Coconut water solutions for the preservation of spleen, ovary, and skin autotransplants in rats.

BACKGROUND: The purpose of this study was to evaluate the efficacy of coconut water in the preservation of spleen, ovary, and skin autotransplantations in rats. METHODS: Fifty female Wistar rats were divided randomly into 5 groups on the basis of the following tissue graft preservation solutions: group 1, lactated Ringer's; group 2, Belzer's solution; group 3, mature coconut water; group 4, green coconut water; and group 5, modified green coconut water. In group 5, the green coconut water solution was modified to obtain the same electrolyte composition as Belzer's solution. The spleen, ovaries, and a skin fragment were removed from each animal, stored for 6 hours in one of the solutions, and then re-implanted. The recoveries of tissue functions were assessed 90 days after surgery by means of spleen scintigraphy and blood tests. The implanted tissues were collected for histological analyses. RESULTS: Higher immunoglobulin G levels were observed in the animals of group 5 than in the animals of group 1. Differences in follicle-stimulating hormone levels were observed between groups 1 and 2 (P < .001), between groups 4 and 2 (P = .03), and between groups 5 and 2 (P = .01). The spleen scintigraphy results did not differ among the groups. The ovarian tissue was better preserved in the mature coconut water group (P < .007). CONCLUSIONS: Solutions containing coconut water allowed for the preservation of the spleen, ovaries, and skin for 6 hours, and the normal functions of these tissues were maintained in rats.

Phospholipase A2 activity in platelets of patients with primary thrombocythemia.

Patients with primary thrombocythemia (PT) have both, bleeding and thrombotic events. Although platelet aggregation tests are usually abnormal, synthesis of thromboxane B2 (TxB2) by platelets is increased. This feature could be the consequence of an increased phospholipase activity or a facilitated metabolism of arachidonate by prostaglandin synthetase pathway. We studied the activity of phospholipase A2 as well the arachidonate metabolism in platelets of patients suffering from PT. Eleven patients and 11 controls were included. Platelets were labelled with [14C]arachidonic acid ([14C]AA). Lost of radioactivity from phospholipids and new radioactive prostanoids were evaluated in calcium ionophore A23187 activated platelets, to explore phospholipase A2 activity. This assay was also carried out in aspirin-incubated platelets. We also studied the formation of prostanoids in platelets activated by radioactive free arachidonic acid. Platelet aggregation studies of patients were abnormal. [14C]AA incorporation in platelet phospholipids was normal. Ionophore activated platelets from patients and controls lost 26.1 +/- 8.3% and 24.1 +/- 10.5% of radioactivity, respectively, mainly from phosphatidylcholine. The main arachidonate metabolite was 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which comprised 14.1 +/- 5.1% of the radioactivity released from phospholipids in patients, and a similar amount in the controls (14.4 +/- 7.5%). Formation of TxB2 was also similar in patients (5.5 +/- 1.2%) and controls (4.9 +/- 2.9%). Formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was also normal. Ionophore A23187 activation of aspirinized platelets of patients released 19.5 +/- 7.4% of radioactivity from phospholipids, which was completely metabolized to HETE. Formation of prostanoids HETE, HHT and TxB2 by arachidonic acid activated platelets of patients was normal. Phospholipase A2 activity as well both cyclooxygenase and lipoxygenase activities in platelets of patients with PT were found to be normal.

Síndrome de Hermansky-Pudlak / Hermansky-Pudlak syndrome

Se describe un caso de síndrome de Hermansky-Pudlak (SHP) en un enfermo con albinismo conocido y que fue descubierto por sangrado intenso postamigdalectomía. En los estudios de hemostasia realizados se comprobó la existencia de enfermedad de von Willebrand tipo I junto con alteraciones de la agregación plaquetaria. El paciente tuvo que ser intervenido por colelitiasis con colecistectomía, para la cual se administró profilaxis con desmopresina, sin que aparecieran sangrados patológicos ni otras complicaciones (AU)

Hereditary thrombocythaemia is a genetically heterogeneous disorder: exclusion of TPO and MPL in two families with hereditary thrombocythaemia.

