Editorial

Interferon-Induced Thrombocytopenia:Is It Time for Thrombopoietin?

SEE ARTICLE ON PAGE 1424

In 1988, Souyri et al. discovered a novel hematopoieticcytokine receptor fused to the envelope protein of the murinemyeloproliferative leukemia virus. The viral receptor, termedv-mpl, was found to be constitutively activated and capable ofinducing a myeloproliferative-like syndrome in mice.1 Thenative murine homologue, termed c-mpl, was later found tobe essential for megakaryocyte development. This observa-tion suggested that the ligand for c-mpl was an importantregulator of megakaryopoiesis. Using c-mpl as a guide, manyinvestigators raced to identify this novel protein. In 1994, fiveindependent groups successfully cloned the gene for thempl-ligand otherwise termed thrombopoietin (TPO).2-6 Nu-merous in vivo and in vitro studies have since shown that TPOis the most potent and specific cytokine for megakaryocytegrowth and platelet production.7

The gene is located at chromosome 3 p26-27. It encodes a332-amino acid protein with a molecular weight of approxi-mately 35,000 kd. The amino-terminal region has 23%identity to erythropoietin and the carboxy-terminus showsno known homology. The amino-terminus is highly con-served between species and contains the receptor bindingsite. The C-terminus has several potential N-linked glycosyla-tion sites suggesting a role in protein kinetics. TPO messen-ger RNA (mRNA) transcripts have been found predominantlyin the liver with lesser amounts also detected in the kidneys,spleen, and bone marrow. Sungaran et al.8 recently showed byin situ hybridization the precise cell types responsible for TPOexpression. In the liver, TPO transcripts were detected in thehepatocytes but not in the phi-sinusoidal cells. In the bonemarrow, stromal cells but not hematopoietic cells showedTPO mRNA expression. In the kidney, the proximal convo-luted tubules showed TPO staining.8 TPO receptors havebeen found on hematopoietic stem cells, megakaryocytes,and platelets.9

The current model suggests that TPO is regulated solely atthe level of ligand binding to receptor. In this model, TPO isconstitutively produced in the liver and released into circula-tion. TPO then binds to specific high affinity platelet recep-

tors (c-mpl). Platelet receptors act as a ‘‘sink’’ and have theability to bind, internalize, and degrade TPO. When plateletcounts are normal (.140,000/µL), most TPO is bound toreceptors on platelets and plasma levels are low. Whenplatelet counts are low (,140,000/µL), few receptors areavailable for TPO binding and serum levels increase. ElevatedTPO levels stimulate megakaryopoiesis, which results inincreased platelet production. This model establishes aninverse relationship between TPO levels and platelet counts.10

Animal and human studies evaluating TPO levels and plateletcounts in chemotherapy-induced thrombocytopenia, aplasticanemia, and bone marrow transplantation have supportedthis inverse relationship model.11,12

The liver appears to be the most important organ for TPOproduction. Consequently, several groups have attempted toinvestigate whether thrombocytopenia associated with cir-rhotic liver disease may be a consequence of altered TPOproduction. Although there are some conflicting reports,most studies have shown inappropriately low endogenousTPO levels in cirrhotic patients with thrombocytopenia.13-15

An inverse relationship between TPO levels and plateletcounts has not been found in cirrhotic patients with thrombo-cytopenia. Several studies have also evaluated serial plasmaTPO levels in cirrhotic patients undergoing liver transplanta-tion.13,15 In 16 of 17 patients undergoing liver transplantationat the University of California, San Francisco, the TPO levels,which were low or undetectable before transplantation,became detectable within 3 days post-transplantation. PeakTPO levels occurred by approximately 1 week post-transplantation and all patients had normalization of theirplatelet counts. Restored TPO production likely accounts forthe rapid resolution of thrombocytopenia after transplanta-tion. These and other data support the hypothesis that alteredTPO production contributes to the thrombocytopenia incirrhosis.13

Despite strong evidence that TPO production is altered incirrhosis, some controversy remains regarding the relativeimportance of altered TPO production versus hypersplenism.Hypersplenism certainly contributes to the thrombocytope-nia in cirrhosis. Nuclear medicine studies have clearly shownsplenic pooling of platelets.16,17 Unfortunately, noninvasiveprocedures have limited use in evaluating the extent to whichhypersplenism is a cause of thrombocytopenia in cirrhosis.Furthermore, in the studies discussed above, one cannotexclude the possibility that low TPO levels are a direct resultof TPO binding to a normal number of platelets sequesteredin the spleen, i.e., pseudo-thrombocytopenia caused byhypersplenism. Consequently, additional studies are neededto further evaluate the relative importance of altered TPOproduction versus hypersplenism in the thrombocytopeniaassociated with cirrhosis.

