The coagulation system in humans.

Complex, interrelated systems exist to maintain the fluidity of the blood in the vascular system while allowing for the rapid formation of a solid blood clot to prevent hemorrhaging subsequent to blood vessel injury. These interrelated systems are collectively referred to as haemostasis. The components involved in the haemostatic mechanism consist of vessel walls, platelets, coagulation factors, inhibitors, and the fibrinolytic system. In the broadest sense, a series of cascades involving coagulation proteins and enzymes, as well as cell surfaces (platelets and endothelial cells), work together to generate thrombin, the key enzyme in coagulation, subsequently leading to the formation of a fibrin clot. However, there also exist direct and indirect inhibitors of thrombin to ensure that clot formation does not go uncontrolled. Once the fibrin clot is formed, the fibrinolytic system ensures that the clot is lysed so that it does not become a pathological complication. Taken together, the systems exist to balance each other and maintain order. The balance of coagulation and fibrinolysis keeps the haemostatic system functioning efficiently.

The role of the vessel wall.

The role of the vessel wall is complex and its effects are wide-ranging. The vessel wall, specifically the endothelial monolayer that lines the inner lumen, possesses the ability to influence various physiological states both locally and systemically by controlling vascular tone, basement membrane component synthesis, angiogenesis, haemostatic properties, and immunogenicity. This is an overview of the function and structure of the vessel wall and how disruption and dysfunction in any of these regulatory roles can lead to disease states.

Binding of heparin to metals.

Heparin is a major prophylactic and treatment agent for thrombosis. Structurally, this anticoagulant is a polydisperse, highly negatively charged polysaccharide mixture that contains a variable density of sulfate group substituents per molecule. Previous study has shown that heparin molecules have a high affinity for a wide range of metal ions with varying oxidation states. However, reports in literature on binding of heparin to metals have investigated only a small sampling of heparin-metal ion interactions. Since interaction of heparin with fluid phase and cell surface macromolecules in vivo is dependent on the heparin structure when bound in a metal ion complex, a survey of the physical parameters for heparin binding to metals is imperative. Atomic absorption and spectrophotometry experiments were performed for metal quantification, and in this study, the relative values for affinity constants and number of binding sites for heparin binding to several alkaline, alkaline earth, main group, and transition metals in their most common oxidation states are reported. We found an overall trend for heparin-metal affinity to be Mn(2+) > Cu(2+) > Ca(2+) > Zn(2+) > Co(2+) > Na(+) > Mg(2+) > Fe(3+) > Ni(2+) > Al(3+)> Sr(2+), with the trend in N (b) being opposite compared with the K (a).

Antithrombin-heparin covalent complex reduces microemboli during cardiopulmonary bypass in a pig model.

Transcranial Doppler-detected high-intensity transient signals (HITS) during cardiopulmonary bypass (CPB) surgery have been associated with postoperative neurocognitive dysfunction, suggesting microemboli in the brain could be a contributing factor. HITS occur despite administration of unfractionated heparin (UFH). This study was done to determine whether antithrombin-heparin covalent complex (ATH), a more potent anticoagulant than heparin, can reduce HITS during CPB. In a pig CPB model, ATH, UFH, or UFH + antithrombin (AT) was intravenously administered to female Yorkshire pigs after sternotomy. Twenty minutes later, hypothermic CPB was initiated and continued for 1.25 hours, then normothermia was re-established for 45 minutes. Protamine sulfate was given to neutralize the anticoagulants, and pigs were allowed to recover. HITS were monitored using an arterial flow probe placed over the carotid artery. Compared with UFH (300 or 1000 U/kg), ATH reduced the number of HITS during CPB in a dose-dependent manner. AT (3 mg/kg) + UFH (300 U/kg) resulted in an intermediate HITS rate between UFH and ATH (2 mg/kg in terms of AT). Examination of brain sections for emboli formation confirmed that, similar to HITS, number of thrombi decreased in direct proportion to ATH dosage. These results support the hypotheses that the majority of HITS represent thromboemboli and that ATH reduces emboli formation during CPB.

Management of myocardial infarction in children with Kawasaki disease.

Kawasaki disease is an acute, systemic vasculitis of unknown cause affecting mainly neonates (infants) and young children. Despite treatment during the acute phase with intravenous immunoglobulin and aspirin, up to 5% of those affected will develop coronary aneurysms, predisposing them to thrombotic complications that could result in myocardial infarction and/or death. There are treatment protocols in place for the management of myocardial infarction in adults, but the practical nature of medication is unclear in children. To date, there are no clinical trials or specific recommendations on the dosing of thrombolytic therapy for the treatment of myocardial infarction in Kawasaki pediatric patients. However, there are reports of the use of thrombolytic agents, including streptokinase, urokinase and tissue plasminogen activator, as well as the monoclonal platelet glycoprotein (GP)IIb/IIIa receptor inhibitor, abciximab, that have been used to treat myocardial infarction in children with Kawasaki disease. The outcomes in these reports are varied. This review provides a summary of the available data on the management of children with Kawasaki disease suffering from myocardial infarction or thrombotic complications that can potentially lead to myocardial infarction.

Effect of heparins on thrombin generation in hemophilic plasma supplemented with FVIII, FVIIa, or FEIBA.

Central venous catheters assist infusion of coagulation factors in hemophiliacs but can be problematic due to mechanical dysfunction, infection, and thrombosis. The effect of low molecular weight heparin, unfractionated heparin, or covalent antithrombin-heparin complex on thrombin generation in factor concentrate-supplemented hemophilic plasma were studied. Thrombin levels were similar to normal plasma after the addition of factor eight inhibitor bypassing agent to hemophilic plasma. At 0.2 U/ml of heparin, covalent antithrombin-heparin inhibited free thrombin generation to a greater degree than heparin and low molecular weight heparin. Covalent anti-thrombin-heparin may give a greater anticoagulant response in hemophilic plasma supplemented with factor VIII or factor VIIa than with factor eight inhibitor bypassing agent. Requirements for heparin in hemophilic patients with thrombosis may depend on the procoagulant treatment used.

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

Isoform composition of antithrombin in a covalent antithrombin-heparin complex.

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.

Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombin-heparin complex.

Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although approximately 30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT + H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (

The effects of chemotherapeutic agents on the regulation of thrombin on cell surfaces.

Thromboembolic disorders are common in cancer patients. Two major contributing factors are central venous catheters for drug delivery and the use of l-aparaginase, which decreases the plasma antithrombin level, but the causes of the hypercoagulable state in these patients are not fully understood. In this study, the T24/83 cell line was used as a model to investigate the effects of chemotherapeutic agents on cell surface thrombin regulation. Plasma thrombin generation and prothrombin consumption was increased in most of the treated cells, particularly vincristine- and adriamycin-treated cells (P < 0.05), compared with controls. However, no free thrombin generation or prothrombin consumption was observed in factor VII (FVII)-depleted plasma. No significant differences in the levels of thrombin-alpha2-macroglobulin (IIa-alpha2M) and thrombin-anti-thrombin (TAT) were observed between controls and any of the treatments, except for vincristine- and adriamycin-treated cells, which showed a significant difference in TAT production (P < 0.05). Also, there was an upregulation in tissue factor (TF) mRNA expression in etoposide-, methotrexate- and vincristine-treated monolayers compared with controls, as well as an upregulation in TF protein production in vincristine-treated cells. The data suggests that thrombin generation occurs via the extrinsic (TF-dependent) coagulation pathway on cell surfaces and that some chemotherapeutic agents are able to upregulate TF mRNA and protein expression in T24/83 cells.