Antirabbit Platelet Research Articles

Enhanced Inhibition of in-Vitro Platelet Aggregation by a Targeted Antibody Conjugate Containing an Anti-Rabbit Platelet Glycoprotein Iib/Iiia Antibody

Enhanced Inhibition of in-Vitro Platelet Aggregation by a Targeted Antibody Conjugate Containing an Anti-Rabbit Platelet Glycoprotein Iib/Iiia Antibody

Assessment of the hemostatic effectiveness of human platelets treated with aminomethyltrimethyl psoralen and UV A light using a rabbit ear bleeding time technique

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.

Assessment of the Hemostatic Effectiveness of Human Platelets Treated With Aminomethyltrimethyl Psoralen and UV A Light Using a Rabbit Ear Bleeding Time Technique

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.

Effect of vitamin E on arachidonic acid-release in rat peritoneal macrophages

We have previously reported on suppression of the PGE 2 production in PMA- and calcium ionophore A23187-stimulated macrophages isolated from vitamin E-treated rats. To further study the mechanism, we examined the effect of vitamin E on phospholipase A 2 activity in both intact macrophages and cell-free homogenates measuring the release of [ 14C]arachidonic acid. In macrophages from vitamin E-treated rats, arachidonic acid release in intact cells as stimulated with PMA and calcium ionophore A23187 was hardly detected. In the cell-free homogenates, increase in phospholipase A 2 activity of cytosol and particulate fractions by PMA and A23187 was partially suppressed. In unstimulated macrophages, most of phospholipase A 2 was recovered in the cytosol fraction. The partially purified cytosolic phospholipase A2 showed a molecular mass 95 kDa on TSK gel G3000SW gel-filtration and on Western blot analysis using anti-rabbit platelet phospholipase A 2 monoclonal antibody RHY-5. The activity of cytosolic 95 kDa phospholipase A 2 was not inhibited in vitro by vitamin E. From these results, it was suggested that vitamin E needs intact macrophages to suppress arachidonic acid release.

Effect of Thrombocytopenia on the Onset of Immune Complex Glomerulonephritis

The effect of platelets on the development of immune complex glomerulonephritis (GN) was examined using bovine serum albumin (BSA) GN with platelet depletion. To clarify the role of platelets in the initial stage of BSA GN, thrombocytopenia was induced before BSA infusion. In 18 New Zealand white rabbits, BSA was intravenously injected twice after the presensitization. Eight of these BSA GN rabbits were injected daily with goat anti-rabbit platelet antiserum to induce thrombocytopenia, and platelet counts were maintained below 5 x 10(4)/microliters throughout the experiment. In the thrombocytopenic group, the degree of proteinuria was significantly decreased compared to the control group. Glomerular polymorphonuclear leukocyte infiltration, mononuclear cell proliferation, exudation and glomerular enlargement were significantly suppressed in the thrombocytopenic group. The results suggest that platelets may be quite important in the initiation and development of immune complex GN.

The Requirement for Platelets in Allergen-induced Late Asthmatic Airway Obstruction: Eosinophil Infiltration and Heightened Airway Responsiveness in Allergic Rabbits

In these studies, we have used an allergic rabbit model to investigate the role of platelets in the late asthmatic response (LAR) by depleting platelets with a guinea pig antirabbit platelet antiserum (APAS). Allergen exposure of immunized rabbits pretreated with normal guinea pig serum (NGPS) to serve as a control resulted in an early- and late-phase obstructive airway response that persisted for 6 h. When the immunized animals were pretreated with APAS, the magnitude of the LAR in terms of dynamic compliance was reduced by 86.2% (p less than 0.03), but there was no difference in the early response curve. Allergen challenge of animals treated with NGPS resulted in an increased bronchial responsiveness to inhaled histamine: PD50 Cdyn geometric mean +/- SEM before, 2.36 mg/ml (3.43-1.64); after, 0.60 mg/ml (0.67-0.54) (p less than 0.01). PD50 RL before, 1.78 mg/ml (2.4-1.32); after, 0.58 mg/ml (0.81-0.47) (p less than 0.05). In contrast, when animals were treated with APAS, there was a significant inhibition of allergen-induced airway hyperresponsiveness to inhaled histamine: PD50 Cdyn geometric mean +/- SEM before, 1.42 mg/ml (2.06-0.98); after, 1.10 mg/ml (1.41-0.86) (p less than 0.4). PD50 RL before, 1.62 mg/ml (2.22-1.39); after, 1.05 mg/ml (1.35-0.82) (p greater than 0.4). Analysis of bronchoalveolar lavage fluid revealed an increase in the number of neutrophils and eosinophils after allergen exposure in control animals (p less than 0.01). However, in animals rendered thrombocytopenic, the number of eosinophils, but not neutrophils, was significantly reduced (p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

PAF‐induced bronchial hyperresponsiveness in the rabbit: contribution of platelets and airway smooth muscle

