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.

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.

Lactate dehydrogenase isoenzymes in patients with essential thrombocythemia.

Various abnormalities have been described in platelets of patients with Essential Thrombocythemia (ET), including alteration in the glycolytic pathway and increased lactate production. Lactate dehydrogenase (LDH) isoenzymes were studied in 15 of these patients. Relative activity of LDH-5 isoenzyme was higher comparing with normal platelets (p < 0.05), while LDH-3 activity was diminished (p < 0.05). LDH-1, LDH-2 and LDH-4 isoenzymes were unchanged. We also found an altered profile of LDH isoenzymes in serum. These findings can contribute to improve the knowledge of platelet defects in ET and be useful in the diagnosis of these patients.

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.

[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.

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.

[Studies of aggregation using pairs of agonists in platelet concentrates stored for 5 days. Influence of autologous fresh plasma]. / Estudios de agregación con pares de agonistas en concentrados plaquetarios almacenados durante 5 días. Influencia del plasma fresco autólogo.

Aggregation was studied with synergistic agents on platelets stored for 5 days. Fixed concentrations of agonist agents, capable of inducing maximum response upon platelet concentrate (PC) preparations were used, and the results were compared with those achieved from samples previously incubated in fresh autologous plasma. Platelet aggregation on day 0 ranged between 77 +/- 3% with the combination epinephrine/ADP and 72 +/- 7% with collagen/ADP, these figures decreasing by the fifth day to 14 +/- 5% and 10 +/- 4%, respectively. The other paired aggregation agents were epinephrine/collagen and arachidonic acid (AA)/ADP; with these agents, similar values were attained on day 0, along with an analogous derangement of the response by the fifth day. With the last combination, the aggregation value attained on days 3 was 14 +/- 10% in experiments with the PC plasma, as opposed to 49 +/- 15% in assays with fresh plasma (p less than 0.001). Such difference persisted on day 5 and was thought to be due to competence of the high concentration of fatty acids generated in the PC plasma by AA. No differences induced by fresh plasma on the other agonist combinations were detected. These results suggest that some derangement of platelet function, conditioned by nonreversible alterations, and measured by paired agonist aggregation, may derive from storage.

Arachidonic acid metabolism in platelets stored for 5 days.

(C-14)-Arachidonic acid [(C-14)-AA] metabolism was studied in platelet concentrates (PCs) stored for 5 d. There was a gradual decrease in uptake of radioactivity from day 0 to 3 (P less than 0.01). On day 0, distribution of radioactivity in platelet phospholipids (PLs), and formation of phosphatidic acid, HETE and cyclooxygenase products, when platelets were exposed to thrombin (5 U/ml), were similar to that reported for fresh platelets. On day 3 there was a change in the distribution of (C-14)-AA in platelet PLs which consisted of an increase in the percentage of radioactivity bound to phosphatidylserine, from 5.3 +/- 0.9% on day 0 to 8.8 +/- 1.5% on day 3 (P less than 0.001), and a decrease in (C-14)-AA in phosphatidylinositol (PI), from 12.4 +/- 1.5% on day 0, to 7.9 +/- 0.9% on day 3 (P less than 0.001). Phosphatidic acid generated by thrombin-stimulated platelets on day 0, comprised 2.6 +/- 0.5% of total radioactivity, but dropped to 1.4 +/- 0.3% on day 3 (P less than 0.001), and 0.9 +/- 0.2% on day 5 (P less than 0.01). These values showed a good correlation with the percentage of (C-14)-AA released from PI on the same days (r = 0.9). On day 0, 13.4 +/- 4.4% of platelet radioactivity was released from phosphatidylcholine by thrombin, but this amount was reduced to 6.8 +/- 3.4% on day 5 (P less than 0.05). Generation of radioactive 12-hydroxy-5,8,10,14-eicosatetraynoic acid (HETE) also dropped from 7.2 +/- 2.9 on day 0, to 2.1 +/- 1 on day 5 (P less than 0.01). We could not detect changes in cyclooxygenase metabolites. In conclusion, we suggest that various enzymatic pathways implicated in AA metabolism by platelets are impaired by storage.

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.

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.

[Measurement of phospholipids in erythrocytes, plasma and platelets in a patient with hereditary spherocytosis]. / Determinación de fosfolípidos en hematíes, plasma y plaquetas en una enferma con esferocitosis hereditaria (EH).

The amount and distribution of phospholipids (PLs) were studied in erythrocytes, plasma and platelets of one patient with hereditary spherocytosis (HS). Phosphatidylethanolamine (PE) was always decreased. Total PLs amount was remarkably reduced in erythrocytes but normal in platelets. Since platelets do not contain those proteins which have been implicated in the red cell abnormalities of patients with HS, our findings reinforce the theory that a defective protein-lipid interaction causes the abnormal lipid loss in the erythrocytes of these patients.

Phospholipid determination in platelets, plasma and red cells of patients with chronic myeloproliferative disorders.

Red cell phospholipids (PLs) were assessed in 11 patients with essential thrombocythemia and 5 patients with polycythemia vera. Platelet and plasma PLs were also determined in 10 of these patients, and the results were compared with studies performed in 16 healthy volunteers. The amount of platelet PLs in patients was similar to controls (556 +/- 90 mmol/10(9) cells, versus 481 +/- 91 mmol/10(9) cells), as was the percentage of the main specimens of these compounds, including phosphatidylserine (11.1 +/- 0.8%), which is relevant for platelet procoagulant activity. We did not find differences between red cell PLs of patients (300 +/- 60 nmol/10(9) cells), versus controls (289 +/- 71 nmol/10(9) cells), and the sphingomyelin/phosphatidylcholine ratio in these cells was the same in both groups (0.75 +/- 0.1). Finally, we did not detect any alteration in the amount of plasma PLs specimens.

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.

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.

[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.

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.