Novel exomic rare variants associated with venous thrombosis.

Exomic rare variant polymorphisms (c. 300 000) were analysed in the Scripps Venous Thrombosis (VTE) registry (subjects aged <55 years). Besides coagulation factor V (F5) single nucleotide polymorphisms (SNPs), family with sequence similarity 134, member B (FAM134B; rs78314670, Arg127Cys) and myosin heavy chain 8 (MYH8; rs111567318, Glu1838Ala) SNPs were associated with recurrent VTE (n = 34 cases) (false discovery rate-adjusted P < 0·05). FAM134B (rs78314670) was associated with low plasma levels of anticoagulant glucosylceramide. Analysis of 50 chr17p13.1 MYH rare SNPs (clustered skeletal myosin heavy chain genes) using collapsing methods was associated with recurrent VTE (P = 2·70 ×10-16 ). When intravenously injected, skeletal muscle myosin was pro-coagulant in a haemophilia mouse tail bleeding model. Thus, FAM134B and MYH genetic variants are plausibly linked to VTE risk.

Acylcarnitines are anticoagulants that inhibit factor Xa and are reduced in venous thrombosis, based on metabolomics data.

In many patients with deep vein thrombosis and pulmonary embolism (venous thromboembolism, VTE), biomarkers or genetic risk factors have not been identified. To discover novel plasma metabolites associated with VTE risk, we employed liquid chromatography-mass spectrometry-based untargeted metabolomics, which do not target any specific metabolites. Using the Scripps Venous Thrombosis Registry population for a case-control study, we discovered that 10:1 and 16:1 acylcarnitines were low in plasmas of the VTE patient group compared with matched controls, respectively. Data from targeted metabolomics studies showed that several long-chain acylcarnitines (10:1, 12:0, 12:2, 18:1, and 18:2) were lower in the VTE group. Clotting assays were used to evaluate a causal relationship for low acylcarnitines in patients with VTE. Various acylcarnitines inhibited factor Xa-initiated clotting. Inhibition of factor Xa by acylcarnitines was greater for longer acyl chains. Mechanistic studies showed that 16:0 acylcarnitine had anticoagulant activity in the absence of factor Va or phospholipids. Surface plasmon resonance investigations revealed that 16:0 acylcarnitine was bound to factor Xa and that binding did not require the γ-carboxy glutamic acid domain. In summary, our study identified low plasma levels of acylcarnitines in patients with VTE and showed that acylcarnitines have anticoagulant activity related to an ability to bind and inhibit factor Xa.

Inhibition of thrombin generation in human plasma by phospholipid transfer protein.

BACKGROUND: Plasma phospholipid transfer protein (PLTP) transfers lipids between donors and acceptors (e.g., from HDL to VLDL) and modulates lipoprotein composition, size, and levels. No study has reported an assessment of the effects of PLTP on blood clotting reactions, such as reflected in thrombin generation assays, or on the association of venous thrombosis (VTE) risk with PLTP activity. METHODS: The in vitro effects of PLTP on blood coagulation reactions and the correlations between plasma PLTP activity levels and VTE were studied. RESULTS: Recombinant (r) PLTP concentration-dependently inhibited plasma thrombin generation and factor XII-dependent kallikrein generation when sulfatide was used to stimulate factor XII autoactivation in plasma. However, rPLTP did not inhibit thrombin generation in plasma induced by factor XIa or tissue factor, implicating an effect of PLTP on contact activation reactions. In purified systems, rPLTP inhibited factor XII autoactivation stimulated by sulfatide in the presence of VLDL. In surface plasmon resonance studies, purified factor XII bound to immobilized rPLTP, implying that rPLTP inhibits factor XII-dependent contact activation by binding factor XII in the presence of lipoproteins. Analysis of plasmas from 40 male patients with unprovoked VTE and 40 matched controls indicated that low PLTP lipid transfer activity (≤25th percentile) was associated with an increased risk of VTE after adjustment for body mass index, plasma lipids, and two known thrombophilic genetic risk factors. CONCLUSION: These data imply that PLTP may be an antithrombotic plasma protein by inhibiting generation of prothrombotic factor XIIa in the presence of VLDL. This newly discovered anticoagulant activity of PLTP merits further clinical and biochemical studies.

Plasma skeletal muscle myosin phenotypes identified by immunoblotting are associated with pulmonary embolism occurrence in young adults.