1. Aerosol administration of platelet activating factor (PAF) to normal rabbits induced an enhanced airway responsiveness to inhaled histamine, 6 and 24 h after exposure. Following exposure to bovine serum albumin (BSA) as the carrier molecule for PAF, there was an increase in airway responsiveness to histamine 6 h after challenge, although by 24 h this was not significantly different from the responsiveness of airways to histamine before BSA. 2. PAF-induced bronchial hyperresponsiveness at 24 h was associated with a substantial increase in the number of neutrophils and mononuclear cells and a small, but significant increase in the number of eosinophils in the lungs as assessed by bronchoalveolar lavage. BSA exposure failed to alter the total number of cells in the lungs, although there was a significant increase in the number of neutrophils in the bronchoalveolar lavage fluid. 3. Selective platelet depletion with a guinea-pig anti-rabbit platelet serum inhibited PAF-induced bronchial hyperresponsiveness. In addition, there was an attenuation of PAF-induced airway inflammation in animals rendered thrombocytopenic. 4. The contractile potency to histamine, methacholine and carbachol was similar in intrapulmonary bronchi taken from rabbits exposed to an aerosol of BSA or PAF. Furthermore, the relaxant potency to the non-selective beta-adrenoceptor agonist isoprenaline, was unaltered in PAF-treated rabbits. In contrast, there was a 2.58 fold reduction in the relaxant potency to theophylline in rabbits exposed to PAF compared with rabbits exposed to BSA. 5. These results suggest that in the rabbit, PAF-induced bronchial hyperresponsiveness at 24 h is associated with airways inflammation and is dependent upon platelet activation, but is not related to changes in airway smooth muscle function.

Measurement of platelet-associated IgG in animal models of immune and nonimmune thrombocytopenia

Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In-labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

Measurement of Platelet-Associated IgG in Animal Models of Immune and Nonimmune Thrombocytopenia

AbstractPlatelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125l-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 ± 168, mean ± 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111ln-labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 ± 55 molecules of SpA per platelet, mean ± SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system.

The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platelet factor 4, using heparin agarose affinity chromatography and immunoprecipitation with specific goat anti-rabbit platelet factor 4 antisera. SDS PAGE of the washed immunoprecipitates demonstrated a [3H]leucine-containing peak which migrated identically with purified homogeneous rabbit platelet factor 4. A second, slightly larger molecular-weight protein was identified in the gels also, suggesting that rabbit platelet factor 4 may be synthesized as a larger molecular-weight precursor in rabbit megakaryocytes. These results provide direct evidence that the platelet-specific protein, platelet factor 4, is synthesized in rabbit megakaryocytes before it is packaged into alpha-granules for release in circulating platelets.

Structural aspects of [formula omitted] aging rabbit blood platelets

The structural and density changes that occur in aging blood platelets may help to evaluate the age distribution of platelets in a blood sample. Separation of normal rabbit platelets on human serum albumin discontinuous density gradient (HSAG), provided four bands of cells, usually in the 18,19,22 and 23% solutions of HSA. Large and small platelets were found in each of the four bands. No significant differences were found in the mean size of the cells taken from the light fractions (18% and 19%) when compared to cells taken from the heavier fractions (22% and 24%). However cells of the light fractions usually contained less Dense Bodies (DB) than cells from the heavier fractions. Newly formed platelets obtained from rabbits recovering from induced thrombocytopenia after injection of goat antirabbit platelet serum or neuraminidase, are generally large and found in the solution of 21% HSA (S.G. 1.0606). These platelets contain glycogen particles and practically no DB. After several days of maturation and regeneration, smaller platelets containing higher number of DB, are found in the circulation. On the 10th day, after the beginning of regeneration, platelets are distributed above and below the solution of 21% HSA, similarly to the normal platelet population. The in vivo aging of platelets, seems to be associated with accumulation of DB within these cells.

The effect of thrombocytopenia on experimental arteriosclerotic lesion formation in rabbits. Smooth muscle cell proliferation and re-endothelialization.

This study was designed to investigate the mechanisms involved in fibromusculoelastic lesion formation produced by selective de-endothelialization by the intra-arterial balloon catheter technique in thrombocytopenic rabbits. Thrombocytopenia was induced and maintained for up to 30 days by daily injections fo highly specific sheep anti-rabbit platelet sera (APS). Evidence for re-endothelialization was obtained by i.v. Evans blue dye 30 min before sacrifice. Rabbits received daily injections of APS, which reduced the mean platelet count to 5,600/cm3; control animals received identically treated normal sheep sera on the same schedule, and had mean daily platelet counts of 363,000/cm3. Evaluation of intimal thickness was assessed by counting cell layers in semithin sections. Intimal thickening in aortae from rabbits treated with APS was strikingly suppressed, in contrast to those from normal sheep sera-treated animals which showed a mean intimal thickness of 18 cell layers within 28 days often after de-endothelialization. Re-endothelialization was not affected by APS treatment. These results indicate that the proliferation of smooth muscle cells is dramatically inhibited by reduction of platelets.

