Ivan Stevic

Carotid artery dissections: thrombosis of the false lumen.

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.

Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.

Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin–heparin complex

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.

Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets

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.

Interactions of heparin and a covalently-linked antithrombin-heparin complex with components of the fibrinolytic system.

Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with limitations, such as the inability to inhibit surface bound coagulation factors. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties compared with UFH. Advantages of ATH include enhanced inhibition of surface-bound coagulation enzymes and the ability to reduce the overall size and mass of clots in vivo. The interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Our study utilised discontinuous second order rate constant (k₂) assays to compare the rates of inhibition of free and fibrin-associated plasmin by AT+UFH vs ATH. Additionally, we evaluated the effects of AT+UFH and ATH on plasmin generation in the presence of fibrin. The k₂ values for inhibition of plasmin were 5.74 ± 0.28 x 10⁶ M⁻¹ min⁻¹ and 6.39 ± 0.59 x 10⁶ M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. In the presence of fibrin, the k₂ values decreased to 1.45 ± 0.10 x 10⁶ M⁻¹ min⁻¹ and 3.07 ± 0.19 x 10⁶ M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. Therefore, protection of plasmin by fibrin was observed for both inhibitors; however, ATH demonstrated superior inhibition of fibrin-associated plasmin. Rates of plasmin generation were also decreased by both inhibitors, with ATH causing the greatest reduction (approx. 38-fold). Nonetheless, rates of plasmin inhibition were 2-3 orders of magnitude lower than for thrombin, and in a plasma-based clot lysis assay ATH significantly inhibited clot formation but had little impact on clot lysis. Cumulatively, these data may indicate that, relative to coagulant enzymes, the fibrinolytic system is spared from inhibition by both AT+UFH and ATH, limiting reduction in fibrinolytic potential during anticoagulant therapy.

Phenotype Presentation for a Novel Mutation Affecting a Conserved Cysteine Residue in Exon 63 of Fibrillin-1 (Cys2633Arg)

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).