BACKGROUND: Purified skeletal muscle myosin (SkM) binds factor Xa and is procoagulant. The molecular forms of SkM in human plasma have not been characterized. METHOD: Human plasma SkM heavy chain (HC) isoforms of different molecular weights were detected by a newly developed immunoblotting protocol. In this pilot study, the distribution of SkM HC antigen isoforms in plasmas of healthy subjects and young adult patients with venous thrombosis was analyzed. RESULTS: Multiple SkM HC antigen bands were detected in human plasmas, corresponding to full-length SkM HC, heavy meromyosin, or the S1 fragment. Plasma immunoblots of healthy subjects displayed three major phenotypes: Type I with two primary bands for full-length SkM and heavy meromyosin, and two lesser bands including S1 fragment (54%); Type II with bands primarily for full-length SkM HC (34%); and Type III with only a band for the S1 fragment (12%). Plasma SkM HC antigen Type II phenotype was associated with an increased occurrence of isolated pulmonary embolism in younger patients, respectively (≤50 years old). CONCLUSIONS: Three SkM HC antigen phenotypes were identified in human plasma by immunoblotting, and Type II phenotype was correlated with the occurrence of isolated pulmonary embolisms in younger patients.

Serum amyloid A4 is a procoagulant apolipoprotein that it is elevated in venous thrombosis patients.

BACKGROUND: Serum amyloid A4 (SAA4) is an apolipoprotein that is in the SAA family and it is constitutively translated. Previously, acute-phase SAA1 and SAA2 levels were associated with venous thromboembolism (VTE). OBJECTIVE: We investigated the association of plasma SAA4 with VTE and the role of SAA4 in coagulation. PATIENTS AND METHODS: The association of SAA4 with VTE in a case-control study of adult VTE subjects (N = 113 each group) and the effects of recombinant SAA4 on plasma blood coagulation assays and prothrombin activation initiated by factor Xa were evaluated. RESULTS: Plasma SAA4 levels in VTE subjects were higher vs. controls (48.1 vs. 38.4 µg/mL; P < .001). Elevated plasma SAA4 level (above the 90th percentile of controls) was associated with increased VTE occurrence (odds ratio, 3.8; 95% confidence interval, 1.8-8.0). This association remained significant after the adjustment for acute-phase SAA level, suggesting that SAA4 associated with VTE is independent of acute-phase SAA. Two isoforms of SAA4, that is, glycosylated and nonglycosylated SAA4 isoforms, were each higher in VTE patients. When recombinant SAA4 was added to plasma, it shortened factor Xa-1-stage clotting times, showing that it enhances clotting in plasma. In reaction mixtures containing purified factors Xa and Va and prothrombin, recombinant SAA4 increased prothrombin activation, showing that it enhances prothrombinase activity. CONCLUSION: Elevated plasma constitutive SAA4 levels were linked to VTE in adults, and SAA4 can enhance thrombin generation in plasma. Our data highlight a previously unknown procoagulant activity of SAA4 that appears to be related to risk of venous thrombotic events.

An engineered factor Va prevents bleeding induced by direct-acting oral anticoagulants by different mechanisms.

Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs.

Elevated plasma fibronectin levels associated with venous thromboembolism.

Elevated plasma fibronectin levels occur in various clinical states including arterial disease. Increasing evidence suggests that atherothrombosis and venous thromboembolism (VTE) share common risk factors. To assess the hypothesis that high plasma fibronectin levels are associated with VTE, we compared plasma fibronectin levels in the Scripps Venous Thrombosis Registry for 113 VTE cases vs. age and sex matched controls. VTE cases had significantly higher mean fibronectin concentration compared to controls (127% vs. 103%, p < 0.0001); the difference was greater for idiopathic VTE cases compared to secondary VTE cases (133% vs. 120%, respectively). Using a cut-off of >90% of the control values, the odds ratio (OR) for association of VTE for fibronectin plasma levels above the 90(th) percentile were 9.37 (95% CI 2.73-32.2; p < 0.001) and this OR remained significant after adjustment for sex, age, body mass index (BMI), factor V Leiden and prothrombin nt20210A (OR 7.60, 95% CI 2.14-27.0; p = 0.002). In particular, the OR was statistically significant for idiopathic VTE before and after these statistical adjustments. For the total male cohort, the OR was significant before and after statistical adjustments and was not significant for the total female cohort. In summary, our results suggest that elevated plasma fibronectin levels are associated with VTE especially in males, and extend the potential association between biomarkers and risk factors for arterial atherothrombosis and VTE.

Minor Plasma Lipids Modulate Clotting Factor Activities and May Affect Thrombosis Risk.

