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© 2002 Schattauer GmbH, Stuttgart Thromb Haemost 2002; 88: 389–401

Platelet and Coagulation Defects Associated with HIV-1-Infection

Simon Karpatkin, Michael Nardi, David Green

New York University School of Medicine, New York, USA

Platelet Defects

HIV-1 seropositive individuals: homosexuals (1), intravenous nar-cotic addicts (2), hemophiliacs (3), and their heterosexual partners (4)develop chronic immunologic thrombocytopenic purpura (ITP). Thethrombocytopenia can present early in the course of HIV-1-infection aswell as in association with AIDS. The early onset disease (emphasizedin this review) is clinically indistinguishable from classic autoimmunethrombocytopenic purpura (ATP) seen predominantly in females, withrespect to increased megakaryocytes in the bone marrow, peripheral destruction of antibody-coated platelets and/or impaired platelet production, negative antinuclear antibody, and response to prednisone,i.v. � globulin and/or splenectomy. The disease is different fromclassic ATP with respect to the markedly increased platelet-associatedIgG, C3C4, the presence of circulating immune complexes and the male predominance.

Historical Descriptions

Homosexual ITP (HSITP)

Shortly after the first recognition of Kaposi’s sarcoma in sexually-active homosexuals at the Bellevue Hospital Oncology Clinic of NewYork University Medical Center (5), an “epidemic” of thrombocytope-nia was recognized in the same cohort of patients at the same instituti-on in November 1980. The syndrome was clinically indistinguishablefrom classic ATP, which is seen predominantly in females (11). Elevensevere cases were first described with a mean platelet count of 16,000 ± 3,000/�L, as well as two mild cases. All patients had increa-sed megakaryocytes in the bone marrow, no splenomegaly, negativeantinuclear antibody titers, and no clinical disorders known to causethrombocytopenia. Other findings included: decreased helper/suppres-sor T cell ratios, elevated platelet bound IgG, elevated circulating immune complexes, relative lymphopenia and elevated � globulin levels. Both the patient group as well as a control homosexual group(normal complete blood count) had similar increased exposure to her-pes simplex virus, cytomegalovirus, Epstein-Barr virus (EBV), hepati-tis A and B viruses; and similar histories for the use of recreationaldrugs: marijuana, amyl nitrate, and cocaine. HLA-typing for A-, B-, C-and D-loci revealed no significant associations.

Narcotic Addict ITP (NITP)

In November 1982 (2 years later), a similar epidemic of chronic ITPwas noted in 70 intravenous (IV) narcotic addicts treated at BellevueHospital (female to male ratio: 1 to 3), with mean platelet counts of53,000 ± 4,000 (range 13,000 to 140,000/�L) (2). These patients wereor had been chronic intravenous abusers of heroin, cocaine, or both fora mean duration of 10 years. Thirty-three patients had stopped takingIV drugs for an average of 21 months, indicating that the thrombocyto-penia was not caused by acute exposure to narcotics. Other possiblecauses of thrombocytopenia such as classic ATP, hypersplenism, chronic active hepatitis, or AIDS had been ruled out. Elevated plateletbound IgG, C3C4 and serum circulating immune complexes were alsonoted.

Hemophiliac ITP (HITP)

In 1983, Ratnoff et al. (3) reported on the development of ITP in five patients with hemophilia (AHF deficiency) who were multi-ply-transfused with lyophilized AHF concentrates for 5 to 9 years witha mean platelet count of 34,600 ± 29,000 (range 8,000 to 82,000/�L).This syndrome was clinically indistinguishable from classic ATP. Allfive had elevated platelet-bound IgG levels. These patients also presented with hypergammaglobulinemia, elevated circulating immunecomplexes, lymphopenia, decreased helper/suppressor T-cell ratios andpositive serology for HIV-1.

Heterosexually Transmitted HIV-1-Related ITP

In 1987 six cases of HIV-1-related ITP were recognized in “non-risk” individuals who had acquired the disorder through heterosexualcontact (4). One such patient was a 64-year-old white woman whosecontact was her husband who had received an HIV-1-contaminatedblood transfusion for a coronary bypass. The patient developed thrombocytopenia approximately 2 years after resumption of sexual intercourse. Thus, HIV-1-related ITP can be disguised as classic ATP,and is therefore part of the differential diagnosis of unexplained thrombocytopenia. A careful social-sexual history is mandatory for diagnosing patients.

Thrombotic Thrombocytopenic Purpura (TTP)

In 1988, eight cases of TTP associated with HIV-1 infection were reported (6-8). All 8 cases had been recognized since 1985, relativelylate in the development of the HIV-1 epidemic. Five cases reportedfrom NYU Medical Center (7) from 1985 to 1987 represented one thirdof all cases of TTP diagnosed at the institution. When this rate of inci-

Correspondence to: S. Karpatkin, New York University School of Medici-ne, 550 First Avenue, New York, N.Y. 10016, USA – Tel.: 212 263-5609, Fax: 212 263-0695; E-mail: [email protected]

Review Article

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Thromb Haemost 2002; 88: 389–401

dence, 5 of 15 patients, was compared with the incidence of patientshospitalized with AIDS during the same period (3560 of 171,210), thedifference was significant by chi square analysis, indicating that the apparent association is not caused by ascertainment bias.

