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SUMMARY BACKGROUND: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. OBJECTIVE: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. METHODS: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. RESULTS: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. CONCLUSIONS: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.

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BACKGROUND: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. RESULTS: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. CONCLUSIONS: This study presents the first patient with a proven genetic GPVI defect.

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The laboratory diagnosis of lupus anticoagulants (LA) remains difficult despite internationally accepted guidelines for its detection. Several interlaboratory surveys have shown poor agreement between laboratories. Further standardization of LA testing will to a large extent depend on better insights on the mechanisms by which LA affect phospholipid-dependent coagulation assays as well as on the availability of well characterized and internationally accepted reference materials and control specimens. We recently raised a series of murine monoclonal antibodies against human beta2GPI (anti-beta2GPI moabs), a phospholipid-binding protein directly involved in the interaction between certain lupus anticoagulants and phospholipids. In this study we report on the use of LA positive anti-beta2GPI moabs as easy to handle reference and control material. The relative LA responsiveness of various phospholipid-dependent clotting assays was determined on plasmas spiked with such moabs and compared well with that determined on LA positive plasma samples. Plasmas spiked with LA positive anti-beta2GPI moabs were also used as control specimens to study the interlaboratory precision of LA testing. With these control specimens, low interassay coefficients of variation were obtained. Our results indicate that LA positive anti-beta2GPI moabs have potential for the production of control specimens that could be made available to routine hemostasis laboratories to assess intra-laboratory precision of LA testing and to manufacturers to control batch-to-batch variability of their reagents.

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The precise mechanism by which Beta-2-glycoprotein I (beta2-GPI-) dependent lupus anticoagulants lengthen phospholipid-dependent clotting reactions is still poorly understood. In order to study this, murine monoclonal antibodies (moabs) against human beta2GPI were raised. Eight of the 21 anti-beta2GPI moabs, obtained from 2 fusions, fulfilled the criteria for lupus anticoagulant (LA) activity as tested with a variety of sensitive screening assays and confirmatory tests. Seven moabs did not influence any clotting test. The LA positive moabs were found to compete for similar or closely spaced epitopes on immobilized beta2GPI. Two moabs with potent LA activity (moabs 22 F 6 and 22 B 3) and 1 moab without LA activity (moab 16 B 3) were selected to study the interaction between antibody, beta2GPI and phospholipid. Interactions were investigated by real-time biospecific interaction analysis (BIA) based on plasmon surface resonance technology on a BIA-core instrument using a sensor chip coated with phospholipid. When 22 F 6, the moab with the most pronounced LA activity, was allowed to interact with the phospholipid surface at concentrations between 0 and 400 nmol/l, no appreciable binding could be detected. Likewise, no binding could be measured when beta2GPI at concentrations between 0 and 400 nmol/l was passed over the phospholipid coated sensor chip. Combinations of beta2GPI and 22 F 6 resulted in significant binding. Similar results were obtained with 22 B 3, another moab with LA activity. A LA negative Moab, 16 B 3, did not cause binding of antibody-beta2GPI complexes. Fab' fragments, derived from moab 22 F 6, inhibited the binding of beta2GPI-22 F 6 and beta2GPI-22 B 3 in a concentration dependent way, indicating that only bivalent beta2GPI-antibody complexes bind with high affinity to phospholipids. Fab' fragments, derived from moab 22 F 6, also inhibited the LA effect of moabs 22 F 6 and 22 B 3 in diluted plasma. In summary, these experiments indicate that the beta2GPI-dependent LA effect depends on the formation of bivalent beta2GPI-antibody complexes on phospholipid surfaces.