Hereditary thrombocythaemia (HT) is an autosomal dominant disorder with clinical presentation and complications resembling sporadic essential thrombocythaemia (ET). Mutations in the thrombopoietin (TPO) gene causing overproduction of TPO and elevated TPO serum levels have been found previously in three families with HT. Here, we present evidence for genetic heterogeneity by demonstrating that HT in a Spanish and a US family is caused by genes other than TPO. Affected family members in both families had normal TPO serum levels. Genetic linkage analysis with TPO microsatellite markers excluded TPO as the disease gene in the Spanish HT family, and sequencing of the TPO gene revealed no mutations in the propositus of the US family. To test a role for MPL, the gene for the TPO receptor, we identified three single nucleotide polymorphisms (SNP) and a novel polymorphic CA microsatellite marker. By linkage analysis, we excluded MPL as the cause of HT in the Spanish family. Interestingly, mapping of the CA microsatellite marker to a region 40.5 kb upstream of MPL revealed the presence of sequences from the TIE gene, which encodes a tyrosine kinase receptor expressed on megakaryocytes and endothelial cells. Thus, MPL and TIE are in close physical proximity, and the CA microsatellite described here will be a useful genetic marker for both genes.

Prevention of alphaII(b)beta3 activation by non-steroidal antiinflammatory drugs.

We have studied the effect of non-steroidal antiinflammatory drugs (NSAIDs) on alphaII(b)beta3 integrin activation and platelet aggregation. NSAIDs such as meloxicam, piroxicam, indomethacin and aspirin, but not aceclofenac or diclofenac interfered with the activation state of alphaII(b)beta3. NSAIDs that inhibited alphaII(b)beta3 activation were also able both to partially inhibit platelet primary aggregation and to accelerate platelet deaggregation. These effects of NSAIDs were not dependent on cyclooxygenase inhibition. The results obtained indicate that some NSAIDs exert a specific action on alphaII(b)beta3 activation, and provide an additional mechanism that accounts for their beneficial effects in diseases in which platelet activation is involved.

[Von Willebrand disease: characteristics and response to desmopressin. Study of 103 cases]. / Enfermedad de Von Willebrand: características y respuesta a desmopresina. Estudio de 103 casos.

BACKGROUND: To describe the main characteristics and response to desmopressin infusion in 103 patients suffering from von Willebrand disease (vWD). PATIENTS AND METHODS: The criteria for diagnosis were (except for type 2N) the coexistence of von Willebrand factor ristocetin cofactor (vWF:RCo) activity < 50 U/dl with bleeding disease or one of the following data: von Willebrand factor antigen (vWF:Ag) activity < 50 U/dl, factor VIII (FVIII) activity < 50 U/dl or the existence of a increased bleeding time (BT). Multimeric studies of vWF were performed in 51 cases and ristocetin induced platelet aggregation (RIPA) was also performed. RESULTS: Spontaneous bleeding was found in 36 patients, while in 18 cases the diagnosis was done after surgical bleeding. Thirteen patients (6 presenting with mild bleeding) were studied for abnormalities in the routine preanestesic tests. Other 22 patients were diagnosed with vWD by familial studies. There were 3 patients with type 2B, 1 case with type 2N and other patient with type 3. BT was found increased in 26 out of 58 patients. The activities of vWF:CoR and vWF:Ag were 38.4 (9.4) U/dl and 45.8 (23.2) U/dl, respectively, while the activity of FVIII was 49.9 (20.8) U/dl. Prophylactic DDAVP (desmopressin) was infused in 32 patients. After 1 h, basal activities of vWF:CoR and vWF:Ag were increased by 3.1 (3.2) and 3.4 (3.1) times, respectively, and maintained for 3 h. FVIII activity increased 3.6 (2.3) times the basal levels decreasing after 3 h (2.9 [2.1]; p < 0.01). The BT was corrected in 8 out of ten patients. CONCLUSIONS: vWD is a major cause of surgical bleeding. Preanestesic anamnesis and coagulation tests can be useful to identify vWD. Many patients with vWD have normal BT. A failure in the response to desmopressin infusion is unusual.

Lipid composition of platelets in patients suffering from migraine without aura.

Patients with migraine have a platelet hyperaggregability. As this alteration could be the consequence of an abnormal lipid composition of platelet membranes, we studied the phospholipid specimens and the cholesterol/phospholipid ratio in platelet of neuron patients suffering from migraine. The cholesterol/phospholipid ratio was 0.7 +/- 0.1 (normal 0.6 +/- 0.1, molar ratio). The proportion of five main platelet phospholipids components including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol, were also normal. These data suggest that platelet hyperactivity in patients with migraine is not due to an altered lipid content of those cells.

Platelets isolated by albumin gradient retain normal activation pathways.

