The platelet defect in acute myeloid leukaemia

In a study of platelets from 13 patients with acute myeloid leukaemia abnormal aggregation and release reactions were found. A previously unrecognised quantitative defect of thromboxane B2 production may, at least in part, explain these findings. In contrast to a previous report, we were unable to show a convincing storage pool defect in these platelets. The platelet membrane glycoproteins were largely normal.     

Ultrastructural Findings in Storage Pool Disease and Aspirin-Like Defects of Platelets

Previous studies have shown that abnormalities in collagen-induced platelet aggregation may be due to an impaired release of storage pool ADP, the agent ultimately responsible for platelet aggregation. In some patients and in normal subjects who ingest aspirin, the storage pool of ADP is present in normal amounts, but the mechanism for releasing it appears to be defective (“aspirin-like” defect). In these subjects, the centripetal reorientation of the platelet granules, which may be early structural changes of the release reaction, failed to occur. In one preleukemic patient with an aspirin-like defect, the elements of the open-channel system were also increased. In a second group of patients the impairment of aggregation is due to a deficiency of storage pool ADP. In these patients with storage pool disease, the initial ultrastructural changes associated with the collagen-induced release reaction were normal. The most striking abnormality was a marked decrease in the number of platelet dense bodies. Since the platelets of these patients are deficient in both serotonin and the storage pool of adenine nucleotides, the findings suggest that, in human platelets, these substances are normally stored in the dense bodies. A defect in the formation or function of the dense bodies may account for the abnormalities of platelet aggregation in storage pool disease.     

Platelet function in beta-thalassaemia major.

Abnormal platelet aggregation was found in eight (44%) of 18 patients with beta-thalassaemia major and transfusional iron overload. The aggregation defect bore no correlation with the degree of hepatic fibrosis, liver function tests, whether or not splenectomy had been performed, the degree of iron overload, haematocrit, platelet count, serum vitamin E level, or leucocyte ascorbate concentration. Only three of the 18 patients showed prolonged bleeding times as well as abnormal platelet aggregation, and only one of these suffered clinically significant haemorrhage. The results show that a proportion of patients with beta-thalassaemia major have abnormal platelet function. It is possible, however, that the in vitro abnormality might be due partly to artefacts induced by manipulations required to remove the abnormal thalassaemic red cells, and this may explain the much lower incidence of significant haemorrhage.     

Effects of Normal and Aspirin Platelets on Defective Secondary Aggregation in the Hermansky-Pudlak Syndrome: A Test for Storage Pool Deficient Platelets

Platelets from patients with the Hermansky-Pudlak syndrome (albinism, accumulation of ceroid-like pigment in bone marrow macrophages and mild hemorrhagic symptoms) and aspirin-treated normal platelets fail to develop irreversible secondary waves of aggregation when exposed to aggregating agents on the platelet aggregometer. The present study demonstrates that equal volumes of Hermansky-Pudlak and aspirin-treated platelets, when mixed together, respond in a biphasic manner comparable to normal cells. Correction of the release defect of aspirin platelets by storage pool deficient Hermansky-Pudlak cells indicates that different mechanisms are involved in the failure of the release reaction in the two cell types. The simple test may serve to distinguish other patients with storage pool deficient platelets from individuals who have taken aspirin or other drugs with an aspirin-like effect on platelet function.     

A clinical and experimental study of platelet function in chronic renal failure

Coagulation and platelet function studies were performed on 24 normal subjects and 29 patients with chronic renal failure due to various causes. Thrombocytopenia was uncommon in the uraemic patients but there was reduced platelet retention in glass bead columns and platelet aggregation with adenosine diphosphate (ADP) and thrombin was slower and less complete than normal. The rate of platelet disaggregation in uraemic patients was significantly reduced. The abnormalities tended to be more severe in more uraemic subjects. In normal subjects no inter-relationships were observed between the various measurements of platelet activity. In patients there were significant interrelationships between the measurements of platelet aggregation with ADP and thrombin and between the measurements of aggregation and retention in glass bead columns. It is suggested that if a common pathway is involved in these reactions it is adversely affected in uraemia.Plasma coagulation defects were uncommon and present in only five of the uraemic subjects. Impaired prothrombin consumption apparently due to defective platelet function was present in half the patients but was not detected by a kaolin activation method. Although platelet coagulation function was activated during ADP aggregation and disaggregation in normal and uraemic subjects, it did not correlate in the latter with impairment of aggregation. It is suggested that aggregation and activation of platelet coagulant activity are not necessarily related aspects of platelet function. An effect of uraemic plasma on normal platelets was demonstrated by mixing experiments consistent with a humoral cause for the uraemic platelet defects.     

