Michael Nardi

On the Mechanism of Thrombocytopenic Purpura in Sexually Active Homosexual Men

Thrombocytopenic purpura has recently been noted in sexually active homosexual men. To elucidate the pathogenesis of thrombocytopenic purpura in this population, we compared the disorder in 33 homosexual men with that in 23 patients (15 women and 8 men) thought to have classic autoimmune thrombocytopenic purpura. The homosexual group had 3.8-fold higher levels of platelet-bound IgG and 4.2-fold higher levels of platelet-bound complement than the patients with autoimmune thrombocytopenic purpura. Eluates from the platelets of only 1 of 10 homosexual patients reacted in vitro with normal platelets, as compared with those from the platelets of 12 of 15 patients with autoimmune thrombocytopenic purpura. Twenty-one of 24 homosexual patients (88 per cent) had elevated serum levels of immune complexes that were capable of binding to platelets, whereas none of 5 patients with autoimmune thrombocytopenic purpura had circulating immune complexes. The IgG fraction of positive serum samples from three homosexual patients did not bind to normal platelets, whereas that from the positive serum of two patients with autoimmune thrombocytopenic purpura and one woman in whom isoimmune antiplatelet antibody developed during pregnancy (studied as a positive control) did bind to normal platelets. We conclude that, whereas classic autoimmune thrombocytopenic purpura involves antiplatelet IgG directed against platelet antigenic determinants, the thrombocytopenic purpura that occurs in sexually active homosexual men is usually caused not by antiplatelet IgG but probably by the nonspecific deposition of complement and immune complexes on platelets.

Complement-Independent, Peroxide-Induced Antibody Lysis of Platelets in HIV-1-Related Immune Thrombocytopenia

Immunologic thrombocytopenia is seen commonly in HIV-1 infection. The pathogenesis of this problem has been unclear, but it is associated with circulating immune complexes that contain platelet membrane components and anti-platelet membrane GPIIIa49-66 IgG antibodies. These antibodies cause acute thrombocytopenia when injected into mice. We now show that purified anti-GPIIIa49-66 causes platelet fragmentation, in vitro in the absence of complement, and in vivo in wild-type and C3-deficient mice. The mechanism of complement-independent platelet lysis is shown to be caused by the antibody-induced generation of H202, as indicated by in vitro experiments with inhibitors of reactive oxygen species, and in vivo studies carried out with p47phox-deficient mice. Thus, a novel mechanism of immunologic platelet clearance is described in which an anti-platelet IgG causes platelet fragmentation via the induction of reactive oxygen species.

Thrombocytopenic Purpura in Narcotics Addicts

Since November 1982 we have seen an association of thrombocytopenic purpura with chronic narcotic addiction in 70 patients with a mean platelet count of 53000 +/- 4000 (SE); 33 had stopped taking intravenous drugs for an average of 21.2 +/- 4.7 months; 13 of 15 had elevated antibody titers for a virus related to the acquired immunodeficiency syndrome. Platelet-bound IgG, IgM, and complement levels were 16.7-, 5.6-, and 3.1-fold greater than control values, respectively, and 2.6-, 1.9-, and 2.4-fold greater than values in 25 patients with classic autoimmune thrombocytopenic purpura studied at the same time. Thirty-three of thirty-six addicts had elevated circulating immune complexes, whereas 8 patients with autoimmune thrombocytopenia had no elevation. Eleven of eighteen addicts had positive serum platelet-reactive IgG titers, compared to 5 of 19 patients with classic autoimmune thrombocytopenia. The platelet-reactive IgG in sera of addicts was composed of 7S IgG antibody as well as high molecular weight (immune complex) IgG. Thus, chronic addicts appear to have a new immunologic platelet disorder associated with the presence of 7S IgG antiplatelet antibody, like patients with classic autoimmune thrombocytopenic purpura, and immune complex associated nonspecific platelet IgG, like male homosexual patients with thrombocytopenia.

Aspirin Attenuates Platelet Activation and Immune Activation in HIV-1-Infected Subjects on Antiretroviral Therapy: A Pilot Study

Background:Mechanisms for increased cardiovascular risk in HIV-1-infected adults are incompletely understood, but platelet activation and immune activation leading to a prothrombotic state have been proposed as significant contributors. Aspirin has antiplatelet and immunomodulatory properties. We explored whether 1 week of low-dose aspirin attenuates platelet activation and immune activation in HIV-1-infected and virologically suppressed adults on antiretroviral therapy. Methods:Platelet activation and immune activation were measured in HIV-1-infected subjects virologically suppressed on antiretroviral therapy and controls before and after 1 week of low-dose aspirin. Results:Compared with control subjects, HIV-1-infected subjects had increased platelet activation, as measured by spontaneous platelet aggregation and aggregation in response to adenosine diphosphate, collagen, and arachidonic acid. After aspirin therapy, percent aggregation decreased similarly in both HIV-1-infected and control subjects to all platelet agonists tested except aggregation in response to arachidonic acid, which remained elevated in the HIV-1-infected group. HIV-1-infected subjects exhibited increased markers of T-cell activation (CD38 and HLA-DR) and monocyte activation (sCD14), which decreased after 1 week of aspirin therapy. Moreover, leukocyte responses to Toll-like receptor stimulation were enhanced after 1 week of aspirin therapy. In vitro studies showed that HIV-1 plasma could activate healthy platelets, which in turn activated monocytes, implicating a direct role for activated platelets in immune activation. Conclusions:Our data demonstrate that heightened platelet activation and immune activation in treated HIV-1 disease are attenuated by 1 week of aspirin therapy. Aspirin should be further studied for its antithrombotic and immunomodulatory benefits in treated HIV-1 disease.

Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C-related immunologic thrombocytopenia.

Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1-seropositive drug abusers are more prone to develop immune thrombocytopenia than non-drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non-drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti-GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r(2) = 0.7, P < .01). Ab raised against peptide PHC09 in GPIIIa(-/-) mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti-GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P < .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.

Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway

Antiplatelet GPIIIa49-66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS.

Prothrombin and protein C in early childhood: Normal adult levels are not achieved until the fourth year of life

Prothrombin and protein C are vitamin K--dependent proteins with important roles in hemostasis. During the neonatal period, levels of both proteins are lower than in the adult. TM However, the normal range in later infancy and the age at which adult levels are reached have not been established for either protein. We report values for protein C and prothrombin in 148 healthy children ranging in age from 1 month to 4 years. METHODS The children attended the Family Care Clinic at Belle- vue Hospital Center. All were born at term after uncom- plicated pregnancies, were well at the time of study, and had no significant previous illness. Capillary blood speci- mens were obtained by finger or heel stick into heparinized microhematocrit tubes. The tubes were centrifuged imme- diately, and the plasma stored at -70 ~ C until tested. from I 1 healthy adults obtained from venous blood drawn into dry heparin. Protein C level in this pool was 115.5%, and prothrombin was 116% expressed as a percentage of pooled plasma obtained from George King. Assuming a dilution factor of 1:6 in the George King plasma related to the anticoagulant, these values are 101.5% and 101%, respectively. RESULTS Mean protein C levels in plasma obtained from citrated blood of 28 healthy adults were 102% _+ 12.5% in venous samples and 118% + 14% in capillary samples. Assuming a dilution factor of 1:6 in the venous samples because of the anticoagulant, the mean level in the capillary samples was 103% + 14%. There was a significant correlation between the venous and capillary values in the individuals studied (r -- 0.995, P <0.001). The mean values for prothrombin in the same samples were venous 98.3%-+ 11.5% and capillary 113.0% _+ 13.0%. When dilution of the venous samples with antico- agulant was taken into account, the capillary values were 98.9% _+ 13.0%. The correlation between capillary and venous sample was also highly significant (r--0.995, P <0.001 ). Levels of protein C and of prothrombin in relation to age from 1 month to 4 years are shown in the Figure and the Table. At 36 months, mean values for both proteins were still significantly lower than the adult mean level, and did not reach adult values until sometime during the fourth year of life. The initial rise was relatively rapid, but became progressively slower (Figure). Levels of prothrom- bin were higher than those of protein C for the first 8 months, and the ratio of protein C to prothrombin was significantly lower than in the adult. By 1 year of age the ratio had reached the normal adult value of I, and remained there subsequently (Table).

Low protein C in the neonatal period

Summary. Protein C was measured by electroimmunoassay in 47 infants within 24 h of delivery. Gestational age ranged from 28 to 43 weeks. The mean level was 27% (range < 10–67%) of the normal adult mean. In the 22 infants who had no clinical problems, protein C levels correlated significantly with gestational age. In the 25 who were sick there was no correlation, and the mean level was significantly lower than that of the healthy infants. Postnatal rise was slow; on day 7 the mean was 32% and on day 28, 31%. Levels of protein C correlated significantly with prothrombin in both the healthy and sick infants. Crossed immunoelectrophoresis in the presence of calcium ions gave one protein C peak of the same electrophoretic mobility as is seen in plasma of healthy adults, indicating that the infants’ protein C is gamma carboxylated. It is concluded that: (1) Protein C in neonates is in or below the range associated with thromboembolism in patients congenitally deficient in this protein; (2) protein C levels correlate with gestational age; and (3) the low levels during the neonatal period are not due to decreased gamma carboxylation but may reflect decreased synthesis when compared to the older child or the adult.

Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1½-year-old girl: Report of a case and review of the literature

Acquired inhibitors of coagulation causing bleeding manifestations are rare in children, particularly without an associated underlying disorder such as autoimmune disease. We describe an otherwise healthy 1½‐year‐old girl who had extensive spontaneous bruising and prolonged bleeding from venipuncture sites. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, with evidence of an immediate‐acting inhibitor. Thrombin clotting time, fibrinogen, and platelets were normal. Biologic assay of factors II, V, VII, and X were all low, with increasing values at higher dilutions. However, by immunoassay and/or chromogenic assays, only factor II was reduced. An antibody which failed to neutralize prothrombin activity in vitro was detected against radiolabeled prothrombin. Coagulation studies normalized in parallel with clinical recovery and disappearance of the antibody. This case demonstrates acute hypoprothrombinemia‐lupus anticoagulant syndrome as a rare presentation of bleeding diathesis in a healthy young child.

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.