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A patient with a history of habitual abortion, deep venous thrombosis, thrombocytopenia, high titer IgG anticardiolipin antibodies and a clearly positive lupus anticoagulant, was treated during her seventh pregnancy with high-dose intravenous immunoglobulins (IVIg) from the third month onwards. Every month, a daily infusion of 400 mg immunoglobulins per kg body weight was given during five consecutive days. The patient's pregnancy ended preterm with a live birth, delivered by caesarian section because of a placental abruption. The 1070 g (P20-P25) weighing girl was in good health, apart from a bradycardia, due to dysfunction of the atrioventricular conduction. Each treatment with IVIg resulted in a slight reduction of both anticardiolipin antibodies and lupus anticoagulant levels and in an increase in platelet count. During the six-month observation period, a gradual decline in antiphospholipid antibodies and an increase in platelet count was found. The potential role of anti-idiotypic antibodies, present in the IVIg used for treatment, was studied. In vitro, IVIg were able to reduce the binding of the patient's anticardiolipin antibodies to cardiolipin coated microtiter plates. The presence of anti-idiotypic antibodies in IVIg was further documented by affinity chromatography and by realtime biospecific interaction analysis (BIA) on a BIA-core instrument. Affinity purified anticardiolipin antibodies were retarded on a column of insolubilized IVIg and a weak interaction was found between IVIg and affinity purified antiphospholipid antibodies, coupled to the BIA-core biosensor. In addition, the same technology revealed increased levels of anti-antiphospholipid antibodies in the patient's plasma following IVIg therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

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Treatment of intact human umbilical vein endothelial cells with NaF results in a dose-dependent biphasic response in both prostacyclin and inositol phosphate production: the stimulation observed with 10-20 mM NaF decreases with higher concentrations. High concentrations of NaF furthermore reduce thrombin- or A23187-stimulated prostacyclin production. Direct assay of phospholipase C activity in cell homogenates shows a similar biphasic response to NaF, also after chelation of Ca2+; addition of AlCl3 shifts the inhibition toward lower NaF concentrations. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) also causes a dose-dependent biphasic response in inositol phosphate formation in permeabilized cells and homogenates; a higher inhibitory concentration of GTP gamma S abolishes the stimulation of inositol phosphate production by low NaF concentrations. A high concentration of NaF furthermore inhibits the non-G-protein-dependent activation of phospholipase C by deoxycholate. NaF also induces a dose-dependent biphasic response in cyclic AMP formation in intact cells, indicating that the inhibition of phospholipase C at higher NaF concentrations does not result from a rise in cyclic AMP. The data are compatible with the existence of a guanine nucleotide-dependent, cyclic AMP-independent, phospholipase C-inhibitory pathway in endothelial cells.

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Human mammary tumor cells in continuous culture (MCF-7 cells) are hormone- and radiosensitive. The interaction of both factors is analyzed. Ionizing irradiation lowers the concentration of both the estradiol and progesterone receptors per cell. The reduction is dose dependent. However, the effects on the cytoplasmic and nuclear forms of the receptors are not similar. For the estradiol receptor, an accumulation in the nuclear fraction is observed 48 hr after irradiation when no appreciable amounts of estrogens are present. After administration of 10(-8) M estradiol, the cytoplasmic clearance is comparable to the unirradiated controls. However, nuclear accumulation is impaired. The processing of the nuclear estrogen receptor remains identical. Nuclear progesterone receptor is not significantly increased due to irradiation in the absence of progestins. Cytoplasmic decrease after incubation with progestins is unaffected. Again, nuclear accumulation is impaired in contrast to the unchanged processing of the nuclear form of the progesterone receptor. A decrease in "nuclear acceptor sites" for both receptors after irradiation may be an explanation for these observations. No significant effects of ionizing irradiation are observed in the initial steps of steroid hormone action.

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Phorbol derivatives (phorbol-12,13-diacetate, phorbol-12,13-dibenzoate, phorbol-12,13-dibutyrate, and phorbol-12-myristate-13-acetate) interact with cortisol on a molecular basis. These molecular interactions are demonstrated via dexamethasone receptor assays and by changes in spectrophotometric characteristics when equimolar solutions of these phorbol compounds together with cortisol are compared to those of the pure solutions. The phorbol compounds characterized by modifications in the molecular ring structure and by substitution at position C20, lose the capacity to bind cortisol. The tumor promoting effects of phorbol compounds are apparently achieved, in addition to other well-known mechanisms, by neutralizing cortisol, thus suppressing its regulatory effect on cell proliferation.

