Presence Of Normal Plasma Research Articles

ENPP1 Enzyme Replacement Prevents the Osteomalacia and Paradoxical Mineralization in the Enpp1asj/asj mouse model of Autosomal Recessive Hypophosphatemic Rickets Type‐2.

A paradoxical feature of several disorders of the skeleton involves deficits in bone mineral density yet progressive increases in vascular and/or soft tissue calcifications. ENPP1 loss of function (LOF) mutations represent this combination of defects, in that both life threatening vascular calcifications in Generalized Arterial Calcification of Infancy (GACI) and a severe rickets in Autosomal Recessive Hypophosphatemic Rickets Type-2 (ARHR2) are observed with this mutation. Disease in ARHR2 is severe and symptomatic, leading to repeated fractures of the long bones, rachitic deformities, and impaired growth and development. ENPP1 LOF mutations in mice induces paradoxical mineralization characterized by increased cardiovascular and periarticular calcifications and an osteopenic bone phenotype, reflecting important aspects of both GACI and ARHR2. Here, we report that the osteopenic phenotype in Enpp1asj/asj mice results from an osteomalacia, the histologic hallmark of rickets. The osteomalacia present in Enpp1asj/asj mice occurs in the presence of normal plasma [Pi] in mice fed a high phosphate diet. Treatment of Enpp1asj/asj mice with ENPP1 enzyme replacement therapy (ERT) eliminates the osteomalacia, increases bone mineral density, and markedly improves bone strength and ductility. Our work demonstrates that ENPP1 ERT addresses the pathologic etiology of ARHR2, but phosphate supplementation does not. Combined with our previous studies demonstrating the efficacy of ENPP1-Fc suppressing lethal vascular and connective tissue calcifications, this study establishes a role for ENPP1 in paradoxical mineralization disorders and suggests a therapeutic strategy for treating these diseases. Support or Funding Information The studies were financially supported by research grants from Inozyme Pharmaceuticals Inc., and The Connecticut Bioscience Innovation Fund (to DTB). Funding to the George M O'Brien Kidney Center at Yale, NIH grant P30DK079310, supported the evaluation of plasma analytes. Histology of the proximal tibias of 5-week old mice on the high phosphate diet are displayed from each experimental cohort to compare histologic changes. Deficiency in ENPP1 resulted in morphologically apparent reductions in trabecular bone and markedly thinner growth plates in the Enpp1asj/asj mice. Treating Enpp1asj/asj mice with mENPP1-Fc markedly increased trabecular bone volume and growth plate thickness. WT and Enpp1asj/asj mice treated with vehicle or ENPP1-Fc were loaded to failure in three-point bending. a. Stiffness – slope of the load vs. displacement curve; Max Load – highest load reached during the test.; Total Work – The energy needed to fracture the bone. b. Post-yield deflection of experimental cohorts – defined as the amount of deformation after the yield point. *p < 0.05, **p< 0.01, ***p<0.001, ANOVA. Histomorphometry, 5 week females, High Phosphate Diet, osteoid. Genotypes/Treatment WT/PBS N=6 asj-asj/PBS n=7 asj-asj/mENPP1 n=6 BV/TV 19.23 ± 3.42 24.61 ± 6.45 20.63 ± 4.58 OV/TV 1.38 ± 0.31 2.35 ± 0.57** 1.78 ± 0.50 OV/BV 6.99 ± 1.76 9.72 ± 1.57* 8.60 ± 1.45 OS/BS 27.89 ± 4.66 42.83 ± 4.52*** 33.68 ± 6.41 OTh 3.01 ± 0.29 3.53 ± 0.29** 3.30 ± 0.12 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chitosan treatment abrogates hypercholesterolemia-induced erythrocyte’s arginase activation

This study aimed to evaluate the protective effect of chitosan (CS) against hypercholesterolemia (HC) induced arginase activation and disruption of nitric oxide (NO) biosynthesis using erythrocytes as cellular model. Human erythrocytes were isolated and classified into eight groups. Next, cells were treated with l-arginine (l-ARG), Nω-nitro-l-arginine methyl ester (l-NAME), CS or CS+l-ARG in the presence of normal plasma or cholesterol enriches plasma. Then, erythrocytes were incubated at 37°C for 24h. The present results revealed that, HC induced significant increase of cholesterol inclusion into erythrocytes membrane compared to control. Moreover, HC caused significant decrease in nitric oxide synthase (NOS) activity similar to l-NAME; however, arginase activity and arginase/NOS ratio significantly increased compared to control. On contrast, treatment of HC with, l-arginine, CS or CS plus l-arginine prevents HC induced cholesterol loading into erythrocytes membrane, NOS inhibition and arginase activation. This study suggested that CS could be protective agent against HC induced disruption of erythrocyte’s oxidative status and arginase activation.

