Walter Jeske

Acute Onset Human Atrial Fibrillation Is Associated With Local Cardiac Platelet Activation and Endothelial Dysfunction

OBJECTIVES The purpose of this study was to determine whether acute onset atrial fibrillation (AF), independent of other risk factors, predisposes to an early prothrombotic state. BACKGROUND Several risk factors predispose to the hypercoagulable state in human AF, but whether acute onset AF alone is prothrombotic remains unclear. METHODS Patients with paroxysmal AF (n = 22) underwent radiofrequency catheter ablation. All patients presented in sinus rhythm. Baseline blood samples were obtained simultaneously from the femoral vein (systemic sample) and the coronary sinus (local cardiac sample). The AF was induced by burst atrial pacing in 14 patients (AF group). A control group (n = 8) underwent atrial pacing at 120 beats/min. Blood samples were recollected after 15 min. Platelet P-selectin expression (CD62) was measured using flow cytometry. Markers of thrombin generation (thrombin antithrombin complex, prothrombin fragment 1.2), inflammation (C-reactive protein, interleukin-6), and nitric oxide were measured using enzyme-linked immunosorbent assays. RESULTS Neither local nor systemic platelet activation changed in the control group. In the AF group, local cardiac platelet activation (percent P-selectin [+] platelets) increased significantly (2.2 +/- 0.6% to 2.8 +/- 1.0%, p = 0.007); however, systemic platelet activation did not change. The AF group had increased local thrombin generation (thrombin antithrombin complex: 8.5 +/- 7.6 ng/ml to 33.2 +/- 17.4 ng/ml, p = 0.003; prothrombin fragment 1.2: 95.6 +/- 45.6 micromol/l to 243.8 +/- 120.1 micromol/l, p = 0.003), decreased nitric oxide production (25.2 +/- 10.8 micromol/l to 22.3 +/- 10.0 micromol/l, p < 0.02), and no change in inflammatory markers. CONCLUSIONS Human AF causes local cardiac platelet activation within minutes of onset. The results demonstrate how AF alone, independent of other risk factors, may contribute to the hypercoagulable state.

Low-molecular-weight heparins: pharmacologic profile and product differentiation

The interchangeability of low-molecular-weight heparins (LMWHs) has been the subject of discussion since these products were first introduced for the prophylaxis of deep vein thrombosis. Experimental evidence now exists to show that LMWHs differ from each other in a number of characteristics. Products have been differentiated on the basis of molecular weight and biologic properties, but only limited information derived from the clinical setting is available. Potency has been described on the basis of anti-Factor Xa activity, but at equivalent anti-Xa activities, the anti-Factor IIa activity of different products shows marked variations. At the relatively small doses used for the management of postsurgical deep vein thrombosis, the effect of these interproduct differences may be relatively minor, but as LMWHs are developed for therapeutic use at much higher doses, such differences may become clinically important. Variations in safety and efficacy reported in clinical trials of LMWHs may reflect the known differences in their molecular composition and pharmacologic properties.

Laboratory Diagnosis of Heparin-Induced Thrombocytopenia

The major cause of morbidity and mortality in pa tients with type 1 diabetes mellitus is vascular disease and the death rate in this group of patients can be up to six times that of the general population. Elevated levels of blood glucose can cause endothelial cell damage, and markers of endothelial dam age such as von Willebrand factor (vWF) and thrombomodulin (TM) have been reported to increase in adult diabetic patients. Growth factors are strongly linked to smooth muscle cell pro liferation that contributes significantly to the vascular occlusive process and it has been shown that vascular endothelial cell growth factor (VEGF) stimulates release of vWF from endo thelial cells. Vascular endothelial cell growth factor levels have been shown to be increased in vitreous fluid from the eyes of diabetic patients with proliferative retinopathy compared to those without. In this study we have shown that plasma levels of both TM and VEGF were significantly increased in juvenile diabetic patients with no clinical evidence of vascular disease compared to normal age and sex-matched control subjects. Me dian TM levels were 45.5 ng/mL (I.Q.R. 34 to 56 ng/mL) and 61 ng/mL (I.Q.R. 41 to 72 ng/mL) in the control group and in the diabetic patients respectively (p = .0005) and median lev els of VEGF were 19.6 pg/mL (I.Q.R, 15.9 to 28.1 pg/mL) in the control group and 37.1 pg/mL (I.Q.R. 22.1 to 50.3 pg/mL) in the diabetic patients (p = .027 Mann-Whitney U test). This suggests that microvascular disease begins in childhood and can be detected using laboratory tests before any clinical changes are apparent. Key Words: Diabetes mellitus— Thrombomodulin—Vascular endothelial cell growth factor.

