This month in J Lab Clin Med

The Journal of LABORATORY and CLINICAL MEDICINE Copyright © 1997 by Mosby-Year Book, Inc.

VOLUME 129

FEBRUARY 1997

NUMBER 2

THIS MONTH IN J Lab Clin Med Issue Highlights for February 1997

Why stop making platelets? Is thrombospondin the yang to thrombopoietin's yin? The regulation of platelet production has come under closer scrutiny over the last several years as new tools have emerged for its study. Since the late 1950s, it has been recognized that a plasma factor recovered from patients or animals with thrombocytopenia could promote platelet production when injected into platelet-replete recipients. The long-postulated thrombopoietin has recently been isolated and its gene has been cloned; its clinical use appears to be on the near horizon. The negative feedback remains mysterious: how does an organism "know" that it has enough platelets and thus shut off thrombopoietin production or make the marrow less sensitive to its stimulatory effects? In a number of other contexts--generally involving malignant transformation--adhesive or adhesogenic proteins have been shown to be involved in the dysregulation of cell proliferation. Dr. Yuan Zhong Chen and colleagues from the University VII of Paris and H6pital St. Louis (also in Paris) therefore explored the possibility that thrombospondin (TSP), a platelet granule constituent, might be part of the regulatory feedback on platelet production. As described on page 231, the authors used TSP and a number of recombinant TSP fragments and fusion proteins, exploring their effects on murine megakaryopoiesis in vitro. In the simplest systems, they found that TSP did not inhibit growth of CFU-MK (megakaryocyte colonies in serum-flee agar) or CFU-GM (granulocyte-and-macrophage colonies in plasma clot). The story was quite a bit different when thrombopoietin was present. Plasma from patients with aplastic anemia was used to create a high-thrombopoietin plasma clot system. Under these conditions, TSP inhibited megakaryocyte growth in a dose-dependent manner. No inhibition was seen if a TSP derivative lacking the heparin-binding domain was used instead of the intact molecule. The authors then repeated the study, using recombinant thrombopoietin, interleukin-3, or basic fibroblast growth factor (bFGF) (a fibroblast growth factor with known effects on megakaryocytes); they also studied additional stimulatory substances in a methylcellulose culture system. The findings were analogous, indicating that the inhibitory effect was on stimulated megakaryocyte growth generally rather than on thrombopoietin specifically. The apparent importance of the heparin-binding domain was further explored by adding a low-molecular-weight heparin to the system; it blunted the effect of thrombospondin as a growth inhibitor. 169

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J Lab Clin Med February 1997

The authors conclude that thrombospondin inhibits megakaryocyte growth in response to a variety of stimuli in vitro; because it is released from platelet a-granules, its level may be a marker of platelet sufficiency (or excess); it is thus an attractive candidate to be part of the negative feedback of platelet production in vivo.

But maybe it's something more general: Thrombospondins inhibit endothelial cell mitogenesis A number of growth factors have been shown to induce angiogenesis in rabbit corneas; among these are several tyrosine kinase receptor ligands, including bFGF (the same growth factor studied by Chen et alii, [vide supra]). Thrombospondin-1 (TSP1) inhibits this process and is believed to be an important modulator of angiogenesis in wound healing, tumor metastasis, etc. Dr. Tracee S. Panetti and coworkers from the University of Wisconsin asked whether this inhibition involved direct effects on the growth of endothelial cells and whether it involved regulation of transforming growth factor [3 (TGF[3). Uptake of tritiated thymidine by bovine aortic endothelial cells increased in response to either of two growth factors, bFGF or lysophosphatidic acid (LPA). These two mitogens work through distinct receptors that have convergent signaling pathways. Both the baseline mitogenic activity and the increment provoked by either bFGF or LPA were inhibited in a dose-response manner by human TSP1 (either recombinant or purified from platelets). Recently it has been reported that TSP2 does not activate latent TGF[3--thus a similar effect with that mediator would argue against TSP's inhibition being worked through regulation of TGF[3. Indeed, recombinant TSP2 (murine) inhibited mitogenesis with a similar dose-response profile. The authors conclude that structural motifs common to TSP1 and TSP2 inhibit endothelial cell proliferation in a manner not dependent on TGF[3 and not highly specific for an individual mitogen receptor. Their report may be found beginning on page 208.