Abbreviations: TPO, thrombopoietin; mRNA, messenger RNA; IFN, interferon alfa;PEG-rHuMGDF, pegylated recombinant human megakaryocyte growth and develop-ment factor; rHuTPO, recombinant human thrombopoietin.

Received September 8, 1998; accepted September 14, 1998.From the Division of Hematology/Oncology, University of California San Francisco

Medical Center, San Francisco, CA.Address reprint requests to: Thomas G. Martin, M.D., Department of Medicine, Box

1270, University of California, San Francisco, CA 94143. Fax: (415) 476-4943.Copyright r 1998 by the American Association for the Study of Liver Diseases.0270-9139/98/2805-0036$3.00/0

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In this issue of HEPATOLOGY, Peck-Radosavljevic et al. (seepage 1424) analyzed serial TPO levels in patients receivinginterferon alfa (IFN) therapy for chronic hepatitis C virusinfection. Approximately one third of the patients hadevidence of cirrhosis before starting IFN therapy. Baselineplasma TPO levels were similar in patients with or withoutcirrhosis; however, cirrhotic patients had significantly lowerinitial platelet counts. Both groups of patients experiencedapproximately a 32% to 35% decrease in platelet count whileon IFN therapy. As expected, plasma TPO levels increased inresponse to IFN-induced thrombocytopenia in patients with-out cirrhosis. In contrast, plasma TPO levels remainedunchanged in cirrhotic patients with IFN-induced thrombo-cytopenia. These data provide further evidence for alteredTPO production in liver disease. The lack of TPO response toIFN-induced thrombocytopenia in the cirrhotic patientssuggests that these patients are already producing maximalTPO.

IFN is the only therapeutic agent known to have lastingbeneficial effects on either hepatitis B or C virus infections.18

Unfortunately, the side effects of IFN are considerable. Asdepicted by Peck-Radosavljevic et al., one of the most significanthematologic side effects of IFN is a dose-dependent decreasein platelet count. The thrombocytopenia usually occurs withinthe first several weeks of IFN therapy and often leads tosuboptimal dosing. The thrombocytopenia is caused by adirect inhibitory effect on megakaryocytes. Peck-Radosavl-jevic et al. also investigated whether IFN can directly effecthepatic TPO mRNA expression, protein synthesis, or proteinexcretion. They used an in vitro culture system with thehepatoma cell line HepG2. Overall, IFN had no effect on TPOmRNA expression but a significant inhibitory effect on thecells’ ability to secrete TPO into the culture medium. Al-though this finding is provocative, it requires confirmation intissue.

Perhaps the issue of greatest importance is whether or notthrombopoietin administration can facilitate the use of IFN inhepatitis C infection or ameliorate the numerous complica-tions of thrombocytopenia associated with liver disease. Atpresent there are two molecules, pegylated recombinanthuman megakaryocyte growth and development factor (PEG-rHuMGDF or MGDF) and recombinant human thrombopoi-etin (rHuTPO) being manufactured by for clinical use byAmgen Inc. (Thousand Oaks, CA) and Genentech Inc. (SouthSan Francisco, CA), respectively. PEG-rHuMGDF is a trun-cated molecule containing the ligand binding domain and apolyethylene glycol molecule attached to the amino termi-nus. Pegylation improves protein stability and prolongs thehalf life (T1/2 approximately 30-40 hours). The protein ismanufactured in Escherichia coli. rHuTPO is the full-lengthnative hormone with a half life of approximately 18 to 32hours. The hormone is produced in a mammalian cell line topreserve C-terminal glycosylation.

