Acute intraoperative heparin-induced thrombocytopenia (HIT) and thrombosis during coronary artery bypass grafting: Two case reports providing evidence for the role of preoperative LMWH in triggering sensitization.

INTRODUCTION: Systemic anticoagulation is necessary during cardiac surgery. To date, the only well established anticoagulation protocol involves the use of heparin. However, heparin can cause heparin-induced thrombocytopenia (HIT) a potentially life threatening immune-mediated thromboembolic syndrome. Until now, devastating consequences of HIT syndrome in patients undergoing heart surgery have been described, but only postoperatively. Here we report the development of HIT syndrome during cardiac revascularization by intra-operative heparin administration in two patients previously exposed to LMWH. PATIENTS/METHODS: We report on two patients who developed rapid and profound intravascular coagulation with severe thrombocytopenia (platelet count decreased from ≥250×109/L to 50×109/L) due to HIT development caused by heparin administration during coronary artery bypass graft surgery. In addition we report that fondaparinux, given intra-operatively in association with antithrombin, may be a suitable alternative anticoagulant for successfully preventing the devastating consequences of intra-operative HIT development. CONCLUSION: To our knowledge, this is the first report describing the development of acute intra-operative HIT, secondary to high-dose UFH administered for coronary revascularization, in which the unexpected presence of platelet-activating anti-PF4/heparin antibodies at surgery was explained by preoperative administration of a one-week course of LMWH but without any preoperative evidence for HIT.

Heart transplantation in a low-organ-donation environment: a single center experience.

OBJECTIVE: Heart transplantation is the "gold standard" for treating patients in end-stage heart failure who satisfy strict selection criteria. However, infrequent transplant performance, eg, less than nine per year, may be associated with suboptimal results. METHODS: We reviewed our 13-year clinical experience (1996-2008) with 73 orthotopic heart transplants performed under strict selection criteria and followed closely thereafter at the only accredited center in Greece, a country with an annual rate of only seven donors per million population. RESULTS: Low perioperative (5.47%) and long-term (7.5%) mortality rates were responsible for a 94% survival rate in the first year, 92% at five years, and 70% at ten years-similar to those reported worldwide-along with excellent functional recovery. CONCLUSION: Strict recipient and donor selection criteria, combined with a rigorous multidisciplinary follow-up, yield excellent results despite the existing shortage of available grafts.

Heparin-induced thrombocytopenia thrombosis after cardiac surgery. A case report.

We report a rare case of cerebral infarct resulting in brain death due to heparin-induced thrombocytopenia thrombosis (HITT), manifested in the immediate postoperative period following aortic valve replacement in a 46-year-old woman whose only prior exposure to heparin was during catheterization four months prior to surgery. The diagnosis of HITT was suspected after a significant decrease of the platelet count and it was confirmed by a heparin-induced platelet activation assay showing platelet aggregation in the presence of heparin.

Design and synthesis of thrombin receptor-derived nonpeptide mimetics utilizing a piperazine scaffold.

Focal thrombus formation and vasoconstriction serve to defend vessels when vascular damage occurs, but may be detrimental when an atherosclerotic plaque is disrupted. Recently, the identification of the platelet thrombin receptor opened a new area in the development of agents that may selectively inhibit the effects of thrombin on cells, without affecting fibrin formation. In this regard, we have synthesized a number of 1,4-disubstituted piperazines which are designed to be analogues of thrombin receptor activating peptides (TRAP) and carry the pharmacophoric features of Phe and Arg residues present in the active pentapeptide SFLLR. These compounds were tested in the rat aorta relaxation assay and in platelet aggregation studies and their biological activity was consistent with a direct action on thrombin receptor. Furthermore, the structure activity relationships confirmed the importance of Phe and Arg for receptor activation and the molecular modeling revealed an intriguing relationship between their amphipathic similarity with SFLLR and their biological activity.

Occurrence of aflatoxin M1 in commercial pasteurized milk determined with ELISA and HPLC.

Eighty-one samples of commercial pasteurized milk from Athens market were analysed for the presence of aflatoxin M1 (AFM1). A combination of a commercial ELISA kit and a modified HPLC method was applied for the rapid and reliable determination of AFM1. AFM1 concentrations in milk extracts were initially estimated by ELISA. Samples found to contain more than 5 ng/l were further quantitated by HPLC. Determination was performed after derivatization of AFM1 to its hydroxylated product AFM2a. The recovery of the HPLC method used was found to be close to 100%. Thirty-two samples contained aflatoxin M1 at levels of 2.5-5 ng/l, none contained more than 5 ng/l, while 31 contained only traces of aflatoxin (0.5-1 ng/l). In nine samples no AFM1 was detected. There was no seasonal influence on the aflatoxin content of the milk samples analysed.