Fondaparinux: a synthetic heparin pentasaccharide as a new antithrombotic agent

Fondaparinux (Arixtra®, Sanofi-Synthélabo/Organon) is the first of a new class of antithrombotic agents distinct from low molecular weight heparins (LMWHs) and heparin. It is a chemically synthetic pentasaccharide mimicking the site of heparin that binds to antithrombin III (AT). It exhibits only factor (F) Xa (FXa) inhibitor activity via binding to AT, which in turn inhibits thrombin generation. In contrast to heparin and LMWH, plasma anti-Xa activity corresponds directly to levels of fondaparinux. It does not release tissue factor pathway inhibitor (TFPI). There is nearly complete bioavailability by the sc. route, rapid onset of action, a prolonged half-life in both iv. and sc. (14 - 20 h) dosing regimens and no metabolism preceding renal excretion. Phase IIb clinical studies have identified a dose of 2.5 mg once-daily for prophylaxis of venous thrombosis. Four Phase III studies (n > 7000) have demonstrated a combined 50% relative risk reduction of venous thromboembolic events in orthopaedic surgery patients in comparison to the LMWH, enoxaparin. Haemmorrhagic complications for fondaparinux were either comparable to or higher than those for LMWH. The activated partial thromboplastin time (aPTT) is not affected by fondaparinux. At present, laboratory monitoring is not recommended. Clinical trials for treatment of established thrombosis, coronary syndromes and adjunct to thrombolytic therapy are in progress.

Pharmacodynamic and pharmacokinetic properties of enoxaparin : implications for clinical practice.

Enoxaparin is a low-molecular-weight heparin (LMWH) that differs substantially from unfractionated heparin (UFH) in its pharmacodynamic and pharmacokinetic properties. Some of the pharmacodynamic features of enoxaparin that distinguish it from UFH are a higher ratio of anti-Xa to anti-IIa activity, more consistent release of tissue factor pathway inhibitor, weaker interactions with platelets and less inhibition of bone formation. Enoxaparin has a higher and more consistent bioavailability after subcutaneous administration than UFH, a longer plasma half-life and is less strongly bound to plasma proteins. These properties mean that enoxaparin provides a more reliable anticoagulant effect without the need for laboratory monitoring, and also offers the convenience of once-daily administration. Clinical studies have confirmed that these pharmacological advantages translate into improved outcomes. There are important pharmacokinetic and pharmacodynamic differences between enoxaparin, other LMWHs and UFH, and therefore these molecules cannot be regarded as interchangeable.

Synthetic pentasaccharides do not cause platelet activation by antiheparin-platelet factor 4 antibodies.

A synthetic selective inhibitor of factor Xa, the pentasaccharide SR90107A/Org31540 is in clinical develop ment for the prophylaxis of postsurgical deep vein thrombosis. Another synthetic pentasaccharide with even more sustained inhibition of factor Xa, SanOrg34006, has also been developed. Both of these agents were tested in comparison to unfraction ated heparin and a low molecular weight heparin (enoxaparin) for their relative platelet activation potential in heparin-induced thrombocytopenia assays. Sera from patients (n = 30) with heparin-induced thrombocytopenia were pooled and validated for heparin-dependent aggregation responses. Using heparin- platelet factor 4 Sepharose columns, antibodies to heparin- platelet factor 4 were purified from the same pool. The effects of heparin, enoxaparin, SR90107A/Org31540, and San Org34006 were evaluated in a platelet aggregation assay using platelet donors (n = 10). At comparable concentrations, hep arin and enoxaparin consistently produced platelet activation, whereas both pentasaccharides failed to produce a response at a concentration up to 100 μg/mL (∼50 μM). Similarly, in the 14C-serotonin release and flow cytometric assays, heparin and enoxaparin produced positive responses (n = 30), whereas the two pentasaccharides consistently failed to produce any effect. These observations suggest that the two pentasaccharides with highly selective anti-Xa activity are devoid of generating anti heparin-platelet factor 4 antibody, do not produce heparin- induced thrombocytopenic responses and may inhibit active heparin-induced thrombocytopenia antibody platelet activation.

