Identification of N-linked glycosylation and putative O-fucosylation, C-mannosylation sites in plasma derived ADAMTS13.

BACKGROUND: Acquired deficiency of ADAMTS13 causes a rare and life-threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases.N-linked glycosylation and putative O-fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13. OBJECTIVES: Here we investigated the presence of putative O-fucosylation, C-mannosylation and N-linked glycosylation sites on plasma derived ADAMTS13. METHODS/RESULTS: Sites of N-linked glycosylation were determined by the use of peptide N-glycosidase-F (PNGase F), which removes the entire carbohydrate from the side chain of asparagines. Nine of the 10 predicted N-linked glycosylation sites were identified in or near the metalloproteinase,spacer, thrombospondin type 1 repeat (TSR1) and the CUB domain of plasma ADAMTS13. Moreover, six putative O-fucosylated sites were identified in the TSR domains of plasma ADAMTS13 by performing searches of the tandem mass spectrometry (MS/MS) data for loss of hexose (162 Da), deoxyhexose (146 Da), or hexose deoxyhexose(308 Da). The use of electron transfer dissociation (ETD) allowed for unambiguous identification of the modified sites. In addition to putative O-fucosylation and N-linked glycosylation, two putative C-mannosylation sites were identified within the TSR1 and TSR4 domains of ADAMTS13. CONCLUSIONS: Our data identify several glycosylation sites on plasma derived ADAMTS13. We anticipate that our findings may be relevant for the initiation of autoimmune reactivity against ADAMTS13 in patients with acquired TTP.

Does activated protein C-resistant factor V contribute to thrombin generation in hemophilic plasma?

In this study we assessed the role of factor V (FV) inactivation in hemophilic plasma with particular reference to the activated protein C (APC)-resistant variants FV-R506Q (FV Leiden) and FV-R306T (FV Cambridge). Purified recombinant full-length FV carrying these single substitutions and FV-R306T/R506Q were used in thrombin generation experiments. Plasma was first immunodepleted of FV, and subsequently of factors VIII, IX, or combinations thereof. Thrombin generation was initiated by low concentrations of recombinant tissue factor. Recombinant soluble thrombomodulin (TM) was used to trigger the APC system. Surprisingly, TM concentrations that reduced thrombin generation in normal plasma by no more than 50% virtually abolished thrombin formation in plasma deficient in the factor VIII/IX complex. This was already apparent at TM levels as low as 0.1 nmol L(-1). By varying the concentrations of purified (activated) protein C to plasma that was additionally depleted of protein C, we confirmed that impaired thrombin generation indeed was the result of the action of APC. In contrast, this did not occur when FV-depleted plasma had been reconstituted with FV-R306T/R506Q. Addition of FV-R306T or FV-R506Q partially reduced prothrombin activation, demonstrating the involvement of both APC cleavage sites. FV inactivation also occurred on the surface of human microvascular endothelial cells. Apparently, these cells express sufficient TM to down-regulate thrombin production via the APC pathway. We further conclude that in hemophilic plasma this pathway can induce a secondary defect because of premature FV inactivation. It therefore seems conceivable that APC-resistant FV has the potential of alleviating hemophilic bleeding.

Adolescent with occluded left main coronary artery.

Here we report unexpected findings in a 17-year-old female patient referred for coronary angiography and percutaneous intervention. During the angiography we observed a complete occlusion of the left main coronary artery (LMCA). The occurrence of coronary abnormalities at this age is extremely rare and mostly caused by congenital abnormalities. The diagnosis of premature atherosclerosis at this age is unlikely unless the patient suffers from severe lipoprotein disease. Here we describe a rare case of LMCA occlusion, the most likely cause of the disease and the potential implications for therapy.

Pharmacodynamics and safety of lefradafiban, an oral platelet glycoprotein IIb/IIIa receptor antagonist, in patients with stable coronary artery disease undergoing elective angioplasty.

