Clinical and Applied Thrombosis/Hemostasis

Although both argatroban and lepirudin are used for the management of heparin-induced thrombocytopenia (HIT), data comparing these agents are lacking. The objective of this project was to compare the clinical outcomes of lepirudin vs argatroban therapy. Patients who received a direct thrombin inhibitor (DTI) from January 2000 to December 2001 were identified. Medical charts were retrospectively reviewed and relevant data extracted. The primary efficacy outcome was effective anticoagulation and the primary safety outcome was major bleeding. Data were analyzed using the t test and Fisher’s exact test. Sixty-one lepirudin patients and 29 argatroban patients received a DTI during the study period. A new diagnosis of HIT was the indication for DTI therapy in 44.8% of argatroban patients and 57.4% of lepirudin patients. Effective anticoagulation was achieved in 77.8% of argatroban patients and 69.5% of lepirudin patients (p = .61). Major bleeding occurred in 10.3% and 11.5% of argatroban and lepirudin patients, respectively (p = 1.0). Argatroban and lepirudin demonstrated comparable safety and efficacy outcomes.

We prospectively assessed whether thrombophilia and hypofibrinolysis, amplified by thrombophilic hormone replacement therapy (HRT), were associated with retinal vein occlusion (RVO). We studied 44 cases (18 men, 26 women), ≥ 3 months after RVO, 42 with central RVO, 2 with branch RVO, in the consecutive order of their referral by 2 community-based ophthalmologists. PCR and serologic coagulation assays were compared to 83 and 40 healthy adult normal controls, respectively. The 4G allele frequency of the plasminogen activator inhibitor-1 (PAI-1) gene, associated with hypofibrinolysis, was 56 of 88 (64%) in cases vs 79 of 166 (48%) in controls, X2 = 5.95, p = .015. The PAI-1 gene product, plasminogen activator inhibitor activity (PAI-Fx), was higher in cases than controls (age-race-sex-adjusted mean 12.2 U/mL vs 6.3, p = .013). By stepwise logistic regression, the PAI-1 gene 4G allele was associated with RVO, odds ratio 1.94, 95% CI 1.12-3.34, p = .018. Thrombophilic resistance to activated protein C (RAPC) was present in 6 of 32 (19%) of cases vs 0 of 40 (0%) controls, Fisher’s p [p_f] = .006. Thrombophilic high factor VIII (>150%) was present in 3 of 30 (10%) cases vs 0 of 40 (0%) controls, p = .041, p_f = .07. Comparing 23 RVO cases ≤ age 55 and controls ≤ age 55 (n = 44 for PCR, n = 40 for serologic measures), RAPC was present in 17% of cases vs 0% controls (p_f = .026), high Factor VIII in 17% vs 0% (p_f = .026), heterozygosity for the G1691A Factor V Leiden mutation in 13% vs 2% (p_f = 0.11), and the 4G allele frequency of the PAI-1 gene 74% vs 39% (p = .0001). PAIFx was higher in cases than controls (age-race-sex adjusted mean 12.7 U/mL vs 6.7, p = .016). The case-control odds ratio for the PAI-1 4G allele was 5.54, 95% CI = 1.86-16.7, p = .002. Of the 26 women, 9 (35%) took HRT; 4 of the 9 had PAI-1 gene 4G4G homozygosity, 2 had thrombophilic high anticardiolipin antibody (IgG), 1 was heterozygous for the G1691A Factor V Leiden mutation, and 2 were heterozygous for the thrombophilic PL A1/A2 mutation of the platelet glycoprotein IIb/IIIa gene. Associations between heritable coagulation disorders and RVO, most marked in cases ≤ age 55, and often amplified in women by thrombophilic HRT, are, speculatively, causal.

