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Summary.  Background: Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis‐prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. Methods: Severe hemophilia A (factor VIII‐deficient [FVIIIo]) mice were crossed with mice lacking apolipoprotein E (ApoE−/−) or mice lacking the LDL receptor (LDLR−/−), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. Results: ApoE−/−/FVIIIo mice showed a time‐dependent protective effect against the development of atherosclerosis, beginning after 22 diet‐weeks and persisting to 37 diet‐weeks in both the aorta sinus and whole aorta as compared with ApoE−/− mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIIIo/LDLR−/− model as compared with controls at early or late time points. Conclusions: Hypocoagulability ameliorates vascular disease in the ApoE‐deficient model in a lipid‐independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR−/− mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.

A polymorphism at codon 129 of the prion protein gene has been shown to confer genetic susceptibility to prion diseases, and to influence the epidemic course of variant Creutzfeldt-Jakob disease. We employed a PCR-endonuclease digestion-based assay to investigate this genetic trait in Brazil, and then compared our results to previously published data from several European and Asian countries.

Platelet membrane glycoprotein (GP) Ibα is a critical component of platelet adhesion complex to subendothelium structures following tissue injury or pathological surfaces, such as atherosclerotic plaques. Polymorphisms of the GPIbα gene have been associated with a high risk for occlusive vascular disease, and its distribution varies considerably among distinct populations. These polymorphisms comprise the human platelet antigen (HPA)‐2 system, the −5C/T dimorphism of the Kozak sequence, and the variable number of tandem 39‐bp repeats (VNTR). Here we report the prevalence of the GPIbα gene polymorphisms among Brazilians, a highly ethnically diverse population. We analyzed 492 subjects of European, African, or Indigenous origin. It was possible to determine ten distinct haplotypes. The most common (∽40%) haplotype was the Kozak‐TT/HPA‐2aa/VNTR‐CC for both Caucasian and African descent. However, among Indigenous, Kozak‐TT/HPA‐2aa/VNTR‐CC and Kozak‐TC/HPA‐2aa/VNTR‐CC were equally present. Although a strong linkage disequilibrium between VNTR and HPA‐2 polymorphism had also been observed, here we determined incomplete linkage disequilibrium in 10% of subjects from all ethnic groups. VNTR‐E, a rare variant lacking the 39‐bp repeat, was identified in two unrelated subjects, and functional platelet studies revealed no abnormalities. The VNTR‐A allele, the largest variant containing four copies of the repeats, was not identified in this population. However, homozygosity for the VNTR‐A allele (Kozak‐TT/HPA‐2aa/VNTR‐AA) was determined in two distinct species of nonhuman primates. These results suggest a greater complex evolutionary mechanism in the macroglycoprotein region of the GPIbα gene and may be useful in the design of gene–disease association studies for vascular disease. Am. J. Hematol. 77:107–116, 2004. © 2004 Wiley‐Liss, Inc.

For several years, coagulation has been implicated in the pathogenesis of sepsis. However, results from clinical trials with natural anticoagulants, as well as studies with knock-out mice for specific coagulation factors yielded conflicting results on the role of coagulation in the pathogenesis of sepsis. The aim of this study was to evaluate the impact of severe The factor VIII:C (FVIII:C) and factor IX:C (FIX:C) deficiency on a lipopolysaccharide (LPS)-induced murine model of sepsis. FVIII:C and FIX:C deficient mice, and their haemostatic normal littermate controls were challenged with LPS, and several parameters of the host response were evaluated: seven-day survival experiments were performed using two dose levels of LPS; biochemical and histological markers of tissue damage, coagulation parameters, and pro-inflammatory cytokines were evaluated at baseline and after 3 h and 6 h after an injection of LPS. Severe FVIII and FIX deficiency were compatible with normal survival in experimental sepsis. In addition, LPS-induced tissue damage and coagulation activation were similar in FVIII or FIX deficient mice compared to their respective controls. A lower release of pro-inflammatory cytokines was observed in FIX but not in FVIII deficient mice. Severe FIX or FVIII deficiency does not protect mice from mortality or from tissue damage in the endotoxemia model, supporting the hypothesis that FVIII and FIX are not critical to the pathogenesis of experimental sepsis.

