Clinical and Applied Thrombosis/Hemostasis

The purpose of this study was to determine the in vitro effects of different anticoagulant drugs on fibrinopeptide A (FPA) generation inhibition and to identify whether there is any correlation between FPA generation, Hemochron ACT, global clotting as says, and chromogenic assays. Unfractionated heparin is a conventionally used anticoagulant. New anticoagulant drugs such as low molecular weight heparins (LMWHs), pentasaccharide, and antithrombin drugs are now approved for various indications. Anti-Xa drugs are in various phases of clinical development. The influence of different anticoagulant agents has been studied on fibrinopeptide A generation, Hemochron celite ACT, global clotting assays, and chromogenic anti-Xa and anti-Ila assays. Different LMWHs (Clivarin, Dalteparin, Enoxaparin, and Tinzaparin), anti-Xa agents (Pentasaccharide, DX-9065a and unfractionated heparin), and anti-Ila agents (PEG-Hirudin, Hirudin, Efegatran and Argatroban) were studied. The blood from healthy volunteers (n=4) was drawn for each drug. Imuclone FPA enzyme-linked immunosorbent kit assay, Hemochron celite ACT assay, global clotting assays (PT, APTT, Heptest-HI, thrombin time), and Loyola chromogenic anti-Xa and anti-Ila assays were studied. Pentasaccharide demonstrated minimal effects on the whole blood clotting time such as ACT and on inhibition of FPA generation (IC₅₀ > 25 /g/mL). DX-9065a exhibited a significant prolongation of ACT and marked inhibition of FPA generation (IC50 = 4.12 Ag/mL). Unfractionated heparin showed a marked inhibition of FPA generation (IC,, = 5.16 Aog/mL). Pentasaccharide, DX-9065a and UFH showed a marked correlation between ACT and inhibition of FPA generation. LMWHs demonstrated concentrationdependent inhibition of FPA generation. LMWHs studied showed good correlation between FPA generation inhibition and ACT test. Similar correlation was seen between FPA generation inhibition and the APTT, anti Xa (heptest-HI assay) and anti-IIa activity. Anti-IIa drugs demonstrated concentration-dependent inhibition of FPA generation. Their FPA generation inhibition potency is correlated with the ACT assay. A strong correlation between Hemochron ACT and FPA generation inhibition was observed. Based on this significant correlation, the FPA generation inhibition can be predicted by point-of-care ACT assay.

This commentary briefly reviews the controversies of therapeutic and generic interchangeability, as they apply to the antithrombotic drug class called low-molecular-weight heparin (LMWH). Recommendations are prepared for the generic LMWH approval process by various regulatory bodies.

Genetic and acquired factors may influence phenotypic expression of inherited thrombophilia. Hypofibrinolysis due to excess PAI-I can be found in patients with deep vein thrombosis (DVT) and 4G/5G polymorphism of the PAI-1 gene may modulate the inhibitor's synthesis. In 149 patients with inherited thrombophilia, the possible thrombotic contribution of both 4G/5G polymorphism and PAI-1 plasma levels was evaluated. Sixty-seven patients with idiopathic DVT and 98 normal subjects were also studied. By comparison with controls, a significantly higher prevalence of 4G/4G genotype was seen in idiopathic DVT and in thrombophilia patients, although in this latter group the difference only remained significant in cases symptomatic for thrombosis (p=0.01). The 4G/4G genotype was associated with a greater risk of thrombosis both in symptomatic thrombophilia patients (OR 2.85, 95% CI 1.26-6.46) and in idiopathic DVT patients (OR 3.1, 95% CI 1.26-7.59). The greater frequency of 4G allele in symptomatic thrombophilia patients with respect to controls was statistically significant (p=0.04). Compared to healthy subjects, PAI-i:Ag levels were higher in symptomatic thrombophilia patients and related to the 4G/5G polymorphism, with significantly higher values in the 4G/4G carriers. In conclusion, PAI-1 4G/5G polymorphism may influence PAI-i expression and thrombotic risk in patients with inherited thrombophilia.

Platelet activation markers (platelet-derived microparticles and P-selectin on activated platelets), chemokines (monocyte chemotactic peptide and regulated on activation normally T-cell expressed and secreted), and soluble markers (sP-selectin, sE-selectin, sVCAM-1, and sCD14) were measured and compared in patients with pulmonary embolism (PE). These substances are thought to participate in the pathogenesis of PE. Levels of all of the platelet activation markers, chemokines, and soluble markers were higher in the patients with PE than in normal controls. Levels of platelet activation markers were also significantly increased postoperatively after total knee arthroplasty. Anti-platelet therapy significantly inhibited the elevation of platelet activation markers after total knee arthroplasty. These findings suggest that antiplatelet therapy may be useful for PE-related interaction of platelets, leukocytes, and endothelial cells.

