Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal

Alpha2-antiplasmin (alpha2AP) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Two forms of alpha2AP circulate in human plasma: a 464-residue protein with methionine as the amino-terminus (Met-alpha2AP) and an N-terminally-shortened 452-residue form with asparagine as the amino-terminus (Asn-alpha2AP). Human plasma alpha2AP concentration is 1 micro M and consists of approximately 30% Met-alpha2AP and approximately 70% Asn-alpha2AP. The major form (Asn-alpha2AP) is rapidly crosslinked to fibrin during blood clotting by activated coagulation factor XIII and as a consequence, fibrin becomes more resistant to fibrinolysis. It is apparent that alpha2AP is important in modulating the effectiveness and persistence of fibrin with respect to its susceptibility to digestion and removal by plasmin. Hence, the physiologic role of alpha2AP suggests that it may be a useful target for developing more effective treatment of thrombotic diseases. Research on alpha2AP appears to be moving in two main directions: (1) efforts to use variant forms of alpha2AP to reduce bleeding secondary to thrombolytic therapy while not slowing thrombolysis; and (2) efforts to use variant forms to diminish the activity of alpha2AP as a plasmin inhibitor so that fibrinolysis becomes enhanced. Methods to accomplish these two goals mostly involve manipulation of defined functional domains within the molecular structure of alpha2AP, or inhibition of a newly described novel plasma proteinase, termed antiplasmin-cleaving enzyme, that generates the more favorable form of alpha2AP, Asn-alpha2AP, for crosslinking to fibrin. The antiplasmin-cleaving enzyme has similarity in primary structure and catalytic properties to fibroblast activation protein/seprase. This review summarizes recent studies that may hold promise for modulating alpha2AP activity and its interactions with certain proteins as new therapeutic strategies for preventing and treating thrombotic disorders.     

Colectomy for acute colitis: is it safe to close the rectal stump?

We report 62 operations for acute colonic inflammatory bowel disease in which the rectal stump was closed. Operative findings were of severe colitis in 46, toxic megacolon in 8 and faecal peritonitis in 8 patients. Histology showed ulcerative colitis in 48, Crohn's disease in 9 and indeterminate colitis in 5 patients. Clinical evidence of stump leakage occurred in only one of 53 patients with a long rectal stump in contrast to 3 of 9 patients who had a short rectal stump. Leaving a very short stump also led to difficulty at subsequent proctectomy in 3 patients and at restorative proctocolectomy in 1 patient. This suggests that careful closure of the rectum above the peritoneal reflection can be a safe means of dealing with the rectal stump after total colectomy and ileostomy for acute colitis.

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Résumé Nous rapportons l'expérience de 62 résections coliques pour poussées inflammatoires aiguës du côlon chez lesquelles le moignon rectal a été fermé. Les constatations opératoires mettaient en évidence une colite sévère chez 46 patients, un mégacôlon toxique chez 8 patients et une péritonite fécale chez 8 patients. L'examen histologique a mis en évidence une colite ulcéreuse chez 48 patients, une maladie de Crohn chez 9, une colite indéterminée chez 5. Un lâchage clinique du moignon rectal ne s'est produit que chez un des 53 patients chez lesquels le moignon rectal était long comparativement à 3 sur 9 patients dont le moignon rectal était court. Un moignon rectal court a, par ailleurs, entraîné des difficultés opératoires lors d'une proctectomie subséquente chez 3 patients et d'une procto-colectomie avec rétablissement de la continuité chez 1 patient. Ces observations suggèrent qu'une fermeture soigneuse du rectum au-dessus de la ligne de réfection du péritoine peut être un moyen sûr de traiter le moignon rectal apprès colectomie totale et iléostomie en cas de colite aiguë.

     

CD4 memory T cells divide poorly in response to antigen because of their cytokine profile

Immunological memory is a hallmark of adaptive immunity, and understanding T cell memory will be central to the development of effective cell-mediated vaccines. The characteristics and functions of CD4 memory cells have not been well defined. Here we demonstrate that the increased size of the secondary response is solely a consequence of the increased antigen-specific precursor frequency within the memory pool. Memory cells proliferated less than primary responding cells, even within the same host. By analyzing the entry of primary and memory cells into the cell cycle, we found that the two populations proliferated similarly until day 5; after this time, fewer of the reactivated memory cells proliferated. At this time, fewer of the reactivated memory cells made IL-2 than primary responding cells, but more made IFNγ. Both these factors affected the low proliferation of the memory cells, because either exogenous IL-2 or inhibition of IFNγ increased the proliferation of the memory cells.     

A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.     

Radioactive choline metabolism in guinea pig gallbladder

Acetylcholine may be released from gallbladder intrinsic nerves in response to cholecystokinin stimulation. This study characterized metabolites of [¹⁴C]choline produced in the gallbladder and released during incubation, with or without cholecystokinin-octapeptide. Radiolabeled [¹⁴C]choline was applied to the mucosal or muscle surface of intact guinea pig gallbladders in an organ bath. After radiolabeling, gallbladders were incubated with or without the contractile agonist cholecystokinin-octapeptide. Metabolites of [¹⁴C]choline were identified in gallbladder tissue and incubation buffers using HPLC and thin-layer chromatography. The major metabolites of [¹⁴C]choline were betaine and phosphocholine. [¹⁴C]Phosphocholine was incorporated slowly into [¹⁴C]phosphatidylcholine. [¹⁴C]Choline was released into buffers during incubation. [¹⁴C]Acetylcholine constituted less than 1% of radiolabel in the gallbladder. There was no identifiable [¹⁴C]acetylcholine released in buffers. Cholecystokinin-octapeptide did not affect choline metabolism. These studies showed that choline in the gallbladder is metabolized along pathways similar to those in the liver. Gallbladders released mostly choline, rather than acetylcholine, even during hormonally induced contraction.This project was supported by the Research and Development Office of the Department of Veterans Affairs.     

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.     

A radioreceptor assay for quantitating plasma factor VIII/von Willebrand's protein

A sensitive and precise radioreceptor assay for determining plasma levels of human factor VIII/von Willebrand's factor (FVIII/vWF) has been developed by taking advantage of the FVIII/vWF receptor sites on human platelets. Paraformaldehyde-fixed platelets, which were processed and then stored, retained FVIII/vWF binding activity and therefore could be used as a convenient source of receptors. The human plasma samples to be tested were initially filtered on 4% agarose columns to concentrate the FVIII/vWF protein in the void volume and to remove the factor(s) that interferes with the assay. The percent recovery of FVIII/vWF in the pooled eluent was measured by the recovery of added trace 125I-FVIII/vWF. The coefficients of intra- and interassay variation were 6% and 10%, respectively. The plasma FVIII/vWF concentrations determined by the assay for pooled normal plasma, hemophilia A plasma, and plasmas from two patients with von Willebrand's disease were 16.3 +/- 0.5, 52.6 +/- 1.5, 6.8 +/- 0.8, and 3.2 +/- 0.2 microgram/ml, respectively. The range of plasma FVIII/vWF concentrations varied between 8.3 microgram/ml and 24.9 microgram/ml for 10 normal adults. The plasma FVIII/vWF concentrations determined by the radioreceptor assay correlated well with levels measured by the ristocetin-induced platelet aggregation method, thus demonstrating the functional relevancy of the radioreceptor assay for plasma FVIII/vWF.