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Complications of atherosclerosis are the most common causes of death in Western societies. The knowledge that atherosclerosis is an inflammatory disease and coagulation affects the disease’s complications offers new opportunities for prevention and treatment. Population studies demonstrated that the risk for myocardial infarction is reduced by 80% among hemophilia A (HA) men compared to age and gender-matched controls. However, early atherosclerotic lesions were readily identified in small cohorts of adults HA and hemophilia B (HB) by ultrasonography of carotid and femoral arteries in a similar fashion to male controls. Here we sought to determine the role of FVIII and FIX on the development of atherosclerosis in two different mouse models. We compared a group of FVIII (HA) or FIX deficient mice (HB) lacking the low-density lipoprotein receptor (LDLR−/−) or apolipoprotein E (apoE−/−) with hemostatically normal littermate controls lacking either LDLR (LDLR−/−) or apoE (apoE−/−). All mice were on C57Bl/6 background and all groups were matched for gender and age. When mice were 8 weeks old, they were placed on atherogenic diet. Analyzes of whole aortic (Sudan IV staining) or aortic sinus (Oil Red O) revealed that HA/apoE−/− mice exhibit less atherosclerotic lesions in a time-dependent manner. At weeks 22 (n=9) and 37 (n=9), the rate of atherosclerosis was 2-3-fold lower in HA/apoE when compared to age and gender-matched apoE (−/−) control group (p=0.001, and p<0.001, respectively), but not at week 8. In contrast, no differences were noted in the rates of vascular lesions between HA/LDLR(−/−) (n=9) and controls (n=9) at all time points. Overall, HB/LDLR (−/−) developed atherosclerosis lesions at aortic sinus with similar rates as the controls at 8 and 22 weeks. However, at week 37, these mice exhibit more extensive tissue damage than the control (p=0.02). Surprisingly, the analyses of whole aorta revealed that atherosclerotic area was more wide-spread in FIX deficient mice in both models, i.e. HB/apoE(−/−) and HB/LDLR(−/−), compared to matched controls. At week 22, 16% and 11% (p=0.04) of the aorta (HB/LDLR[−/−] vs LDLR[−/−]) was covered by atherosclerosis, whereas the rates were 37% vs 21% (p=0.001) at week 37. Moreover, in the apoE(−/−) model the lack of FIX enhances the vascular lesions (14% vs 7%, p=0.02 at week 22) and (36% vs 22%, p=0.04 at week 37). In another model, we tested whether male transgenic (Tg+) mice for high levels of human FIX (∼ 200% of activity above the normal) would influence the development of atherosclerotic lesions in the apoE(−/−) model. After 20 weeks, the rates of arterial vascular lesions were not different among Tg+FIX mice and controls after weeks 20 or 40 on atherogenic diet. In addition, Tg+FIX mice kept on regular chow diet up to 52 weeks showed no enhanced atherosclerosis but they presented high risk for venous thrombosis, as documented in 3/6 Tg+FIX mice and none of controls (0/6). These data demonstrate that coagulation activation in atherogenesis exhibits a rather heterogenous role in the disease evolution which suggest that clotting factors may have functions other than hemostasis in the development of vascular disease. Further studies in hemophilia B subjects are warranted to define the FIX effect on the onset and progression of occlusive vascular disease which may raises concerns on the onset cardiovascular risks on an aging hemophilia population.

Background: Myeloproliferative disorders are a risk factor for deep venous thrombosis. The most common venous event is deep vein thrombosis of the lower limbs, cerebrovascular thrombosis is one of the major causes of morbidity and mortality among patients with MPD and splanchnic vein thrombosis are frequent in patients with latent MPD. The recently identified Janus kinase (JAK2) V617F somatic mutation is an acquired mutation frequently found in MPD patients and has been related to a higher risk of vascular events among those patients. Objective: Determine the presence of JAK 2 V617F mutation among patients with deep venous thrombosis of the lower limbs, splanchnic veins and central nervous systems without overt MPD. Patients and methods: We accessed the medical histories of 240 adult patients with the diagnosis of deep vein thrombosis who have been treated in the Haematology Department of the State University of Campinas, 63 patients with splanchnic vein thrombosis, 126 patients with deep vein thrombosis of the lower limbs and 51 patients with central nervous system vein thrombosis. The DNAs obtained at the diagnosis of all those 240 patients were than tested for the JAK 2 V617F mutation by polimerase chain reaction followed by restriction enzyme analysis. None of them fulfilled criteria for diagnosis of MPD at the time of the thrombosis. Results: The three groups were similar with regard to age of the thrombosis, platelets and hemoglobin values at the diagnosis. There was also no diference between the three groups regarding the presence of hereditary thrombofilia and antiphospholipid antibodies. Females accounted for 47% of the patients with splanchnic vein thrombosis, 72.2% in thrombosis of the lower limbs and 74% in central nervous system (p values of 0.0013 and 0.042 respectively). The median neutrophils values at diagnosis were significantly lower among patients with splanchnic vein thrombosis compared with patients with thrombosis of the lower limbs or central nervous system (p= 0.007 and p= 0.004 respectively). The thrombotic event was provoked in 23.8% of patients with splanchnic vein thrombosis versus 57.8% in thrombosis of the lower limbs (95% CI 0.12–0.45, p<0,001) and versus 43% in central nervous system thrombosis (95% CI 0.18–0.92, p =0,043). The number of provoked events was similar between patients with thrombosis of the lower limbs and central nervous system. No homozygous JAK 2 V167F mutation was found. The heterozygous JAK 2 V167F mutation was detected in 5 patients (7.9%) with splanchnic vein thrombosis versus none in deep vein thrombosis of the lower limbs (95%CI 1.29–437.6, p=0.003) and versus none in central nervous system vein thrombosis (95% CI 0.52–179.5, p= 0,06). Patients carrying the JAK 2 V167F mutation were all negative for Factor V Leiden, Factor II 20210A and antiphospholipid antibodies. Two out of the 5 patients had a provoked thrombosis, all related to conjugated strogens intake. Within a median follow up of 3.6 years, only one patient developed a Myeloproliferative disorder (Primary Myelofibrosis). Conclusion: The JAK2 V167F mutation in frequently present in cases of splanchnic vein thrombosis without overt MPD but not in case of deep vein thrombosis of the lower limbs and probability not in cases of central nervous system vein thrombosis. The further development of MPD in the positive JAK2 V167F mutation cases is yet to be determined.