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10 Myeloproliferative and metabolic causes
Polycythaemia vera (PV) was first described in 1892 by Vazquez [1] in a case report describing a patient with ruddy cyanosis, splenomegaly, and an increased red cell count not associated with a congenital form of heart disease. A decade later in 1903 it was defined more clearly by Osler [2], and the disease then became known as Vasquez–Osler disease until this eponymous term was superseded by PV. PV together with essential thrombocythaemia (ET) and primary myelofibrosis (PMF) (Chap. 12) represent an overlapping spectrum of clonal haematological disorders called the human myeloproliferative neoplasms (MPN). The MPNs were first grouped together, along with chronic myeloid leukaemia (CML), by Dameshek in his seminal paper of 1951 [3]. CML is now generally considered as a distinct entity, but shares several features with the other MPNs. All of these disorders result from acquired genetic changes in the haematopoietic stem cell compartment and are characterized by proliferation of various cells of the myeloid lineages. They also all share the propensity to develop into acute myeloid leukaemia (AML), albeit with varying incidence, and, as will be described next, the majority demonstrate abnormalities of intracellular signalling. Taking all of these characteristics together, the MPN and CML therefore provide in vivo model systems to study the multistep development of AML. In addition, as MPNs are associated with full terminal differentiation of myeloid lineages, they allow the study of the effects of oncogenic mutations on normal myeloid homeostasis before this is complicated by cooperating mutations which block differentiation.
Background: The symptoms primarily described in Morbus Moschcowitz included thrombocytopenic purpura and hemolytic anemia. In addition it presents with a variety of clinical manifestations depending on the organs involved (i. e. neurological, renal, gastrointestinal, cardiac involvement). It is a rare disease and the pathogenesis still remains unclear. The efficacy of derived therapeutical concepts can hardly be assessed in controlled trials. Conclusion: Currently the main option seems to be plasma therapy. In nonresponders surgical procedures (splenectomy) may be of benefit to the patient.
Zusammenfassung
□ Hintergrund Die Symptome des Morbus Moschcowitz waren bei Erstbeschreibung charakterisiert durch eine thrombozytopenische Purpura und
hämolytische Anämie. Daneben umfaßt der Morbus Moschcowitz ein klinisch buntes Bild von Organmanifestationen (neurologische,
renale, aber auch gastrointestinale oder kardiale Beteiligung). Auch aufgrund der Seltenheit der Erkrankung sind die Überlegungen
zur Pathogenese uneinheitlich. Die abgeleiteten Therapieversuche sind kaum durch kontrollierte Studien zu untersuchen, und
ihre Bewertung bleibt schwierig.
□ Schlußfolgerung Neben vielen weiteren Therapieansätzen scheint derzeit die Plasmatherapie (Gabe von Fresh-frozen-Plasma oder Austauschtherapie)
die wichtigste konservative Behandlungsmöglichkeit. In therapierefraktären Situationen kann die chirurgische Option einer
Splenektomie hilfreich sein.
La question du « risque génétiqueremonte au début des années 1970, alors que la biologie moléculaire donnait naissance aux biotechnologies modernes. Qu'est-elle devenue depuis, et comment le public a-t-il ét'e amené à donner son avis?
Thromboembolic complications are common in patients with malignant disease. We studied the activation of coagulation in 106 patients with solid tumours and 72 healthy volunteers by measuring plasma levels of tissue factor, factor VIIa, factor XIIa, thrombin-antithrombin complex, and prothrombin fragments 1 + 2. Tissue factor was 67% higher in cancer patients (median 582 vs 349 pg/mL, p = 0.0006) and factor VIIa was 46% higher (100 vs 69 mU/mL, p = 0.0002), indicating extrinsic pathway activation. Modest activation of the intrinsic pathway (elevated factor XIIa) was seen only in patients with advanced disease or those receiving chemotherapy. Excess thrombin generation was manifested by elevations in thrombin-antithrombin complex and prothrombin fragments 1 + 2. Tissue factor pathway is clearly implicated in the hypercoagulable state of cancer.
