Mirko Zuzel

Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases.

Local and systemic haemorrhage are common consequences of crotaline and viperine envenoming. Several studies carried out using purified toxins have indicated that local haemorrhage can be attributed to a distinct class of venom metalloproteinases. Analyses of their cDNAs predict multi-domain enzymes, with an N-terminal metalloproteinase domain, a disintegrin-like domain and a Cys-rich C-terminus. Haemorrhagic metalloproteinases are responsible for degrading proteins of the extracellular matrix and they also have cytotoxic effects on endothelial cells. However, to date very few investigations have been carried out on the effects of venom haemorrhagic metalloproteinases on components of the haemostatic system. We describe here the effects of a high molecular weight haemorrhagic metalloproteinase, jararhagin, from the venom of a South American pit viper Bothrops jararaca, on platelet and plasma components involved in haemostasis. Jararhagin, which is not inhibited in plasma, causes the loss of the platelet collagen receptor alpha 2 beta 1 integrin (gpIa/IIa or VLA-2) and degrades the adhesive plasma protein von Willebrand factor. Alterations of these haemostatic components are known to result in bleeding. This suggests that venom haemorrhagic metalloproteinases, in addition to causing local bleeding, may also contribute to systemic haemorrhage.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, α2-macroglobulin

Observations that a haemorrhagic metalloproteinase (jararhagin) from Bothrops jararaca venom had less effect on platelets suspended in plasma than in washed platelet suspensions, suggested that plasma contains naturally occurring inhibitor(s) of this enzyme. By using radiolabelled jararhagin and crossed immunoelectrophoresis, we have demonstrated the binding of this enzyme to alpha 2-macroglobulin in plasma. SDS-PAGE analysis of this binding revealed the presence of radioactivity in four bands with relative molecular masses of 640, 570, 520 and 410 kDa; in addition a small amount of 47 kDa free enzyme was demonstrable. Reduced samples showed an additional non-complexed 90 kDa fragment of alpha 2-macroglobulin generated by jararhagin. These results are compatible with a model in which, upon multiple cleavages of alpha 2-macroglobulin, the enzyme becomes covalently bound to the inhibitor, and the two halves of the inhibitor become crosslinked. However, jararhagin activity was not completely inhibited even after long incubation (60 min) with a large (10-fold) molar excess of alpha 2-macroglobulin either in plasma or a purified alpha 2-macroglobulin preparation. Kinetic studies showed that inhibition was comparatively slow, although jararhagin readily cleaved alpha 2-macroglobulin in the bait region. Therefore, the ineffectiveness of the inhibition could have resulted from a low tendency of this proteinase to form covalent complexes with the inhibitor. We conclude that the pronounced haemorrhagic activity of jararhagin can be attributed to prolonged access of this enzyme to high molecular weight substrates, even in the presence of a large molar excess of alpha 2-macroglobulin.

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming.

Because of uncertainties regarding the comparability of granulocyte‐macrophage and granulocyte colony‐stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl‐leucylphenylalanine (fMLP) was not influenced by G‐CSF (0.1–100 ng/ml), whereas GM‐CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM‐CSF and G‐CSF in combination was not different from that of PMNs treated with GM‐CSF alone. GM‐CSF (10 ng/ml) increased the rate of O2 ‐ production by 79%, caused a fivefold increase in fMLF‐induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G‐CSF (50 ng/ml) only slightly increased O2 ‐ production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM‐CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G‐CSF on mature PMNs is negligible compared with that of GM‐CSF. Our results are in conflict with previous reports of much more pronounced G‐CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM‐CSF. We therefore suggest that the primary role of G‐CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in viva J. Leukoc. Biol. 55: 612–616; 1994.

WHAT IS THE NATURE OF THE HAIRY CELL AND WHY SHOULD WE BE INTERESTED

Hairy-cell leukaemia (HCL) has always attracted an interest out of all proportion to its frequency. Initially, this interest arose from the striking appearance of the pathognomonic hairy cell (HC) and centred on the diagnostic features of the disorder. More recently, interest has continued as the disease first became treatable with interferon (IFN) and then became potentially curable with nucleosides. However, now that questions of diagnosis and treatment seem largely answered, why should we continue to be interested in the disorder? There are, we believe, two major reasons. First, the very distinctive appearance that initially attracted attention to the HC is likely to be an outward reflection of the basic oncogenic event. Thus, by more precisely understanding the unique features of the HC, it may be possible to elucidate the nature of the underlying malignant change. Second, it seems likely that the high degree of sensitivity of the HC to IFN and nucelosides is also related to the ‘uniqueness’ of the cell. For this reason, an understanding of the nature of the HC may well provide important insights relevant to the use of nucleosides in chronic lymphoproliferative disorders in general. The nature of the HC has not been specifically reviewed for a number of years. During this time, not only has the understanding of HCs improved, but substantial new insights have been gained into the development of normal and abnormal lymphocytes. For all these reasons, a reconsideration of the nature of the HC in the light of B-cell development seems timely.

A function of CD10 on bone marrow stroma

Summary. Bone marrow (BM) stromal cells express CD10 (cALLA), a surface antigen now known to be a neutral endopeptidase (NEP‐24.11). The function of CD10 in BM stroma is unknown, although purified NEP‐24.11 is known to degrade different substrates including interleukin 1β (IL‐1β).