Evidence that abnormal platelet functions in human Chédiak-Higashi syndrome are the result of a lack of dense bodies

The structure and functions of platelets from three patients with the Chédiak-Higashi syndrome were examined. Electron-microscopic observations revealed a large reduction in the number of serotonin-storage granules or dense bodies but otherwise normal ultrastructure and normal amounts of alpha-granules and catalase-positive granules. The number of mepacrine-labeled granules was also reduced. Platelets contained normal amounts of beta-thromboglobulin and Platelet Factor 4. The platelet release reaction studied with thrombin as the inducer was impaired. The serotonin uptake by the patients' platelets was low and not inhibited by reserpine, and its metabolism was increased. These findings clearly show that platelets from human Chédiak-Higashi syndrome are deficient in the storage pool of dense granule substances and suggest that this granule defect has an influence on the release mechanism of other granule constituents.     

Megakaryocytopoiesis in mice with platelet storage pool deficiency

Megakaryocytopoiesis was evaluated in beige and pallid mice, two strains with platelet storage pool deficiency (SPD). The purpose was to determine if the known deficiency of platelet dense body contents would be associated with evidence for abnormalities of the production or regulation of megakaryocytes. Platelet number and size were normal. Megakaryocyte number and ploidy were normal. Mature megakaryocytes in beige mice were slightly larger than those of control C57BI/6J mice. Megakaryocyte size was normal in pallid mice. No other haemopoietic abnormality was detected in either strain. These findings indicated that there is a mild abnormality of megakaryocyte growth in beige mice. However, this could not be attributed solely to the SPD as it was not present in pallid mice.     

Analysis of platelet shape change, inositol metabolism, and Ca mobilization in patients with platelet dysfunction]

Agonists-induced platelet shape change, inositol metabolism, and Ca mobilization were investigated in patients with various platelet dysfunctions. The platelet shape change determined by our method revealed that arachidonate-induced platelet shape change was completely defective in patients with cyclo-oxygenase (CO) deficiency (A). STA2-induced platelet shape change was also defective in one of five patients with impaired aggregation to STA2 (B). Thrombin-induced platelet shape change was weak in patients with Bernard-Soulier syndrome. In patient with Hermansky-Pudlak syndrome, the platelets did not respond normally to STA2, arachidonate or PMA. These findings suggested that the determinations of platelet shape change by our method was useful in diagnosing platelet dysfunctions. Inositol metabolism and Ca mobilization in response to thrombin, STA2, or NaF were also investigated in patient A,B, and impaired aggregation to A23187 in patient C. The responses were normal in patient A, suggested that CO activity did not affect them. Inositol metabolism was also normal in patient C, although Ca mobilization in response to A23187 was delayed, and that in response to thrombin was defective in the absence of extracellular Ca2+. This suggests that the patient's platelets have a defective IP3-induced Ca mobilization pathway. STA2 selectively failed to induce IP3 formation and Ca mobilization in patient B, although 3H-labelled thromboxane ligand (3H-U46619) bound to the patient's platelets, normally. These findings suggested that the patient's platelets have a defect in postreceptor signal transduction, especially thromboxane receptor-mediated phospholipase C activation pathway.     