Thrombotic microangiopathy (TMA) is a heterogeneous disorderthat includes both classic thrombotic thrombocytopenic purpura (TTP)and hemolytic uremic syndrome (HUS). Intravascular platelet aggrega-tes are characteristic of the pathologic lesions with sparing of the liverand lung (9) and von Willebrand factor expression is a prominent feature (10). TMA has been subsequently described in over 150 HIV-1cases (6, 7, 11-14).

Incidence and Prognosis

HIV-1-ITP. In the early description of the epidemic, sexually-activehomosexual patients presented with ITP in the presence as well as absence of AIDS. Although many patients developed ITP early afterHIV-1 infection, at least 11 other ITP patients had not developed AIDS4-7.5 years (mean 5.2 years) after HIV-1 seropositivity (15). Severalpatients studied (9 patients) reverted to normal platelet counts 5-27months (median 10 months) after the diagnosis of thrombocytopenia in the absence of treatment (i.e. splenectomy or steroids). One of thesepatients presented with a platelet count of 11,000/�L.

Thrombocytopenia can occur without clinical symptoms and increa-ses in incidence with duration of illness and the development of AIDS.For example, the incidence of thrombocytopenia in an asymptomaticgroup of 26 seropositive homosexual men was 0 percent in 1987 (16).This incidence rose to 8 percent in 59 patients with non-AIDS HIV-1-related symptoms and 30 percent in 20 patients with AIDS. Theincidence of thrombocytopenia (<100,000/�L) in 679 HIV-infected i.v. drug abusers was 21-37% (17, 18); 5% with platelet counts< 30,000/�L (18). The incidence of thrombocytopenia in 1711 HIV-1-infected patients with hemophilia varied from 5-19% (19, 20). The incidence rose in the first 5 years, with the risk increasing to 50% in older patients 1 year after the development of AIDS (20).

An analysis of 44 HIV-1-ITP patients studied for a period of 844person-months revealed no greater risk for the development of AIDSthan a similar non-thrombocytopenic seropositive cohort over a periodof 36 months (21).

HIV-1-TTP. While complete remissions are observed with appro-priate treatment in HIV-associated TTP, unlike classic TTP the repor-ted long-term survival is poor, with no survivors reported beyond 2 years (11, 13). HIV-associated TTP may differ from classic TTP in itsgradual onset, presence of concurrent medical illness, occasional spon-taneous improvement and inferior response to treatment (11).

Treatment

The rationale for treatment of HIV-1-ITP has been to use the lowestpossible corticosteroid dose capable of raising the platelet count to safelevels, generally > 25,000/�L with absence of significant purpura.Prednisone is administered at a dose of 30 to 40 mg/day for 1 to 2 weeksand rapidly tapered to a maintenance dose of 10 to 15 mg/day. The patient is then observed for 10 months, if possible, which is the mediantime for spontaneous reversion to a normal platelet count in 18% of ourpatients studied. If the patient has not obtained a safe platelet count bythis time, or requires more than 10 to 15 mg of prednisone per day tomaintain such a platelet count, splenectomy is performed. In our studyof 41 homosexual patients, 35 had a moderate (>50,000/�L) to excel-lent (>100,000/�L) response while on corticosteroids (30 relapsed

following cessation of corticosteroids). An excellent response tosplenectomy was noted in 27 of 32 patients (84%) with a mean follow-up time of 14 months (15, 22, 23). Of our original cohort of homosexualpatients, 26 of 41 (63%) did not require treatment for their throm-bocytopenia. There is no rigorous evidence that corticosteroid treat-ment or splenectomy contributes to the development of AIDS. Predni-sone therapy has been complicated, however, by the development oforal candidiasis as well as activation of latent herpes simplex virus.

The efficacy of azidodeoxythymidine (AZT) in the treatment of ITPin homosexuals is often dramatic (24, 25). There is an exponential risein platelet count within the first 1 to 2 weeks of AZT treatment (200 mgevery 6 h), with the maintenance of normal or higher counts for 7 weeks(duration of study). Hematocrits decreased in an exponential mannerduring the same period of time. After discontinuation of AZT, the platelet count remained elevated for more than 4 weeks in 3 of 5 pa-tients. These observations on the relatively rapid rise in platelet countfollowing AZT treatment may therefore suggest that HIV-1 infection ofbone marrow precursor cells, megakaryocytes or stromal cells may beinhibiting platelet production (see below). Similar results have been reported with the new anti-retroviral drugs: Indinavir, Saquinavir andritonavir. Twenty-three patients treated without AZT increased theirplatelet counts from 63,000 to 125,000/�l and decreased their viral load 74 fold during 6 months of observation with this treatment. Nocorrelation was noted with CD4 count (26).

Intravenous gamma globulin (1 to 2 g/kg for 2 to 5 days) is usuallyeffective in the treatment of HIV-related thrombocytopenia; however,its duration is transient, 7 to 10 days (27-30). Six of eight homosexualpatients had a significant rise in their platelet count, as did all of 3 drugabusers and all of eight hemophiliacs (27). Another group reported agood to excellent initial response in 12 of 17 patients (71%) (28). Pa-tients refractory to prednisone will occasionally respond to gamma globulin given with steroids.

Intravenous anti-D treatment for Rh+ nonsplenectomized patients isalso effective in the treatment of the disease. HIV-1-infected childrenhave been reported to have the best results with an effect lasting 21 daysin 50% of the responders (31).