New synthetic direct and indirect factor Xa or factor Ila inhibitors are increasingly used for the prevention and treatment of thrombotic disorders, including patients suffering from antiphospholipid syndrome. In this study, the effects of the synthetic direct factor Xa inhibitor DX-9065a, the indirect synthetic heparinomimetic pentasaccharide, and the direct factor IIa inhibitor Argatroban were studied. These two widely used assays for the detection of lupus anticoagulant, namely the tissue thromboplastin inhibition (TTIT) and the dilute Russell viper venom tests (DRVVT) proved useful. The drugs were added to a normal human plasma pool ranging in concentration from 0.04 to 10 Ag/mL. Using the two tests named above, DX-9065a and Argatroban showed a dose-related prolongation of TTIT and DRVVT in the concentration range from 0.04 to 5 Amol/mL, but the pentasaccharide only slightly prolonged the clotting times of these assays even at high concentrations. Argatroban had the more pronounced effect on both tests when compared with DX-9065a (p<0.001). The most responsive assay for DX-9065a up to a concentration of 2.5 Amol/mL was the DRVVT. For Argatroban both TTIT and DRVVT were equally responsive. Patients whose plasma was tested for suspected lupus anticoagulant and who have been given DX-9065a or Argatroban may have false-positive results with the TTIT tests and DRVVT. This effect should be considered during patient management. These results indicate that these assays could be used for the effective quantitation of the direct factor Xa or factor Ila inhibitors when suitable controls are used.

Infection with human immunodeficiency virus (HIV) may lead to hemostatic imbalances. Forty-nine consecutive patients with acute opportunistic infections were screened for thrombophilic parameters. A follow-up investigation was performed after 10 8 weeks in 26 patients. In acutely ill patients, the incidence of protein S deficiency was 67% (33/49) and of protein C deficiency 25% (12/49), while at the follow-up visit the incidences were 54% (14/26) and 8% (2/26), respectively. Protein S and protein C levels increased significantly from initial to follow-up visit (p < 0.05). Lupus anticoagulants were not detected and anticardiolipin IgG antibodies were present in 11.4% (5/44). Three patients presented with deep venous thrombosis on admission; in two, protein S or protein C deficiency was observed. In conclusion, an acquired protein S and protein C deficiency often develop in patients with HIV and acute illness; this may be reversible after treatment for opportunistic infections.

PAI-2 is one of the regulators of the fibrinolytic system. The importance of the fibrinolytic cascades in the pathogenesis of myocardial infarction has been demonstrated by many investigators. Recently, some investigators have shown that two variants of PAI-2, designated A and B, are associated with the formation of large molecular PAI-2 complexes. This polymorphism is therefore present a genetic predisposition for the development of coronary artery disease and multiple sclerosis. Therefore, the prevalence of this polymorphism among 45 patients with MI and 20 control subjects was investigated. The AA genotype of the PAI-2 gene was found to be more frequent among those subjects with MI. These data provide evidence that a polymorphism of the PAI-2 gene is associated with an increased risk of MI.

The green pit viper (Trimeresurus albolabris and Trimeresurus macrops) is a common venomous snake in Thailand. The green pit viper venom has major effects on the hematologic system. The venom is found to have a thrombin-like effect in vitro but cause a defibrination syndrome in vivo. Unlike other pit viper snakes (such as Malayan pit viper and Russel's pit viper), the venomous effect on red blood cells is rarely mentioned. Clinical features of venomous snake bites vary from asymptomatic to fatal bleeding. In this study, an attempt was made to discern hematologic parameters in green pit viper envenomation and to characterize the changes in blood cells. The study included data from 40 patients who were hospitalized at the King Chulalongkorn Memorial Hospital during 1995 to 1999 and 40 normal, healthy subjects as control. Concerning platelet determination, significantly decreased (p < 0.05) total platelet count and mean platelet volume (MPV) were demonstrated in envenomous blood. The changes in blood cells may be partly due to the effect of green pit viper toxin on the platelet morphology. However, the explanation of this phenomenon requires further investigation for the specific pathophysiology. The components of green pit viper venom that have these hematologic effects in humans are interesting and should be further identified.

A patient with systemic lupus erythematosus and anticardiolipin antibodies and antibodies to platelet factor 4/heparin complexes suffered an acute myocardial infarction caused by delayed heparin-induced thrombocytopenia after heparin administration given to treat pulmonary hypertension. Furthermore, additional heparin administration for emergency coronary angiography appeared to have led to an acute immunoreaction, which might have resulted in acute coronary occlusion during coronary angiography and to a decreased platelet count. The present findings suggest that one must suspect delayed-type HIT in rare cases of induction of thrombosis after the cessation of heparin treatment, and avoid re-exposure to heparin in such cases.

Congenital absence of the inferior vena cava (AIVC) has been reported as a risk factor of deep vein thrombosis (DVT) in young people. DVT is caused by an interaction between congenital coagulation abnormalities and acquired risk factors. We observed an 18-year-old patient with AIVC who developed recurrent deep vein thrombosis at the left leg. Molecular studies showed an etherozigousity for FV Leiden gene (G1691A) and a homozigousity for methylenetetraidrofolate reductase gene (C677T) in absence of folate and vitamin B12 deficiency. After the second DVI episode, the patient has been treated with heparin and oral anticoagulant without discontinuation.