Low-molecular-weight heparin versus warfarin for prevention of recurrent venous thromboembolism: a randomized trial.

A group of 105 consecutive patients with venographically proved major acute deep vein thrombosis (DVT) were randomized in an open prospective study to evaluate the comparative efficacy and safety of a fixed dose of subcutaneous low-molecular-weight heparin (LMWH) and warfarin for the prevention of recurrent venous thromboembolism. Four patients developed venographically proved recurrent DVT during the 3 months of treatment: three in the LMWH group and one in the warfarin group. Nonfatal pulmonary embolism occurred in two patients in the LMWH group and in one in the warfarin group. Five of the 55 patients (10%) in the warfarin group and none of the 50 patients in the LMWH developed bleeding complications (two-tailed Fisher exact test, p = 0.06). A preliminary assessment of the costs indicated that treatment with LMWH was less expensive by Pounds 900 per patient than warfarin. In conclusion, the fixed daily dose of LMWH and the adjusted dose of warfarin therapy were of similar efficacy in preventing recurrence of DVT. However, warfarin therapy, despite strict laboratory control, is associated with more frequent side effects and is expensive. Another study with a higher dose of LMWH is recommended.

Long-term administration of heparin and heparin fractions and osteoporosis in experimental animals.

To test whether heparin-induced osteoporosis is influenced by the molecular weight of heparin, 24 male rabbits received single daily subcutaneous injections of either physiological saline (controls, n = 5), low molecular weight heparin (LMWH, n = 7), conventional heparin (UFH, n = 7) or high molecular weight heparin (HMWH, n = 6). Heparin was administered in supratherapeutic daily dosages for 120 days (750 anti-FXa units/kg for 90 days and 1500 anti-FXa units/kg for another 30 days). Studied variables were: serial analysis of serum calcium, albumin, phosphate and alkaline phosphatase, measurement of the cortical thickness of the femur (radiographically), tibial and trabecular bone density (both by cross-sectional analysis) and femoral fragility. Observed changes in blood biochemistry associated with bone metabolism were not correlated to any of the treatments. Compared with the controls, a reduction in cortical and trabecular bone density was seen with UFH (P < 0.05) and HMWH (P < 0.01) but not with LMWH. Femoral fragility was also significantly increased (P < 0.002) by HMWH. In conclusion, LMWH did not cause toxic skeletal effects as opposed to HMWH which clearly did, and UFH which induced some osteoporotic changes.

Antithrombin-TRI (Ala382 to Thr) causing severe thromboembolic tendency undergoes the S-to-R transition and is associated with a plasma-inactive high-molecular-weight complex of aggregated antithrombin.

An antithrombin (AT) variant Ala382 to Thr (AT-TRI) was identified by mass spectrometric techniques. The variant behaved as a substrate rather than a thrombin inhibitor, but, contrary to previously described P12 AT variants, AT-TRI, expressed as a heterozygous dominant trait, caused severe thromboembolic tendency beginning in their teens in affected members of an English family. In addition, it underwent the S-to-R conformational state transition as evidenced by an increased resistance to thermal denaturation on active centre cleavage, but did not react with a monoclonal antibody, 4C9, directed against a neoepitope that is present on complexed and cleaved normal AT. Antithrombin-TRI, in plasma, was also associated with an abnormal high molecular weight (M(r)) 194,000) component composed of non-covalently-linked antithrombin molecules. This component (D194) showed low affinity for heparin and was devoid of antithrombin progressive activity. D194, isolated by ammonium sulphate precipitation and three chromatographic steps (heparin Sepharose, ion exchange and immunoaffinity), migrated as a single band of M(r) 60,000 on SDS-PAGE under both reducing and non-reducing conditions and was recognized by monospecific anti-human antithrombin antibodies, but did not immunoreact with antibodies raised against a number of proteins including albumin and thrombin. The above data and the fact that the 15 N-terminal amino acids of this M(r) 60,000 band were identical to that of normal antithrombin indicated that the inactive D194 component was composed of aggregated antithrombin molecules, possibly antithrombin trimers. In conclusion, early adulthood severe thromboembolic tendency, failure to expose the 4C9 epitope, and presence of aggregated AT molecules in the plasma are characteristic features of AT-TRI not previously described in other ALA-382 THR mutations.

Cleaved antithrombin (ATc): a new marker for thrombin generation and activation of the coagulation system.

