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Treatment of venous thromboembolism with concomitant thrombocytopenia is challenging. The platelet threshold for safe administration of anticoagulants is under debate, with minimum platelet count of 50 × 109/l being recommended as the safe cutoff. However, some evidence suggests administration of anticoagulants may still be safe at platelet levels of 30 × 109/l. Therefore, we developed an in-vitro thromboelastography (TEG) study to examine the effect of therapeutic or prophylactic levels of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) on the clotting profile of platelet-reduced whole blood. Using magnetic bead-based antibody chromatography, platelets were removed to achieve platelet-depleted blood (<10 × 109/l of platelets). Platelet-depleted blood was then mixed with whole blood to produce blood samples with platelet counts of 30 × 109, 50 × 109 and 150 × 109/l. These blood samples were incubated with therapeutic or prophylactic levels of UFH or LMWH in disposable TEG cups. Clotting was initiated with 10 mmol/l calcium and optimized tissue factor levels for each anticoagulant used (2.25 pmol/l for UFH and 2.05 pmol/l for LMWH). Clotting was monitored by TEG at 37 °C for 180 min. The following TEG parameters were evaluated: R (time to clot), maximum amplitude (strength of clot) and area under the curve in 15 min (overall speed and strength of the clot at 15 min of clotting). No statistically significant differences were observed between platelet counts of 30 × 109 and 50 × 109/l for R, maximum amplitude or area under the curve in 15 min for most of the therapeutic and prophylactic doses of UFH and LMWH tested in this study. Use of anticoagulants compromised all of the TEG parameters relative to a normal platelet count of 150 × 109/l, in a dose dependent manner. The current study demonstrates that in-vitro clotting is impaired with use and increasing doses of anticoagulants. Despite this observation, we did not observe a significant difference in clotting between platelet levels of 30 × 109 and 50 × 109/l. Overall, this work provides further insight in the debated use of anticoagulants in patients with venous thromboembolism and concomitant thrombocytopenia, and provides support for possible use of anticoagulants at lower platelet thresholds.

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Rivaroxaban after total knee arthroplasty (TKA) is used to prevent postoperative venous thromboembolism (VTE); however, despite thromboprophylaxis, some patients still develop postoperative VTE. To determine whether tourniquet time, time to initiate rivaroxaban (TTIRIV), or Body Mass Index (BMI) was associated with postoperative VTE. A retrospective case-control study was conducted. Those patients that developed VTE despite prophylaxis (cases) were compared to controls (no VTE). A univariate analysis was conducted (p < 0.05 statistically significant). Seven VTE cases were identified from 234 TKA-patients. Patients with and without VTE had BMI of 40.1 ± 9.1 and 32.8 ± 7.5, respectively (p = 0.064). TTIRIV in VTE and control group was 28.2 ± 4.7 hours and 26.4 ± 4.2 hours, respectively (p = 0.39). Mean tourniquet time in VTE and control group was 65.0 ± 8.7 minutes and 49 ± 8.8 minutes, respectively (p = 0.0007). Statistically significant differences in tourniquet times were noted between VTE and non-VTE group but not for TTIRIV and BMI. Prolonged tourniquet use could pose a potential risk factor for postoperative VTE. Thromboprophylaxis management may need to be adjusted, based on patient-specific factors that could include increasing doses of oral anticoagulants and/or mechanical prophylaxis. However, further large-scale studies are required to establish pathophysiology.

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: Thromboelastography (TEG) is a global assay used for evaluating features of clot formation in vitro. Dabigatran is a reversible direct inhibitor of thrombin that has not been studied in neonates using a sophisticated global assay, such as TEG. Neonatal hemostasis differs from adult hemostasis in both quantitative and qualitative characteristics. Our aim was to compare the TEG clotting profile of neonatal and adult platelet-poor plasma when exposed to different concentrations of dabigatran. We used commercially collected adult pooled plasma and neonatal cord blood collected from placentas of healthy full term newborns. Platelet-poor plasma was isolated, pooled, and frozen. Prior to experiment, plasma was thawed and filtered. A reaction mixture of CaCl2, corn trypsin inhibitor, tissue factor, and dabigatran in imidazole buffer was mixed with plasma in a TEG cup. Time to clot initiation (R-time), speed of clot strengthening (α-angle), and maximum clot strength (maximal amplitude) were measured. Scanning electron microscopy was performed to evaluate fibrin clot structure. Without dabigatran, there was no significant difference in TEG measurements between neonatal and adult samples. However, neonatal plasma clotting with dabigatran had slower onset, slower speed, and weaker clots that were more porous with thicker fibers, compared with adult plasma clotting. Thus, neonatal plasma may be more sensitive to dabigatran as assessed by our in-vitro TEG study.