Different minor abundance plasma lipids significantly influence thrombin generation in vitro and significant differences in such lipids are linked to risk for venous thrombosis. Some plasma sphingolipids including glucosylceramide, lyso-sulfatide and sphingosine have anticoagulant properties whereas, conversely, some plasma phospholipid derivatives, including certain lyso-phospholipids and ethanolamides, have procoagulant properties. Plasma metabolite profiling of venous thrombosis patients showed association of venous thrombosis with decreased plasma long-chain acylcarntines, leading to discovery of their anticoagulant activity as inhibitors of factor Xa. Inhibition of factor Xa by acylcarnitines does not require the protein's Gla-domain, emphasizing an expanded framework for the paradigm for lipid-clotting factor interactions. Overall, whether by genetics or environment, alterations in the dynamics of lipid metabolism linked to an altered lipidome may contribute to regulation of blood coagulation because imbalances between physiologic procoagulant and anticoagulant lipids may contribute to excessive thrombin generation that augments risk for thrombosis.

Low level of the plasma sphingolipid, glucosylceramide, is associated with thrombotic diseases.

BACKGROUND: One previous pilot study suggested the association of low plasma glucosylceramide (GlcCer) levels with venous thrombosis (VTE) risk. OBJECTIVE: We aimed to confirm and evaluate the association of low plasma GlcCer levels with VTE and myocardial infarction (MI) occurrence, respectively. PATIENTS AND METHODS: We evaluated the association of GlcCer in two independent case-control studies of Caucasian VTE populations (N = 210 and 636) and one case-control study of Caucasian MI patients (N = 345). RESULT: Plasma GlcCer levels in VTE patients were lower compared to controls in two independent VTE populations (5.0 vs 5.8 µg/mL, p = 0.003 for the Scripps registry, and 5.6 vs 6.0 µg/mL, p = 0.001 for the Valencia registry, respectively). A low plasma GlcCer level (below 10th percentile of controls) was associated with increased VTE occurrence [odds ratio (OR) = 3.7 (95%CI, 1.8-7.9) for Scripps registry and OR = 2.1 (95%CI, 1.3-3.3) for Valencia registry, respectively). For the MI study, the median GlcCer plasma level was lower in MI patients than in controls (4.3 vs 5.6 µg/mL, p<0.001), and a low level of GlcCer (below 10th percentile of control) was associated with higher MI occurrence [OR = 7.7, (95%CI, 4.3-13.8)]. CONCLUSION: Lower concentration of GlcCer was associated with VTE occurrence in two independent studies and also with MI occurrence in one study.

High-density lipoprotein deficiency and dyslipoproteinemia associated with venous thrombosis in men.

BACKGROUND: Although dyslipoproteinemia is associated with arterial atherothrombosis, little is known about plasma lipoproteins in venous thrombosis patients. METHODS AND RESULTS: We determined plasma lipoprotein subclass concentrations using nuclear magnetic resonance spectroscopy and antigenic levels of apolipoproteins AI and B in blood samples from 49 male venous thrombosis patients and matched controls aged <55 years. Venous thrombosis patients had significantly lower levels of HDL particles, large HDL particles, HDL cholesterol, and apolipoprotein AI and significantly higher levels of LDL particles and small LDL particles. The quartile-based odds ratios for decreased HDL particle and apolipoprotein AI levels in patients compared with controls were 6.5 and 6.0 (95% CI, 2.3 to 19 and 2.1 to 17), respectively. Odds ratios for apolipoprotein B/apolipoprotein AI ratio and LDL cholesterol/HDL cholesterol ratio were 6.3 and 2.7 (95% CI, 1.9 to 21 and 1.1 to 6.5), respectively. When polymorphisms in genes for hepatic lipase, endothelial lipase, and cholesteryl ester transfer protein were analyzed, patients differed significantly from controls in the allelic frequency for the TaqI B1/B2 polymorphism in cholesteryl ester transfer protein, consistent with the observed pattern of lower HDL and higher LDL. CONCLUSIONS: Venous thrombosis in men aged <55 years old is associated with dyslipoproteinemia involving lower levels of HDL particles, elevated levels of small LDL particles, and an elevated ratio of apolipoprotein B/apolipoprotein AI. This dyslipoproteinemia seems associated with a related cholesteryl ester transfer protein genotype difference.

Elevated CETP Lipid Transfer Activity is Associated with the Risk of Venous Thromboembolism.