ADAMTS-13 Activity and Antigen in Commercial Von Willebrand Factor Concentrates.

The plasma metalloprotease ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type I motif - 13) cleaves the Von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1606 and thereby controls the hemostatic activity of the VWF. We tested three commercial VWF concentrates with respect to their ADAMTS-13 activity and antigen contents. ADAMTS-13 activity in the VWF concentrates was tested by measuring the capacity of the concentrates for auto-proteolysis both in the presence and in the absence of neutralising ADAMTS-13 auto-antibodies. To achieve this, the concentrates were reconstituted to a final VWF concentration of 100 I.E. U VWF:Ristocetin Cofactor activity (VWF:RCo). The VWF solutions were diluted with 5 mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples or Imidazole buffer as plasma replacement.The residual VWF:RCo was subsequently measured using the BC von Willebrand Reagent from Dade Behring (Marburg, Germany). The residual VWF:RCo in the presence of plasma from a patient with acquired TTP (ADAMTS-13 activity: <6.25% due to high levels of neutralising ADAMTS-13 auto-antibodies) was then compared to the residual VWF:RCo in the mixtures containing normal plasma, Imidazole buffer or plasma from a patient with congenital TTP (ADAMTS-13 activity: <6.25% due to mutations in the ADAMTS-13 gene). ADAMTS-13 antigen was measured by a commercial ADAMTS-13 ELISA from American Diagnostica (Stamfort, USA).Residual VWF:RCo after proteolysis of VWF concentrates in the presence of Normal plasma, Imidazole buffer and plasma from patients with congenital and acquired TTP, respectivelyVWF concentrateNormal plasmaImidazole bufferCongenital TTPAcquired TTPA7%106%111%138%B10%11%16%104%C10%32%36%63%ADAMTS-13-Antigen in VWF concentrates given as ng ADAMTS-13 Antigen per I.E. Units VWF:RCoVWF concentrateADAMTS-13 Antigen [ng/I.E. U VWF:RCo]Anot detectableB6.7C0.8The loss in VWF:RCo in the samples containing either Imidazole buffer or congenital TTP plasma compared to the loss in the samples containing acquired TTP plasma hints at auto-proteolysis due to specific action of ADAMTS-13. All concentrates seem to contain some ADAMTS-13 activity, but ADAMTS-13 activity in the solutions of 100 I.E. U/ml VWF:RCo used here is always lower than the activity in normal plasma (which contain per definition 1U/ml ADAMTS-13 activity). The ratio of ADAMTS-13 activity/VWF:RCo is thus always lower than 1:100 in all the investigated concentrates. However, concentrate B seems to show the highest ADAMTS-13 activity as well as the largest amount of ADAMTS-13 antigen. Concentrate B is followed by concentrate C. Concentrate A only shows traces of ADAMTS-13 activity and non detectable levels of ADAMTS-13 antigen. The results demonstrate that concentrates B and C should not be used as VWF substrates in ADAMTS-13 activity assays. The ADAMTS-13 content in the various VWF concentrates may influence stability, recovery and half-lives of the concentrates.

ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions

Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibalpha, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm(2)) and arterial (20 and 50 dyne/cm(2)) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.

Heparin attenuates cytotoxic and inflammatory activity of Alzheimer amyloid-beta in vitro.

Amyloid-beta protein (Abeta) is implicated in the pathogenesis of Alzheimer's disease because of its neurotoxicity and the ability to trigger local inflammation. Compounds that interact with the amino acids of the N-terminal region or interfere with aggregation can reduce the Abeta biologic activity. We evaluated the effect of heparin on Abeta (1-42) neurotoxicity and on its ability to activate complement and contact system. On differentiated PC12 cells, a reliable model of neuronal cells, heparin at the doses of 10 and 20 microg/ml significantly counteracted Abeta cytotoxicity as assessed by measuring MTT conversion. We then explored the effect of heparin on Abeta (1-42)-induced complement and contact system activation. Abeta (1-42) was incubated with heparin in presence of normal plasma as the source of complement and contact system factors. Heparin reduced, in a dose-dependent manner, complement and contact system activation, assessed by measuring the degree of C4 and high molecular weight kininogen cleavage. The present data show that heparin can attenuate neurotoxic and pro-inflammatory activity of Abeta and suggest that this drug could represent a new strategy to reduce the progressive neurodegeneration in AD.

