Platelet function testing: state of the art.

Platelet function testing has evolved from crude tests, such as the bleeding time, to tests that permit a relatively sophisticated evaluation of platelet activity. Nonetheless, these tests are hampered by lack of specificity and sensitivity, and poor standardization of methods and techniques. The bleeding time, which has long been a staple of hemostasis testing, has been dropped from the test menu at many laboratories. In its place, tests such as the Platelet Function Analyzer-100 are increasingly used to screen patients with possible bleeding disorders. Older tests, such as platelet aggregometry and lumiaggregometry, are still used frequently because they provide insight into receptor, signaling pathway and granule release mechanisms. Flow cytometry is available in some specialized laboratories and allows for quantitative and qualitative assessment of some platelet functions, although the expense of testing is often prohibitive. Finally, the wider availability of platelet function testing has stimulated interest and demand for monitoring the effect of platelet inhibitory drugs, such as aspirin and clopidogrel. As platelet function pathways become better understood, the demand for these type of monitoring tests is likely to increase.

Diagnosis and management of heparin-induced thrombocytopenia.

Heparin use is ubiquitous, wherein 1 to 5% of patients exposed to standard unfractionated heparin develop thrombocytopenia due to antibodies to a complex of heparin and platelet factor 4. Classic features include onset of thrombocytopenia after 5 to 10 days of ongoing heparin exposure, a 50% fall in the platelet count from baseline, resolution of the thrombocytopenia 5 to 10 days after cessation of heparin and a high risk of thrombosis noted in 30 to 75% of patients with heparin-induced thrombocytopenia (HIT) in terms of every-other-day platelet-count monitoring in patients on standard unfractionated heparin. And those patients developing thrombocytopenia necessitate an accurate, readily accessible diagnostic test for HIT. Diagnosis has been recently facilitated by the development of an enzyme-linked immunosorbent assay (ELISA) test for the heparin-P4 antibody complex, although this test carries a relatively low specificity. Widespread use of the ELISA demonstrates a relatively high prevalence of the antibody in patients exposed to heparin in certain settings, such as cardiopulmonary bypass, wherein a quarter of patients have a positive ELISA of unclear significance. Once HIT is diagnosed, the high risk of thrombosis necessitates empiric anticoagulation with an antithrombin such as argatroban or lepirudin, or the heparinoid danaparoid. Additional agents under further study include the antithrombin bivalirudin and the pentasaccharide fondaparinux. Future issues in HIT include increasing awareness for HIT, improving the specificity of HIT testing and the development of new anticoagulants for HIT that will enable out-patient management.

Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease.

Mus81-Mms4/Eme1 is a conserved structure-specific endonuclease that functions in mitotic and meiotic recombination. It has been difficult to identify a single preferred substrate of this nuclease because it is active on a variety of DNA structures. In addition, it has been suggested that the specificity of the recombinant protein may differ from that of the native enzyme. Here, we addressed these issues with respect to Mus81-Mms4 from S. cerevisiae. At low substrate concentrations, Mus81-Mms4 was active on any substrate containing a free end adjacent to the branchpoint. This includes 3'-flap (3'F), regressed leading strand replication fork (RLe), regressed lagging strand replication fork (RLa), and nicked Holliday junction (nHJ) substrates. Kinetic analysis was used to quantitate differences between substrates. High Kcat/Km values were obtained only for substrates with a 5'-end near the branchpoint (i.e., 3'F, RLe, and nHJ); 10-fold lower values were obtained for nicked duplex (nD) and RLa substrates. Substrates lacking any free ends at the branch point generated Kcat/Km values that were four orders of magnitude lower than those of the preferred substrates. Native Mus81-Mms4 was partially purified from yeast cells and found to retain its preference for 3'F over intact HJ substrates. Taken together, these results narrow the range of optimal substrates for Mus81-Mms4 and indicate that, at least for S. cerevisae, the native and recombinant enzymes display similar substrate specificities.

Slx1-Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1-Top3.

The RecQ DNA helicases human BLM and yeast Sgs1 interact with DNA topoisomerase III and are thought to act on stalled replication forks to maintain genome stability. To gain insight into this mechanism, we previously identified SLX1 and SLX4 as genes that are required for viability and for completion of rDNA replication in the absence of SGS1-TOP3. Here we show that SLX1 and SLX4 encode a heteromeric structure-specific endonuclease. The Slx1-Slx4 nuclease is active on branched DNA substrates, particularly simple-Y, 5'-flap, or replication fork structures. It cleaves the strand bearing the 5' nonhomologous arm at the branch junction and generates ligatable nicked products from 5'-flap or replication fork substrates. Slx1 is the founding member of a family of proteins with a predicted URI nuclease domain and PHD-type zinc finger. This subunit displays weak structure-specific endonuclease activity on its own, is stimulated 500-fold by Slx4, and requires the PHD finger for activity in vitro and in vivo. Both subunits are required in vivo for resistance to DNA damage by methylmethane sulfonate (MMS). We propose that Sgs1-Top3 acts at the termination of rDNA replication to decatenate stalled forks, and, in its absence, Slx1-Slx4 cleaves these stalled forks.

The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10.

Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae. We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.