Background:
The symptoms primarily described in Morbus Moschcowitz included thrombocytopenic purpura and hemolytic anemia. In addition it presents with a variety of clinical manifestations depending on the organs involved (i.e. neurological, renal, gastrointestinal, cardiac involvement). It is a rare disease and the pathogenesis still remains unclear. The efficacy of derived therapeutical concepts can hardly be assessed in controlled trials.
Conclusion:
Currently the main option seems to be plasma therapy. In non-responders surgical procedures (splenectomy) may be of benefit to the patient.
Severe pulmonary embolism with cardiac arrest occurred half an hour after the 5th spontaneous delivery in a 34-year-old multipara. Standard resuscitation for 30 minutes remained ineffective. Only after Actilyse rt-PA infusion the patient's state improved. She regained consciousness on the third day. After 32 days of hospital treatment the patient was discharged in a generally good condition. Today, 5 years after the described event her state of health is very good.
The management of pregnant patients with chronic myeloproliferative disorders (MPD) is a difficult problem. Patients with essential thrombocythaemia (ET), and, less frequently, those with chronic myeloid leukaemia (CML) or polycythaemia vera (PV), present at a childbearing age. Pregnancy itself does not appear to affect adversely the natural course and prognosis of the MPD. However, fertility might be reduced, and an adverse outcome of pregnancy due to thrombotic or bleeding complications is a matter of concern. In ET, first-trimester abortion is the most frequent complication but increased perinatal mortality and premature delivery are also observed. Placental infarction due to thrombosis seems to be the most consistent event, Maternal thrombotic or haemorrhagic complications are rare but are more common than seen in normal pregnancy. The outcome of pregnancy seems to be positively influenced by aspirin, at least in some cases. The value of cytoreduction and/or heparin prophylaxis has not been established but may have a role in selected cases. In CML, the potential adverse effects of hyperleukocytosis, and sometimes thrombocytosis, generally make myelosuppressive treatment essential. In PV, the number of reported pregnancies is low. Maintaining the PCV below 0.45 is of the utmost importance relating to the outcome of pregnancy. Although cytoreductive drugs should generally be avoided, if possible, until at least after the first trimester of pregnancy, interferon-alpha seems to be the drug of choice when myelosuppression is indicated.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane- binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b- 9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9- treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.
Important complications in sickle cell anemia occur secondary to vascular occlusion, which is postulated to be initiated by interactions of erythrocytes with vascular endothelial cells. In patients with sickle cell anemia, up to 25% of reticulocytes express the alpha 4 beta 1-integrin complex. Furthermore, erythrocytes from patients with sickle cell anemia bind to endothelial cells activated by tumor necrosis factor alpha via (TNF alpha) via interactions between erythrocyte alpha 4 beta 1 and endothelial cell vascular cell adhesion molecule-1 (VCAM- 1). Thus, binding of alpha 4 beta 1-expressing reticulocytes to cytokine-activated endothelial cells may initiate vascular complications in sickle cell anemia and perhaps other hemolytic anemias during episodes of infection and inflammation.
To determine the role of von Willebrand factor (vWF) in adhesion of sickle (SS) erythrocytes in microvascular flow conditions, we have perfused the ex vivo mesocecum vasculature of the rat with desmopressin, an analogue of vasopressin that causes the release of endothelial vWF. Analysis of vWF in the venous effluent of the isolated vasculature showed mainly the presence of extra-large molecular weight forms characteristic of endothelial vWF, which in the presence of desmopressin showed an average increase of 54%. Also, desmopressin induced a significant increase in adhesion of washed oxygenated (oxy) unseparated SS erythrocytes, accompanied by a persistent microvascular obstruction and a pronounced increase in the peripheral resistance (PRU). In contrast, infusion of SS deformable discocytes (SS2) in desmopressin-perfused vasculature resulted in a significant adhesion but not in persistent vasoocclusion, showing that SS2 discocytes alone are not sufficient for microvascular obstruction. Furthermore, SS4 erythrocytes (dense discocytes and irreversibly sickled erythrocytes) caused a persistent microvascular blockage and a significantly higher PRU than SS2 discocytes. However, the increase in PRU for SS4 erythrocytes following desmopressin treatment was 50% less compared with a corresponding increase for SS2 discocytes over the control values, which showed a smaller effect of desmopressin on the hemodynamic behavior of SS4 dense erythrocytes. Incubation of desmopressin-treated vasculature with anti-vWF antibodies resulted in a pronounced decrease in adhesion and significantly improved hemodynamic behavior of SS cells. Also, in untreated vasculature, similarly incubated with anti-vWF antibodies, there was almost complete inhibition of adhesion. Under the described perfusion conditions, antibodies to fibronectin and thrombospondin, as well as incubation of SS erythrocytes with anti-vWF antibodies did not affect adhesion. These results are compatible with a model for SS vasoocclusion in which extra- large vWF-mediated adhesion of deformable SS erythrocytes is the first step followed by an accelerated entrapment of dense SS erythrocytes.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.