Plasma levels of granulocyte-derived elastase (GE-XDP), D-dimer, and soluble fibrin (SF) were examined in 177 patients with disseminated intravascular coagulation (DIC) of various etiologies. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with sepsis and solid cancer. The ratio of GE-XDP/D-dimer was significantly high in patients with trauma, burn, and sepsis, suggesting that fibrinolysis due to GE-XDP may be dominant in DIC. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with overt DIC and correlated with DIC score. Plasma levels of GE-XDP, but not SF, correlated significantly with D-dimer. Plasma levels of D-dimer, but not SF, correlated significantly with plasmin plasmin inhibitor complex (PPIC). Plasma levels of GEXDP and D-dimer, but not SF, were significantly high in nonsurvivors. Plasma levels of GE-XDP, but not SF, correlated significantly with sepsis-related organ failure assessment (SOFA) score. These results suggest that GE-XDP is a potentially useful marker for the diagnosis of overt-DIC and as a predictor of organ failure-related outcome.

This paper presents an analysis of 24 cases in which recombinant factor VIIa (rFVIIa) was used in the management of hemorrhage in patients with thrombocytopenia associated with hematologic malignancies. This is the largest case aggregation to date and focuses on preliminary experience in the off-label use of this hemostatic agent. Data were extracted from the international, Internet-based registry, www.haemostasis.com, accessed in September 2003. The search results were manually cross-checked against monthly summary reports. The physicians providing the cases were contacted individually to approve the use of their cases, supply any information missing from the database, and validate the data already held. Patients with acute myeloid leukemia, acute lymphoblastic leukemia, Hodgkin’s disease, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, B-cell or T-cell lymphoma, or aplastic anemia received rFVIIa at total doses of between 18 and 1040 μg/kg body weight. Bleeding stopped in 11 of 24 (46%) patients, markedly decreased in 8 of 24 (33%) patients, and decreased in 4 of 24 (17%) patients. In most patients, the response was achieved within 2.5 hours of administration of rFVIIa. The use of rFVIIa was generally well tolerated—1 case of ischemic stroke was considered to be possibly related to rFVIIa administration, but this has yet to be confirmed. A review of these 24 cases submitted to the www.haemostasis.com database suggests that rFVIIa is beneficial in the management of hemorrhage in patients with thrombocytopenia and hematologic malignancies. This warrants further investigation in rigorously controlled clinical trials.

Despite of several lines of evidence indicating a pathophysiologic role of platelets in pulmonary hypertension, the occurrence of chronic endogenous platelet activation has been a matter of debate. It was hypothesized that the pattern of tyrosine phosphorylation of platelet proteins examined ex vivo could provide information on the state of platelet activation. This was examined in 10 patients with pulmonary arterial hypertension aged 18 to 53 years. Phosphotyrosine density and the amounts of specific proteins were analyzed in resting platelets after reaction with anti-phosphotyrosine, anti-pp60^c-src, anti-pp125^FAK, and anti-αIIbβ3 antibodies. There was a 79% increase in protein-associated phosphotyrosine in patients in comparison to levels in controls (p<0.05). In particular, phosphorylation on tyrosine residues of pp120 and pp125^FAK increased 24% and 57%, respectively (p<0.05). Although pp60^c-src-associated phosphotyrosine was not altered in the patient group as a whole, it was clearly decreased in three subjects. Platelet content of β3 integrin, pp60^c-src, and pp125^FAK, was not altered. This pattern of phosphorylation suggests an ongoing process of platelet activation. Because phosphorylation of pp125^FAK is a late, integrin-dependent event, results suggest that platelet activation and aggregation occur in vivo in these patients.

Hydroxymethyl-glutaryl-CoA-reductase inhibitors (statins) reduce cardiovascular mortality by decreasing cholesterol as well as by non-lipid-related actions. Oxidized low-density lipoproteins (ox-LDL) are pro-atherogenic molecules and potent platelet agonists. CD36 and lectin-like ox-LDL receptor-1 (LOX-1) are specific ox-LDL receptors also expressed in platelets. This study was planned to address whether treatment with atorvastatin 10 mg/day, pravastatin 40 mg/day or simvastatin 20 mg/day could affect platelet CD36 and LOX-1 expression. Twenty-four patients for each treatment were evaluated after 3, 6, and 9 days and at 6 weeks for complete lipid profile (chromogenic), ox-LDL (ELISA), platelet P-selectin (P-sel), CD36, LOX-1 (FACS), and intracellular citrullin recovery (iCit) (HPLC). Data show hyperactivated platelets (P-sel absolute values, percent variation in activated cells, all p < 0.001), and CD36 and LOX-1 overexpression (all p < 0.001) in patients at baseline. P-sel, CD36, and LOX-1 were significantly decreased by atorvastatin and simvastatin (all p < 0.01) and related with iCit increase (r = 0.58,p < 0.001) and platelet-associated ox-LDL (r = 0.51, p < 0.01) at 9 days. Pravastatin reduced LOX-1 and P-sel (p < 0.05) at 6 weeks in relation with decreased LDL and ox-LDL (r = 0.39, p < 0.01 and r = 0.37, p < 0.01, respectively). These data suggest that atorvastatin and simvastatin reduce platelet activity by exposure of CD36 and LOX-1 before significant LDL reduction, whereas pravastatin action is detected later and in relation with LDL and ox-LDL lowering. Rapid and consistent reduction of CD36 and LOX-1 could be considered a direct anti-atherothrombotic mechanism related to the role of ox-LDL in platelet activation, platelet-endothelium interactions, and NO synthase activity.