Mechanisms of Venous and Arterial Thrombosis in Heparin-Induced Thrombocytopenia

Since the reports by Weismann and Tobin in 1958 and Roberts et al. in 1964 called attention to paradoxical thrombosis in patients treated with heparin, the thrombotic aspect of the heparin-induced thrombocytopenia syndrome (HIT) has been emphasized. Yet to this day, the mechanism of thrombosis associated with HIT (HITT) is unclear. It is important to understand the etiology of HITT because of its devastating clinical consequences. We believe one rational approach to understand the mechanism underlying HITTS is to invoke Virchows triad: stasis, vascular injury and a hypercoagulable state. A hypercoagulable state exists in all HIT patients due to platelet activation by heparin antibody binding. Thrombin generation from platelet microparticles and exposed platelet phospholipid, coupled with stasis (elderly bedridden or otherwise sedentary ill patients who comprise the majority of the HIT population), provide two risk factors that can lead to venous thrombosis. A hypercoagulable state coupled with endothelial cell dysfunction due to injury from heparin antibody, activated platelets, leukocytes, platelet microparticles, complement, atherosclerosis or medical intervention can lead to arterial thrombosis. Of patients with HIT, HITT occurs in about 25%, suggesting that a second set of patient specific risk factors, in addition to the generation of pathological heparin antibodies, determine whether HITT will develop. Interaction between activated platelets and other platelets, and with endothelial cells, leukocytes, neutrophils, monocytes and cytokines are areas of research that may provide more specific characterization of the hypercoagulable state and vascular damage. Nuances involving genetic variation in platelets, endothelial cells and immune function are also likely to be a major component of the observed variability of this disease spectrum. Virchows triad may explain the different manifestations of HITTS.

Affinity purification of heparin‐dependent antibodies to platelet factor 4 developed in heparin‐induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H‐PF4) complexes were purified from the plasma of three patients with heparin‐induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 m NaCl at pH 7·5 (peak 1) and the remainder was obtained using 0·1 m glycine/0·5 m NaCl at pH 2·5 (peak 2). In an enzyme‐linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H‐PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H‐PF4 complexes. Finally, IgG in patient 3 only bound to H‐PF4 and was unreactive with PF4 alone. Using [14C]‐serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H‐PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.

Rivaroxaban - an oral, direct Factor Xa inhibitor - has potential for the management of patients with heparin-induced thrombocytopenia

Rivaroxaban is an oral, direct activated Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. Currently available anticoagulants include unfractionated heparin (UFH) and low molecular weight heparins (LMWHs); however, their use can be restricted by heparin‐induced thrombocytopenia (HIT). HIT is usually caused by the production of antibodies to a complex of heparin and platelet factor‐4 (PF4). This study was performed to evaluate, in vitro, the potential of rivaroxaban as an anticoagulant for the management of patients with HIT. UFH, the LMWH enoxaparin, fondaparinux and the direct thrombin inhibitor argatroban were tested to enable comparative analyses. Rivaroxaban did not cause platelet activation or aggregation in the presence of HIT antibodies, unlike UFH and enoxaparin, suggesting that rivaroxaban does not cross‐react with HIT antibodies. Furthermore, rivaroxaban did not cause the release of PF4 from platelets and did not interact with PF4, unlike UFH and enoxaparin. These findings suggest that rivaroxaban may be a suitable anticoagulant for the management of patients with HIT.

The role of tissue factor pathway inhibitor in the mediation of the antithrombotic actions of heparin and low-molecular-weight heparin.

&NA; It is widely accepted that antithrombin III (ATIII) mediated anti‐Xa and anti‐IIa effects are the sole determinant of the antithrombotic actions of unfractionated heparin (UFH) and low‐molecular‐weight heparins (LMWHs). However, there are several unexpected observations such as the greater than 100% bioavailability of subcutaneously administered LMWH as measured by a chromogenic based anti‐Xa method. The authors have proposed that, besides ATIII mediated antiprotease actions, additional endogenous factors may be responsible for the observed therapeutic and prophylactic actions of heparins. With the identification of tissue factor pathway inhibitor (TFPI) some of the unexpected effects of heparins can now be clarified. To investigate the role of heparin‐releasable TFPI on LMWHs the anti‐Xa and TFPI antigen levels after prophylactic and therapeutic administration of UFH and LMWHs have been studied in defined clinical trials. Regardless of the dosage designation (mg/kg or units/kg) each LMWH followed a distinct TFPI release profile. Similarly, in the intravenous studies these LMWHs produced an instantaneous increase in the TFPI antigen level. The anti‐Xa effects did not always follow the same pattern as the TFPI antigen levels. These data suggest that the anti‐Xa potency of a given LMWH is not the sole determinant of the antithrombotic actions of heparin and LMWH. In addition to pharmacologic agents, the effect of sequential compression devices (SCD) on the release of TFPI was also studied. A two‐fold increase in TFPI antigen levels was observed in normal volunteers undergoing long leg compression for 1 h. These data suggest that, in addition to LMWH and heparin, manipulation of the vascular endothelium by SCD results in the release of TFPI.