Mechanisms of Thrombocytopenia

The mechanism of thrombocytopenia in patients with homosexual,i. v. narcotic addict, and hemophiliac chronic ITP (HSITP, NITP, andHITP, respectively) have been studied extensively by our group (1, 2,32-39) in patients with early onset HIV-1-infection in the absence ofAIDS where the mechanism appears to be increased platelet destruction(40) and compared with those of classic ATP. Myelodysplastic featureswith hypocellularity have been reported in patients with long-termHIV-infection and hemophilia (median 12.5 yrs from seroconversion)(41). In another study in children, TPO levels were 5 fold elevated insevere thrombocytopenic patients who did not respond to i.v. gammaglobulin suggesting marrow failure (42).

Increased Platelet Destruction

Autologous platelet survival studies performed in 13 HSITP patientswith a mean platelet count of <33,000/�L demonstrated a platelet survival of less than 1 day (normal survival time 8 to 10 days). Plateletsequestration was exclusively to predominantly splenic in 85 percent ofthe patients studied. Calculated blood flow and mean transit time werenormal, ruling out hypersplenism (43). Autologous platelet survivalstudies performed in 19 i.v. drug abuser HIV-1-ITP patients with a

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Karpatkin et al.: Platelet and Coagulation Defects Associated with HIV-1-Infection

mean platelet count of <31,000/�L were < 1 day with no evidence ofsplenic sequestration (43). In another study mean platelet lifespan in 7 patients was 3 ± 4 h with a marked increase in platelet production butinsufficient to maintain a normal platelet count. Five patients with nor-mal platelet counts had a compensated thrombocytolytic state (44). Theabove data support an increased peripheral platelet destruction mechanism for the pathophysiology. Indeed the rapid improvement following treatment with steroids and/or splenectomy in most early-onset HIV-1-infected patients is strongly suggestive of this pattern. However, others have reported impaired platelet production, as well asdecreased platelet survival (see below).

Decreased Platelet Production

Kinetic data on the rapid rise in platelet count (as early as 1 week)following AZT administration suggested that impaired megakaryocyteproduction, platelet release, or both may also be involved (24, 25). Therapid rise in platelet count following the use of AZT precludes a priorchange in anti-platelet Ab levels and suggests that this drug eitherblocks reticuloendothelial function or prevents HIV-1-induced damageof megakaryocytes, which otherwise results in ineffective thrombo-poiesis; or stimulates platelet production. Three platelet kinetic reportssuggest that impaired thrombopoiesis may be operative in patients withHSITP. In one study, platelet production was normal or decreased in 7of 14 patients despite a decreased platelet survival (45). Another groupstudied platelet survival and turnover in 19 homosexual patients withITP treated with and without AZT (46). Both cohorts had a decreasedplatelet survival of about 50 percent of normal. Platelet turnover (pro-duction) was significantly decreased (approximately half the normal rate) in the untreated group (13 patients), compared with normal or increased turnover in the treated group (6 patients). Two patients werestudied before and after AZT administration. Their rise in platelet countwas associated with a three-to-six-fold rise in platelet turnover, with nochange in platelet survival. Thus, AZT appears to exert its effect by enhancing platelet production, suggesting that the thrombocytopeniamay also be related to impairment of the production or function of megakaryocytes. A third study of 6 patients revealed a combination ofshortened autologous platelet survival by 2/3 normal, doubling of sple-nic platelet sequestration and ineffective delivery of platelets to the pe-ripheral blood despite a 6 fold increase in TPO and a 3 fold increase inmegakaryocyte mass (47). These observations are supported by a studyof hemopoietic progenitor cells in 15 patients with AIDS or HIV-1 in-fection, 3 of whom were thrombocytopenic (48). These investigatorsreported a significant impairment of growth of bone marrow mega-karyocyte colony-forming units, as well as granulocyte, erythocyte, andmacrophage precursors in comparison with normal controls, which wascorrected by depletion of T cells and reversed by readdition of autolo-gous T cells. Because most patients have normal to increased mega-karyocytes in their marrow, it is likely that the impairment is caused byineffective thrombopoiesis (platelet production and release). The me-chanism of inhibition of thrombopoiesis is currently unresolved.

An unlikely possibility is the direct infection of megakaryocyteswith HIV-1 (49, 50). Megakaryocytes have CD4 (51, 52) as well asCXCR4 (53, 54), known receptors for HIV-1gp120 and various mega-karyocyte lines are infectable with HIV-1 (52, 55). However reports ofHIV-1 infection of wild-type human megakaryocytes reveal little to noinfectivity (1-5% of cells) compared to T cells or macrophages withquestionable clinical significance (49).

There is now abundant evidence from several laboratories thatCD34+ stem/precursor hematopoietic cells are incapable of being infec-

ted in vivo or in vitro with HIV-1 (56-65). It is therefore logical to suggest that HIV-1-infected stromal cells may be responsible for the well-documented impaired megakaryocytopoiesis of HIV-1-infected patients (48, 58, 65, 66). This could be via induction of inhibitory cyto-kines. This is supported by impaired CFU-MK in 15 HIV-1-seropositivepatients compared to control bone marrows (47, 48), the morphologicalabnormalities detected in the MK of HIV-1-ITP patients (67) decreasedMK precursors (48, 58, 65, 66), and impaired MK survival due to in-creased apoptosis (66). Several cytokines have been shown to inhibitMk growth and differentiation: TGF�-1 (68-72), PF-4 (73), TNF� (74,75), � and � IFN (76) and IL-4 (77). Of interest is the observation thatHIV-1 tat protein, released by infected cells stimulate macrophage pro-duction of TGF�-1 (72), one of the most potent negative regulators ofhematopoiesis, resulting in a dose-dependent inhibition of CFU-MK. Analternative and/or additional mechanism could be autoimmune destruc-tion of MK’s or their precursor by anti-platelet/MK Ab.