Antagonists of PAF-acether do not suppress thrombin-induced aggregation of ADP-deprived and aspirin-treated human platelets

Four chemically distinct PAF-acether antagonists were used to test the hypothesis that the cyclooxygenase and ADP-independent thrombin-induced aggregation of human platelets is due to PAF-acether. The compouds 48740 RP, CV-3988, BN 52021 and Ro 19-3704 inhibited aggregation by PAF-acether whereas 48740 RP also interfered with aggregation by arachidonic acid, U 46619, collagen and thrombin. Aspirin-treated platelets aggregated in response to PAF-acether and to 0.25 U/ml thrombin as much as control platelets in absence of detectable thromboxane A₂, and were less responsive to 0.05–0.1 U/ml. Thrombin-induced aggregation of aspirin-treated platelets was unaffected by the PAF-acether antagnists BN 52021, CV-3988 and Ro 19-3704. In separate experiments, platelets were exposed for five min to convulxin, a glycoprotein extracted from a snake venom which depletes granular ADP and ATP. A combination of PGI₂, aspirin and anticrotalid serum used to disaggregate allowed the recovery of approximately 80% free platelets, which failed to respond to PAF-acether but still aggregated in presence of thrombin. This residual ADP and cyclooxygenase-independent aggregation is not accountable for by the platelet formation of PAF-acether, since it was not modified by the latters' antagonists nor by platelet exposure to convulxin. Our results do not support the proposal that PAF-acether mediates a third pathway of human platelet aggregation.     

Change in activity of factor XIII in the plasma during platelet aggregation

During platelet aggregation induced by ADP or thrombin, a reduction in the activity of factor XIII is observed in platelet-enriched rat plasma. On the addition of active factor XIII (XIIIa) to this plasma, besides the increased ADP-induced aggregation, activity of factor III in the plasma is reduced. In the case of thrombin-induced platelet aggregation, addition of factor XIIIa is accompanied by a marked decrease in its activity in the plasma. The degree of aggregation under these circumstances is lower than in control samples. The observed differences in the character of aggregation taking place in the presence of factor XIIIa, when different aggregants (ADP and thrombin) are used, are evidently due to interaction between active factor XIII and thrombin added to the plasma as the aggregant.     

Platelet function and structure in myeloproliferative disease, myelodysplastic syndrome, and secondary thrombocytosis

Platelet function and morphologic characteristics were evaluated in 43 patients with myeloproliferative disease (MPD), 5 patients with myelodysplastic syndrome (MDS), and 7 patients with secondary thrombocytosis (ST). Platelet Factor IV (PF4) and B-thromboglobulin (BTG) showed slight elevation in ST but significant elevation in all MPDs. They were either normal or slightly elevated in MDS. Defective platelet aggregation with one or more inducers was seen in 62% of all patients. An epinephrine-induced defect was the most consistent aggregation abnormality. Hyperaggregation and spontaneous aggregation were seen in 15% of patients. Of the eight patients who showed increased bleeding tendency, all eight showed defective aggregation with two or more inducers, five showed decreased surface activation response, as well as decreased or abnormal granules and dense tubular disarray in the transmission electron microscope (TEM) study. Seven patients had clinical evidence of recurrent arterial and venous thromboses. Five of these patients showed hyperaggregation response to adenosine diphosphate and collagen and abnormal Wu and Hoak platelet aggregate ratio. Four patients showed spontaneous aggregation on aggregometer. Surface activation response was significantly increased in five patients, and an increase in platelet granules by TEM study was seen in four patients. Primary thrombocythemia could be differentiated from secondary thrombocytosis (ST) by the presence of abnormal aggregation response and significantly increased PF4 and BTG in the former, and greatly elevated plasma fibrinogen and Factor VIII, as part of acute phase reactant response, in the latter.     

Platelet dysfunction induced by parenteral carbenicillin and ticarcillin. Studies of the dose-response relationship and mechanism of action in dogs

Sequential studies of platelet function were performed in dogs receiving continuous intravenous carbenicillin (CARB) or ticarcillin (TIC). Dose- and time-dependent platelet dysfunction was uniformly observed during the administration of CARB or TIC, 250 to 1000 mg/kg/24 hr. ADP-induced primary and secondary platelet aggregation was markedly inhibited within 24 to 48 hours in dogs receiving 750 or 1000 mg/kg/24 hr, but maximum impairment of aggregation did not occur until 3 to 5 days in dogs receiving 250 or 500 mg/kg/24 hr. Platelet glass bead column retention was abnormal in all dogs studied, and platelet factor 3 availability was impaired in 91%. Collagen-induced platelet aggregation was consistently impaired and the bleeding time was prolonged only during the infusion of greater than or equal to 750 mg/kg/24 hr. Plasma fibrinogen concentrations and thrombin times remained normal. CARB and TIC infusions resulted in inhibition of 14C-serotonin release and slightly decreased platelet ADP, while serotonin, ATP, and ultrastructure remained unchanged. The mutual correction of abnormal platelet aggregation by mixing CARB or TIC platelets with aspirin-treated platelets suggested that CARB and TIC inhibited the platelet release reaction by a mechanism other than inhibition of platelet cyclo-oxygenase. The platelet inhibitory properties of CARB and TIC demonstrated in this study suggest that they may be useful antithrombotic agents.     

