Heparin induced thrombocytopenia: position paper from the Italian Society on Thrombosis and Haemostasis (SISET).

Heparin induced thrombocytopenia (HIT) is a rare immune mediated adverse drug reaction occurring after exposure to heparin. It is a serious and potentially fatal condition, which may be associated with the development of arterial or venous thrombotic events. Although known for many years, HIT is still often misdiagnosed. Pre- test clinical probability, screening for anti-PF4/heparin antibodies and documentation of their platelet activating capacity are the cornerstones of diagnosis. However, both clinical algorithms and test modalities have limited predictive values and limited diffusion so that the diagnosis and management is challenging in the clinical practice. For this reason, there is an unmet need for novel rational non-anticoagulant therapies based on the pathogenesis of HIT.The present paper reports the position of the Italian Society on Haemostasis and Thrombosis (SISET) in order to increase awareness of HIT among clinicians and other health care professionals and to provide information on the most appropriate management.

Platelet function tests: a comparative review.

In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation - adhesion, shape change, release reaction, and aggregation - have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT) dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]). POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well-tried and innovative platelet function tests and their methodological features and clinical applications are considered.

Hyperhomocysteinemia in patients with pulmonary embolism is associated with impaired plasma fibrinolytic capacity.

Hyperhomocysteinemia (HHcy) affects haemostasis and shifts its balance in favour of thrombosis. In vitro and in vivo studies suggested that HHcy may impair fibrinolysis either by influencing the plasma levels of fibrinolytic factors or by altering the fibrinogen structure. We investigated the influence of mild HHcy levels on plasma fibrinolytic potential by using clot lysis time (CLT) and fibrin susceptibility to plasmin-induced lysis in 94 patients with previous pulmonary embolism and no pulmonary hypertension. CLT was measured as lysis time of tissue factor induced clots exposed to exogenous tissue plasminogen activator (t-PA). The rate of in vitro plasmin-mediated cleavage of fibrin ß-chain was assessed over a 6-h period on fibrin clots, which were obtained by exposition to thrombin of purified fibrinogen. Homocysteine plasma levels were measured by Abbott Imx immunoassay and we considered as altered the values above 15 µmol/L according to the literature. In 68 patients homocysteine levels were below 15 µmol/L (NHcy) and in 26 they were above (HHcy). Significant differences were observed between the two groups regarding plasma fibrinolytic potential (p = 0.016), TAFIact (expressed as clot lysis ratio) (p = 0.02), t-PA (0.008) and PLG (0.037), but not for the other assessed components. The HHcy-patients had a threefold higher risk to have an impaired fibrinolysis. Instead, a multivariate logistic regression analysis adjusted for significances of univariate showed that HHcy (OR 5.2 95% CI 1.7-15.9; p = 0.003) and BMI (OR 5.0 95% CI 1.6-15.9; p = 0.006) resulted independently associated with impaired fibrinolytic activity. HHcy affects TAFI-mediated hypofibrinolysis but not fibrin(ogen) structure or function as documented by fibrin degradation analysis.

Stress-induced hyperviscosity in the pathophysiology of takotsubo cardiomyopathy.

Takotsubo cardiomyopathy (TC) is characterized by transient hypokinesis of the left ventricular apex or midventricular segments with coronary arteries without significant stenosis. It is often associated with emotional or physical stress; however, its pathophysiology is still unclear. In the present study, we analyzed the alterations in blood viscosity and markers of endothelial damage induced by sympathetic stimulation in patients with previous TC. Seventeen women (mean age 71 years) with previous TC, included and investigated in the TC Tuscany Registry, were compared to a control group of 8 age- and risk factor-matched women with chest pain and coronary arteries free of stenosis. All subjects underwent the cold pressor test (CPT). Before and after the CPT, the hemorheologic parameters (whole blood viscosity at 0.512 s(-1) and 94.5 s(-1), plasma viscosity, erythrocyte deformability index, and erythrocyte aggregation), catecholamines, plasminogen activator inhibitor-1 (PAI-1), and von Willebrand factor levels were assessed. The patients with TC had significantly greater baseline PAI-1 levels (p <0.01) and lower erythrocyte deformability index values (p <0.01). After CPT, both the patients with TC and the controls had a significant increase in several hemorheologic parameters, catecholamines, and von Willebrand factor levels and a decrease in erythrocyte deformability index. However, the PAI-1 levels were significantly increased only in the patients with TC. Compared to the controls, the patients with TC had significantly greater values of whole blood viscosity at 94.5 s(-1) (p <0.05), PAI-1 (p <0.01), von Willebrand factor (p <0.05) and lower erythrocyte deformability index values (p <0.01) after CPT. In conclusion, the results of the present study suggest that in patients with TC, the alterations in erythrocyte membranes and endothelial integrity induced by catecholaminergic storm could determine microvascular hypoperfusion, possibly favoring the occurrence of left ventricular ballooning.