Impaired leukocyte phagocytosis in patients undergoing hemihepatectomy for liver metastases.

Patients undergoing partial hepatectomy have an increased susceptibility to infection. To investigate whether this increased risk is related to impaired leukocyte function, we studied polymorphonuclear leukocyte (PMN) phagocytosis in patients undergoing a hemihepatectomy because of liver metastasis (LM, n = 11) and in patients undergoing major abdominal surgery because of abdominal malignancy (AM, n = 8). Eight healthy volunteers (HVs) served as controls. Leukocyte suspensions were incubated with fluorescein isothiocyanate-labeled Staphylococcus aureus, and phagocytosis was measured by flow cytometry. Preoperative PMN phagocytosis, in the presence of autologous plasma, was significantly less in patients with LM compared with patients with AM or HVs. This impaired phagocytosis was potentially restored in the presence of normal plasma. The decreased phagocytic capacity of PMNs from patients with LM was not related to levels of known plasma opsonins or phenotypic changes of PMNs. Rather, it was related to a deficiency of unidentified plasma factors. After surgery, the phagocytic capacity of PMNs of patients with AM decreased by approximately 30%, which correlated with decreasing levels of immunoglobulin G and C3. In conclusion, patients with LM had a decreased PMN phagocytic capacity before surgery. This impairment in phagocytosis disappeared 1 week after surgery. We propose that the presence of LM leads to a deficiency of factor(s) in the blood that impairs PMN phagocytic capacity.

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34-55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the alpha-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the alpha-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.

Quantitative defect of glycoprotein Ib in severe cirrhotic patients.

A decrease of platelet agglutination induced by ristocetin has been described in cirrhotic patients. In order to investigate the relationship of such phenomenon with a putative defect on platelet membrane glycoprotein Ib, we have studied the quantitative and functional status of Gp Ib in eleven severe alcoholic cirrhotic patients, and the ability of their formalin-fixed platelets to agglutinate in the presence of normal plasma plus ristocetin. Interestingly, we found a significant decrease of immunoreactive GP Ib molecules (9,978 +/- 1,534 vs. 17,064 +/- 404 molecules per platelet) and ristocetin-dependent binding of vWF (9,113 +/- 1,338 vs. 13,992 +/- 1,968 molecules per platelet) (P < 0.01) in comparison to the levels found in a control group of healthy subjects. Immunoblotting analysis of platelet lysates confirmed the reduction of GP Ib level in cirrhotic patients, but showed no modification on the precipitation pattern of this glycoprotein. The ristocetin-induced platelet agglutination (light transmission %) was also significantly lower in patients with cirrhosis (48 +/- 7.6 vs. 92 +/- 3.6, P < 0.01), and correlated with the binding of normal vWF (r = 0.863, P = 0.0013). Patients with bleeding times longer than 7 min and/or clinical history of bleeding episodes showed the lowest values of platelet agglutination and GP Ib level. In conclusion, our present results indicate that liver cirrhosis is associated with a relevant decrease of functional GP Ib molecules on the platelet surface. This reduction might be related to the prolongation of bleeding time and to the bleeding diathesis observed in cirrhotic patients.

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.

In Vitro Effects of Hematopoietic Growth Factors on the Proliferation, Endoreplication, and Maturation of Human Megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.

In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.

Anemia in alcoholics.