Hemostatic evaluation in bleeding disorders from native blood. Clinical experience with the hemostatometer

Primary hemostasis (PH), i.e., hemostatic platelet plug formation, and the subsequent coagulation were recorded and quantified from the same nonanticoagulated venous blood sample with the use of the Haemostatometer. In addition, platelet thrombus formation induced by interaction of flowing native blood with a collagen fiber under low shear rates (450 s-1) was simultaneously analyzed by this device. The effect of monoclonal antibodies (MoAbs) directed against von Willebrand's factor antigen (vWF:Ag), platelet glycoprotein Ib (GPIb) and the GPIIb/IIIa complex, and fibrinogen were studied. PH was significantly inhibited by MoAbs against vWF:Ag, GPIIb/IIIa, and fibrinogen but was unaffected by antibody against GPIb. Collagen-induced thrombosis was prevented by MoAbs against vWF:Ag and GPIb, slightly inhibited by antifibrinogen, and unaffected by blockage of platelet membrane GPIIb/IIIa. The effect of a single 600-mg dose of aspirin was monitored, and abnormal PH was still detectable five days later. From the 13 hemophiliacs tested, 7 showed significantly prolonged PH. In von Willebrand's disease, a characteristic defect of PH with significant inhibition or absence of collagen-platelet interaction was observed in all the 11 patients. PH was greatly prolonged in both of the two patients with storage pool deficiency. The technique detected improvement of platelet function, i.e., PH in all of six patients with bleeding disorders after replacement therapy or DDAVP infusion. The authors conclude that the Haemostatometer technique is a sensitive test for determining platelet dysfunction and monitoring efficacy of factor-replacement or DDAVP therapy.     

血管内皮细胞与血小板聚集、释放和超微结构变化的相互关系研究

实验用培养的牛主动脉内皮细胞(EC)在体外条件下与采自兔血的富含血小板血浆(PRP)孵育,观察EC对血小板聚集的影响,用生物发光法测定血小板聚集时ATP的释放量,并观察这一反应过程中血小板的超微结构变化。结果表明血小板聚集强度与加入的EC量呈负相关性,且血小板ATP释放量和形态学变化与聚集程度相符合,即随血小板聚集受抑,其变形减轻,颗粒物质及ATP释放减少。形态观察中发现血小板对EC的粘附及颗粒释放现象,即使在血小板聚集完全为EC抑制时仍可见这一现象,其机理尚不明了。     

Mechanisms of platelet aggregation disturbances and their relation to treatment in patients with chronic uremia

We have examined platelet aggregation in patients with chronic uremia using ADP, thrombin and calcium ionophore A23187 as inducers. The study was performed on patients treated conservatively, by hemodialysis and peritoneal dialysis. Platelet aggregation was most significantly depressed in patients treated conservatively and by hemodialysis. Different mechanisms are responsible for platelet dysfunction.     

Inherited heterogenous defect in platelet aggregation selectively with ADP and epinephrine--a series of 25 cases

Inherited heterogeneous defects of platelet function caused by impairment of platelet responses to weak agonists as ADP, epinephrine and others as low concentration collagen and platelet activating factor (PAF) have been described, though quite rarely. We describe here 25 cases of this defect with impairment in response to ADP and epinephrine. Subjects with a history of generalized bleeding and a prolonged bleeding time, PF3 availability or prothrombin consumption index and a normal platelet count, prothrombin time, activated partial thromboplastin time and clot solubility were subjected to platelet aggregation. Those of these which showed a normal aggregation with collagen and arachidonic acid and an absent or reduced aggregation with ADP and epinephrine were included in our study group. Subjects with history or findings suggestive of antiplatelet drug intake or any acquired condition affecting platelet functions were excluded from this study. 76% of the patients had onset of recurrent bleeding manifestations since childhood with a mean age at onset of 9.2 years. A positive family history was present in 36% of the patients. Majority of the patients (88%) presented with mild bleeding manifestations, the commonest symptom being appearance of recurrent ecchymotic spots. We present here a series of patients with a hereditary platelet aggregation defect selectively with ADP and epinephrine.     