Isolated platelets from samples with low counts produce technical problems. Albumin gradient (AG) has been shown to be useful for this purpose, preserving the aggregating response of these cells. The influence of this method in the enzymatic pathways that regulate the platelet activation is studied. Platelets were isolated by either AG or conventional centrifugation methods and labelled with C-14-arachidonic acid (C-14-AA). Isolated platelets were activated with thrombin (5 U/ml) and lipids were extracted according to Bligh and Dyer. Platelet phospholipids and prostanoids were resolved by TLC. The incorporation of C-14-AA by platelets was similar by both methods (31.7 +/- 18% versus 47.2 +/- 6.9%), as well as the distribution of C-14-AA in the five major platelet phospholipids. Formation of radioactive thromboxane B2, hydroxyheptadecatrienoic acid and hydroxyeicosatetraenoic acid by activated platelets was also similar by both methods. These findings suggest that platelet isolation by albumin gradient preserves the enzymatic pathways responsible for the activation of these cells.

Plasma phospholipids and platelet function in uremic patients.

An increased activity of phospholipase A2 has been observed in the plasma of patients with uremia. This enzyme converts phosphatidylcholine to lysophosphatidylcholine (LPC), an inhibitor of platelet aggregation. We measured the levels of plasma phospholipids including LPC, and platelet aggregation in 7 patients with uremia. Platelet response to agonists was defective, mainly with collagen (p < 0.001). The patients' levels of LPC in plasma were similar to those of controls (109.7 +/- 41.6 vs. 80.4 +/- 16.8 nmol/ml) and did not correlate with the platelet response to adenosine diphosphate (r = -0.51). The amount of phosphatidylcholine was increased with respect to normal plasma (1,041.0 +/- 201.8 vs. 760.8 +/- 142.7 nmol/ml, p < 0.01), while the levels of other phospholipids were normal. These results do not suggest a participation of plasma LPC in the genesis of the platelet defect observed in patients with uremia.

Arachidonic acid metabolism in platelets of patients with essential thrombocythaemia.

Platelet aggregation tests and studies comprising [14C] arachidonic acid [14C]AA incorporation, release and metabolism were performed in resting and thrombinstimulated platelets of 11 patients with essential thrombocythaemia (ET) and 11 normal subjects. Nine patients had abnormal aggregation tests. Incorporation and distribution of [14C]AA in main platelet phospholipids (PLs) was similar in both groups. Activated platelets of patients with ET released more radioactivity from PLs that controls (13.7 +/- 5.4% versus 8.2 +/- 1.9%, p < 0.01). The formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was also increased (3.3 +/- 1.4% of total radioactivity versus 1.6 +/- 0.4% in controls (p < 0.0001). The same results were obtained for the generation of thromboxane B2 (p < 0.01). We did not detect differences in the formation of 12-L-hydroxy- 5,8,10,14-eicosatetraenoic acid (3.3 +/- 1.7% in patents versus 2.0 +/- 0.5% in controls). These results indicate that platelets of patients with ET have an increased activity of phospholipases and suggest a facilitated metabolism of arachidonate by the prostaglandinsynthetase pathway. Our results also demonstrate that impairment of aggregation tests in these patients was not due to a defective activity of the enzymes involved in the release and metabolism of AA by platelets.

Increased levels of plasma von Willebrand factor in migraine crisis.

INTRODUCTION: To evaluate the participation of the vessel wall in the pathogenesis of migraine attack, we measured the plasma levels of von Willebrand factor (vWF), a protein secreted from the endothelial cells. MATERIAL & METHODS: 17 patients suffering from migraine without aura and 25 healthy volunteers were studied. von Willebrand factor and platelet aggregation tests were studied by conventional methods. RESULTS: The levels of vWF:antigen increased from 72.4 +/- 29 U/dl in the intercrisis to 130.2 +/- 75 U/dl during the attack (p < 0.01). We did not detect difference in the platelet aggregability in both phases. Plasma vWF activity measured as ristocetin cofactor (vWF:RCo) was similar in intercrisis and crisis (100.6 +/- 31 U/dl vs 94.5 +/- 44 U/dl). CONCLUSIONS: There is a plasma release of vWF molecules during the migraine crisis. This feature is not platelet dependent and is probably a consequence of endothelial stress.

[Checking oral anticoagulation in capillary blood]. / Control de la anticoagulación oral en sangre capilar.