Continuing the theme: Lipoproteins and mitogenesis in the kidney Another article in this month's issue also deals with the regulation of mitogenesis. Glomerular injury can be associated with hyperlipidemia, and this appears to involve interactions of low-density lipoproteins (LDLs) and oxidized LDLs with mesangial cells. It is also known that endothelin-1 is produced by mesangial cells, is a potent vasoconstrictor, and may work as an autocrine/paracrine mitogen for mesangial cells. Mian-Shin Tan and associates from the Kaohsiung Medical College (Taiwan) asked whether the effects of lipoproteins on mesangium might involve endothelin production. In an article beginning on page 224, the authors describe their use of competitive reverse transcription polymerase chain reaction to explore endothelin-1 mRNA expression in cultured rat mesangial cells. Such expression was increased after incubation with oxidized LDLs but not after incubation with native LDLs. Oxidized LDLs also increased the amount of assayable endothelin-1 released into the culture medium, in a time- and dose-dependent manner. At very high concentrations, the oxidized LDLs were actually cytotoxic. The authors conclude that the effect of oxidized LDLs on endothelin-1 production by mesangial cells may contribute to glomerular injury in hyperlipidemia. Their report adds to the many now showing that oxidized lipids are nastier than their native counterparts.

J Lab Clin Med Volume 129, Number 2

In this issue

Collagen binding to von Willebrand factor: An interesting wrinkle in type 2a vWd Von Willebrand factor (vWf) occupies a key position at the intersection between platelets and the plasma coagulation cascade. It acts as the stabilizing carrier for plasma factor VIII and it also acts as a bridge between activated platelets and extracellular matrix proteins such as collagen. When this protein is deficient or dysfunctional, patients suffer a bleeding disorder that has clinical features both of platelet-type bleeding (failed primary hemostasis) and of plasma-factor-deficiency-type bleeding (failed secondary hemostasis). The function of vWf is highly dependent on its state of polymerization; in fact, one type of von Willebrand disorder (type 2a) is associated with a normal level of vWf in plasma but a relative lack of the largest multimers. Plasma from such patients has long been known to be ineffective at promoting the adherence of stimulated platelets to collagen in vitro. vWf is made and stored in endothelial cells, from which it may be released by epinephrine, vasopressin, or a number of other stimuli. High-molecular-weight multimers are well represented in the stored vWf, even in patients with type 2a vWd. This is because most such patients actually make the multimers; the multimers are simply unstable once released to the plasma. One can exploit this peculiarity clinically; the administration of desmopressin brings about a transient rise in vWf levels and may improve clinical hemostasis in patients with v W d - including those with 2a disease. Clinical response to desmopressin would suggest that the vWf of patients with type 2a vWd works pretty well, even if it is unstable. That, in turn, suggests that its collagen-binding domain is probably normal. Dr. A. Casonato and several coauthors from the University of Padua tested this concept in patients, and they present their findings beginning on page 251. Four patients with type 2a vWd were treated with desmopressin, and their plasma vWf was examined before and after this manipulation. As expected, the level rose within 30 minutes, and more high-molecular-weight (HMW) multimers appeared. If the vWf was collected into a medium containing protease inhibitors, it was found to have normal binding to collagen in microtiter wells. If there were no protease inhibitors present, the collagen-binding activity rapidly decayed. Finally, vWf from these patients' platelets--relatively protected from proteolysis--showed better collagen-binding activity and more HMW multimers than did vWf from their plasma samples. The findings of this study are in some ways what one would have predicted; the confirmation is nonetheless useful. Moreover, the demonstration that theplatelet vWf in patients with vWd 2a has enough multimers to behave essentially normally may be an important insight: this may be telling us that plasma vWf is far more important than platelet vWf in maintaining hemostasis.

The Ethics Forum: Scientific and academic misconduct This month we are pleased to present another in our occasional series of essays on topics in research ethics. At issue on this occasion is the attempt to define what does and does not constitute scientific misconduct. Although there are many egregious behaviors that most if not all of us would identify as wrong, it's not such a simple matter to establish ground rules and definitions that will reliably separate ovine from caprine behavior in less obvious cases. Dr. Frederick Grinnell, on page 189, presents his response to a definition proposed by the Commission on Research Integrity, arguing that it is insufficient to serve as an adequate moral or legal standard. Guest Editor Arthur Caplan adds his comments on page 172. For the editors DALE E. HAMMERSCHMIDT, MD Senior Editor

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