These apparently contradictory conclusions regarding decreasedproduction vs increased destruction of platelets can be reconciled,perhaps by the observations of Najean and coworkers who performedplatelet kinetic studies on HIV-1-ITP patients with and without AIDS.They concluded that patients with AIDS are more likely to have decrea-sed platelet production, whereas patients with early onset HIV-1 infec-tion are more likely to have increased peripheral destruction of platelets(40).

Increased Platelet Consumption

TTP is associated with an absent VWF-cleaving Zn metalloproteaseof the ADAMTS13 family (a disintegrin and metalloprotease) in the familial form (78) and the presence of an IgG inhibitor of the proteasein the non-familial form (79, 80). In HUS (81) and in bone marrowtransplant associated TMA normal protease levels are detected (82).Absent VWF-cleaving protease and associated inhibitor have also beendocumented in a case report of HIV-associated TTP (83). The availa-bility of a standardized VWF-cleaving protease assay may allow forimproved classification and management of TMA and elucidate theprecise role of HIV in pathogenesis. In light of its proposed mechanism,TTP may be viewed similarly to ITP, as a manifestation of the clinicalspectrum of HIV-related autoimmunity.

Immunologic Measurements of Platelet Antibody and Immune Complexes

Immunologic measurements of platelet associated IgG, C3C4 andserum circulating immune complexes (CIC), determined by the PEGmethod in homosexual ITP (HSITP), narcotic addict ITP (NITP) andhemophiliac ITP (HITP) reveal strikingly elevated values compared tothe values found in classic ATP patients, i.e. 2.6-3.8 fold greater IgG,2.2-3.9 fold greater C3C4, 2.4-7.3 fold greater PEG-IC. Intermediateelevations of these measurements were noted in non-thrombocytopenicHIV-1-seropositive individuals of the same cohorts (2, 32, 38). Thesedata suggested that circulating immune complexes may be responsiblefor the elevated platelet levels and that no relationship existed betweenthe platelet count and the platelet associated IgG or C3C4. This provedto be the case. Isolated CIC were shown to bind to normal platelets in aconcentration-dependent manner; indeed the level of serum CIC corre-lated with platelet bound IgG, r = 0.5, p <0.05, n = 17. However, un-like the situation with classic ATP, there was no inverse relationship between platelet count and platelet associated IgG, r = -0.2, p >0.1, n = 16 (similar results were reported by others); nor was the platelet

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count inversely related to CIC’s, r = –0.2, p >0.1, n = 20. There-fore, although the platelet immunologic/serum CIC profile is of valuediagnostically in differentiating the thrombocytopenia from classic ATP,it is of no value in predicting platelet count or response to therapy.

Serum anti-platelet binding has been reported employing an indirectsemiquantitative platelet suppression immunofluorescence test in ho-mosexual patients, with impaired binding to Glanzmann’s throm-basthenia platelets [devoid of platelet GPIIb/IIIa (84)]. However, theseauthors did not prove F(ab’)2 binding. A second group, although unableto elute anti-platelet Ab from HIV-1-ITP narcotic addict patients wereable to detect plasma anti-platelet Ib/IX and IIb/IIIa Ab utilizing theMAIPA technique on normal platelets preincubated with patient plasma in 43% of 45 patients (85). Our group has been unable to detectserum F(ab’)2 binding in the homosexual cohort of HIV-1-ITP patients.However we have detected F(ab’)2 binding in HIV-1 seropositive i.v.drug abusers and hemophiliacs (2, 32). The reasons for these differen-ces are likely to be the presence of anti-idiotype blocking Ab (see below).

Composition of Immune Complexes

Anti-F(ab’)2 Antibodies

Studies designed to analyze platelets and immune complexes for viral antigens suggested the presence of anti-antibodies. Fixed washedplatelets or eluates of HSITP patients revealed absence of EBV, cyto-megalovirus (CMV), herpes simplex virus (HSV), or adenovirus(ADE) viral antigens on their platelets, despite the presence of viral an-tibodies for these antigens in their sera. Similar findings were noted infixed immune complexes of these patients. Viral antibodies againstEBV, CMV, HSV, and rubeola virus were noted in these complexes,and correlated with their presence in sera. No such correlation was noted with preparations from healthy control subjects whose immunecomplexes were negative in the presence of positive antiviral serum titers (39).

Following this lead, anti-F(ab’)2 antibodies were sought and foundin 9 of 12 HSITP and 6 of 6 i.v. drug abusers, which correlated with im-mune complex levels, r = 0.83, p <0.01, n = 16. In contrast, anti-F(ab’)2

antibodies were not detectable in 6 patients with classic ATP againstF(ab’)2 fragments of subjects from autologous, homologous ATP ornormal control groups (39).

Anti-HIV-1gp120 and Anti-idiotype Antibodies

Analysis of serum PEG-CIC as well as platelet acid eluates of HIV-1-ITP, HSITP and NITP patients has revealed the presence of anti-HIV-1gp120 Ab and its anti-idiotype (33, 86). HIV-1 antigen or provi-ral DNA was not detectable. Approximately 50% of eluted platelet IgGcontained anti-HIV-1gp120 Ab (33).