The thrombophilic pattern of different clinical manifestations of venous thromboembolism: a survey of 443 cases of venous thromboembolism.

Although pulmonary embolism (PE) and deep vein thrombosis (DVT) share many risk factors, it is uncertain whether thrombophilic abnormalities may impact differently on the development of these two clinical manifestations of venous thromboembolism (VTE). To give further insight into this issue, we estimated the association of PE with different types of thrombophilia and evaluated whether these abnormalities have a different prevalence in patients presenting with PE, alone or associated with DVT, as compared with those with isolated DVT. In this study 443 consecutive patients with a first episode of VTE and 304 matched healthy controls underwent laboratory screening for thrombophilia, including natural anticoagulants, factor V Leiden and prothrombin G20210A polymorphisms, antiphospholipid antibodies, homocysteine, factor VIII, and lipoprotein(a). Of the 443 patients, 224 patients had isolated DVT, 144 had combined DVT/PE, and 75 had isolated PE. At least one thrombophilic abnormality was detected in 72.8% of DVT, 66% of DVT/EP, and 60% of isolated PE patients. A high prevalence of hyperhomocysteinemia and elevated lipoprotein(a) levels was found in all patients with no significant differences among the three groups. The prevalence of prothrombin G20210A polymorphism and of elevated factor VIII levels was significantly higher in patients with DVT and DVT/PE than in controls, but not in those with isolated PE, whereas factor V Leiden polymorphism was associated with isolated DVT but not with DVT/PE or isolated PE. In conclusion, the thrombophilic burden seems different in isolated PE versus DVT with or without PE, suggesting that PE may encompass a different pathophysiological process of thrombosis to DVT.

Assessment of fibrinolytic activity by measuring the lysis time of a tissue-factor-induced clot: a feasibility evaluation.

A clot lysis time assay in which a tissue factor-induced fibrin clot is lysed by exogenously added tissue plasminogen activator has been recently reported. We evaluated the feasibility of clot lysis time in a routine hemostasis laboratory, and its correlation with thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 levels and changes with aging in 185 healthy participants. Clot lysis time was assessed by monitoring changes in turbidity during clot formation and subsequent lysis using a computerized kinetic spectrophotometric microtiter plate. After preliminary experiments, 100 and 160 ng/mL tissue plasminogen activator concentrations were chosen for the study. Clot lysis time was calculated by a new mathematical analysis of the lysis curve based on discrete derivative. Clot lysis time, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor-1 plasma levels showed a normal distribution. For both concentrations of tissue plasminogen activator, clot lysis time progressively increased with increase in age (P < .0001) and was significantly correlated with thrombin activatable fibrinolysis inhibitor antigen, thrombin activatable fibrinolysis inhibitor activity, and plasminogen activator inhibitor-1 antigen (at least P < .01). During linear regression analysis, thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 antigen were found to significantly influence clot lysis time (at least P < .01). Clot lysis time determination has a good laboratory performance. Our new method of calculation is independent of the time of reading and allows a more accurate and consistent detection of both short and prolonged lysis times. Our data suggest the feasibility of the use of this test in the work of routine hemostasis laboratory.

Cardiovascular and thrombophilic risk factors in patients with retinal artery occlusion.

This article evaluates the prevalence of cardiovascular and thrombophilic risk factors in patients with retinal artery occlusion. Forty-one patients with a first episode of a retinal artery occlusion underwent complete ophthalmic examination, routine blood testing and specific laboratory tests for thrombophilia, such as fasting and postmethionine homocysteine, lipoprotein(a), plasminogen activator inhibitor-1, factor VIII, factor V Leiden, factor II G20210A polymorphism, lupus anticoagulant and anticardiolipin antibodies. The control population consisted of 100 healthy individuals comparable as regards age and sex. At univariate analysis, hypertension, smoking, dyslipidaemia (both high cholesterol and triglyceride levels), antiphospholipid antibodies, hyperhomocysteinaemia, elevated factor VIII and lipoprotein(a) levels were significantly associated with retinal artery occlusion; at multivariate analysis, adjusted for age, sex, traditional and thrombophilic risk factors, smoking, hypercholesterolaemia, elevated homocysteine and lipoprotein(a) levels confirmed their independent role as risk factors for retinal artery occlusion. In conclusion, the results of the present pilot study demonstrate that the prevalence of hypercholesterolaemia and smoking and the 'thrombophilic burden' are increased in patients with retinal artery occlusion. Our findings may have implications for the management of these patients, suggesting the need for an intensive and tailored secondary prevention and new therapeutic approaches.

High rate of recurrence in renal transplant recipients after a first episode of venous thromboembolism.