In order to develop a diagnostic approach to the common problem of anemia associated with alcoholism, 121 chronic alcoholics admitted to a general medical service with a low hematocrit were evaluated. Multiple contributing causes of anemia were present in most patients. Megaloblastic marrow change was found in 33.9% of patients, sideroblastic change in 23.1%, absent iron stores in 13.2%, aggregated macrophage iron in 81.0%, and acute blood loss in 24.8%. The MCV was of little value in predicting the presence of megaloblastic change unless markedly elevated (greater than 110 fl). In 15 of 41 patients with megaloblastic marrow morphology (36.6%) the MCV was normal or low. Among 40 patients with MCV values between 100 and 110 fl, megaloblastic change was not present in the bone marrow smears of 24 (60.0%). Neutrophil hypersegmentation was 95% specific but only 78% sensitive for megaloblastic change; in contrast, the presence of macroovalocytosis was 90% sensitive but only 68% specific. Serum lactic dehydrogenase, plasma folate, and erythrocyte folate levels had such low sensitivities and specificities for megaloblastic change as to be of little predictive value. Hematologic responses to folic acid were often inadequate in patients with megaloblastic morphologic changes, apparently because of associated acute and chronic illness. Our findings are consistent with the hypothesis that 2 mechanisms account for the development of megaloblastic hematopoiesis in alcoholics: induction of folate deficiency and a direct toxic effect of alcohol on erythroid precursors independent of folate depletion, as reflected by the presence of normal plasma and erythrocyte folate levels in several patients with megaloblastic change. In no patient was sideroblastic change the sole apparent cause of anemia. Megaloblastic hematopoiesis and aggregated macrophage iron frequently accompanied sideroblastic change. Examination of the blood smear revealed siderocytes in one-third of patients with sideroblastic marrows and dimorphic erythrocyte morphology in the majority. Dimorphic blood smears, however, were neither sensitive nor specific for sideroblastic change. Serum iron concentrations were usually not elevated in the group with sideroblastic abnormalities. In predicting marrow iron stores, serum iron and iron-binding capacity concentrations were often non-diagnostic or misleading. Serum ferritin levels less than 100 ng/ml, however, showed 100% sensitivity and 95% specificity for absent marrow iron stores despite the frequent presence of abnormal liver function. On the basis of our findings, practical guidelines have been formulated for the evaluation and therapy of anemia in alcohol

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

AbstractVon 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.

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.

Polybrene-Induced Platelet Agglutination and Reduction in Electrophoretic Mobility: Enhancement by von Willebrand Factor and Inhibition by Vancomycin

It has recently been reported that the polycation Polybrene (hexadimethrine bromide), like ristocetin, agglutinates platelets more extensively in the presence of normal plasma than von Willebrand plasma. Since we have previously proposed that ristocetin may initiate agglutination by reducing platelet surface charge, I investigated the correlation between Polybrene's ability to induce agglutination and alter platelet electrophoretic mobility. In the absence of plasma, low concentrations of Polybrene produced small platelet aggregates and reduced the electrophoretic mobility. Higher concentrations were needed to produce small platelet aggregates in the presence of von Willebrand plasma; these same concentrations produced more rapid agglutination and much larger aggregates in the presence of normal plasma. The reductions in electrophoretic mobility were also greater in the presence of normal than von Willebrand plasma. Both agglutination and the reduction in mobility could be partially reversed with citrate. Vancomycin, an antibiotic that inhibits ristocetin-induced agglutination with some specificity, inhibited both Polybrene-induced agglutination and the reductions in platelet mobility. These data are consistent with our electrostatic model and support the view that reductions of platelet surface charge may be necessary (but perhaps not sufficient) to initiate the interaction between platelets and von Willebrand factor.

Polybrene-induced platelet agglutination and reduction in electrophoretic mobility: enhancement by von Willebrand factor and inhibition by vancomycin

It has recently been reported that the polycation Polybrene (hexadimethrine bromide), like ristocetin, agglutinates platelets more extensively in the presence of normal plasma than von Willebrand plasma. Since we have previously proposed that ristocetin may initiate agglutination by reducing platelet surface charge, I investigated the correlation between Polybrene's ability to induce agglutination and alter platelet electrophoretic mobility. In the absence of plasma, low concentrations of Polybrene produced small platelet aggregates and reduced the electrophoretic mobility. Higher concentrations were needed to produce small platelet aggregates in the presence of von Willebrand plasma; these same concentrations produced more rapid agglutination and much larger aggregates in the presence of normal plasma. The reductions in electrophoretic mobility were also greater in the presence of normal than von Willebrand plasma. Both agglutination and the reduction in mobility could be partially reversed with citrate. Vancomycin, an antibiotic that inhibits ristocetin-induced agglutination with some specificity, inhibited both Polybrene-induced agglutination and the reductions in platelet mobility. These data are consistent with our electrostatic model and support the view that reductions of platelet surface charge may be necessary (but perhaps not sufficient) to initiate the interaction between platelets and von Willebrand factor.