The role of platelet factor (PF-4) in the haemostatic-coagulation System

(1) Platelet factor 4 (PF-4) wich shows antiheparin activity was purified from the human platelets. After homogenization by sonifier and Al(OH)3 gel absorption, crude PF4 was further purified by the Heparin-Sepharose affinity column chromatography and G-75 sephadex gel filtration. Final PF-4 activity was 186% and purification fold was 500. The purified PF4 neutralized 200u/ml of heparin. The molecular weight of final PF4 was found to be about 20, 000.(2) In normal rabbits a single injection of 2000r endotoxin, extracts of gastric cancer tissue, anti-rabbit platelet immune guinea pig serum and a subsequent infusion of normal saline induced disseminated intravascular coagulation (DIC). The remarkable changes of platelet count and PF4 levels were observed. In contrast to these facts, in the thrombocytopenic rabbits treated with Busulfan DIC was not induced by the infusion of the same trigger substances and PF4 levels did not change dominantly.(3) The relationship of PF4 to haemorrhagic tendency in the patients with DIC, hypoplastic anemia and ITP was investigated. PF4 levels increased markedly in the haemorrhagic patients with DIC and hypoplastic anemia, but it decreased in the patients with ITP. In all cases it was observed that when platelets counts increased and haemorrhage was relieved, PF4 levels became to normal. PF4 would be a good parameter to reflect the platelet function in haemorrhagic disorders and thromboembloic diseases.

Ultrastructural changes of endothelium associated with thrombocytopenia

In a study of the relationship between thrombocytopenia and increased vascular fragility, changes in the endothelium of capillaries and postcapillary venules of the tongue were examined by electron microscopy. Adult male albino rabbits (4 kg) were maintained thrombocytopenic (platelets less than 20,000/cu mm) up to 24 hr by one to three injections of guinea pig antirabbit platelet serum. Within 6 hr the normal projections and folds of the lumenal surface of the endothelial surface were largely effaced. In addition, the endothelium became thinner. In places, pores and membranous diaphragms were observed. Endothelial junctions appeared normal. Identical findings were observed if rabbits were made thrombocytopenic by administration of intraperitoneal busulfan. Intravenously administered Thorotrast was observed in endothelial cells and in the extravascular spaces within 3 min after injection into thrombocytopenic animals, while it was seen only intravascularly in control rabbits. With the spontaneous restoration of circulating platelets, the endothelium reverted to normal.

Ultrastructural Changes of Endothelium Associated With Thrombocytopenia

In a study of the relationship between thrombocytopenia and increased vascular fragility, changes in the endothelium of capillaries and postcapillary venules of the tongue were examined by electron microscopy. Adult male albino rabbits (4 kg) were maintained thrombocytopenic (platelets less than 20,000/cu mm) up to 24 hr by one to three injections of guinea pig antirabbit platelet serum. Within 6 hr the normal projections and folds of the lumenal surface of the endothelial surface were largely effaced. In addition, the endothelium became thinner. In places, pores and membranous diaphragms were observed. Endothelial junctions appeared normal. Identical findings were observed if rabbits were made thrombocytopenic by administration of intraperitoneal busulfan. Intravenously administered Thorotrast was observed in endothelial cells and in the extravascular spaces within 3 min after injection into thrombocytopenic animals, while it was seen only intravascularly in control rabbits. With the spontaneous restoration of circulating platelets, the endothelium reverted to normal.

Experimentally Induced Alterations of the Bactericidal Property of Rabbit Serum for Bacillus Subtilis

Summary The bactericidal property of rabbit serum consists of two protein components derived from rabbit platelets. These are designated components 1 and 2. Total body x-ray irradiation of 700 r causes a striking reduction of platelets and of components 1 and 2. As little as 100 r of x-ray irradiation produce a decrease of component 1 without causing a decrease of the platelets. Specific antiserum for rabbit platelets causes a rapid decrease in platelets and in components 1 and 2, when injected intravenously into rabbits. Studies in vitro reveal that normal rabbit serum loses its bactericidal property when antiplatelet serum is added. Highly bactericidal rat serum is not inhibited by anti-rabbit platelet antibody. Intravenous injection of thorotrast into rabbits causes a precipitous decrease in platelets and of components 1 and 2 without producing any decrease in leukocytes or hematocrit.

Antirabbit platelet and red-cell factors in normal blood.

Abstract Normal human, dog, and sheep plasma or serum contains antirabbit platelet and erythrocyte factors which produce thrombocytopenia and anemia in rabbits following intravenous injection. These factors are relatively stable to heat, but labile on aging. Their effect is not opposed by ACTH, cortisone, antihistaminics, or splenectomy. The antiplatelet factor can be separated from the plasma by chemical means that also remove prothrombin.