A modified form of antithrombin (AT) cleaved at the active site by thrombin (ATc) has been shown to be generated in vivo, corresponding to 1-4% of the circulating AT. An enzyme immunoassay has been developed for measuring ATc following plasma treatment with ammonium sulphate and heat denaturation of native AT. ATc plasma levels were found to be significantly higher (P = 0.003) in patients developing venous thromboembolism when compared to patients without such events or healthy controls (age and sex matched). In addition, ATc levels correlated with thrombin generation markers: thrombin-AT complex (r2 = 0.66, P = 0.005) and prothrombin fragment 1 + 2 (r2 = 0.58, P = 0.018), but, in contrast to both these markers, elevated ATc levels were detected for at least 2 weeks after the thromboembolic event. In conclusion, ATc appears to be a new marker for thrombin generation and overall activation of the coagulation system, having the advantage of being detected in the circulation for a longer period than other thrombin generation markers.

A dose ranging study to evaluate dermatan sulphate in preventing deep vein thrombosis following total hip arthroplasty.

Dermatan sulphate catalyses thrombin inhibition by heparin cofactor II; it has a lower haemorrhagic to antithrombotic ratio than that of heparin in animal models. Consecutive patients aged forty years or more, electively undergoing total hip replacement under general anaesthesia, were randomly allocated to one of three dosage regimens of dermatan sulphate (MF701, Mediolanum Farmaceutici) given intramuscularly. These were 200 mg once daily (n = 50), 200 mg twice daily (n = 52) and 300 mg twice daily (n = 51), administered from twenty-four hours pre-operatively until the tenth postoperative day. The overall incidence of DVT assessed by bilateral venography was 53%, 51% and 34% respectively (Chi-square test for trend p = 0.06). The incidence of major proximal DVT was 10.6%, 8.5% and 2.1% respectively. Pulmonary embolism (PE) and bleeding were assessed in all 153 patients. There was one case of PE in each dose group. The incidence of bleeding episodes, volume of blood lost and blood transfusion requirements were low and showed no increase with increasing dose. The patients were followed up 4-8 weeks after discharge. We conclude that the two lower doses were subtherapeutic in this population, however dermatan sulphate given 300 mg twice daily, proved to be efficacious with an incidence of proximal major DVT of 2.1% and a low incidence of bleeding complications. A trial of dermatan sulphate 300 mg twice daily compared to standard prophylactic agents is needed.

Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis.

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80-->Thr and His120-->Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251-->Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158-->Cys) was also found to occur in several individuals of Scandinavian origin.

The influence of tamoxifen in vivo on the main natural anticoagulants and fibrinolysis.

The long-term effects of tamoxifen on alterations in haemostasis which could lead towards thrombosis were investigated in 149 women who were disease-free for at least 5 years following mastectomy for breast cancer. All participants were randomized to receive tamoxifen as a post-surgical adjuvant treatment (89 patients, treated group) or not (60 patients, controls) for at least 2 years. 5.62% of the cases treated with tamoxifen suffered a venous thrombosis, while no thromboembolism was reported in the control group. No significant differences were observed between groups in the global clotting times, fibrinogen, fibrinolytic factors, or in the concentration of the main natural anticoagulants, antithrombin III (AT-III), protein C(PC) and protein S (PS). However, when the treated group was sub-divided, current users (n = 18) of the drug (median treatment duration 72 months) had significantly lower AT-III (P < 0.05) and PC (P < 0.05) activities, together with higher levels of plasminogen activity (P < 0.01) and tissue plasminogen activator antigen (P < 0.01), compared with 71 ex-users (who mostly received treatment for 2 years) and controls. We conclude that long-term treatment with tamoxifen for 2 or more years tends to reduce both AT-III and PC, a situation possibly predisposing towards thrombosis. Monitoring haemostasis in tamoxifen-treated breast cancer patients is therefore advisable.

Use of low molecular weight heparin in pregnancy.

In a controlled study of 15 pregnant patients undergoing therapeutic termination of pregnancy, seven received subcutaneously 5,000 anti-FXa units of low molecular weight (LMW) heparin 15 and 3 h prior to the termination, and eight patients acted as controls. Paired maternal and fetal blood samples were taken (before or immediately after the termination) for assay of heparin activity by a chromogenic anti-FXa method sensitive to levels of 0.02 anti-FXa U/ml. LMW heparin was detected in all maternal samples of the test patients but was not detected in any of the fetal samples. The use of LMW heparin as a thromboprophylactic agent was then evaluated in 11 patients who were known to have a severe thromboembolic tendency, had suffered recurrent miscarriages and had responded poorly to conventional anticoagulation (oral anticoagulant, conventional heparin). All patients receiving LMW heparin in thromboprophylactic doses completed uneventful pregnancies and gave birth to healthy babies (three for the first time) without complication. Bone density scans performed in all patients shortly after the delivery showed normal mineral mass. We conclude that LMW heparin does not cross the placental barrier, and in addition offers satisfactory antithrombotic protection for both maternal and placental circulation. In addition, this study provides preliminary data from 11 patients suggesting LMWH may not give rise to maternal osteoporosis, a finding that now needs further investigation.