AIM: Cholesteryl ester transfer protein (CETP) is an important lipid transfer factor in plasma that enhances prothrombinase activity in purified systems. This study was conducted to test the association of plasma CETP activity with venous thrombosis (VTE) and to address the procoagulant mechanism of CETP activity in prothrombinase assays. METHODS: We measured CETP lipid transfer activity in plasmas of 49 male VTE patients and in plasmas of matched controls. CETP procoagulant activity was tested in purified prothrombinase systems. RESULTS: CETP lipid transfer activity levels were significantly higher in VTE patients than in controls (p=0.0008). A subset of patients carrying the CETP mutations Ala373Pro and Arg451Gln, which were also linked to the VTE risk, showed significantly higher plasma CETP activity than the noncarriers. The plasma CETP activity negatively correlated with APTT, suggesting that the CETP activity is associated with plasma coagulability. Recombinant (r) CETP bound to both factor Xa (Kd=15 nM) and Gla-domainless factor Xa (Kd=59 nM), whereas rCETP enhanced prothrombin activation by factor Xa, but not by Gla-domainless factor Xa. rCETP also required factor Va for enhancement of prothrombinase activity. When we addressed the effects of mutations in CETP on prothrombinase activity, Gln451-rCETP was found to have five-fold higher thrombin generation activity than wt-rCETP or Pro373-rCETP. CONCLUSIONS: Elevated CETP lipid transfer activity in plasma was associated with the risk of VTE. Gln451-CETP, which is linked to VTE, has much higher procoagulant activity than wt-CETP. CETP might act as a physiologic procoagulant by mechanisms that involve its direct binding to factor Xa.

Prevalence and risk factors for hypertension in hemophilia.

Hypertension (HTN) is a major risk factor for intracranial hemorrhage. We, therefore, investigated the prevalence, treatment, and control of HTN in adult patients with hemophilia (PWH). PWH≥18 years (n=458) from 3 geographically different cohorts in the United States were evaluated retrospectively for HTN and risk factors. Results were compared with the nationally representative sample provided by the contemporary National Health and Nutrition Examination Survey (NHANES). PWH had a significantly higher prevalence of HTN compared with NHANES. Overall, the prevalence of HTN was 49.1% in PWH compared with 31.7% in NHANES. At ages 18 to 44, 45 to 64, 65 to 74, and ≥75 years, the prevalence of HTN for PWH was 31.8%, 72.6%, 89.7%, and 100.0% compared with 12.5%, 41.2%, 64.1%, and 71.7% in NHANES, respectively. Of treated hypertensive PWH, only 27.1% were controlled, compared with 47.7% in NHANES (all P<0.05). Age, body mass index, diabetes mellitus, and renal function were independently associated with HTN. Among patients with moderate or severe hemophilia there was a trend (≈1.5-fold) for higher odds of having HTN compared with patients with mild hemophilia. On the basis of these results, new care models for adult PWH and further studies for the causes of HTN in hemophilia are recommended.

Engineered virus-like nanoparticles reverse heparin anticoagulation more consistently than protamine in plasma from heparin-treated patients.

Heparin is widely used for anticoagulation, often requiring the subsequent administration of a reversal agent. The only approved reversal agent for heparin is protamine sulfate, which induces well described adverse reactions in patients. Previously we reported a novel class of heparin antagonists based on the bacteriophage Qß platform, displaying polyvalent cationic motifs which bind with high affinity to heparin. Here we report heparin reversal by the most effective of these virus-like particles (VLP) in samples from patients who were administered heparin during cardiac procedures or therapeutically for treatment of various thrombotic conditions. The VLP consistently reversed heparin in these samples, including those from patients that received high doses of heparin, with greater efficiency than a negative control VLP and with significantly less variability than protamine sulfate. These results provide the first step towards validation of heparin antagonist VLPs as viable alternatives to protamine.

Association of Apo(a)isoform size with dyslipoproteinemia in male venous thrombosis patients.

BACKGROUND: Lp(a) is a proatherogenic lipoprotein that may also be prothrombotic. Apo(a) size isoforms have differential effects on fibrinolysis. Whereas Lp(a) concentrations have been linked to venous thromboembolic disease (VTE) risk, apo(a) polymorphisms in VTE have not been studied. METHODS: We used a standardized high resolution agarose gel electrophoresis technique to determine apo(a) isoform size, and a Lp(a) immunoassay insensitive to apo(a) size to measure Lp(a) concentration in 46 men with VTE and 46 age-matched healthy controls. RESULTS: Apo(a) isoform distribution in VTE cases and controls was bimodal and VTE patients tended to have more medium-sized isoforms K(4)-(19-27) (54.3% vs. 34.8%, p=0.06). Cases and controls had the same median predominant apo(a) size isoform (23.5 K(4) repeats) and comparable Lp(a) concentrations. However, subgroup analysis based on apo(a) isoform size (K(4)< or =23 or K(4)> or =24) revealed that cases in the K(4)> or =24 subgroup had higher Lp(a) concentrations than the controls in this isofrom subgroup (14.5 mmol vs. 6.6 mmol, p=0.029). Also, dyslipoproteinemia (smaller LDL and HDL particles, higher LDL and lower HDL parameters) was strongly associated with VTE only in this larger apo(a) isoform group. CONCLUSIONS: These observations provide the first evidence that determination of apo(a) isoforms may provide useful novel insights into VTE risk.