Antithrombin

A 20-year-old male with asymptomatic inherited type 1 antithrombin deficiency and a family history of thrombosis started injecting himself with testosterone 250 mg intramuscularly twice weekly for 5 weeks. He presented to the hospital with progressive dyspnea on exertion, chest pain and hemoptysis. Workup revealed bilateral submassive pulmonary embolism and proximal right lower extremity deep vein thrombosis. He was treated with intravenous (IV) unfractionated heparin and underwent catheter-directed thrombolysis with alteplase to the main pulmonary arteries. Postprocedure, he remained on IV alteplase infusion for 24 h and unfractionated heparin in the intensive care unit. Concomitantly he received plasma-derived antithrombin concentrate. He was transitioned to subcutaneous enoxaparin twice daily and discharged from the hospital on oral rivaroxaban 15 mg twice a day. This case highlights the heightened thrombogenic effect of anabolic steroids in the setting of underlying thrombophilia especially in younger subjects.

A study of coagulation factors in humans from bite of Asian medicinal leech confirms that bleeding from a leech bite bleeds for hours but after 15 minutes coagulation is normal; The bleeding time for Hirudo nipponia is on average 210 minutes, compared to 490 minutes from Hirudo medicinalis. Total blood loss in H. medicinalis is 10 times that of H. nipponia.

The objective of this study was to investigate the effect of freezing of normal plasma samples on protein C, free protein S (FPS) and antithrombin levels in order to determine its potential impact on the interpretation of the results of similarly frozen patients' samples. Protein C, FPS and antithrombin levels were measured by clotting-based test, by sandwich ELISA and by chromogenic assay, respectively, in 50 normal plasma samples prior to freezing, and after 2 and 4 weeks in parallel aliquots frozen at S25-C. The mean levels of the three proteins dropped significantly after a fortnight's freezing, protein C: 130.7-122.8% (P < 0.0246); FPS: 105.9-94.1% (P < 0.0016); antithrombin: 103.2-95.8% (P < 0.0001). The corresponding inter-assay coefficient of variances of the two sets of results were 8.9, 6.6 and 9.3%. Thereafter, only FPS declined significantly (84.3%) (P < 0.0001). In two of 48 and five of 48 cases at the end of 2 and 4 weeks, respectively, the levels of FPS values went below the lower limit of the normal range established from the 50 plasma samples. Freezing of plasma at S25-C for 24 h per se did not alter the levels of protein C and antithrombin and caused only a negligible change in FPS levels. Since 6, 4 and 14% of normal plasma samples would have been labeled as antithrombin, protein C and protein S deficient, respectively, had the tests been performed after 4 weeks of freezing, it is recommended that for correct interpretation of the results, laboratories should establish their reference ranges on normal samples frozen for the same period of time as the patients' samples. Blood Coagul

We report 10 patients with congenital deficiencies of the natural anticoagulant proteins S, C and antithrombin III. Thirteen of a total of 30 pregnancies were managed at the perinatal branch of our department. We discuss the mechanism of action of these proteins and their role in thrombotic events. We analyze the most frequent thrombotic complications and we discuss the general guidelines for the investigation of a patient with a suspected congenital thrombophilia with special regard to its management during pregnancy, delivery and perinatal outcome.

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the Cterminal portion of the molecule which is replaced by aspartic acid. Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported. The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides. HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene. Hirudin, the most potent thrombin inhibitor known, is a small polypeptide discovered over 100 years ago in the saliva of the medicinal leech Hirudo medicinalis [l]. It was first characterized biochemically by Markwardt [2, 31 and its structure was lately elucidated by Dodt et al. [4]. It is a single-chain polypeptide composed of 65 amino acids, including 6 cysteine residues, and a molecular mass of about 7000 Da. Hirudin produces its action by binding directly to thrombin

ABSTRACT: In this paper the effectiveness of the so-called American medicinal leech Macrobdella decora in overcoming human haemostasis is compared to that of the European medicinal leach Hirudo medicinalis. Thrombin-clotting times indicated that M. decora prevents coagulation of human blood by means of anti-thrombin activity. Our findings suggest that its role is similar to that of hirudin. In human volunteers, the duration of bleeding from the bite of M. decora (mean = 73 min; n = 18) is significantly shorter than H. medicinalis (mean = 600 min; n = 15). The mean concentration of anti-thrombin units in each specimen of M. decora and H. medicinalis is 100 and 285 AT-U, respectively. These findings support the concept that H. medicinalis is more advanced in terms of haematophagous predation. Despite similar feeding durations by both species of leech (means = 68 min and 70.5 min by M. decora and H. medicinalis, respectively), the mean increase in body weight of M. decora was only 58% compared to 460% in H. medicinalis.

Deficiencies of natural anticoagulants protein C, protein S, antithrombin and activated protein C resistance are components of inherited thrombophilia. Inherited thrombophilia was defined as a genetically determined tendency towards venous thromboembolism, which characteristically occurs in young patients (before 40 to 45 years old), without apparent causes, and tend to recur. There have been many debates about the implication of these defects in arterial thromboses (peripheral artery disease, myocardial infarction, cerebral infarction) in the recent years. The screening for thrombophilia is recommended for young patients with spontaneous thromboses, arterial infarctions, family history of thromboses, personal history of recurrent abortions, with thrombosis of venous dural sinuses or strokes or myocardial infarctions, in patients with venous thrombosis in unusual sites, because the diagnosis of such a disease leads to a treatment that is lifesaving [1,2].

I1 modified by pyridoxal 5"phosphate; rHC, recombinant HC; HPLC, high-performance liquid chromatography; TPCK-trypsin, tosylphenylalanylchloromethyl ketone-treated trypsin; Chromozym TH, P-ptosyl-Gly-Pro-Arg-p-nitroanilide. Portions of this paper (including "Experimental Procedures" and Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The antithrombin A384S mutation has a relatively high frequency in the British population but has not been identified in other populations. This variant has been associated with cases of thrombotic disease, but its clinical relevance in venous thrombosis remained unclear. We have conducted a secondary analysis of the prevalence of the mutation in a large case-control study, including 1018 consecutive Spanish patients with venous thromboembolism. In addition, we evaluated its functional consequences in 20 carriers (4 homozygous). This mutation, even in the homozygous state, did not affect anti-Xa activity or antigen levels, and it only slightly impaired anti-IIa activity. Thus, routine clinical methods cannot detect this anomaly, and, accordingly, this alteration could have been underestimated. We identified this mutation in 0.2% of Spanish controls. Among patients, this variant represented the first cause of antithrombin anomalies. Indeed, 1.7% patients carried the A384S mutation, b...

ABSTRACT: Prolongation of haemostatic parameters in the host achieved by hatchling Hirudo medicinalis differs quantitatively but not qualitatively from those achieved by adult leeches. The duration and the rate of blood loss from the bite wounds, the gain in body weight of leeches and the duration of feeding each increase with successive feeding episodes. The mean duration of bleeding from bite wounds of hatchling leeches feeding for the first time on human volunteers was 43 min compared to 6 min from control incisions and 600 min in adult leeches. The mean ‘whole body’ concentration of hirudin in 2-week-old hatchlings was 10 antithrombin units (AT-U) compared to 285 AT-U in the head of each adult leech.