Prostaglandins (PGs) and thromboxane (TX) produced by cyclooxygenase (COX) have a great influence on vascular systems and platelet functions. The serum levels of epidermal growth factor (EGF) and PGs were measured in patients with lung cancer treated with gefitinib, and the influence of EGF on platelet aggregation was investigated. Twenty patients were investigated. The serum level of TXB₂ increased significantly in all patients who received gefitinib for 2 weeks (before vs. after = 94.1 ± 47.3 vs. 190.9 ± 54.3, p<0.01). TXB₂ also increased significantly in responders without concurrent chemotherapy (before vs. after = 79.3 ± 35.5 vs. 194.5 ± 58.1, p<0.05), but not in non-responders (before vs. after = 106. 5 ± 65.8 vs. 162. 2 ± 52.8, N.S.). PG 6-keto F1α and PGE₂ did not exhibit significant changes. Furthermore, EGF showed no significant change (after vs. before = 234 ± 35 vs. 276 ± 72, N.S.). Although there was no correlation between the levels of EGF and TXB₂ (N.S.), the PG 6-keto F2α/TXB₂ ratio decreased significantly (before vs. after = 0.054 ± 0.018 vs 0.033 ± 0.015, p<0.05). The secondary platelet aggregation observed after high-dose adenosine diphosphate stimulation was inhibited after a 1-minute preincubation with EGF. Platelet aggregation in patients after gefitinib administration tended to accelerate and secondary aggregation was observed after low-dose adenosine diphosphate stimulation. We conclude that careful observation is needed for patients with chronic obstructive pulmonary disease, pulmonary fibrosis, and thromboembolic diseases receiving gefitinib. Furthermore, measurement of prostanoids may be a good predictor of the beneficial and adverse effects. Moreover, the combination of gefitinib with a COX inhibitor that regulates TXA₂/PGI₂ balance should be evaluated.

Pulmonary hypertension is common in patients with chronic obstructive pulmonary disease (COPD), but the precise mechanism of vascular impairment in these patients is unknown. We, therefore, decided to investigate whether endothelial cell dysfunction is present in patients with COPD with a wide range of chronic airflow obstruction before the development of severe pulmonary hypertension. Selected plasma markers of endothelial cell activity were studied: nitrate+nitrite (NO-₂/NO-₃), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), soluble selectins (endothelium sES, leukocyte sLS, platelet sPS), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1). Twenty-five patients with COPD (forced expiratory volume in one second/vital capacity [FEV₁/VC] < 88% predicted) and 29 healthy control subjects were recruited to the study. Among patients nine had a pulmonary artery systolic pressure (PASP) between 15 and 30 mmHg, 13 between 32 and 38 mmHg, 2 had a PASP of 41 and 42 mmHg, respectively. One patient had severe pulmonary hypertension with a PASP of 70 mmHg. The average FEV₁ of patients with COPD was 46 ± 4% predicted. As compared to control subjects, patients with COPD showed a significant increase in plasma levels of TM and TFPI, indicating that their endothelial cells are still able to produce potent coagulation inhibitors. Levels of NO-₂/NO-₃were similar in the two groups of subjects examined, further suggesting preserved endothelial function in patients with COPD. In regard to adhesion molecules, patients with COPD showed a reduction in sLS, sPS, and sPECAM-1, and an increase in sICAM-1. This study shows that endothelial cell activity is largely preserved in patients with COPD without severe pulmonary hypertension, suggesting that these patients, despite quite severe airway obstruction, retain reasonably normal endothelial function until they develop severe pulmonary hypertension.