Anti-GPIIIa Ab In Vitro and In Vivo

PEG-IC’s of HIV-1-ITP patients were purified on protein A col-umns, dissociated in acid, separated by gel filteration on an acidified G200column and affinity-purified on anti-IgG and anti-IgM columns. BothIgG and IgM reactivity was found against HIV-1gp120, HIV-1gp24CD4 receptor, and human platelets. Only the IgM reacted with purifiedFc fragments, indicating rheumatoid factor (RF) reactivity. The IgG an-ti-platelet reactivity was shown to be specific for platelets by demon-strating reactivity with F(ab’)2 fragments (34) (Fig. 1). Anti-plateletIgG reactivity was affinity purified on fixed platelets and the eluate in-cubated with platelet lysates containing 125I-labeled surface plateletmembrane. The immune complex precipitate was then analyzed onSDS-PAGE in the presence and absence of reducing agent. An ~100 Kdpredominant band was noted, which paradoxically increased its mwupon reduction demonstrating the physical characteristics of plateletGPIIIa. This was proven with specific MoAb’s against platelet GPIIIa,simultaneously incubated with platelet lysate (Fig 2) (34). Further stu-dies revealed specificity for amino acids 49-66 on GPIIIa, with an inverse curvilinear relationship between patient anti-platelet IgG serum(as well as anti-GPIIIa49-66) and platelet count (Fig. 3). The in vivo pathophysiologic relevance of these in vitro observations was substan-tiated by demonstrating that ~25 �g of affinity-purified anti-plateletIgG1 of HIV-1-ITP PEG-IC’s induced dramatic thrombocytopenia inrecipient mice (which have 83% homology with human GPIIIa and Fc receptors for human IgG1). In vivo specificity was substantiated by demonstrating prevention or reversal of the anti-GPIIIa-inducedthrombocytopenia with an albumin conjugate of GPIIIa49-66 (Fig. 4)(37).

The presence of this immunodominant autoimmune GPIIIa49-66epitope appears to be unique, since classic ATP appears to have multi-ple epitopes within platelet membrane receptors, GPIIb/IIIa and GPIb.

Fig. 1 Anti-platelet reactivity of disso-ciated IgG and IgM of immune complexescompared to HIV-1-ITP patient serumIgG. Washed platelets (1�107) were ap-plied to plastic microtiter wells in PBS for1 h at room temperature, blocked withPBS/1% BSA, and then assayed by ELISAfor IgG or IgM. (A) Anti-platelet reactivityof dissociated IgG and serum IgM. (B) An-ti-platelet reactivity of dissociated F(ab’)2

fragments of IgG vs. serum IgG (n = 4)

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The observations in HIV-1-ITP could be related to cross-reactivity withHIV-1 antigens. This is suggested from the work of Bettaieb et al whonoted cross-reactivity with HIV-1gp120 and platelet GPIIIa (87); ourlaboratory which found anti-HIV-1gp120 on platelets of HIV-1-ITP patients (33); Gonzalez-Conejero et al. who noted cross-reactivity withHIV-1gp120 of serum-platelet reactive eluates with GPIIb/IIIa andGPIb/IX in 20% of 45 drug abusers (85); and Chia et al. (88) who described cross-reactive HIV-1p24 and HIV-1gp120 Ab’s in alkalineand acid eluates of serum Ab’s bound to an HIV sepharose 4B affinitycolumn. Indeed 48% of alkaline-eluted Ab bound to platelets as well asp24.

Rheumatoid Factor Antibodies

Elevated CD5+ B cells have been reported in patients and mice withautoimmune diseases as well as in HIV-1-ITP (89). CD5+ B cells gene-rally produce low affinity antibodies, which are predominantly RF ofthe IgM class against Fc of IgG (90). Because PEG-IC’s of these pati-ents contain high concentrations of IgM, RF-IgG complexes weresought within their serum PEG-IC’s. Purified PEG-IC IgM was shownto react with purified PEG-IC IgG of these patients, relocating ~50% ofthe IgG preincubated with IgM to the Vo (void volume) region of aG200 gel filtration column (34). Thus PEG-IC’s of HIV-1-ITP patients

Fig. 2 Immunoprecipitation of 125I-platelet surface antigens with anti-platelet IgG. (A) Experiment 1. Lanes 1 and 2 contain platelet lysates of RM (homosexualITP) treated with anti-platelet IgG, unreduced and reduced, respectively; lanes 3 and 4 were treated with control IgG, unreduced and reduced, respectively. (B) Ex-periment 2. Lanes 1, 3 and 5 are unreduced. Lanes 2, 4 and 6 are reduced. Lanes 1 and 2, anti-platelet IgG of MM (intravenous drug abuser ITP); lanes 3 and 4,treated with monoclonal antibody LK3r against GPIIIa; lanes 5 and 6, treated with monoclonal antibody LK7r against GPIIIa. Control IgG (not shown) gave noprecipitation as in experiment 1, lanes 3 and 4. Arrow, 100-kDa band

