Chan K. N. K. Chion

The central role of thrombin in hemostasis

Summary  Following vascular injury, blood loss is controlled by the mechanisms of hemostasis. During this process, the serine proteinase, thrombin, is generated both locally and rapidly at sites of vessel damage. It plays a pivotal role in clot promotion and inhibition, and cell signaling, as well as additional processes that influence fibrinolysis and inflammation. These functions involve numerous cleavage reactions, which must be tightly coordinated. Failure to do so can lead to either bleeding or thrombosis. The crystal structures of thrombin, in combination with biochemical analyses of thrombin mutants, have provided insight into the ways in which thrombin functions, and how its different activities are modulated. Many of the interactions of thrombin are facilitated by exosites on its surface that bind to its substrates and/or cofactors. The use of cofactors not only extends the range of thrombin specificity, but also enhances its catalytic efficiency for different substrates. This explains a paradox (i.e. thrombin is a specific proteinase, and yet one that has multiple, and sometimes opposing, substrate reactions). In this review, we describe the context in which thrombin acts during hemostasis and explain the roles that its exosites and cofactors play in directing thrombin function. Thereafter, we develop the concept of cofactor competition as a means by which the activities of thrombin are controlled.

ADAMTS13 Substrate Recognition of von Willebrand Factor A2 Domain

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605–Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593–1668), VWF115-(1554–1668), VWFA2-(1473–1668)) and one A2-A3 (VWF115-A3-(1554–1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 μm; kcat 0.14 s–1), from which the specificity constant kcat/Km was calculated, 8.70 × 104 m–1 s–1. Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved ∼8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to ∼1.3 μm), compared with VWF115 (KD 20 nm). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.

A functional calcium-binding site in the metalloprotease domain of ADAMTS13

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca(2+) ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca(2+) concentrations, a high-affinity functional ADAMTS13 Ca(2+)-binding site was suggested with K(D(app)) of 80 muM (+/- 15 muM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca(2+)-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca(2+) dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca(2+)-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K(D(app)) for Ca(2+) of ADAMTS13, and for D187A and E212A also reduced the V(max) to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca(2+) revealed an approximately 13-fold reduction in specificity constant, k(cat)/K(m), contributed by changes in both K(m) and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca(2+) upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

Further characterization of ADAMTS-13 inactivation by thrombin

Summary.  Background: The multimeric size and platelet‐tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS‐13). In vitro ADAMTS‐13 is susceptible to proteolytic inactivation by thrombin. Objectives: In this study, we aimed to characterize the inactivation of ADAMTS‐13 by thrombin and to assess its physiological significance. Methods and results: By N‐terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS‐13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS‐13. Proteolysis of ADAMTS‐13 by thrombin caused an 8‐fold reduction in its affinity for VWF that contributed to its loss of VWF‐cleaving function. Intriguingly, thrombin‐cleaved ADAMTS‐13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS‐13, but not added ADAMTS‐13, appeared resistant to coagulation‐induced fragmentation. An estimation of the Km for ADAMTS‐13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS‐13. This was corroborated by the comparatively low affinity of ADAMTS‐13 for thrombin (KD 95 nm). Conclusions: Together, our data suggest that ADAMTS‐13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non‐haem iron containing monooxygenases including benzene monooxygenase, toluene 4‐monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co‐ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α‐helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co‐ordinates of the methane monooxygenase α‐subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non‐haem iron monooxygenases.

BREVIBACTERIUM LINENS PBL33 AND RHODOCOCCUS RHODOCHROUS PRC1 CRYPTIC PLASMIDS REPLICATE IN RHODOCOCCUS SP. R312 (FORMERLY BREVIBACTERIUM SP. R312)

The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312). The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp. R312. They all carry the Tn903 neomycin-resistance-encoding gene (aphI).