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The effects of ionizing irradiation on the sedimentation coefficients of both estrogen receptor (ER) and progesterone receptor (PgR) have been examined in comparison to the effects of proteolysis. DMBA-induced rat mammary tumors were subjected to a treatment of 20 Gy and the ER and PgR concentrations were determined at different time intervals after irradiation. On a 5-20% sucrose gradient the ER sedimented as 9-11 and 4-5 S molecular forms, while PgR sedimented as a small 8-9 S peak and a major 4-5 S peak. Radiotherapy particularly reduced the 4-5 S sedimentation peaks of both receptors but did not initiate any new sedimentation forms. Although the 4-5 S ER receptor concentrations remained low, both progesterone receptor forms appeared to recover by 60 days after treatment. As these effects could be due to the release of proteolytic enzymes following irradiation of tumors, the receptors from untreated tumors were exposed to different concentrations of trypsin. The effects of trypsin were identical for ER and for PgR, and proved to be dependent on the trypsin concentration. Only concentrations of trypsin up to 30 micrograms/ml resulted in a reduction of 9-11 S ER or 8-9 S PgR forms which was accompanied by a simultaneous increase in the 4-5 S peaks, resulting in no change in total binding sites. Still higher trypsinization (300-3000 micrograms/ml) also reduced the 4-5 S ER and PgR fractions. In the presence or the absence of sodium molybdate, a stabilizer of the faster sedimenting forms of the receptor, no alterations were observed in the position of, or the total number of binding sites of, the sucrose gradient fractions from control or irradiated tumors. The irradiation effects appear to be due either to damage of the cytosolic ER receptor, thereby preventing its participation in the induction of de novo synthesis of ER and PgR, or to the non-specific damage of transcription and/or translation systems.

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The determination of estradiol and progesterone receptor concentrations in mammary tumors is useful in predicting the hormone responsiveness. As this assay is carried out on tumor tissue which may have been subjected to radiotherapy, the possibility of an ionizing irradiation affecting the steroid receptor levels in neoplastic tissue should be taken into account. The steroid receptor concentrations are examined in dimethylbenz(a)anthracene-induced tumors of Sprague-Dawley rats. The estradiol and the progesterone receptor titers become reduced significantly after treatment with 20 Gray while an application with 7 Gray does not affect the titer values. After treatment of the tumor with 20 Gray, the steroid receptor concentrations decrease progressively, reaching a maximal reduction 20 to 30 days after exposure. This measured reduction in binding sites seems to be the result of a specific irradiation effect and not due to a possible increase in lytic enzyme levels in the regressing tumors. As radiation treatment affects the receptor concentrations, this should be kept in mind when interpreting the steroid receptor concentrations.

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Interactions between the estradiolreceptor and the progesteronereceptor are known to exist in the uterus. The "priming effect" of estradiol is likely to exist also in human and rat mammary tumors. In detecting also the progesteronereceptors along with the estrogenreceptors, one cannot only demonstrate the presence but also the activity of the estrogenreceptor. This finding should improve the response rate of hormonesensitivity to receptorpositivity. However preoperative irradiation possibly induces negative progesteronereceptortiters in human breast tumors.

PIP: Interactions between the estradiolreceptor and the progesteronereceptor have been demonstrated in many laboratories. The priming effect of estradiol is likely to exist in human and rat mammary tumors. The presence and activity of the estrogenreceptor can be demonstrated in detecting the progesteronereceptors along with the estrogenreceptors. This finding should improve the response rate of hormonesensitivity to receptorpositivity. Nearly 80% of estrogen, and progesterone, receptorpositive human breast tumors are likely to be hormonedependent; only 50% of the estrogenpositive tumors are. 500 mg tissue samples were analyzed for estradiol and progesteronereceptors. The receptor titers for the receptorpositive samples were not affected by irradiation. The amount of progesteronereceptor-positive tumors decreased from 76% for the controls to 25% and 37% for those who received 7 and 20 Gy respectively. Irradiation may reduce progesteronereceptors in human breast tumors.

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