Fig. 3 Correlation of platelet count with anti-platelet IgG and anti-GPIIIa-(49-66). (A) Anti-platelet IgG was extracted from PEG-ICs, affinity-purified with fi-xed platelets, and tested for its reactivity with washed platelets adsorbed to a microtiter plate via a dilution determination of antibody titer, as described. A best-fitcurvilinear power regression curve was selected by computer analysis (Macintosh DELTA GRAPH PRO 3.5) with R2 = 0.61, F(1,25) = 40.2, and P< 0.0001. (B)Anti-GPIIIa-(49-66) concentration was measured in serum by using a standard curve of affinity-purified anti-GPIIIa-(49-66) vs. purified peptide. Ordinate repre-sents nanograms of antibody per ml of sera, diluted 1:4, reacting with GPIIIa-(49-66) adsorbed to microtiter plates. Gray horizontal band represents mean ± 2 SDof 10 control subjects tested. A similar best-fit curvilinear power regression curve was selected with R2 = 0.51, F(1,29) = 30.0, and P< 0.0001

A B

PLATELET COUNT/µl � 103 PLATELET COUNT/µl � 103

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Fig. 4 In vivo induction of thrombocytopenia in mice injected with affinity-purified anti-platelet IgG of HIV-1-ITP patients and its reversal with GPIIIa-(49-66).(A) Thirty micrograms of anti-platelet IgG of patients 6 and 11 or purified control human IgG was injected i.p. into eight experimental and six control mice, respectively. (B) Twelve mice were injected i.p. with 25 �g of anti-platelet IgG of patient 11, as above. Seven of these mice were simultaneously injected in theopposite flank with 226 nmol of GPIIIa-(49-66)-albumin conjugate, whereas five were simultaneously injected with the irrelevant scrambled GPIIIa-albumin conjugate (CGGGARVLEDRP) at the same concentration. (C) Ten mice were injected i.p. with 50 �g of anti-platelet IgG of patients 10 and 11, as above. At 2 h, six of these mice were injected with GPIIIa-(49-66)-albumin conjugate, whereas four were injected with the irrelevant-scrambled GPIIIa-albumin conju-gate

A

C

B

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contain RF-IgG complexes as well as anti-HIV-1gp120-anti-idiotypecomplexes.

Anti-GPIIIa Ab and Anti-idiotype Ab

Serum from HSITP and NITP patients have little anti-platelet Ab aswell as anti-GPIIIa49-66 Ab compared to purified serum IgG sug-gesting the presence of blocking Ab. Indeed no inverse correlation wasnoted between platelet associated IgG and platelet count. This appearedto be different from our findings in hemophiliac HIV-1 seropositivethrombocytopenic patients (HITP) as well as 15 classic ATP patients,in which serum anti-platelet Ab as well as F(ab’)2 fragment binding waseasily detectable. Indeed 6/6 HITP patients had elutable anti-plateletAb from their platelets and an inverse relationship was noted betweenplatelet count and platelet bount IgG, r = -0.8, p <0.001, n = 28 (34). Similar results were obtained with classic ATP patients, similarly studied (34). To investigate this difference in detectable serum anti-GPIIIa49-66 Ab in HSITP and NITP patients, crude serum, purified serum IgG and PEG-IC IgG were isolated and studied for anti-GPIIIa49-66 reactivity. This revealed ~150 fold greater Ab reactivity inpurified serum IgG and ~4000 fold greater reactivity in PEG-IC IgG(Fig. 5). This was explained by the presence of anti-idiotype Ab2 (bothIgG and IgM) sequestered within the PEG-IC.

The IgM anti-idiotype was further studied since it is predominantlya blocking Ab, as demonstrated by specificity for F(ab’)2 fragments ofanti-GPIIIa49-66, and inhibition with peptide GPIIIa49-66, not with acontrol peptide. The IgM anti-idiotype Ab is not polyreactive. In agroup of HIV-1-infected patients (non-thrombocytopenic as well asthrombocytopenic) a positive correlation was noted between plateletcount and anti-idiotype IgM (r = 0.7, p = 0.0001, n = 22). The in vivorelevance of the IgM anti-idiotype Ab was tested by inducing throm-bocytopenia (–30% baseline) in mice with anti-GPIIIa49-66. Preincu-bation of the anti-GPIIIa49-66 Ab with the IgM anti-idiotype (molar ratio1:7) improved the platelet count to 80% of normal and throm-bocytopenia could be reversed, 4 h after induction of thrombocytopenia

(Fig. 6) (36). Thus CIC’s of HIV-1-infected patients contain blockingIgM anti-idiotype Ab against anti-GPIIIa which regulates their serumreactivity in vitro and level of thrombocytopenia in vivo.

These observations along with the presence of platelet reactive idiotype-anti-idiotype complexes (i.e., HIV-1gp120) as well as the presence of RF offer an explanation for the markedly elevated immunecomplex/Ig on platelets of HIV-1-ITP patients. They also offer an explanation for the presence of IgG on platelets of HIV-1-infected patients with normal platelet counts – namely the presence of sufficientanti-idiotype Ab to neutralize the thrombocytopenia.

Fig. 5 Anti-platelet GPIIIa49-66 reactivity of serum, serum IgG, and IC-IgGof one representative HIV-1-ITP patient of five patients studied. Various IgGconcentrations of serum, purified serum IgG, and purified IC-IgG were assay-ed for anti-GPIIIa49-66 reactivity by solid-phase ELISA

