An αIIb ß3 antagonist prevents thrombosis without causing Fc receptor γ-chain IIa-mediated thrombocytopenia.

Essentials FcγRIIa-mediated thrombocytopenia is associated with drug-dependent antibodies (DDAbs). We investigated the correlation between αIIb ß3 binding epitopes and induction of DDAbs. An FcγRIIa-transgenic mouse model was used to evaluate thrombocytopenia among anti-thrombotics. An antithrombotic with binding motif toward αIIb ß-propeller domain has less bleeding tendency. SUMMARY: Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb ß3 . Objective To explore the correlation between αIIb ß3 binding epitopes and induction of DDAb binding to conformation-altered αIIb ß3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb ß3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb ß3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb ß-propeller domain of αIIb ß3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb ß3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb ß3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

Inhibitory effects of polypeptides derived from a snake venom C-type lectin, aggretin, on tumor cell-induced platelet aggregation.

BACKGROUND AND OBJECTIVES: Podoplanin, a transmembrane sialoglycoprotein, is expressed by lymphatic endothelial cells and many tumor cells, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. A recent study found that C-type lectin-like receptor 2 (CLEC-2) is a physiologic receptor for podoplanin. Previous studies showed that aggretin, a snake venom-derived protein, activates platelets by targeting platelet CLEC-2. We hypothesized that the C-terminal fragment of aggretin may bind to platelet CLEC-2 and displace podoplanin, in turn exerting antitumor metastatic effects. METHODS AND RESULTS: Aggretin α-chain C-terminus (residues 106-136; AACT) prolonged the lag phase of platelet aggregation induced by aggretin in human washed platelets, indicating that AACT may target the binding site of CLEC-2. HepG2 cells, which are podoplanin-expressing hepatoma cells, induced platelet aggregation with a lag phase. Pretreatment with AACT inhibited platelet aggregation and prolonged the lag phase induced by HepG2 cells. This inhibitory effect was also found with another hepatocarcinoma cell line, HuH-7. AACT inhibited the interaction between HuH-7 cells and platelets, and a specific binding assay demonstrated that CLEC-2 was the binding site for AACT on platelets. In addition, the invasive ability of HepG2 cells was abolished by AACT in a chick embryo chorioallantoic membrane model. Furthermore, formation of lung metastases after intravenous administration of HuH-7 cells was significantly reduced when mice were treated with AACT. CONCLUSIONS: AACT interacts with CLEC-2 of platelets, leading to interference with platelet aggregation and the subsequent metastatic potential of tumor cells. These results suggest that aggretin AACT is a potential candidate for the treatment of tumor metastasis through CLEC-2 blockade.

Improvements in endotoxemic syndromes using a disintegrin, rhodostomin, through integrin αvß3-dependent pathway.

BACKGROUND AND OBJECTIVES: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg-Gly-Asp-containing snake venom disintegrin, on lipopolysaccharide (LPS)-activated phagocytes in vitro and LPS-induced endotoxemia in vivo. METHODS AND RESULTS: Rn inhibited adhesion, migration, cytokine production and mitogen-activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti-αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP-1, Rn bound to LPS-activated THP-1 and specifically blocked anti-αvß3 mAb binding to THP-1. Binding assays proved that integrin αvß3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS-induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS-induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor-α (TNF-a), interleukin (IL)-6, -1ß and -10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS-induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. CONCLUSIONS: Rn may interact with αvß3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS-induced endotoxemia may be attributed to its anti-inflammation activities in vivo.

Intra-abdominal actinomycosis with hepatic pseudotumor and xanthogranulomatous pyelonephritis in a 6-y-old boy.

We report the case of a 6-y-old boy with actinomycosis, presenting as xanthogranulomatous pyelonephritis (XGP), hepatic pseudotumor and abdominal abscess. Symptoms included intermittent fever, abdominal pain and significant weight loss. Hepatic and renal tumor masses were suspected on sonography and computerized tomography. XGP and actinomycosis were proven by pathology. The patient recovered well with antibiotic alone.

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.

Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells.