The existence of an association between idiopathic intracranial hypertension (IIH) and coagulation disorders in men was assessed prospectively. Microthrombi, associated with thrombophilia-hypofibrinolysis, occlude arachnoid sinus villi, thus reducing resorption of cerebrospinal fluid, leading to IIH. Ten consecutively referred men with IIH, nine whites, one African American, median age 36 years, were 2 to 1 matched by age and race by healthy male controls. Polymerase chain reaction assays were done for four thrombophilic and one hypofibrinolytic gene mutations: G1691A factor V Leiden, G20210A prothrombin, C677T MTHFR, platelet glycoprotein IIb/IIIa (PL A1/A2), and 4G/5G polymorphism of the plasminogen activator inhibitor (PAI-1) gene promoter. Coagulation measures in plasma included dilute Russel’s viper venom time (dRVVT), activated partial thromboplastin time (aPTT), the lupus anticoagulant, factor VIII, factor XI, plasminogen activator inhibitor activity (PAI-Fx), protein C antigenic, protein S total (antigenic), protein S free (antigenic), antithrombin III (functional), and resistance to activated protein C (RAPC). Tests performed on serum included anticardiolipin antibodies, homocysteine, and Lp(a). The body mass index was 40 kg/m² or greater (extremely obese) in two men, 30 to 40 kg/m²(obese) in three, and was 25 to 30 kg/m² in five (overweight). Cases differed from controls for inherited 4G4G homozygosity of the PAI-1 gene, four of 10 (40%) vs. one of 20 (5%), Fisher’s p [p_f]= .031, and for high levels (>21.1 U/mL) of the hypofibrinolytic PAI-1 gene product, PAI-Fx, 5 of 10 (50%) vs. one of 18 (6%), p_f = .013. Thrombophilic factor VIII was high (≥ 150%) in three of 10 (30%) cases vs. zero of 16 (0%) controls, p_f=. 046. The thrombophilic lupus anticoagulant was present in two of 10 (20%) cases vs. zero of 32 (0%) controls, p_f = .052. Heritable hypofibrinolysis and heritable and acquired thrombophilia appear, speculatively, to be treatable etiologies of IIH in men. Understanding contributions of hypofibrinolysis and thrombophilia to the development of IIH should facilitate development of novel new approaches to treat this often-disabling neurologic disorder.

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of thrombin-activatable fibrinolysis inhibitor (TAFI) activity, the inhibitor of fibrinolysis, and also prothrombin time (PT), active partial thromboplastin time, and D-dimer and fibrinogen levels in 21 patients affected with clonal thrombocytemia as compared with 21 patients with reactive thrombocytosis and 21 healthy controls. In the clonal thrombocytemia group, plasma levels of TAFI activity were significantly higher than in both the reactive thrombocytosis and the control group. Plasma levels of leukocyte and platelet counts were significantly higher in the clonal thrombocytemia group than in the other two groups and also higher in the reactive thrombocytosis group than in the control group, which was also significant. Fibrinogen and D-dimer levels were higher in patients than in the control group but showed no significant difference between the clonal and secondary thrombocytosis groups. Plasma levels of PT and aPTT were higher in secondary thrombocytosis group than the clonal thrombocytosis group. The results of this study showed for the first time that TAFI activity is increased in patients with clonal thrombocytosis. These increased levels in clonal thrombocytosis can be considered a factor to explain the thrombotic tendency in myeloproliferative disorders.