Fig. 6 Prevention and reversal of in vivo thrombocytopenia induced by anti-GPIIIa-(49-66) induced in mice with IgM anti-idiotype Ab against anti-GPIIIa-(49-66). (A) Prevention. Four to seven mice in each group were treated intrape-ritoneally with either anti-GPIIIa-(49-66) Ab, Ab plus control IgM (125 �g).Ab plus anti-idiotype IgM (125 �g). Ab plus anti-idiotype IgM (875 �g), or Abplus anti-idiotype IgG (875 �g). Platelet counts were obtained at various timeintervals for 24 h. The mean platelet count ± SEM for all four groups was similar (1.72 ± 0.56 � 106/�l). Pt, patient. (B) Reversal. 16 mice were treatedintraperitoneally with 25 �g of anti-GPIIIa-(49-66), and 4 mice were treated intraperitoneally with saline. At 4 h, after induction of thrombocytopenia, the16 antibody-treated mice were treated (4 mice in each group) with 875 �g ofeither control IgG, patient (Pt) anti-idiotype IgG, control IgM, or patient anti-idiotype IgM, and were followed for 24 h. Note reversal of thrombocytopeniawith IgM anti-idiotype at 6, 8, and 12 h compared with control IgM (P =0.0008, 0.0015, and 0.0137, respectively; paired Studient’s t test), not with IgGanti-idiotype or control IgG

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Complement-Independent, Peroxide-induced Anti-GPIIIa49-66 Platelet Lysis

A recent report that recognized the presence of platelet fragmentswithin the serum PEG-IC’s stimulated a search for antibody-dependentplatelet lysis. This was found to occur in vitro with gel-filtered platelets(Fig. 7 and 8) as well as in vivo in association with the induction ofthrombocytopenia. The same results were noted with F(ab’)2 fragmentsas well as intact IgG in complement-deficient mice, indicating the absence of complement-induced lysis. This new pathophysiologic mechanism of platelet destruction was shown to be induced via peroxi-de generation from a platelet-associated NADPH oxidase pathway. Invitro platelet lysis could be blocked by scavengers of reactive oxygenspecies (catalase, superoxide dismutase, diphenyleneniodonium) (Fig. 9) and platelet lysis did not occur in NADPH oxidase-deficientmice (p47phox-/-). Thrombocytopenia was significantly improvedcompared to its induction in wild-type mice by 60% (Fig. 10).

Coagulation Abnormalities

A number of coagulation abnormalities have been described in HIV disease including acquired deficiency states of the physiologic anticoagu-lants: protein C (91), protein S (92) and heparin cofactor II (93). Of these,protein S deficiency is the most consistently observed and is present in upto 73% of HIV-infected men (92, 94, 95). Most commonly there is reduc-tion in free protein S and coordinate reduction in functional protein S activity. C4bBP levels are normal indicating that reduced protein S is not aconsequence of a shift to the bound form, which is typically seen in in-flammatory states. There is no correlation of free protein S deficiency withclinical thrombosis in HIV-infected men (96). Protein S is reduced in numerous other conditions including pregnancy, use of oral contraceptives,DIC, acute thrombosis and liver disease. The clinical significance of acqui-red protein S deficiency associated with HIV disease is unclear. High levelsof plasma von Willebrand factor have been reported in HIV disease andmight be indicative of activated endothelium (97).

Fig. 7 In Vitro Platelet Fragmentation Induced by Anti-GPIIIa49-66 Ab. A. Immunoblot of PEG-IC of control and HIV-1-ITP patients employing anti-GPIIIa,anti-GPIba and control mouse monoclonal Ab, MoPC. CTL refers to control. B. Flow Cytometry of Platelet In Vitro Particle Formation. Gel filtered platelets were labelled with anti-GPIIb-FITC monoclonal Ab, washed and then treated with various agents: buffer, control IgG and patient anti-GPIIIa49-66 for 4 h at 37° C. Three panels represent: CTL, buffer alone; CTL IgG, IgG isolated from control PEG-IC; PT IgG, IgG isolated from HIV-1-ITP patient PEG-IC. X axis refers to fluorescence, Y axis to forward scatter. Numbers in left upper quadrant refer to the % Ab-induced particles with decreased fluorescence attributed to platelet fragmentation. A highly-reactive anti-GPIIIa49-66 IgG is shown. C. Distribution of % platelet particle formation in control vs. HIV-1-ITP vs. ATP patients at 1 h incubation. Results shown for IgG isolated from PEG-IC’s of 12 control, 16 HIV-1-ITP and 5 ATP patients. Box Plot. Mean is shown by the solidblack box; median by the horizontal line in the large open box; 25th and 75th percentiles by the lower and upper border of the large open box from which spread ofthe data from the position of the median can be assessed. Whiskers include 99% of a Gaussian distribution

A

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Anticardiolipin antibodies (aCL) and lupus anticoagulants (LA) arefrequent incidental laboratory findings in HIV-infected patients. Thepresence of LA is an established risk factor for both venous and arteri-al thrombosis in patients with SLE and is detected by a prolonged par-tial thromboplastin time (aPTT) that fails to correct with mixing, or al-ternatively by prolongation of the Kaolin clotting time, diluted tissuethromboplastin time or dilute Russell viper venom time. Elevated aCLare found in 20-70% of HIV patients (98) and are predominantly IgG(99). LA activity is prevalent in HIV-infected patients and consistsmainly of IgM antibody (100, 101). Despite the high prevalence of aCLand LA in HIV-infected patients, the clinical manifestations of the clas-sic antiphospholipid antibody syndrome (APS) such as stroke or TIA,recurrent venous thrombosis, cutaneous lesions and miscarriage or themanifestations of catastrophic APS are distinctly unusual. Only a singlecase report links APS to HIV infection (102). Anti-beta-2-glycoproteinI (-�2GPI) and anti-prothrombin antibodies are seen less commonly inHIV infected patients than in APS/SLE or primary APS patients. Thebiological false positive test for syphilis is rarely detected in HIV-in-

