Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

Isolation and characterization of the gene encoding a novel factor Xa-directed anticoagulant from the yellow fever mosquito, Aedes aegypti.

Mosquito salivary glands secrete a number of proteins that inhibit mammalian hemostasis and facilitate blood feeding. We have isolated the protein product and corresponding cDNA of a gene designated Anticoagulant-factor Xa (AFXa), that encodes the factor Xa (FXa)-directed anticoagulant of the yellow fever mosquito, Aedes aegypti. The protein was purified partially by cation exchange chromatography and shown by enzyme activity profiles and SDS-polyacrylamide gel electrophoresis analysis to have an Mr = 54, 000. The protein was purified further by preparative SDS-polyacrylamide gel electrophoresis and subjected to internal protein sequencing, and the sequence of five peptides was determined. Degenerate oligonucleotides were designed based on three of the peptide sequences, and these were used to screen an adult female salivary gland cDNA library from A. aegypti. A 1.8-kilobase pair cDNA was isolated and shown to encode a 415-amino acid conceptual translation product with a predicted molecular mass of 47.8 kDa that contains the five sequenced peptides. Hydrophobicity analysis predicts a 19-amino acid signal peptide typical for secreted proteins. Northern analysis demonstrated that AFXa is expressed only in female salivary glands. Baculovirus-expressed AFXa protein has the appropriate size and expected FXa-directed anticoagulant activity. Analysis of the primary amino acid sequence shows that the AFXa gene product has similarities to the serpin superfamily of serine protease inhibitors and may represent a novel, highly diverged member of this family.

Anticoagulants in vector arthropods.

Arthropod-borne diseases cause significant morbidity and mortality worldwide. Mosquitoes alone may account for as many as three million deaths annually via the transmission of malaria. Because these diseases are transmitted to humans and to other vertebrates as a result of the ability of arthropods to feed on blood, the study of the biochemical mechanisms and adaptations that arthropods have evolved to facilitate hematophagy may provide insight into how this feeding behavior contributes to the transmission of disease. In this review, Kenneth Stark and Anthony James examine the diversity of arthropod anticoagulants and their role in hematophagy and potential implications for parasite transmission.

Salivary gland anticoagulants in culicine and anopheline mosquitoes (Diptera:Culicidae).

Female salivary gland extracts from 9 mosquito species representing the 2 hematophagous subfamilies delayed coagulation of human plasma. All extracts significantly delayed recalcification time, prothrombin time, and activated partial thromboplastin time, indicating an effect on the common coagulation pathway. Chromogenic assays to determine the coagulation inhibition site were performed for coagulation factor Xa (FXa) and thrombin, the major components of the common pathway. Although variation existed in the degree of inhibition, all anophelines have thrombin-directed anticoagulants, and culicine mosquitoes have FXa-directed anticoagulants. Differences in the site of action of the anticoagulants most likely reflected the long period of independent adaptation of the 2 subfamilies to the challenges presented by vertebrate hemostasis.

A factor Xa-directed anticoagulant from the salivary glands of the yellow fever mosquito Aedes aegypti.

Salivary gland extracts from the yellow fever mosquito Aedes aegypti contain a factor Xa-specific anticoagulant detectable in vitro by clotting assays. The anticoagulant is found only in female salivary glands, which is consistent with its role in bloodfeeding. Extracts of adult female glands significantly delay both the prothrombin time and the activated partial thromboplastin time, but have no effect on the thrombin clotting time. The anticoagulant has been shown to result from the specific inhibition of coagulation factor Xa based on a FXa clotting assay and the inhibition of FXa-directed cleavage of the synthetic chromogenic substrate, chromozym X. The inhibition of FXa by female salivary gland extracts exhibits noncovalent, noncompetitive inhibition kinetics and is reversible. The anticoagulant is destroyed by boiling for 10 min or heating at 56 degrees C for 30 min, has a 4 mM calcium optimum with no magnesium requirement, and has a pH optimum of 8.0. The anticoagulant activity has an apparent molecular weight of 35.5 kDa, as determined by molecular sieve. We propose that the A. aegypti anticoagulant is a novel, proteinaceous serine protease inhibitor specific for FXa.

Poliovirus translation initiation: differential effects of directed and selected mutations in the 5' noncoding region of viral RNAs.

We have analyzed the translational defects of a number of mutations in the 5' noncoding region of poliovirus type 1 RNA. These mutations fall into three categories: (1) two mutations which resulted in temperature sensitive (ts) viruses, (2) the second-site mutations responsible for the reversion of the two ts viruses, and (3) mutations which were lethal to virus production. RNAs containing either of the ts mutations translated in vitro at levels significantly lower than wild-type levels. RNAs containing the respective second-site reversions had corrected these translational defects to levels corresponding to their viral growth potentials. Unlike in vitro translation of wild-type poliovirus RNA, translation of the RNAs which gave rise to ts mutant viruses was not stimulated by the addition of an S10 fraction from an uninfected HeLa cell extract to a rabbit reticulocyte lysate (RRL). In vitro translation of the mutant RNAs (corresponding to the ts viruses) in a RRL was stimulated by factors present in a ribosomal salt wash (RSW) from a HeLa extract, although the levels of stimulation were only half those seen for wild-type. These results suggest that the stimulatory factors present in the RSW have a decreased affinity for the mutant RNA templates but can, to some extent interact, with such RNAs if provided in high enough concentration. The in vitro translation of RNAs containing either of the lethal mutations was not stimulated by factors present in the S10 or the RSW. Taken together, our data suggest a correlation between the ability of a genetically altered RNA to respond to translation stimulatory factors in vitro and the ability of that mutation to be recovered in infectious virus. In addition, we have identified the in vivo-selected reversion of translational defects for two different ts viruses.

Spontaneous clostridial myonecrosis in a man with drug-induced agranulocytosis.

A middle-aged man had spontaneous clostridial myonecrosis during a period of neutropenia caused by drug-induced agranulocytosis. This complication in the absence of any prior cytotoxic chemotherapy or any evidence of an ileocecal neoplasm argues for the central role of neutropenia in the development of this rare syndrome. Survival of the patient was dependent upon prompt, aggressive medical and surgical management and the spontaneous recovery of the neutrophil count.

Improved operative exposure of infrapopliteal vessels in combined vascular and orthopedic injuries.

In a series of six patients, a single-bar anterior external fixator was used to treat combined vascular and orthopedic injuries of the leg. This method of external fixation facilitated operative vascular exposure without sacrificing bony stability or alignment. It may be the preferred method of external fixation for these complex injuries.

Management of common femoral artery occlusion. A report of ten cases.

Complete occlusion or high-grade stenosis of the common femoral artery (CFA) occurs infrequently, whether as an isolated lesion or associated with similar lesions in other arteries. Ten patients with this condition comprise this report. Three had severe claudication, while seven required treatment for limb salvage. Two of the patients with claudication and one with critical ischemia had isolated CFA lesions. The remaining seven had CFA disease in tandem with either inflow aortoiliac disease or associated distal occlusions. Physical examination and vascular laboratory studies failed to suggest the diagnosis in every instance. Angiography was definitive in revealing the location and extent of pathology. Patients with localized disease were treated by patch angioplasty, balloon dilatation, or by graft replacement and profundaplasty. All others required more extensive reconstructions, with important variations from standard technique being necessary because of the CFA lesion. Amelioration of symptoms was achieved in all patients.