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, although angiogenic factor and integrin-extracellular matrix interaction modulate this process. We report here that a snake venom-derived disintegrin, rhodostomin, inhibited distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF), and also suppressed in vivo murine melanoma tumor growth. Rhodostomin dose-dependently inhibited bFGF-induced human umbilical vein endothelial cell (HUVEC) proliferation as examined by cell number count, metabolic activity, and BrdU incorporation assays with submicromolar IC(50) values. However, it apparently did not affect the viability of murine B16F10 melanoma cells, even up to 50 microM. Rhodostomin also inhibited HUVEC migration and invasion evoked by bFGF, and tube formation of bFGF-treated HUVECs in Matrigel. Moreover, rhodostomin selectively inhibited bFGF-, but not vascular endothelial growth factor-associated angiogenesis in the chick chorioallantoic membrane model. Furthermore, rhodostomin blocked both bFGF- and B16F10-induced neovascularization in murine Matrigel plug model and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the rhodostomin-treated C57BL/6 mice. The antiangiogenic effect of rhodostomin on bFGF-treated HUVECs is related to the integrin alpha(v)beta(3) blockade, as evidenced by its selective inhibition on the binding of 7E3, a monoclonal antibody (mAb) raised against alpha(v)beta(3,) but not that of P1F6, an alpha(v)beta(5) mAb toward both naive and bFGF-primed HUVECs. Moreover, 7E3 specifically blocked fluorescein isothiocyanate-conjugated rhodostomin binding to HUVEC, whereas P1F6 and anti-integrin alpha(2), alpha(3), alpha(4), or alpha(5) mAbs did not.

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist.

1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.

Surgical management of complete ureteric duplication abnormalities in children.

BACKGROUND: A variety of clinical problems due to complete ureteric duplication (CUD) in children may be encountered. Surgical management of CUD varies and is controversial. Therefore, we reviewed the cases of 15 children with CUD operated on at our hospital in an effort to evaluate the various facets of this disorder that influenced our surgical management. METHODS: Fifteen children with abnormalities associated with CUD underwent surgery from 1987 to 1998. There were 14 girls and one boy. Their age at surgery ranged from 15 days to 3.5 years (average, 9.5 months). All children underwent ultrasonography, including 10 intravenous urograms and five prenatal examinations. The anatomic abnormalities of all children were noted according to the Weigert-Meyer rule. RESULTS: Six of 15 ultrasonograms were misinterpreted as hydronephrosis only, while the 10 intravenous urograms were interpreted correctly as CUD. Four patients had recurrent urinary tract infections after surgery. Two of them underwent further surgery, one for stone formation in the residual ureter stump and the other for persistent ureterocele. There was one surgical complication after an upper pole nephroureterectomy. It involved ureteric necrosis with urine leakage. This patient underwent ipsilateral lower pole nephrectomy 28 days after the first operation. All children remained symptom-free during follow-up. CONCLUSIONS: Accurate diagnosis of duplex kidney requires careful imaging studies, especially for fetal renal abnormalities. Management of CUD should be individualized. Usually, upper pole nephroureterectomy is performed for a nonfunctioning moiety. For functioning segments, ureteropyelostomy or ureteric reimplantation can be considered. The results are satisfactory, but long-term follow-up is necessary.

Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation.

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.

A new short chain RGD-containing disintegrin, accutin, inhibits the common pathway of human platelet aggregation.

A new short-chain disintegrin, accutin, was purified from the Formosan Agkistrodon acutus venom by using of gel filtration, ion exchanger and reverse phase HPLC. The homogeneous protein is a 47-residue polypeptide with a molecular mass of 5241 Da containing an Arg-Gly-Asp sequence and seven cysteinyl residues at positions highly homologous to other disintegrins. Accutin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, thrombin or the thromboxane analogue U46619 in platelet suspension with IC50 values of 66-267 nM. It was also active in inhibiting platelet aggregation of platelet-rich plasma. However, accutin apparently did not affect the shape change caused by these agonists. Accutin also inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner. Furthermore, accutin dose-dependently inhibited the binding reaction of fluorescein isothiocyanate (FITC)-conjugated arietin, a member of the disintegrin family, to human platelets. In addition, the binding of FITC-conjugated accutin to platelets was almost completely blocked by a monoclonal antibody, 7E3, raised against the platelet glycoprotein IIb/IIIa complex. On the other hand, accutin as well as other disintegrins, rhodostomin and arietin, exhibited an inhibitory effect on 7E3 binding toward platelets and endothelial cells in a dose-dependent manner. It is concluded that accutin, a new platelet aggregation inhibitor belonging to the short-chain disintegrin family, acts specifically on a binding epitope of GPIIb/IIIa overlapping with that of 7E3, leading to the blockade of fibrinogen binding to its receptor.

Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin alphavbeta3 antagonist and inducing apoptosis.

Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin alphaIIbbeta3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as alpha2beta1, alpha3beta1, and alpha5beta1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin alphavbeta3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents.

Congenital megacystis: a case report.

Congenital megacystis without other anomalies is extremely rare. A female newborn, delivered by cesarean section at the gestational age of 36 weeks, was seen. Her prenatal sonogram at 28 weeks' gestation had demonstrated a large cystic lesion in the abdomen. Abdominal distention with a palpable mass, difficult feeding and vomiting were noted after delivery. Abdominal computed tomography scan revealed a large cyst in the middle abdomen without any other anomaly. Exploratory laparotomy was performed on the fourth day of life, and showed a large bladder with normal trigon and normal ureteral insertion. There was no abnormality of other abdominal organs. Reductive cystoplasty was performed and the post-operative course was uneventful. The infant was doing well after an eight-month follow-up. Although congenital megacystis is rare, it should be considered in the differential diagnosis of any intraabdominal cystic lesion demonstrated in a prenatal sonogram. The etiology is obscure, and the benefit of reductive cystoplasty needs more cases to be confirmed.

Interaction of thrombin-activated platelets with extracellular matrices (fibronectin and vitronectin): comparison of the activity of Arg-Gly-Asp-containing venom peptides and monoclonal antibodies against glycoprotein IIb/IIIa complex.

Platelets adhere to fibronectin and vitronectin substrates following activation with physiological concentrations of thrombin. Adhesion of activated-platelets to either substrate is dependent upon the amount of fibronectin and vitronectin, and the duration of the adhesion assay. In this study, we showed that the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, triflavin, trigramin and rhodostomin, synthetic peptides GRGDS, GRGDSPK, GRGDF, and GRGD and monoclonal antibodies, 7E3, 10E5 and AP2, raised against glycoprotein IIb/IIIa complex, inhibited the adhesion of activated-platelets to fibronectin and vitronectin-coated plates in a dose-dependent manner. In fibronectin-coated plates, GRGDF was shown to be much more efficient than GRGDS, GRGDSPK and GRGD at inhibiting the adhesion of activated-platelets to immobilized fibronectin. On the other hand, there were no marked differences in the abilities of these three peptides (GRGDF, GRGDS and GRGDSPK) to inhibit platelet adhesion to immobilized vitronectin. Furthermore, the RGD-containing venom peptide, triflavin was more effective than rhodostomin and trigramin at inhibiting the adhesion of activated-platelets to either substrates. The monoclonal antibodies raised against glycoprotein IIb/IIIa complex (i.e., 7E3, 10E5 and AP2) inhibited platelet adhesion to fibronectin and vitronectin in a similar dose-dependent manner. Interestingly, we found that 7E3 was more efficient than 10E5 and AP2 in this reaction. These studies suggest that the glycoprotein IIb/IIIa complex, present on activated-platelets, may interact with fibronectin and vitronectin substrates through the Arg-Gly-Asp-dependent mechanism. Since fibronectin and vitronectin are present in the subendothelial matrix, they may be involved in platelet-vessel wall interaction. The Arg-Gly-Asp containing peptide, especially triflavin, is an ideal therapeutic agent for inhibiting thrombus formation by interrupting platelet-platelet and platelet-subendothelium interactions.

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits the adhesion of tumor cells to matrix proteins via binding to multiple integrin receptors expressed on human hepatoma cells.

Integrins are a superfamily of cell surface glycoproteins that promote cellular adhesion. The interaction of integrins with extracellular matrices such as fibronectin and vitronectin has been shown to be mediated through an arginine-glycine-aspartic acid (RGD) sequence within adhesive proteins. Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins. Disintegrins have been isolated from the venom of various vipers and have been shown to be potent inhibitors of platelet aggregation. In this study, we found that human hepatoma cell adhesion to immobilized matrix proteins (i.e. fibronectin, collagen, laminin, and vitronectin) was differentially affected by various anti-integrin monoclonal antibodies (mAbs) (i.e., alpha3beta1, alpha5beta1, alpha6beta1, and alpha v beta3) as well as by the peptide GRGDS. Indirect flow cytometric analysis of hepatoma cells with anti-integrin mAbs demonstrated that alpha6beta1 was uniformly expressed at a high density, while alpha3beta1, and alpha5beta1 were moderately expressed and alpha v beta3 was expressed in small amounts on hepatoma cells, consistent with the results obtained from immunofluorescence microscopic analysis. When immobilized on plastic wells, triflavin promoted hepatoma cell attachment; this cell attachment was inhibited by either GRGDS or mAbs against integrins alpha3beta1, alpha5beta1 and alpha v beta3). In addition, the binding of FITC-conjugated triflavin to hepatoma cells was inhibited by GRGDS, anti-alpha3beta1, antialpha5beta1, and anti-alpha v beta3 mAbs. Among these mAbs, anti-alpha5beta1 exerted the most pronounced inhibitory effect (>70%) on the binding of triflavin to hepatoma cells. Taken together, these results suggest that triflavin binds via its RGD sequence to multiple integrin receptors (i.e., alpha5beta1, alpha3beta1, and alpha v beta3) expressed on the surface of hepatoma cells, resulting in inhibition of hepatoma cell adhesion to extracellular matrices (i.e., fibronectin and vitronectin).

Surgical management of the buried penis.

BACKGROUND: Buried penis is a congenital abnormality in which the phallus is concealed within the surface of prepubic skin. It is probably more common than is generally recognized. The objective was to describe the pathophysiology, and search for the best management of this disease. METHODS: Over a period of 6 years, a total of 31 cases receiving surgery for buried penis at this hospital were analyzed. According to their major pathophysiology, patients were divided into three groups: in the first, the major mechanism was poor skin suspension; in the second, prominent suprapubic fat pad was the major cause. In the third group, the dartos fascia was abnormally thickened and attached to the penile shaft. Different surgical techniques were applied in the different groups. RESULTS: All 15 children in the first group had satisfactory results after penile skin fixation. In the second group, nine children underwent adjunctive lipectomy but only five had satisfactory results. Seven patients in the third group needed degloving of the penis and the three cases who underwent preputial unfurling had severe lymphedema of the inner preputial layer. One of them received revision. The end results were good in the other six patients after long-term follow up. CONCLUSIONS: The buried penis occurs in a spectrum. Although, there are three major pathophysiology mechanisms, most of the patients had a combination. Surgical management should be individualized, and results are usually satisfactory. For the obese patient, weight loss is important. Preputial unfurling and using inner preputial layer to cover the defect should be avoided because of severe postoperative lymphedema.

Antenatal diagnosis and early surgery for choledochal cyst: a report of two cases.

Since the advent of routine antenatal sonography to detect fetal abnormalities, two cases of choledochal cyst have been found prenatally in our hospital. The first presented when a choledochal cyst was demonstrated at 31 weeks of gestation, and a firm diagnosis established within 2 days of birth. Technetium 99m disofenin (DISIDA) cholescintigram revealed delayed visualization of the small bowel. The second case was found by ultrasound examination at 21 weeks of gestation to have a choledochal cyst, and diagnosis was confirmed within 3 days of birth. DISIDA scintigram demonstrated complete obstruction of the extrahepatic biliary tree. Both infants received early excision of the cyst at the ages of three and four days respectively. The postoperative course was quite smooth, and there were no abnormal symptoms after follow up of four and two years, respectively. Neonates with distal common bile duct obstruction in association with presumed choledochal cyst should have prompt surgical exploration, and early excision of the cyst is a safe procedure in the newborn.

Triflavin, an Arg-Gly-Asp-containing peptide, prevents platelet plug formation in in vivo experiments.

Triflavin, an Arg-Gly-Asp-containing peptide from Trimeresurus flavoviridis snake venom (M(r) of 7500 Da) inhibits platelet aggregation through the blockade of fibrinogen binding to activated platelets. The present study demonstrated that the intravenous injection of triflavin (0.1 and 0.25 mg/kg) significantly prolonged the bleeding time about 1.8- to 2.4-fold as compared with control (normal saline) of severed mesenteric arteries in rats, whereas the injection of Gly-Arg-Gly-Asp-Ser (GRGDS) (2-8 mg/kg) failed to increase the bleeding time in this model. Continuous infusion of triflavin (0.08 mg/kg/min) significantly increased the bleeding time about 2.6-fold, and the bleeding time returned to normal within 20 min after the cessation of triflavin infusion. Triflavin (10-20 mg/kg) significantly prolonged the occlusion time of platelet plug formation induced by irradiation of mesenteric venules of fluorescein sodium-pretreated mice. In contrast, trigramin (10-20 mg/kg) and GRGDS (500 and 1000 mg/kg) showed no significant effect. These results suggest that triflavin has an effective antiplatelet effect in vivo and this peptide may be a useful therapeutic agent for arterial thrombosis.

Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis.

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC50 (approximately 1.0 micrograms/ml, 0.11 microM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins, e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

Posterior urethral valves: a report of five cases.

From 1985 to 1993, five male patients with posterior urethral valves were treated at our hospital; they included three newborns, a 32-month-old and a 10-year-old child. On initial examination, all patients showed severe bilateral hydronephrosis and hydroureter on sonographic findings, dilatation of the prostate urethra and trabeculation of the bladder wall in the voiding cystourethrogram; only one patient showed vesicoureteral reflux. Type I urethral valves were present in all patients. Retrograde transurethral ablation of the valves was performed in four patients with good preoperative renal function. The postoperative courses were smooth except for vesicoureteral reflux in one patient and systemic fungus infection in another. Reimplantation of the ureter was done for the vesicoureteral reflux, and the fungus infection was treated by amphotericin B. Cutaneous vesicostomy was performed for a newborn since the urethral orifice was small. He presented with poor renal function and ascites. Delayed antegrade ablation of the valves per vesicostomy and closure of the vesicostomy were done three months later after restoration of the renal function. Renal function returned to normal after operation in all patients, but the function of the upper urinary tract and bladder continence need long-term follow-up.