Cellular immune responses in aspergillosis

Diminished platelet aggregation responses to one or more aggregating agents were found in 25 of 32 patients with nasal allergy studied at the peak of the allergy season. Abnormal in-season platelet aggregation induced by epinephrine and collagen was significantly improved when repeated out-of-season, while incomplete platelet aggregation induced by adenosine diphosphate (ADP) and thrombin was unchanged. Recombination of an in-season serum factor with autologous, out-of-season normally aggregating platelets caused marked inhibition of platelet aggregation. Mean bleeding times of 20 symptomatic patients were also prolonged during the height of the pollination season. These data suggest that the allergic diathesis is a model for the study of cyclic, nondrug induction of platelet dysfunction.     

The influence of aspirin and indomethacin on the platelet contractile wave.

Recent evidence suggests that a short-lived product of prostaglandin biosynthesis produced on the addition of arachidonic acid to platelet microsomes is involved in physiologic platelet aggregation and mediates the release reaction by producing the platelet contractile wave. An important corollary of this hypothesis is that aspirin and indomethacin, inhibitors of prostaglandin synthesis, should block the platelet contractile process produced by agents which can initiate platelet aggregation. The present study tested this hypothesis and found that aspirin and indomethacin were, indeed, potent inhibitors of the platelet contractile wave stimulated by collagen and epinephrine, but largely failed to inhibit internal transformation induced by thrombin and ADP. The findings confirm the hypothesis that a prostaglandin produced endogenously by platelets initiates platelet contraction and suggests that ADP and thrombin have the ability to stimulate the platelet contractile apparatus by an alternate mechanism not dependent on prostaglandin synthesis.     

Reactivity of neoplastic cells of hairy cell leukemia with antisera to S-100 protein

Rabbit antibodies to bovine S-100 protein were tested by immunoperoxidase technics against fresh hairy cell leukemia (HCL) cells obtained from nine patients (peripheral blood in six and spleen in three), as well as lymphoblastoid cell lines derived from three patients with HCL. Peripheral mononuclear cells from three normal persons and two patients with chronic lymphocytic leukemia (CLL) and cells from two melanoma lines were used as controls. The melanoma cell lines, cell lines derived from patients with HCL, and fresh HCL cells displayed cytoplasmic and nuclear positivity after exposure to anti-S-100 protein sera. By contrast, normal peripheral blood lymphocytes and CLL cells were negative for S-100 protein. Additional studies were performed by immunoperoxidase technics on representative sections of formalin-fixed splenic tissues from eight patients who had splenectomies. The cause of splenectomy was HCL in three, traumatic rupture in one, CLL in one, Hodgkin's disease in one, and hypersplenism in two patients. Sections from all three HCL patients showed moderate to marked positivity with antisera to S-100 protein. These results strongly suggest the presence of S-100 protein in HCL cells.     

Platelet aggregation in chronic idiopathic thrombocytopenic purpura.

A method for washing platelets by albumin density gradient separation has been modified to prepare platelet rich plasma of thrombocytopenic patients for platelet aggregation studies. The concentration procedure, consisting of centrifuging platelets into a specific gravity gradient between plasma and 40-45% aqueous solution of bovine albumin, does not affect platelet aggregation adversely. Platelet aggregation in eight patients with chronic idiopathic thrombocytopenic purpura was determined by this method. On the basis of the results the patients could clearly be divided into two groups: four patients with normal aggregation and four with a qualitative platelet defect. In contrast to the other patients, the group with an in vitro platelet functional defect all had more prolonged bleeding times and the presence of a serum antiplatelet antibody.