Lymphocyte Blastogenic Response to Autologous Tumor Cells Pretreated with Neuraminidase in Patients with Gastric Carcinoma

The effect of Vibrio cholerae neuraminidase (VCN) treatment of tumor cells on lymphoblastic response was studied using the assay of mixed lymphocyte-tumor cell reaction in 24 patients with gastric carcinoma. Lymphoblastic responses as indicated by stimulation index were significantly augmented in cultures with autologous tumor cells pretreated with mitomycin (MMC) and VCN as compared to those pretreated with MMC alone when cultured in the presence of normal plasma (p < 0.05). In autologous plasma cultures, however, the mean stimulation index in MMC-VCN-treated cell cultures was only slightly higher than that in MMC-treated cell cultures. Accentuation of the response was not observed in any type of cultures using MMC-VCN-treated normal tissue cells. In 20 of these patients, the blastic response to nonviable frozen stored tumor cells was measured simultaneously and significant increase in blastic transformation by pretreatment of the cells with VCN was also observed in normal plasma cultures (p < 0.05). There was significant correlation between the response to nonviable frozen VCN-treated cells and to these, not frozen, viable cells (p < 0.001). These results indicate that lymphoblastic response to autologous tumor cells could be enhanced by pretreating the cells with VCN in patients with gastric carcinoma and the freezing could render the cells nonviable without interfering with the augmented immunogenicity of VCN-treated tumor cells.

The Effects of Ristocetin and von Willebrand Factor on Platelet Electrophoretic Mobility

Ristocetin will induce the agglutination of platelets in the presence of von Willebrand factor. In previous studies, an electrostatic mechanism was proposed for this phenomenon wherein first the platelet's surface charge is reduced by the binding of ristocetin and then the von Willebrand factor acts as a bridge between platelets. To test this hypothesis, the effects of ristocetin and von Willebrand factor, singly and together, on the electrophoretic mobility of normal, trypsinized, and Bernard-Soulier platelets was measured. Ristocetin alone, at concentrations of 0.5 mg/ml or more, produced a statistically significant reduction in the electrophoretic mobility of fresh or fixed platelets. Control experiments showed that the reduction was not due to changes in the ionic milieu of the solution. Therefore, the decrease in platelet mobility is evidence for binding of ristocetin to the platelet surface. Bernard-Soulier and trypsinized platelets also had reductions in mobility with ristocetin, suggesting that ristocetin binds to the platelet at sites other than the binding site for von Willebrand factor. The presence of plasma from a patient with von Willebrand's disease did not alter the reduction in mobility of normal platelets by ristocetin. However, the reduction was markedly enhanced in the presence of normal plasma. This enhancement did not occur with Bernard-Soulier platelets and was inhibited by anti-Factor VIII/von Willebrand factor antiserum or trypsinization of the platelets. Thus, the enhanced reduction appears to be associated with the binding of von Willebrand factor to the platelet surface. These studies indicate that platelets undergo two changes with ristocetin and von Willebrand factor, both of which facilitate agglutination: reduction in net surface charge and binding of von Willebrand factor, a large molecule which can serve as a bridge between platelets. In parallel studies, bovine von Willebrand factor, without ristocetin, agglutinated and reduced the electrophoretic mobility of normal but not Bernard-Soulier or trypsinized platelets; this indicates a similar mechanism of agglutination.

Cellular and Plasma‐Associated Phagocytic Defects Amongst Iranian Blood Donors

Peripheral blood leucocytes from 30 grossly anaemic professional blood donors were investigated for phagocytosis, intracellular killing and chemotaxis. The nitro-blue tetrazolium test was positive in 29 donors and negative in 1. Professional donors' cells in the presence of normal plasma were unable to phagocytose successfully and Candida killing and opsonization were defective when donors' plasma was used in the presence of normal cells. Chemotaxis of professional donors' cells to Ag/Ab-treated normal plasma was reduced as was chemotactic activity of normal cells in both Ag/Ab- and LPS-treated professional donors' plasma.

Cellular and Plasma-Associated Phagocytic Defects Amongst Iranian Blood Donors

Peripheral blood leucocytes from 30 grossly anaemic professional blood donors were investigated for phagocytosis, intracellular killing and chemotaxis. The nitro-blue tétrazolium test was positive in 29 donors and negative in 1. Professional donors’ cells in the presence of normal plasma were unable to phagocytose successfully and Candida killing and opsonization were defective when donors’ plasma was used in the presence of normal cells Chemotaxis of professional donors’ cells to Ag/Ab-treated normal plasma was reduced as was chemotactic activity of normal cells in both Ag/Ab- and LPS-treated professional donors’ plasma.