Increased frequency of thromboembolic events has been recently observed in patients with beta-thalassemia major (TM). Platelet function anomalies including impaired aggregation, increased circulating aggregates, and our finding of shortened platelet survival indicate that platelets may be involved in the hypercoagulability in thalassemia. Consequently, we used a technique based on thin layer chromatography purification and enzyme immunoassay to measure urinary metabolites of thromboxane A2 (TXA2) and prostacyclin (PGI2) in nine splenectomized patients with beta-TM regularly transfused, five non- splenectomized patients with beta-thalassemia intermedia (TI), and 20 healthy individuals. A significant 4- to 10-fold increase was observed in the urinary excretion of 2,3-dinor-TXB2, 11-dehydro-TXB2 and 2,3- dinor-6-keto-PGF1 alpha in patients with TM and TI as compared with healthy controls. No significant differences were found in the concentrations of these metabolites between TM and TI patients. Six TM patients received a very low dose of aspirin (20 mg/day) for 7 days. A significant decrease was observed in the urinary concentrations of 2,3- dinor-TXB2 and 11-dehydro-TXB2 derived from platelets. However, the levels of urinary 2,3-dinor-6-keto-PGF1 alpha reflecting vascular production and TXB2 and 6-keto-PGF1 alpha originating from the kidney were not significantly changed. These results are consistent with those of increased in vivo production of TXA2 because of endogenous platelet activation.
Elevated blood levels of homocysteine are associated with atherosclerosis and thrombotic disease. We previously reported that treatment of cultured endothelial cells with homocysteine increased endogenous factor V activity by activation of the cofactor. Because endothelial cell-associated factor Va would be regulated by the protein C mechanism, the ability of homocysteine-treated arterial and venous endothelial cells to activate protein C was investigated. Both arterial and venous endothelial cells activated protein C; 0.6 mmol/L homocysteine reduced endothelial cell protein C activation by 12%. Maximal inhibition (90%) of protein C activation occurred with 7.5 to 10 mmol/L homocysteine after 6 to 9 hours of incubation. Metabolism of homocysteine was not accelerated by cultured endothelial cells. Investigation of the mechanism(s) by which homocysteine reduced protein C activation indicated that the metabolite did not induce an inhibitor to activated protein C, but in low concentrations acted as a competitive inhibitor to thrombin. These data suggest that perturbation of the vascular endothelial cell protein C mechanism by homocysteine may contribute to the thrombotic tendency seen in patients with elevated blood levels of this metabolite.
We have previously reported the purification of a 37-kd platelet- agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been shown to be inhibited by IgG from normal human adults and the same TTP patient after recovery. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG from normal adults and the same TTP patient after recovery to adsorbed PAP by using an enzyme-linked immunosorbent assay. The binding was specific, concentration dependent, and saturable. IgG purified from a 5-month-old baby and the same TTP patient during active disease did not form complex with PAP p37. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP p37 but not 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP p37 and IgG also revealed the fluid-phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The IgG that was bound to PAP p37 conjugated to Sepharose 4B inhibited the agglutination of washed platelets induced by TTP plasma containing PAP p37, whereas the IgG that was not bound to PAP p37 did not have a significant inhibitory effect. The complex formation between PAP p37 and specific IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and, at least partly, the in vivo successful treatment with specific IgG-containing normal plasma.