PURPOSE: To check out the reproducibility and costs of prothrombin time (PT) determination as a control of oral anticoagulant therapy (OAT) in plasma and capillary blood. PATIENTS AND METHODS: The study was carried out in two phases: along two years, 1,700 patients with OAT were controlled, 700 of them in the hospital outpatient clinic. In 149 patients INR was simultaneously determined in both capillary and venous blood. The 700 patients receiving acenocoumarin who had been controlled in 1991 according to the conventional plasma-sample fashion, were controlled in the second year (i.e., 1992) by means of capillary blood testing, a comparison of the costs of each method and the need for anticoagulant drugs being undertaken. Venous blood PT was assessed with reagent thromboplastin (Tromborel S) in an Electra-1000 (MLA) system. An automated Trombotrack system was used for the capillary blood tests using Thrombotest as current procedure. The results were expressed as INR in both methods. The statistical evaluation of the results was carried out by means of Student's t, variance analysis, and correlation study. RESULTS: No significant differences were found in the anticoagulation intervals attained from venous or capillary blood samples. No significant differences were seen in 87 patients on whom the test was repeated in two samples drawn from a single capillary puncture. The weekly OAT doses of 30 patients along six months were analysed. The need for anticoagulant drugs was similar (17.4 vs 17.2 mg/patient/week). The mean INR in 1991 was 2.82 and the mean drug-need was 15.24 mg/week, whereas in 1992 the mean INR was 2.86 and the need for anticoagulant was 15.49 mg/week. The costs of the conventional method were 103.6 Pta, this being 70 Pta for capillary blood, which means a 32% savings. CONCLUSIONS: OAT control by means of PT performed on capillary blood must be considered a substitutive method for the venous blood assay due to its efficacy, simplicity and lower costs.

Red cell lipid abnormalities in acquired acanthocytosis are extended to platelets.

Acquired acanthocytosis (AA) is an uncommon disease characterized by the presence of abnormal red cells (acanthocytes) in the blood smears of affected subjects. Acanthocyte membrane is enriched in cholesterol by an abnormal plasma lipoprotein. We studied the existence of similar changes in platelets of one patient with AA. Red cell cholesterol/phospholipid (Ch/PL) ratio in the patient was 1.6 (normal 1.1 +/- 0.1). Phosphatidylcholine (PC) comprised 36% of total phospholipid (30.7 +/- 1.8% in controls). Platelets showed aberrant morphology in the blood smears, and the ratio Ch/PL was high in comparison with normal platelets (1.4 v 0.6 +/- 0.1). PC comprised 52% of total PL (39.6 +/- 1.9% in normal platelets). Normal platelets incubated with autologous plasma for 24 h maintained a Ch/PL ratio of 0.7, whereas this value changed to 1.4 when these cells were incubated with plasma from the patient. These results suggest that platelets of patients affected by AA acquire the same biochemical abnormality as red cells.

Von Willebrand factor availability in platelet concentrates stored for 5 days.

Von Willebrand factor (vWF) availability was assessed in platelet concentrates (PCs). After 5 days of storage, 82 +/- 9% of basal levels of ristocetin cofactor activity (vWF:RCo) remained in PCs. vWF antigen (vWF:Ag) increased up to 166 +/- 38% (P < 0.05) in the same period. Autoradiograph pattern of vW:Ag showed an increase in low molecular weight multimers, and fast migrating multimeric forms were visualized by crossed immunoelectrophoresis on day 5. Studies carried out in platelet free plasma stored as PCs showed similar changes in vWF:RCo but increments in vWF:Ag were not detected. These data indicate that PCs maintain vWF:RCo levels of clinical value even after 5 days of storage and suggest that vWF comes out from platelets to plasma during storage.

[Platelet function after allogeneic bone marrow transplantation in Wiskott-Aldrich syndrome]. / Estudio de la función plaquetaria después del trasplante alogénico de medula ósea en el síndrome de Wiskott-Aldrich.

PURPOSE: To assess the platelet characteristics and functionalism in the Wiskott-Aldrich syndrome (WAS) after allogeneic BMT using cyclophosphamide and busulphan for conditioning. MATERIAL AND METHODS: Two WAS patients underwent allogeneic BMT. Platelet aggregation was studied prior to and after BMT, along with the intraplatelet amount of ADP and ATP. RESULTS: Platelet count, size and aggregation wholly recovered after BMT. The post-transplant content of platelet nucleotides was normal. CONCLUSIONS: Platelet function can be totally restored with cyclophosphamide/busulphan conditioned BMT in WAS. Platelet defects in this disease are due to defective thrombopoiesis.