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Congenital nephrotic syndrome (CNS) refers to a disease presenting with massive proteinuria in association with hypoalbuminemia, hyperlipidemia, and edema at birth or within the first three months of life. In the past, most children with CNS had extremely poor prognosis and succumbed to various complications, usually within the first 6 months. Recent advancements in protein supplementation and nutritional support, renal replacement therapy and renal transplantation in infancy, render these patients to have much better outcomes. However, there are still many hurdles in the management of this disease. Thromboembolism is an uncommon, yet important complication which the healthcare givers must be aware of. This article reviews the challenges in the management of the thrombotic complications with special emphasis on the unique characteristics of the newborn hemostasis system and anti-thrombin (AT) depletion in nephrotic syndrome. Due to the relatively low incidence of CNS in children and scarce information in the literature on the optimal management of the thromboembolic complications, most of the recommendations are based on the authors' experience.

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INTRODUCTION: Recombinant activated factor VII (rFVIIa), prothrombin complex concentrate (PCC) and activated PCC (aPCC) are three non-specific haemostatic agents sometimes employed to reverse new, factor-specific oral anticoagulants. METHODS: We conducted a review in the literature to compare the abilities of rFVIIa, PCC and aPCC to reverse factor-specific anticoagulants. MEDLINE and EMBASE databases were searched up to Oct 2013. RESULTS: Eleven animal studies and two human trials met predefined inclusion criteria. To account for dosing variations of anticoagulants among studies, data were interpreted based on standards referenced from human trials at therapeutic doses. In animal studies, inconsistencies in the reversal abilities of rFVIIa, PCC and aPCC can be partly attributed to inter-species differences in the affinity among various clotting factors and tissue factors. Moreover, the differences in the affinity between species-specific clotting factors and anticoagulants that were initially designed to inhibit human factor may impose additional obstacles when comparing single factor rFVIIa with agents that contained multiple clotting factors. In the absence of a common clinical indication for the utilization of rFVIIa, PCC and aPCC, it is difficult, if not impossible, to establish an equivalent dose among these haemostatic agents when comparing their effectiveness in reversing factor-specific oral anticoagulants. Human trials were too few and sub-optimally designed to draw definite conclusions. CONCLUSION: While preclinical studies may hint at a role for these haemostatic agents in reversing the anticoagulant effects of oral, factor-specific anticoagulants, existing trials offer inconclusive evidence to guide a clinical decision among individual agents with respect to potency and thrombosis risk. The mechanistic differences of these hemostatic agents in terms of their interactions with other coagulation factors impose major obstacles for the scientists using animal models to compare the efficacy of these reversal agents.

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Marfan syndrome is an autosomal dominant disease caused by mutations in the gene encoding for fibrillin-1 (FBN1). More than 1,000 FBN1 mutations have been identified, which may lead to multiple organ involvement, particularly of the ocular, skeletal, and cardiovascular systems. Mutations in exons 59-65 have been reported in the past to cause mild Marfan-like fibrillinopathies. We report a family with a mutation in exon 63 that manifests with significant cardiovascular system involvement such as aortic root dilatations, dissection of the aorta, and sudden death at a young age. Genetic analysis revealed that four related individuals are positive for a novel heterozygous Cys2633Arg mutation in exon 63. Their genotype-phenotype profile (based on the revised Ghent nosology) is described. We postulate that the Cys2633Arg mutation may manifest with significant and progressive enlargement of the aortic root, risk of aortic dissections, and minor skeletal abnormalities, without involving the ocular system (i.e., ectopia lentis).

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The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn(2+) in this interaction because Zn(2+) is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn(2+) promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn(2+)-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn(2+)-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn(2+) is present. These results reveal the mechanism by which Zn(2+) augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.

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Factor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ~4-fold faster than AT+UFH. Relative to intact prothrombinase, ratesfor FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.

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BACKGROUND: D-Dimer testing is sensitive but not specific for diagnosing deep venous thrombosis (DVT). Changing the use of testing and the threshold level for a positive test result on the basis of risk for DVT might improve the tradeoff between sensitivity and specificity and reduce the need for testing. OBJECTIVE: To determine whether using a selective D-dimer testing strategy based on clinical pretest probability (C-PTP) for DVT is safe and reduces diagnostic testing compared with using a single D-dimer threshold for all patients. DESIGN: Randomized, multicenter, controlled trial. Patients were allocated using a central automated system. Ultrasonographers and study adjudicators but not other study personnel were blinded to trial allocation. (ClinicalTrials.gov: NCT00157677) SETTING: 5 Canadian hospitals. PATIENTS: Consecutive symptomatic patients with a first episode of suspected DVT. INTERVENTION: Selective testing (n = 860), defined as D-dimer testing for outpatients with low or moderate C-PTP (DVT excluded at D-dimer levels <1.0 µg/mL [low C-PTP] or <0.5 µg/mL [moderate C-PTP]) and venous ultrasonography without D-dimer testing for outpatients with high C-PTP and inpatients, or uniform testing (n = 863), defined as D-dimer testing for all participants (DVT excluded at D-dimer levels <0.5 µg/mL). MEASUREMENTS: The proportion of patients not diagnosed with DVT during initial testing who had symptomatic venous thromboembolism during 3-month follow-up and the proportion of patients undergoing D-dimer testing and ultrasonography. RESULTS: The incidence of symptomatic venous thromboembolism at 3 months was 0.5% in both study groups (difference, 0.0 percentage point [95% CI, -0.8 to 0.8 percentage points]). Selective testing reduced the proportion of patients who required D-dimer testing by 21.8 percentage points (CI, 19.1 to 24.8 percentage points). It reduced the proportion who required ultrasonography by 7.6 percentage points (CI, 2.9 to 12.2 percentage points) overall and by 21.0 percentage points (CI, 14.2 to 27.6 percentage points) in outpatients with low C-PTP. LIMITATION: Results may not be generalizable to all D-dimer assays or patients with previous DVT, study personnel were not blinded, and the trial was stopped prematurely. CONCLUSION: A selective D-dimer testing strategy seems as safe as and more efficient than having everyone undergo D-dimer testing when diagnosing a first episode of suspected DVT. PRIMARY FUNDING SOURCE: Heart and Stroke Foundation of Ontario.