fected patients (103). This antiphospholipid antibody specificity mightexplain the lack of clinically evident thrombosis in the HIV-infectedpopulation. It is suggested that elevated aCL in HIV results from pan-Bcell stimulation/hypergammaglobulinemia or alternatively could resultfrom infection. The presence of aCL and lupus anticoagulant activitydo not correlate with the clinical features of HIV including medicationuse, infections or malignancy or CD4 T-cell count nor with the deve-lopment of thrombosis (96). LAs are not generally considered to pose ableeding risk, however associated thrombocytopenia (104), plateletdysfunction or hypoprothrombinemia (105) may result in unexpectedbleeding particularly after surgery and prophylactic transfusion of pla-telets and plasma may be appropriate in select cases.

HIV and Thrombosis

Venous thromboembolic disease (VTE) is uncommon in AIDS pati-ents but has been described in a number of case reports and in retros-pective reviews (91, 106–108). Thrombosis at unusual sites including

Fig. 8 Electron Microscopy of DamagedPlatelets treated with Anti-GPIIIa49-66 Ab.A: Patient sample at 1 h showing plateletswith a fuzzy material attached to the outersurface of the cell membranes (dotted arro-ws). Gaps are noted in the cell membraneswith leakage of cytoplasmic content (arro-ws). These areas are demonstrated at highermagnification in B and C. E: patient sampleat 4 h showing degenerating platelets and disintegration of the cell membrane (arrow).Swollen platelet (F), platelet fragments (H)and occasional normal platelets (G,I) are alsoseen. None of these changes were present inIgG controls at 4 h (D). Original magnifica-tions: A, D-I: 4,000 X. B: 50,000 X. C:40,000 X

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retinal vein (109) and sagittal sinus (110) have been reported which isalso a characteristic feature of thrombophilic states. The majority ofVTE is associated with advanced HIV (CD4 count <200), underlyingAIDS-related malignancy or opportunistic infection, while idiopathicDVT is less common. It is not known whether the incidence of VTE dif-fers in HIV patients as compared to the general population since thishas not been studied in a prospective case control manner. Given thediagnostic challenge of VTE and the protean clinical manifestations ofHIV disease itself, the incidence of DVT may be underestimated in thispatient population. Further studies will be required to determine thethrombosis risk attributable to HIV and related co-morbidities, as wellas the optimal strategies for DVT treatment and prophylaxis and the utility of thrombophilia screening.

HIV and Hemophilia

AIDS is the leading cause of death in hemophilia. There has not been documented HIV seroconversion since 1986 (111). ITP in HIV-infected hemophiliacs may further worsen the risk of bleeding. The treatment of hemophilia is complicated by the development of high titer factor VIII inhibitors, which may resolve with advanced HIV disease due to loss humoral immunity (112).

A number of reports have suggested that protease inhibitors have thepotential to cause increased bleeding in HIV-infected hemophiliacs(113). Increase in frequency of bleeding episodes, unusual sites of blee-ding and increased factor concentrate requirement have all been noted(114). Ritonavir has received particular attention in this regard, but allthe currently available protease inhibitors are linked to increased blee-ding. The mechanism of bleeding in the majority of cases was not established, although platelet dysfunction was identified as a cause ofbleeding in two hemophiliacs (115). Mucosal bleeding has also been reported in non-hemophiliacs treated with protease inhibitors indicatingthat the effect is not related to factor VIII per se (114, 116).

Fig. 9 Generation of Peroxide and Effect ofPeroxide Inhibitors, Catalase, Diphenylenei-odonium (DPI) and Superoxide Dismutase(SOD) with Platelet Particle Formation Indu-ced by Anti-GPIIIa49-66 Ab or Thrombin. A.Gel filtered platelets were preincubated withinhibitor or buffer for 15 min prior to the ad-dition of rabbit anti-GPIIIa49-66 Ab or con-trol IgG. C refers to control non-immune IgG(similar results with control plus highest inhi-bitor concentration employed), IS refers torabbit anti-GPIIIa49-66 immune serum. Barslabelled 2, 3, 4 after bar 1 refer to doublingconcentrations of inhibitor (50 u/ml catalase,5 nM/ml DPI and 12.5 u/ml SOD), n = 4, SEMis given. B. Generation of peroxide by anti-GPIIIa49-66 at 1 h (panels A, B, C) and 4 h(panels D, E, F). Platelets were loaded withthe intracellular dye, DCFH-DA and thentreated with buffer (panels A and D), controlIgG (panels B and E) or human anti-GPIIIa49-66 (panels C and F). C. Effect of peroxide in-hibitors on thrombin-induced platelet particleformation. Gel-filtered platelets were preincu-bated with inhibitors, as in AA and then treated with 1 u/ml thrombin for 4 h. Bars refer to doubling concentrations, n = 4

Fig. 10 Effect of Anti-GPIIIa49-66 Ab on Platelet Count and Platelet ParticleFormation in Control C57BL/6 and p47phox(-/-) Mice. Control C57BL/6 orp47phox(-/-) mice were injected i.p. with 25 ug of intact anti-GPIIIa49-66 or 16 ug of its F(ab’)2 fragment and platelet count and % platelet particles moni-tored at 2 and 4 h. A. Platelet count. B. % platelet particle. N = 6-8 for eachgroup, SEM is given

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Received May 2, 2002 Accepted June 17, 2002

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