Yama/CPP32β, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase

Caspases, which are the executioners of apoptosis, comprise two distinct classes, the initiators and the effectors. Although general structural features are shared between the initiator and the effector caspases, their activation, inhibition and release of inhibition are differentially regulated. Biochemical and structural studies have led to important advances in understanding the underlying molecular mechanisms of caspase regulation. This article reviews these latest advances and describes our present understanding of caspase regulation during apoptosis.

Molecular mechanisms of caspase regulation during apoptosis

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins.

http://www-heparin.rpi.edu/main/files/papers/4f1623c54d6ab5.21304086.pdf

Heparin-protein interactions

Almost one-third of all proteases can be classified as serine proteases, named for the nucleophilic Ser residue at the active site. This mechanistic class was originally distinguished by the presence of the AspHis-Ser “charge relay” system or “catalytic triad”.1 The Asp-His-Ser triad can be found in at least four different structural contexts, indicating that this catalytic machinery has evolved on at least four separate occasions.2 These four clans of serine proteases are typified by chymotrypsin, subtilisin, carboxypeptidase Y, and Clp protease (MEROPS nomenclature;3 Table 1). More recently, serine proteases with novel catalytic triads and dyads have been discovered, including Ser-His-Glu, Ser-Lys/His, His-Ser-His, and N-terminal Ser.2 Several of these novel serine proteases are subjects of accompanying articles in this issue. This article will review recent work on the mechanism and specificity of chymotrypsin-like enzymes, with the occasional references to pertinent experiments with subtilisin. Chymotrypsin-like proteases are the most abundant in nature, with over 240 proteases recognized in the MEROPS database.3 * E-mail: hedstrom@brandeis.edu; phone: 781-736-2333; FAX: 781-736-2349. 4501 Chem. Rev. 2002, 102, 4501−4523

Serine protease mechanism and specificity

The American Heart Association, in conjunction with the National Institutes of Health, annually reports the most up-to-date statistics related to heart disease, stroke, and cardiovascular risk factors, including core health behaviors (smoking, physical activity, diet, and weight) and health factors (cholesterol, blood pressure, and glucose control) that contribute to cardiovascular health. The Statistical Update presents the latest data on a range of major clinical heart and circulatory disease conditions (including stroke, congenital heart disease, rhythm disorders, subclinical atherosclerosis, coronary heart disease, heart failure, valvular disease, venous disease, and peripheral artery disease) and the associated outcomes (including quality of care, procedures, and economic costs).The American Heart Association, through its Statistics Committee, continuously monitors and evaluates sources of data on heart disease and stroke in the United States to provide the most current information available in the annual Statistical Update. The 2022 Statistical Update is the product of a full year's worth of effort by dedicated volunteer clinicians and scientists, committed government professionals, and American Heart Association staff members. This year's edition includes data on the monitoring and benefits of cardiovascular health in the population and an enhanced focus on social determinants of health, adverse pregnancy outcomes, vascular contributions to brain health, and the global burden of cardiovascular disease and healthy life expectancy.Each of the chapters in the Statistical Update focuses on a different topic related to heart disease and stroke statistics.The Statistical Update represents a critical resource for the lay public, policymakers, media professionals, clinicians, health care administrators, researchers, health advocates, and others seeking the best available data on these factors and conditions. 

https://www.ahajournals.org/doi/pdf/10.1161/CIR.0000000000001052

Heart Disease and Stroke Statistics—2022 Update: A Report From the American Heart Association

Background The β3 integrin [Glycoprotein (GP) IIIa] gene is highly polymorphic and encodes eight of the Human Platelet Antigen (HPA) systems. HPA immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP) and platelet refractoriness. However, the current methods of antibody detection are cumbersome and rely upon the use of platelets from genotyped donors. In the case of the rare HPA antigens, obtaining donor platelets is particularly problematic. We therefore set out to develop an antibody screening assay that uses recombinant, soluble β3-integrin fragments expressing the HPA-1, –4, –6, –7, –10, –11 and –16 epitopes.



Methods  A total of four integrin fragments of different lengths (ΔSDL, ΔβA, PSI-Hybrid and PSI) were designed using structural data and expressed in Drosophila S2 cells. HPA-1a (33) and HPA-1b (P33) forms of all four proteins were expressed as were the fragments ΔSDL-4b and ΔSDL-SuperRare, the latter two for the detection of HPA-4b and the rare HPA antigens (HPA-6, 7, 10, 11 and 16) respectively. The recombinant proteins were then screened for reactivity with monoclonal antibodies (mAb) and polyclonal antisera containing anti-HPA in an ELISA based assay.



Results  The reactivity pattern seen with murine mAbs Y2/51, SZ21 and CRC54 was as expected. Two of three human monoclonal HPA-1a antibodies (19–7, 23–15, both from a PTP patient) reacted with the four L33 fragments but the third (CamTran007, derived from a NAIT patient) only reacted with the ΔSDL-L33 and ΔβA-L33 fragments, defining an epitope split. The reactivity of both potency (NIBSC-03/152) and sensitivity (NIBSC-93/710) standards for anti-HPA-1a detection were comparable or better than those seen by MAIPA. In addition, samples from 132 NAIT patients with anti-HPA-1a [15 with cerebral haemorrhage (CH), 49 with platelet counts <20 × 109 L−1 but no CH, 35 with counts ≥ 20 × 109 L−1 and 33 with antenatal treatment] were tested. Anti-HPA-1a was detected in 126 samples including all CH cases. The reactivity patterns against the four different β3-integrin fragments did not correlate with clinical outcome. Finally, mini-β3 integrins Δ SDL-4b and ΔSDL-SuperRare allowed the detection of 14 of the 15 anti-HPA samples tested (5 HPA-4a, 5 HPA-4b, 1 HPA-6b, 1 HPA-7b and 2 of 3 HPA-11b).



Conclusions  Recombinant fragments of the β3-integrin expressing HPA antigens can be used to successfully detect platelet alloantibodies. These fragments with be of use in the screening and diagnosis of suspected cases of NAIT.

YS04 Recombinant Mini‐Β3 Integrin Fragments for the Detection of HPA Antibodies

Addendum: Kotál et al. Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement. Int. J. Mol. Sci. 2021, 22, 9480

/pdf/mapping-the-prothrombin-binding-site-of-pseutarin-c-by-site-8sz98vmf.pdf

Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation

Anticoagulation therapy is a mainstay of the treatment of thrombotic disorders; however, conventional anticoagulants trade antithrombotic benefits for bleeding risk. Factor (f) XI deficiency, known as hemophilia C, rarely causes spontaneous bleeding, suggesting that fXI plays a limited role in hemostasis. In contrast, individuals with congenital fXI deficiency display a reduced incidence of ischemic stroke and venous thromboembolism, indicating that fXI plays a role in thrombosis. For these reasons, there is intense interest in pursuing fXI/factor XIa (fXIa) as targets for achieving antithrombotic benefit with reduced bleeding risk. To obtain selective inhibitors of fXIa, we employed libraries of natural and unnatural amino acids to profile fXIa substrate preferences. We developed chemical tools for investigating fXIa activity, such as substrates, inhibitors, and activity-based probes (ABPs). Finally, we demonstrated that our ABP selectively labels fXIa in the human plasma, making this tool suitable for further studies on the role of fXIa in biological samples.

https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.2c00845

James A. Huntington

Papers

YS04 Recombinant Mini‐Β3 Integrin Fragments for the Detection of HPA Antibodies

Addendum: Kotál et al. Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement. Int. J. Mol. Sci. 2021, 22, 9480

Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation

Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma