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Biomedical subjects

E C Lian

Publications and source records attributed to E C Lian.

At least 19 recordsLinked to original sources
PubMed1992

Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36).

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAPp37) from the plasma of a patient with Thrombotic Thrombocytopenic purpura (TTP), and have shown recently that p37 causes platelet agglutination through its binding to membrane glycoprotein IV (GPIV). To gain further insight into the mechanism of p37 binding to GPIV, we have studied the interaction between p37 and GPIV. We now demonstrate specific complex formation of p37 with GPIV. In Western immunoblotting p37 binds to purified GPIV and the complex formed between the two proteins was detected by polyclonal antibody to p37 and peroxidase conjugated second antibody. No binding of p37 was noticed with the purified GPIIIa. A solid phase binding assay was developed to study the complex formation. Microtiter wells were coated with GPIV and the control proteins; 125I-p37 was added, allowed to bind and bound radioactivity was measured. Several lines of evidence indicate that the binding of p37 to GPIV was specific. a) GPIV immobilized on Immulon-2 wells bound 10-30 fold more p37 than immobilized fibrinogen, GPIIIa, and BSA. b) Polyclonal antibodies against p37 and GPIV inhibited the binding by 39-68% as compared with control IgG. c) GPIIIa antibody did not inhibit the binding. Molecular sieve chromatography of a mixture of 125I-p37 and GPIV also revealed the fluid phase complex formation ranging in molecular weight from 132,000 to over 350,000 daltons. These results show the specific interaction between p37 and GPIV and suggest that GPIV functions as a p37 receptor during platelet agglutination.

Antigens, CD↗
PubMed1991

Platelet agglutinating protein p37 causes platelet agglutination through its binding to membrane glycoprotein IV.

A 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that 125I-p37 bound to a membrane protein of 97 kDa (red/unred). Furthermore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti-GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti-GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.

Antibodies↗
PubMed1991

Binding of platelet agglutinating protein p37 from the plasma of a patient with thrombotic thrombocytopenic purpura to human platelets.

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding. Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 micrograms/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or GP IIb-IIIa complex.

Antibodies, Monoclonal↗
PubMed1991

Hepatocellular carcinoma in hemophilia.

A questionnaire-based survey involving 11,801 hemophiliacs from 54 hemophilia centers in the USA and Europe documented the occurrence of hepatocellular carcinoma (HCC) in 10 patients. The crude rate of HCC was 3.2/100,000 patients/year, at least 30 times higher than the background incidence of this tumor in the countries of origin of the patients. All patients were Caucasians with hemophilia A, 39 to 74 years of age, and had liver cirrhosis. All had one or more risk factor for cirrhosis and HCC: 5 were positive for serum hepatitis B surface antigen, 4 had the antibody to hepatitis C virus, and 4 had histories of alcohol abuse. Serum alpha-fetoprotein, measured in 6 patients, was significantly elevated in 4 (range: 807-1399 ng/ml), and only moderately elevated in 2 (25 and 171 ng/ml). The onset of HCC was asymptomatic in 5 patients, whereas it was accompanied by jaundice, abdominal pain, or ascites in the remaining patients. Thus, HCC seems to be a more important secondary disease for hemophiliacs than formerly recognized. Since HCC is often asymptomatic, screening hemophiliacs with chronic liver disease with periodic ultrasound scans might increase the changes of detecting HCC at a stage amenable to surgical treatment.

Adult↗
PubMed1989

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein.

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca++ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.

Blood Platelets↗
PubMed1989

Combination immunosuppressive therapy after factor VIII infusion for acquired factor VIII inhibitor.

STUDY OBJECTIVE: To evaluate the effectiveness of combined cyclophosphamide, vincristine, and prednisone (CVP) therapy after antigenic stimulation with factor VIII in the eradication of factor VIII inhibitor. DESIGN: Factor VIII activity and inhibitor titer were measured before and after FVIII-CVP therapy and patients with factor VIII inhibitor were followed for at least 2 years. SETTING: The first course of therapy was carried out in the hospital when nonhemophiliac patients were admitted for bleeding. Otherwise, treatment was administered at the outpatient clinic. PATIENTS: From 1975 to 1986 we studied 12 nonhemophiliac and 5 hemophiliac patients with factor VIII inhibitor treated with FVIII-CVP and followed at our clinic. INTERVENTION: Patients were infused with one dose of factor VIII concentrate, 50 to 100 U/kg body weight, followed by cyclophosphamide, 500 mg on day 1 and 200 mg/d on days 2 to 5; vincristine, 2 mg on day 1; and prednisone, 100 mg/d on days 1 to 5. This regimen was repeated every 3 to 4 weeks. RESULTS: Of 12 nonhemophiliac patients, 11 responded after 1 to 3 courses of FVIII-CVP with complete disappearance of the inhibitor without recurrence. Among 5 patients with hemophilia who were given 3 to 8 courses, only 1 patient responded with a transient disappearance of inhibitor. Mild neutropenia and infection occurred in 3 patients and required antibiotic treatment. CONCLUSION: Factor VIII-CVP therapy is highly effective in the eradication of factor VIII inhibitor in nonhemophiliac patients but not in patients with hemophilia.

Adult↗
PubMed1988

Mechanism of rabbit platelet agglutination induced by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka.

The effects of acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) on rabbit platelets were studied. Using citrated platelet-rich plasma (PRP), washed platelets, and formaldehyde fixed platelets from 10 New Zealand white rabbits, we investigated the effects of platelet inhibitors and various plasma and its fractions on SJAMP-induced agglutination. It was found that the tracing of platelet agglutination induced by SJAMP showed a single phase without a lag period. The lowest concentration of SJAMP required for the agglutination of rabbit platelets was approximately 2 micrograms/ml, and the magnitude of agglutination induced by SJAMP was concentration dependent. In 8 out of 10 rabbits, the platelets in PRP were agglutinated by 10 micrograms/ml of SJAMP. Platelet inhibitors, such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA did not inhibit the agglutination induced by SJAMP. Washed rabbit platelets were not agglutinated by SJAMP even though the concentration of SJAMP was raised up to 50 micrograms/ml. When rabbit plasma, serum, or 50-60% ammonium sulfate saturated plasma fraction was added to the reaction mixture, agglutination of washed platelets by SJAMP was recovered completely. But human plasma or fibrinogen did not have any effect on the reactivity of washed rabbit platelets to SJAMP. From these data we conclude that the SJAMP-induced rabbit platelet agglutination is independent of energy metabolism but requires plasma cofactor(s) other than fibrinogen. The plasma cofactor is present in 50-60% ammonium sulfate saturated plasma fraction.

Animals↗
PubMed1988

Aggregation of human platelets by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka.

The acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) has been shown to cause platelets to aggregate. Using citrated platelet-rich plasma (PRP), washed platelets and formaldehyde-fixed platelets from humans, we investigated the effects of platelet inhibitors and various plasmas and their fractions on SJAMP-induced platelet aggregation. It was found that the lowest concentration of SJAMP required for the aggregation of human platelets was about 0.4 micrograms/ml and the magnitude of aggregation induced by SJAMP was concentration dependent. The platelets were aggregated by SJAMP at 10 micrograms/ml in 25 out of 28 (89%) normal subjects tested. Platelet inhibitors such as PGE1, aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA inhibited by 70 to 100% the aggregation induced by SJAMP. Washed platelets alone were not aggregated by SJAMP. In the presence of fibrinogen, washed platelets were aggregated by SJAMP but formaldehyde-fixed platelets were not. These data indicate that the SJAMP-induced human platelet aggregation requires extracellular calcium, fibrinogen, and energy metabolism. The second phase of aggregation is dependent upon the release of ADP, and cyclooxygenase pathway.

Animals↗
PubMed1988

Thrombotic thrombocytopenic purpura.

Platelet thrombus formation in small vessels is triggered by certain stimuli, including vascular injury, primary platelet agglutination, or both. The formation and dissolution of platelet thrombi is modulated by proteolysis, plasma factors, PGI2 synthesis and stability, and immune mechanisms. Some recent evidence now suggests that there is a subset of thrombotic thrombocytopenic purpura (TTP) patients possessing a specific platelet agglutinating factor that triggers the intravascular platelet agglutination. Identification and classification of TTP patients into subgroups with common triggering agents, and further clarification of the role of modifying factors will enable us to understand the disease better and to improve therapy.

Humans↗
PubMed1988

Platelet-agglutinating protein P37 from a thrombotic thrombocytopenic purpura plasma forms a complex with human immunoglobulin G.

We have previously reported the purification of a 37-kd platelet-agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been shown to be inhibited by IgG from normal human adults and the same TTP patient after recovery. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG from normal adults and the same TTP patient after recovery to adsorbed PAP by using an enzyme-linked immunosorbent assay. The binding was specific, concentration dependent, and saturable. IgG purified from a 5-month-old baby and the same TTP patient during active disease did not form complex with PAP p37. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP p37 but not 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP p37 and IgG also revealed the fluid-phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The IgG that was bound to PAP p37 conjugated to Sepharose 4B inhibited the agglutination of washed platelets induced by TTP plasma containing PAP p37, whereas the IgG that was not bound to PAP p37 did not have a significant inhibitory effect. The complex formation between PAP p37 and specific IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and, at least partly, the in vivo successful treatment with specific IgG-containing normal plasma.

Blood Proteins↗
PubMed1986

Synovectomy of the knee for hemophilic arthropathy.

Synovectomy of the knee for the control of recurrent hemarthrosis was performed in thirteen patients with hemophilic arthropathy. Preoperatively, all patients had experienced an average of three bleeding episodes into the affected joint per month, and had been unresponsive to at least six months of medical management. Radiographically, all knees had either Stage-II or Stage-III hemophilic arthropathy. The average age of the patients at the time of synovectomy was sixteen years and the average length of follow-up was 7.3 years (range, two to eleven years and seven months). Although the motion of the knee remained unchanged postoperatively in two patients, ten patients had an average loss of 41 degrees. One patient eventually required an arthrodesis. Radiographically, there was slight further joint deterioration after synovectomy, and no knee progressed beyond Stage-III hemophilic arthropathy. Only one patient in our series had a recurrent spontaneous hemarthrosis of the synovectomized knee, although two others had traumatic bleeding episodes. The complications included three immediate postoperative hemarthrosis requiring surgical evacuation, isoimmune hemolytic anemia in one patient, patellofemoral adhesions in two knees, and a total fibrous ankylosis that required a knee arthrodesis in one patient. It was concluded from our study that chronic recurrent hemarthrosis and the pain associated with persistent synovitis in the hemophilic knee can be effectively eliminated for as many as twelve years after open synovectomy, although usually with significant loss of motion of the knee. This procedure also appeared to slow the progression of arthropathy, and no patient had been considered for a total knee replacement at the time of writing.

Adolescent↗
PubMed1985

Novel platelet-agglutinating protein from a thrombotic thrombocytopenic purpura plasma.

A novel platelet-agglutinating protein (PAP) was purified approximately 2,000-fold from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) by ammonium sulfate fractionation, DEAE-Sephacel and concanavalin A-Sepharose chromatographies. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with and without reduction, this preparation revealed a major protein band with a molecular weight of 37,000, and a minor band with a molecular weight of 32,000-34,000. After elution from the gel, only the 37,000-mol wt protein corresponding to the major band induced the platelet agglutination. When four normal plasmas and the recovery plasma from the same TTP patient were subjected to the similar purification steps, the 37,000-mol wt major band was absent. The 125I-PAP bound to the platelets in a concentration-dependent manner. The platelet agglutination induced by PAP was not inhibited by hirudin, heparin in the presence of antithrombin III, phenylmethylsulfonyl fluoride, apyrase, aspirin, or prostaglandin I2. However, it was inhibited by IgG from normal adults and from the same TTP patient after recovery. The anti-37,000-mol wt PAP antiserum prepared in the rabbit formed a single precipitin line against the highly purified PAP. Using this antiserum in the Western immunoblotting, the 37,000-mol wt protein band was found in the three TTP plasmas, of which the platelet-agglutinating activity was inhibited by the anti-37,000-mol wt PAP IgG. The 37,000-mol wt immunoprecipitin band was absent in the plasmas obtained from another two TTP patients, two normal subjects, two patients with idiopathic thrombocytopenic purpura, and two patients with disseminated intravascular coagulation. These results suggest that the 37,000-mol wt PAP is present only in certain cases of TTP, and is likely to be responsible for the formation of platelet thrombi in the microcirculation.

Adult↗
PubMed1985

Investigation of the role of von Willebrand factor in thrombotic thrombocytopenic purpura.

Von Willebrand factor (vWF) has been implicated to function as a cofactor in platelet aggregation induced by thrombotic thrombocytopenic purpura (TTP) plasma. To investigate further this role of vWF, we have used rabbit monospecific anti-FVIII/vWF antibodies and a monoclonal antibody to platelet glycoprotein Ib (GP Ib) that blocks the ristocetin-induced platelet aggregation. The monoclonal anti-platelet GP Ib antibody inhibited the platelet aggregation induced by ristocetin in the presence of normal plasma, but not that by any of the five TTP plasma samples. The TTP plasma samples from five patients were incubated with the monospecific antibodies to FVIII/vWF. In all of the samples, the FVIII/vWF:Ag was drastically reduced; however, there was almost no effect on the platelet-aggregating activity. Therefore, it is concluded that vWF is unlikely to play a major role in platelet aggregation induced by majority of TTP plasmas and that the site of platelet GP Ib, to which vWF binds in the presence of ristocetin, is not involved in TTP plasma-induced aggregation.

Antibodies, Monoclonal↗
PubMed1979

Recent therapeutic advances in thrombotic thrombocytopenic purpura.

Whole plasma infusion, in our experience, has been highly effective in the management of patients with thrombotic thrombocytopenic purpura. The effectiveness of plasma infusion in the treatment of this severe disorder implies the deficiency of a factor in the patient's plasma. Furthermore, we have observed that when platelets are suspended in plasma obtained during active, untreated thrombotic purpura, aggregation occurs. This effect is neutralized by preincubation of the thrombotic thrombocytopenic purpura plasma with normal plasma. Thus, there is both a correlation with the clinical pathogenic mechanism, disseminated platelet aggregation, and with the therapeutic response to plasma infusion. Based upon our experience and the concept that thrombotic thrombocytopenic purpura is a plasma factor deficiency state, we recommend initial infusion of a full plasma volume equivalent over the first 24 hours. This should be done under an intensive care setting. After this initial plasma infusion, we advise the infusion of 3 units of plasma daily until a full remission is obtained. When the clinical situation has stabilized, we stop the daily plasma infusions and cautiously observe for recurrence of the manifestations of thrombotic thrombocytopenic purpura. If there is a recurrence, plasma is again infused in substantial quantity during the first 24 hours and then 3 units daily until a full remission is again evident. Whether the plasma requirement might be attenuated or the course of the disease shortened by the concomitant use of antiplatelet agents, corticosteroids or other means remains to be determined.

Adolescent↗
PubMed1979

Presence of a platelet aggregating factor in the plasma of patients with thrombotic thrombocytopenic purpura (TTP) and its inhibition by normal plasma.

Three patients with thrombotic thrombocytopenic purpura (TTP) were treated by infusion of normal plasma with dramatic responses. The plasmas collected from these patients during relapse induced in vitro aggregation of washed platelets from both normal donors and the patients during remission. The platelet aggregating factor was not dialyzable or adsorbable by Al(OH)3 and was not inactivated by diisopropylfluorophosphate, hirudin, or heparin in the presence of normal amounts of antithrombin. In contrast to the platelet aggregation induced by platelet isoantibody, the platelet aggregating activity of TTP plasma diminished as a function of time when it was incubated with normal plasma at 37 degrees C. These observations suggest that at least some instances of TTP appear to be due to deficiency of a plasma inhibitor to counteract a platelet aggregating factor demonstrated to be present in the plasma of these patients.

Adolescent↗
PubMed1979

Synovectomy or total replacement of the knee in hemophilia.

Eighteen major operations on the knee joint were performed in sixteen patients with hemophilia, eight operations being synovectomy and ten being total knee replacement. Synovectomy was performed for recurrent bleeding associated with synovitis. The average age at operation for patients who underwent synovectomy was 16.4 years, and the average follow-up was thirty-one months. No patient had a recurrent hemorrhage following synovectomy. The patients who had total knee arthroplasty for disabling pain had an average age at surgery of 35.7 years and an average follow-up of twenty-three months. The pain was markedly reduced in all eight patients and all achieved full unprotected weight-bearing. Loss of some motion was observed in twelve patients, but the functional gain outweighed the loss of knee mobility. Seven major complications occurred in the sixteen patients, but none of them affected the outcome.

Adolescent↗
PubMed1978

Intermediate syndrome of platelet dysfunction.

We studied eight patients with intermittent bleeding episodes usually following trauma and associated with the ingestion of medicine known to interfere with platelet function. All patients had a normal or minimally prolonged baseline bleeding time. All had a normal platelet count, glass bead retention test, and platelet serotonin content and a variable pattern of abnormalities in prothrombin consumption and platelet factor 3 availability. However, all showed abnormal platelet aggregation reactions using epinephrine, adenosine diphosphate, and collagen. Following the administration of 975 mg aspirin, our patients' bleeding times became prolonged to a greater extent than the bleeding times of normal controls (range 13 to greater than 20 min). Review of the literature showed approximately 5% of "normal" controls had findings similar to those we report. We believe we are describing a group of individuals with an intermediate form of platelet dysfunction. Although their bleeding diathesis is not as severe as that of patients with platelet dysfunction syndromes previoulsy described, they do bleed significantly when subjected to trauma following the ingestion of drugs such as aspirin. We propose that this defect is common and should be screened for. The aspirin tolerance test is a simple test for detecting these patients.

Adolescent↗