length was measured as standing height minus sitting height. Cox regression models were adjusted for age, race, sex, waist circumference, diabetes, and factor VIII. To evaluate whether leg length was associated with VTE risk independent of height we standardized leg length and height per 1 standard deviation (SD), and then included them simultaneously in Cox regression models. A total of 641 incident VTE cases accrued over a median follow-up of 16 yrs. Participants in the highest quintile of leg length were at 59% (95% CI: 22%-108%) greater risk of VTE, relative to the lowest quintile. For height, risk was 45% (12%-88%) greater for those in the highest quintile, compared to the lowest. When leg length and height were modeled simultaneously leg length remained associated with VTE risk (HR per 1 SD: 1.21 (1.04-1.40) while height was unrelated (HR per 1 SD: 1.00 (0.86-1.16). To conclude, participants with longer legs were at greater risk of incident VTE, and leg length explained the relation of height to VTE. It remains to be established whether this finding is due to greater venous surface area, a larger number of venous valves, or greater hydrostatic pressure among individuals with longer legs.

Fibrocytes are bone marrow-derived mesenchymal progenitor cells that express markers of leukocytes, hematopoietic progenitor cells, and fibroblasts. They play a pivotal role in tissue remodeling and fibrosis in both physiologic and pathologic settings. Fibrocytes are unique in that they are capable of differentiating into fibroblasts and myofibroblasts, as well as adipocytes. In this review we will present data supporting the critical role they play in the pathogenesis of chronic inflammatory fibrotic diseases of the lungs, heart and vasculature.

Summary

Chemokines were originally described as cytokines that mediate leukocyte recruitment to sites of inflammation. Members of a subgroup of chemokines, the CXC family, also play a critical role in both physiologic and pathologic angiogenesis, including in the context of chronic inflammation, fibrosis, and malignancy. A unique feature of this family of cytokines is that, on the basis of their structure and receptor binding, individual ligands display either angiogenic or angiostatic biological activity in the regulation of angiogenesis. In this review, we summarize the key literature in this growing field.

Summary

Multiple components of the immune response are involved in the initiation, progression and persistence of atherosclerosis. IL-17A is produced by a broad variety of leukocytes and plays an important role in host defense. IL-17A is also involved in the pathology of several autoimmune diseases mainly via the regulation of chemokine expression and leukocyte migration to the site of inflammation. There is an increasing body of evidence indicating an association between elevated levels of IL-17A and cardiovascular diseases. Interestingly, this IL-17A-dependent response occurs in parallel with the Th1-dominant immune response during atherogenesis. To date, the precise role of IL-17A+ cells in atherosclerosis is controversial. Several studies have suggested a pro-atherogenic role of IL-17A via the regulation of aortic macrophage numbers, Th1-related cytokines and aortic chemokine expression. However, two studies recently described anti-inflammatory effects of IL-17A on mouse plaque burden via possible regulation of aortic VCAM-1 expression and T cell content. Furthermore, an initial study using IL-17A-deficient mice demonstrated that IL-17A affects the immune composition and inflammatory phenotype of the aortic wall; however, no effects were observed on atherosclerosis. Further studies are necessary to fully address the role of IL-17A and other IL-17 family members in atherosclerosis.

Summary

A novel dysfibrinogenemia with a replacement of Tyr by Asn at Bβ41 has been discovered (fibrinogen Caracas VIII). An asymptomatic 39 year-old male was diagnosed as having dysfibrinogenemia due to a mildly prolonged thrombin time (+ 5.8 sec); his fibrinogen concentration was in the low normal range, both by Clauss and gravimetric determination, 1.9 g/l and 2.1 g/l, respectively. The plasma polymerization process was slightly impaired, characterized by a mildly prolonged lag time and a slightly increased final turbidity. Permeation through the patients’ clots was dramatically increased, with the Darcy constant around 4 times greater than that of the control (22 ± 2 ×10−9 cm2 compared to 6 ± 0.5 ×10−9 cm2 in controls). The plasma fibrin structure of the patient, by scanning electron microscopy, featured a mesh composed of thick fibers (148 ± 50 nm vs. 120 ± 31 nm in controls, p<0.05) and larger pores than those of the control fibrin clot. The viscoelastic properties of the clot from the patient were also altered, as the storage modulus (G′, 310 ± 30) was much lower than in the control (831 ± 111) (p ≤ 0.005). The interaction of the fibrin clot with a monolayer of human microvascular endothelial cells, by confocal laser microscopy, revealed that the patients’ fibrin network had less interaction with the cells. These results demonstrate the significance of the amino terminal end of the β chain of fibrin in the polymerization process and its consequences on the clot organization on the surface of endothelial cells.

Summary

The main impediments to clinical application of hematopoietic stem cell (HSC) gene therapy for treatment of hemophilia A are the bone marrow transplant-related risks and the potential for insertional mutagenesis caused by retroviral vectors. To circumvent these limitations, we have adapted a nonmyeloablative conditioning regimen and directed factor VIII (FVIII) protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified HSCs resulted in therapeutic levels of FVIII in the circulation of all transplanted mice for the duration of the study (6 months). Immunostaining of spleen cells showed that the majority of FVIII was synthesized by B220+ B cells and CD138+ plasma cells. Subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII antibody response, demonstrating induction of immune hyporesponsiveness. All transplant recipients exhibited clot formation and survived tail clipping, indicating correction of their hemophilic phenotype. Therapeutic levels of FVIII could be transferred to secondary recipients by bone marrow transplantation, confirming gene transfer into long-term repopulating HSCs. Moreover, short-term therapeutic FVIII levels could also be achieved in secondary recipients by adoptive transfer of HSC-derived splenic B cells. Our findings support pursuit of B cell-directed protein delivery as a potential clinical approach to treat hemophilia A and other disorders correctable by systemically distributed proteins.

Summary

Introduction

By guiding initial warfarin dose, pharmacogenetic (PGx) algorithms may improve the safety of warfarin initiation. However, once INR response is known, the contribution of PGx to dose refinements is uncertain. This study sought to develop and validate clinical and PGx dosing algorithms for warfarin dose refinement on days 6–11 after therapy initiation.

Materials and Methods

An international sample of 2,022 patients at 13 medical centers on 3 continents provided clinical, INR, and genetic data at treatment days 6–11 to predict therapeutic warfarin dose. Independent derivation and retrospective validation samples were composed by randomly dividing the population (80%/20%). Prior warfarin doses were weighted by their expected effect on S-warfarin concentrations using an exponential-decay pharmacokinetic model. The INR divided by that “effective” dose constituted a treatment response index.

Results

Treatment response index, age, amiodarone, body surface area, warfarin indication, and target INR were associated with dose in the derivation sample. A clinical algorithm based on these factors was remarkably accurate: in the retrospective validation cohort its R2 was 61.2% and median absolute error (MAE) was 5.0 mg/week. Accuracy and safety was confirmed in a prospective cohort (N=43). CYP2C9 variants and VKORC1-1639 G→A were significant dose predictors in both the derivation and validation samples. In the retrospective validation cohort, the PGx algorithm had: R2= 69.1% (P<0.05 vs. clinical algorithm), MAE= 4.7 mg/week.

Conclusions

A pharmacogenetic warfarin dose-refinement algorithm based on clinical, INR, and genetic factors can explain at least 69.1% of therapeutic warfarin dose variability after about one week of therapy.

Summary

Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob ‘A’, has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob ‘A’ which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.