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The role of red blood cells (RBCs) in coagulation is not well understood. Overt exposure of phosphatidylserine on surfaces of RBCs provide docking sites for formation of the prothrombinase complex, which further aids in amplification of coagulation leading to subsequent thrombosis. No studies to date have evaluated heparin inhibition of the RBC-prothrombinase system. Therefore, this study examines the ability of heparin and a covalent antithrombin-heparin complex (ATH) to inhibit the RBC-prothrombinase system. Discontinuous inhibition assays were performed to obtain k2 values for inhibition of free or prothrombinase-bound Xa by antithrombin and unfractionated heparin (AT + UFH) versus ATH. In addition, components of the complex (prothrombin, RBCs or Va) were excluded prior to reaction with inhibitors to investigate potential mechanisms involved. Inhibition of thrombin generation, fibrinogen conversion and plasma clotting by the RBC-prothrombinase system was also examined. Protection of Xa was observed for AT + UFH and not for ATH reactions. Inhibition rates for ATH were significantly faster when compared with AT + UFH results. The greatest impact on Xa inhibition was observed from factor Va omission for both inhibitors. ATH inhibited thrombin generation, fibrinogen conversion and plasma clotting better compared with AT + UFH. This study determined potential control of coagulation contributed by RBCs. Moreover, greater control of coagulation is achieved by covalently linking heparin to AT.

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Heparin binds fibrin and, by bridging thrombin onto fibrin, promotes the formation of a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin. Because thrombin binds γ(A)/γ'-fibrin, a variant with an extended γ-chain, with higher affinity than the bulk γ(A)/γ(A)-fibrin, γ(A)/γ'-fibrin affords bound thrombin more protection from inhibition by antithrombin-heparin. We examined the effect of Zn(2+) on heparin-thrombin-fibrin complex formation because Zn(2+) modulates heparin-protein interactions. Zn(2+) increased the affinity of heparin for γ(A)/γ(A)- and γ(A)/γ'-fibrin by 4.3- and 3.7-fold, respectively, but had no effect on the affinity of thrombin for either form of fibrin. In contrast, in the presence of heparin, Zn(2+) increased the affinity of thrombin for γ(A)/γ(A)-fibrin 4-fold (from a K(d) value of 0.8 to 0.2 µM) and slowed the rate of thrombin dissociation from γ(A)/γ(A)-fibrin clots. These findings suggest that Zn(2+) enhances the formation of ternary heparin-thrombin-fibrin complexes with γ(A)/γ(A)-fibrin but does not influence the already high affinity interaction of thrombin with γ(A)/γ'-fibrin. Consistent with this concept, in the presence of Zn(2+), γ(A)/γ(A)-fibrin protected thrombin from inhibition by antithrombin-heparin to a similar extent as γ(A)/γ'-fibrin. Therefore, by enhancing the binding of heparin to fibrin, physiological concentrations of Zn(2+) render fibrin-bound thrombin more protected from inhibition by antithrombin. Because fibrin-bound thrombin can trigger thrombus expansion, these findings help to explain why recurrent thrombosis can occur despite heparin treatment.

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Carotid artery dissections are the second leading cause of stroke in young adults. The hemostatic response to a dissection involves exposure of the subendothelium to the intravascular environment. Platelet activation/aggregation superimposed by secondary coagulation cascade activity attempts to heal the injury. Failure of the hemostatic response to heal the injury may lead to further rupture of the intimal and medial layers, which allows for the blood to penetrate these layers to create a false lumen. Continued hemorrhaging into the false lumen may result in dissection progression or obstruction of blood supply to the true lumen and downstream blood vessels. The effects of thrombosis in the true versus false lumen may lead to opposite consequences. True lumen clotting may lead to ischemic complications of downstream cerebral vasculature, whereas false lumen clotting may lead to dissection healing. Current information on clinical outcomes and degree of false lumen clotting in a carotid dissection model is limited, and most of the available information on this controversial topic has been inferred from aortic dissections. Therefore in this report we summarize the present state of knowledge of the pathophysiology, detailed hemostatic response to the injury, clinical presentation and treatment of carotid dissections. We also emphasize the need for future studies to investigate the degree of false lumen clotting on the clinical outcomes of carotid dissections.

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