BACKGROUND: No data are available about the optimal duration of oral anticoagulant therapy (OAT) after an episode of venous thromboembolism (VTE) occurring in renal transplant (RT) recipients. Our study was undertaken to evaluate the risk of VTE recurrence in patients developing a first episode of VTE after RT. METHODS: Among 484 RT patients, 34 (7%) developed a first VTE: 28/34 VTE patients (Group 1) were prospectively studied, after stopping OAT. Group 1 was compared with a group of 84 patients without history of renal disease who had suffered from a first episode of VTE matched for age, sex and type of thrombotic event (Group 2) and with a matched group of 84 RT recipients with no history of VTE (Group 3). After OAT withdrawal, blood samples were obtained for thrombophilia and clotting activation markers (prothrombin fragment 1+2 (F1+2) and D-dimer plasma levels). RESULTS: During follow-up, 14/28 patients of Group 1 and 8/84 patients of Group 2 experienced VTE recurrence (P < 0.0005). Homocysteine, F1+2 and D-dimer plasma levels were significantly higher in Group 1 than in Group 2 and 3 (P < 0.0001 and <0.05 respectively) for all the three parameters. CONCLUSIONS: Our data outline the high risk of VTE recurrence in RT recipients. Strategies for VTE recurrence prevention are needed; Prolonged OAT, in spite of the high bleeding risk of RT patients, should be considered in this respect.

Culprit factors for the failure of well-conducted warfarin therapy to prevent ischemic events in patients with atrial fibrillation: the role of homocysteine.

BACKGROUND AND PURPOSE: In patients with atrial fibrillation (AF), oral anticoagulant therapy (OAT) is effective in reducing stroke and embolism. However, despite OAT, ischemic events do occur in some patients. Studies specifically addressing the identification of risk factors for ischemic events during well-conducted OAT are not available. In this study, we prospectively investigated the role of classic risk factors and homocysteine levels in the occurrence of ischemic complications in 364 AF patients on OAT. METHODS: The quality of anticoagulation levels and the occurrence of bleeding and thrombotic events were recorded. RESULTS: During follow-up (859 patient years) 21 patients had ischemic complications (rate 2.4 x 100 patient-years). Homocysteine plasma levels were higher in these patients than in patients without ischemic complications during OAT (P<0.01), whereas no difference was observed in relation to the quality of OAT. The presence of a history of previous ischemic events, hypertension, and homocysteine plasma levels over the 90th percentile were all associated with an increased risk of ischemic events during OAT (odds ratio [OR]=7, 4.5, and 4.7, respectively). The coexistence of these risk factors markedly increased the risk (OR=13.1; 95% CI, 3.7 to 45.7; P=0.001). CONCLUSIONS: In conclusion, our results indicate that AF patients with multiple risk factors may not be sufficiently protected by OAT, even when this is well conducted.

High-speed detection of the G894T polymorphism in exon 7 of the eNOS gene by real-time fluorescence PCR with the Light-Cycler.

In endothelial cells nitric oxide (NO) is synthesized by endothelial-nitric oxide synthase (e-NOS), constitutively expressed and encoded by a 26-exon gene, located on chromosome 7q35-36. The prevalence of the T rare variant of the G894T polymorphism in exon 7 of the e-NOS gene (Glu-->Asp amino acid substitution) has been reported to be significantly higher in patients with coronary spasm and coronary artery disease. To date G894T polymorphism detection is performed by PCR-RFLP assay. In order to establish a high-speed genotyping method, we have taken advantage of the Light Cycler instrument, a thermal cycler that combines rapid-cycle DNA amplification with a real-time fluorescence monitoring. This technology is based on hybridization of the adjacent fluorescently labeled probes with PCR products. This methodology is considered more accurate and less time-consuming than conventional PCR-RFLP assay. To validate this technique we genotyped 270 healthy subjects. The results were consistent with those obtained from PCR-RFLP assay.

Increased plasma levels of lipoprotein(a) and the risk of idiopathic and recurrent venous thromboembolism.

PURPOSE: Elevated lipoprotein(a) [Lp(a)] levels are a recognized risk factor for cardiovascular disease; however, little is known about their effects on venous thromboembolism. METHODS: We conducted a case-control study of 603 adult patients with a history of venous thromboembolism (at least 6 months after the acute event) and 430 healthy subjects. We measured Lp(a), homocysteine, and antithrombin levels, factor V Leiden and factor II (prothrombin) polymorphisms, and anticardiolipin antibodies. RESULTS: Lp(a) levels >300 mg/L were found in 24% (n = 146) of the patients and in 13% (n = 58) of the controls (P = 0.005). In a multivariate analysis adjusted for acquired and hemostasis-related risk factors, there was an independent association between elevated (>300 mg/L) Lp(a) levels and venous thromboembolism (odds ratio = 2.1; 95% confidence interval: 1.4 to 3.2; P = 0.002). These results were confirmed in the 341 patients with idiopathic venous thromboembolism, as well as in those with recurrent thromboembolism. CONCLUSION: These results show that Lp(a) is an independent risk factor for venous thromboembolism in adults, suggesting that it may be involved in the pathogenesis of idiopathic and recurrent disease.