Soluble P-selectin and whole blood aggregation (WBA) were measured after percutaneous coronary intervention in patients who then received antiplatelet therapy. One had subacute thrombosis on day 7. This patient’s WBA exhibited time-dependent enhancement. In addition, the accentuation of WBA on day 3 was observed when anti-α_IIbβ₃ antibody was added. The remaining 22 patients were divided according to WBA results on day 3 with anti-α_IIbβ₃ antibody added (A group, WBA enhanced; B group, WBA did not enhance). WBA on day 3 was similar in the two groups. The ratio of WBA with and without anti-α_IIbβ₃ antibody was higher in group A than in group B. A significant time-dependent increase of soluble P-selectin was observed in the A group. These results suggest that enhancement of WBA with anti-α_IIbβ₃ antibody after percutaneous coronary intervention predicts subacute thrombosis.

During percutaneous coronary intervention, slow coronary flow and distal embolization are still important problems, especially in cases with intracoronary thrombus. The aim of this study was to learn the effectiveness and early term results of thrombectomy with the X-SIZER catheter system in acute coronary syndrome. Twenty-nine patients (22 [76%] men; 55.9 ± 11.1 years) with acute coronary syndrome and intracoronary thrombus detected in coronary angiography were included into the study. X-sizer thrombectomy was applied to 14 of the patients, and conventional percutaneous transluminal coronary angioplasty (PTCA) was applied to the others. Baseline characteristics were similar in both groups. Mean thrombolysis in myocardial infarction (TIMI) flow increased from 0.8 ± 0.9 to 2.4 ± 0.6 in X-sizer-treated patients (p<0.001) and TIMI 3 flow was maintained in 71.4% of the patients. Similary, mean TIMI flow increased from 0.36 ± 0.81 to 2.73 ± 0.47 in conventional PTCA-treated patients (p<0.001) and TIMI 3 flow was maintained in 73% of the patients (NS). Mean myocardial blush grade (MBG) increased from 0.7 ± 0.7 to 2.6 ± 0.6 in X-sizer-treated patients (p<0.001) and from 0.27 ± 0.65 to 2.36 ± 0.67 in the conventional PTCA-treated patients (p<0.001). Postprocedural MBG 3 was obtained in 64.3% of X-Sizer-treated patients and in 45% of controls. Although microvascular function in the thrombectomy-applied patients was found better, there was no significant difference between the two groups. Furthermore it was detected that the use of tirofiban yielded no additional improvement in epicardial and microvascular flow. In acute coronary syndromes, use of X-sizer in addition to primary percutaneous coronary interventions is a safe and relatively effective method in the prevention of distal embolization.

Coagulation and fibrinolysis were determined in 67 Indonesian women admitted to the University Hospitals for delivery in Medan. They were diagnosed to be at term gestation (mean 39.3 ± 1.1 weeks) with moderate and severe preeclampsia (n=32) and in labor, and 8 had preterm labor (gestation mean 33.5 ± 2.6 weeks). Twenty-seven normal pregnant women in labor (gestation mean 39.7 ± 1.0 weeks) served as controls. Cord blood from 23 neonates from normal pregnancy and 31 neonates from preeclampsia was also evaluated. Preeclamptic women in labor showed further enhanced coagulation activation (F₁₊₂) with raised urokinase-like plasminogen activator (u-PA) activity and reduced plasminogen activator inhibitor-2 (PAI-2) levels. In preterm preeclampsia, significantly reduced antithrombin III (ATIII) and PAI-2 levels with further elevated tissue-type PA (t-PA) antigen and plasminogen activator inhibitor-1 (PAI-1) antigen were seen compared to normal pregnancy. These would suggest a state of enhanced thrombin generation with elevated fibrinolytic/inhibitor proteins in preterm preeclampsia. The reduced PAI-2 levels seen in preeclampsia have been suggested to be associated with reduced placental function. Neonates born to mothers of either normal pregnancy or preeclampsia at term showed similar hemostatic changes with reduced fibrinogen, ATIII, t-PA, u-PA antigen, PAI-1 levels, and coagulation activation compared to their respective maternal plasma levels. No significant differences in hemostatic parameters studied between the neonates of both cohorts were seen, and this would suggest that the neonates were protected from the adverse effects of preeclampsia and their hemostatic system was physiologically balanced.