OBJECTIVE: Lefradafiban is the orally active prodrug of fradafiban, a glycoprotein IIb/IIIa receptor antagonist. The present phase II study aimed to determine the dose of lefradafiban that provides 80% blockade of the glycoprotein IIb/IIIa receptors by fradafiban, and to study the pharmacodynamics and safety of different doses in patients with stable angina undergoing angioplasty. DESIGN: A double blind, placebo controlled, dose finding study. SETTING: Four academic and community hospitals in the Netherlands. PATIENTS: 64 patients with stable coronary artery disease undergoing elective percutaneous transluminal coronary angioplasty. INTERVENTIONS: 30 mg, 45 mg, and 60 mg of lefradafiban three times daily or placebo was given for 48 hours. MAIN OUTCOME MEASURES: The primary safety end point was the occurrence of bleeding, classified as major, minor, or insignificant according to the thrombolysis in myocardial infarction (TIMI) criteria. Efficacy indices included per cent fibrinogen receptor occupancy (FRO), ex vivo platelet aggregation, and plasma concentrations of fradafiban. RESULTS: Administration of lefradafiban 30, 45, and 60 mg three times daily resulted in a dose dependent increase in median FRO levels of 71%, 85%, and 88%, respectively. Inhibition of platelet aggregation was closely related to FRO. There were no major bleeding events. The 60 mg lefradafiban group had a high (71%) incidence of minor and insignificant bleeding. The incidence of bleeding was 44% in the 30 mg and 45 mg groups, compared with 9% in placebo patients. Puncture site bleeding was the most common event. The odds of bleeding increased by 3% for every 1% increase in FRO. CONCLUSIONS: Lefradafiban is an effective oral glycoprotein IIb/IIIa receptor blocker. The clinical effectiveness of doses up to 45 mg three times daily should be investigated.

Long-term treatment with a platelet glycoprotein-receptor antagonist after percutaneous coronary revascularization. EXCITE Trial Investigators. Evaluation of Oral Xemilofiban in Controlling Thrombotic Events.

BACKGROUND: When administered intravenously at the time of percutaneous coronary revascularization, glycoprotein IIb/IIIa receptor antagonists decrease the incidence of death and nonfatal myocardial infarction and the need for urgent revascularization. We hypothesized that long-term administration of oral glycoprotein IIb/IIIa antagonists, which block the aggregation of platelets, might stabilize intravascular plaque and prevent additional ischemic cardiac events. METHODS: We conducted a prospective, double-blind trial in which 7232 patients were randomly assigned to receive 20 mg of oral xemilofiban or placebo 30 to 90 minutes before undergoing percutaneous coronary revascularization, with maintenance doses of 10 or 20 mg of xemilofiban or placebo administered three times daily for up to 182 days. There were two primary composite end points: one was death, nonfatal myocardial infarction, or urgent revascularization at 182 days, and the other was death or nonfatal myocardial infarction at 182 days. RESULTS: Death, myocardial infarction, or urgent revascularization occurred within 182 days in 324 patients who received placebo (Kaplan-Meier cumulative event rate, 13.5 percent), 332 who received 10 mg of xemilofiban (13.9 percent, P=0.82 for the comparison with placebo), and 306 who received 20 mg of xemilofiban (12.7 percent, P=0.36 for the comparison with placebo). The incidence of death or myocardial infarction was also similar in all three groups. Clinically significant hemorrhagic complications and thrombocytopenia were infrequent. CONCLUSIONS: The administration of the glycoprotein IIb/IIIa antagonist xemilofiban before percutaneous coronary revascularization and for up to six months thereafter does not significantly reduce the incidence of important clinical end points.

Heparin-induced thrombocytopenia and thrombosis: a prospective analysis of the incidence in patients with heart and cerebrovascular diseases.

Heparin-induced thrombocytopenia and/or thrombosis (HITT) are serious complications of heparin treatment. The incidence, as previously reported, varies widely and, in consequence, is not precisely known. Moreover, most reports only concern clinically defined heparin-induced thrombocytopenia. Therefore we carried out a prospective study of the incidence of serologically confirmed HITT. All patients admitted to the Departments of Cardiology and Neurology of our institution with an indication for treatment with therapeutic-dose intravenous unfractionated heparin were enrolled in the study. The patients were examined daily for the occurrence of thromboembolic complications. Regular platelet counts and tests for the presence of heparin-dependent antibodies were carried out using two different tests: a quantitative platelet factor 4/ heparin (PF4/hep) Elisa, and a functional test, the heparin-induced platelet activation assay (HIPAA). HITT was defined as a rapidly occurring (within 5 d) decrease of the platelet count from normal values of > 120 x 10(9)/l to < 60 x 10(9)/l or to < 100 x 10(9)/l if there was a rapid fall of >50% of starting value or >30% with concomitant acute thrombosis. The observed incidence of HITT was 1/358 patients (0.3%, 95% confidence limits 0.01-1.5%). However, Elisa PF4/hep specific IgG antibodies were demonstrated in nine (2.5%) and IgM antibodies in seven (2.0%) of 358 patients. 30/358 patients (8.4%) had platelet activating antibodies in the HIPAA. We conclude that the incidence of serologically confirmed HITT in this study is very low (0.3%) in patients with cardiac and neurologic diseases treated with intravenous unfractionated heparin. The frequency of heparin-dependent antibodies without concomitant occurrence of thrombocytopenia is much higher.