Phase I clinical trials using PEG-rHuMGDF and rHuTPOwere initially performed in patients with advanced metastaticcancer. The hormones were given to patients before chemo-therapy to evaluate safety, pharmacokinetics, and efficacy.PEG-rHuMGDF was initially given at doses ranging from0.03 µg/kg to 1.0 µg/kg daily for 10 doses. The hormone waswell tolerated with only one patient developing superficialthrombophlebitis, which resolved spontaneously. There were

no other substantial alterations in vital signs, temperature,body weight, or performance status. The only significantchanges in laboratory tests were increases in platelet countand number of bone marrow megakaryocytes. PEG-rHuMGDFgiven in doses of 0.1 µg/kg and higher were associated withmarked increases in platelet counts (2 patients with platelets.1.2 million/µL). Platelet counts began to increase onapproximately day 6, peaked between days 12 to 18, andremained elevated for 12 to 26 days. Platelets taken fromMGDF-treated patients were found to be morphologicallyand functionally normal.19 The studies evaluating rHuTPOwere very similar except that rHuTPO was given as a singleinjection. At a dose of 2.4 µg/kg two of three patientsdeveloped platelet counts of approximately 1 million/µL.Toxicities were minimal and the hematologic effects weresimilar to PEG-rHuMGDF.20 Overall, the initial phase Istudies showed that PEG-rHuMGDF and rHuTPO are welltolerated and extremely potent platelet cytokines. Phase IIand III trials in cancer patients are now underway.

Although recombinant thrombopoietins have been studiedpredominantly in oncology, PEG-rHuMGDF has also beenused in transfusion medicine studies. PEG-rHuMGDF givenas a single dose of 3.5 µg/kg to normal platelet pheresisdonors has produced platelet pheresis yields approximatelythree times higher than placebo-treated controls.21 Despitethis initial enthusiasm, Amgen Inc. has recently discontinuedplatelet pheresis clinical trials because of the development ofneutralizing antibodies and prolonged thrombocytopenia in aplatelet pheresis donor. Neutralizing antibodies cross-reactwith endogenous TPO and inhibit the protein’s effects.Further investigations into the cause and effects of neutraliz-ing antibodies are needed. This toxicity has not been reportedin oncology patients who are immunocompromised, perhapsbecause such patients are less likely to develop these alloanti-bodies.

The rHuTPOs are extremely potent megakaryocyte-specific cytokines. The safety profile appears favorable exceptfor the development of neutralizing antibodies in a normalplatelet pheresis donor. Although this rare toxicity deservesserious consideration, expanded investigations in the use ofrecombinant thrombopoietins outside of oncology are war-ranted. Thrombocytopenia in liver disease is associated withsignificant morbidity and mortality. In addition, thrombocyto-penia in cirrhosis is likely a result of impaired TPO produc-tion. Therefore, an interesting and logical initial phase I trialwould involve cirrhotic patients with thrombocytopenia. Thegoals of the study would be to show safety and to ascertainwhether TPO administration could overcome hypersplenismin cirrhosis and result in elevated platelet counts. Once safetyis shown, phase II and III efficacy studies could then beperformed. One potential use for TPO would be in patientsreceiving IFN therapy for chronic viral hepatitis. Manypatients are excluded from IFN therapy because of pretreat-ment thrombocytopenia. In addition, IFN-induced thrombo-cytopenia results in dose reductions or withdrawal of therapyin up to 25% of patients. Thrombopoietins may allow formore therapeutic dosing of IFN resulting in improved effi-cacy. Other potential indications for TPO administrationinclude postliver transplantation thrombocytopenia, recur-rent variceal bleeding with thrombocytopenia, preinvasiveprocedure in cirrhotic patients with thrombocytopenia,

HEPATOLOGY Vol. 28, No. 5, 1998 MARTIN AND SHUMAN 1431

and preliver transplantation to decrease blood product use.rHuTPOs likely will become important therapeutic agents fortreating complications associated with thrombocytopenia inliver disease.

THOMAS G. MARTIN, M.D.MARC A. SHUMAN, M.D.Division of Hematology/OncologyUCSF Medical CenterSan Francisco, CA

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1432 MARTIN AND SHUMAN HEPATOLOGY November 1998