A retrospective analysis of clinico-hematologic parameters of 18 factor XIII-deficient patients was carried out. The hematologic tests included activated partial thromboplastin time (APTT), prothrombin time (PT), and clot solubility. Laboratory diagnosis of FXIII deficiency was made where bleeding time, PT, APTT, and thrombin time were normal and the clot solubility test result with 5M urea was positive. Factor XIII level with family screening was performed using commercially available kits. History of prolonged bleeding from the umbilical stump was present in four (22.2%) patients. The most common site of bleeding was the skin (11 of 18 patients). Three patients were given prophylaxis (FFP in two, factor XIII in one). A high prevalence of recurrent abortion in female pa tients with FXIII deficiency (two of the three patients in this study) was observed.

Plasma viscosity and erythrocyte deformability play a key role in maintaining and regulating microcirculation. In vitro and in vivo studies suggested a role for nitric oxide (NO) in modulating flow-mediated vasodilatation and red blood cell deformability. Impaired NO availability due to mutations in eNOS gene might contribute to the altered haemorheologic state. The aim of this study was to investigate the role of eNOS T-786C, G894T, and 4a/4b polymorphisms in modulating the haemorheologic state in a clinical condition characterized by a microcirculatory disorder. Eighty patients with idiopathic sudden sensorineural hearing loss (ISSHL) and 80 healthy subjects were studied. By using a dominant model of inheritance, we found a significant association between eNOS 894T rare variant and ISSHL (odds ratio [OR] 894TT+GT = 2.08, p = 0.03) after adjustment with traditional vascular risk factors. A higher percentage of altered red cell deformability both in patients and in controls carrying the eNOS rare variants was found in comparison to subjects carrying the wild type. Apart from the disease, eNOS T-786C and G894T polymorphisms independently affected the deformability index (OR,-786CC+TC = 2.81, p = 0.01 and OR, 894TT+GT = 2.5, p = 0.02, respectively), in particular in subjects in whom the contemporary presence of the two rare alleles was observed (OR,-786CC+TC and 894TT+GT combined genotype = 6.9, p<0.0001). Our study documented that eNOS gene affects the red blood cell deformability, so possibly contributing to ISSHL, which may represent a suitable model of microcirculatory disorder.

Autoimmune thrombocytopenic purpura (ITP) is a disease that presents with skin and mucous membrane bleeding due to thrombocytopenia. In the literature, there are a few studies about the effect of high-dose steroid therapy on coagulation tests in different diseases, but their results are still controversial. In this study, coagulation parameters were investigated that might have a role in hemostatic compensation in childhood acute ITP before and after high-dose methylprednisolone (HDMP) treatment. The study includes 21 children age 1.5 to 14 years with acute ITP and 21 healthy age-matched control subjects. All patients with acute ITP received HDMP for 7 days. Before and after HDMP treatment (0 and 8 days) prothrombin time, partial thromboplastin time, fibrinogen, Protein C, Protein S, antithrombin III, and the levels of factor II (FII), FV, FVII, FVIII, FIX, FX, FXI, and FXII were studied in all subjects. The results were compared with those of the control group. Pre-treatment Protein C and Protein S levels in the patient group were significantly lower than those in the control groups (p<0.05). Protein S and Protein C levels were significantly improved after HDMP treatment in patient group. There were lower FV, FVII, FX values in the patient group compared to the control groups on admission. There was no difference in AT III and fibrinogen levels before and after treatment. As a result, some changes in the coagulation system associated with thrombocytopenia were observed in patients with acute ITP. These changes may be accepted as compensatory mechanisms to maintain hemostasis.

Hemophilia A is one of the most common bleeding disorders in man. Approximately half the families with a severe disease have an inversion of the factor VIII gene. The inversion may be detected with a long polymerase chain reaction. This is a simple and reproducible method that may yield satisfactory results in approximately 24 hours. The long-distance polymerase chain reaction amplify three very large amplicons with a very GC-rich region of several kilobases, and is detected with a Expand Long Template DNA polymerase (Roche, Mannheim, Germany). Recently this polymerase has been changed with a new chemical composition. The object of the present method is to standardize the technique using the new Expand Long polymerase. For the new protocol, the cycling conditions, the concentration of nucleotide primers, and the buffer are changed. The need for a rapid response is determined in the case of hemophilia A patients, not only by the desire to reach a proper classification, but also by the urgency to inform the carrier status of the mother or of a female relative.