Microvesicles (diameter ca 200 nm) from the cell-free supernatant of U87MG human glioblastoma cell caused platelet aggregation and coagulation in a manner identical with that previously shown for the intact cells. Both activities were inhibited by dansylarginine -N-(3- ethyl-1,5-pentanediyl) amide (DAPA), confirming the thrombin-dependent nature of both activities. The specific activities per microgram of protein were 2–10 times greater in the microvesicles than in the plasma membrane fraction, suggesting localization in specific membrane domains. Sucrose density centrifugation gave a single protein peak (density 1.14) with congruent procoagulant and platelet aggregating activities. Both activities required the extrinsic pathway, as shown by studies with factor-deficient plasmas, and both were inhibited by heating (60 min/100 degrees C), by reduction and alkylation, and by incubation of the microvesicles with rabbit anti-bovine brain tissue factor antibody. These observations were confirmed using microvesicles from the HL-60 human promyelocytic leukemia cells, which are known to contain tissue factor activity. The results suggest that both procoagulant and proaggregating activities are causally related through the presence of tissue factor in the microvesicles. Studies with the Baumgartner perfusion apparatus showed that U87MG microvesicles increased the size of adherent thrombi nearly tenfold and that these thrombi were associated with nucleated cells from the blood. The increase in adherent thrombi did not occur if perfusion was carried out in the presence of DAPA, confirming the role of thrombin in their formation.
Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.
Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F1+2 and the protein C activation peptide, which measure the cleavage of the prothrombin molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and fragment B beta 1–42, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F1+2 and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta 1–42 in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that prothrombin activation as measured by the F1+2 assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.
Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.
Initiation of vasocclusion in sickle disease pathophysiology may involve abnormal red blood cell (RBC) adhesivity to endothelium, a phenomenon influenced by both RBC and plasma factors. Using human umbilical vein endothelial cells and a gravity sedimentation adherence assay, we have examined thrombospondin (TSP) as a plasma factor in this adhesive event. The already-abnormal adherence of sickle RBCs in buffer/albumin is significantly augmented (P < .001) by the addition of TSP, with half-maximal effect at about 0.3 microgram/mL. This effect is abolished by antibodies to either TSP or glycoprotein (GP) IV (CD36), as well as peptides RGDS and CSVTCG. The even greater adherence (P < .005) of sickle RBCs in autologous platelet-rich plasma (without added TSP) is dramatically inhibited by alpha CD36 antibodies (OKM5 and alpha GPIV) and significantly diminished by alpha TSP, by peptides RGDS and CSVTCG, and by two antibodies to the vitronectin receptor (7E3 and LM609). Studies of density-separated subpopulations and of RBC adhesion to immobilized proteins, as well as analysis of sickle RBCs using fluorescence-activated cell sorting and single cell microfluorometry, show that TSP responsiveness is a feature of the immature sickle “stress” reticulocytes, which carry CD36 (and not GPIIbIIIa-like receptors) as the TSP-receptive moiety. The endothelial cell's participation in this phenomenon appears to be more complex, and the data are consistent with the notion that it involves TSP interaction with other plasma proteins and/or multiple receptor structures. Other potential adhesogenic proteins (plasma von Willebrand factor, vitronectin, fibrinogen, and fibronectin) neither exhibited an affinity for reticulocytes nor supported increased sickle RBC adherence when added to buffer/albumin in these assay systems. In aggregate, our results indicate that TSP may be the major promoter of RBC adhesivity in plasma, and they suggest that therapeutic benefit might derive from interference with sickle reticulocyte CD36, as achieved by antibodies and CSVTCG in these studies.
Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic vascular occlusion in patients with sickle cell disease (SCD). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes. To test the hypothesis that platelet activation contributes to sickle erythrocyte binding, we investigated whether factors released from activated sickle platelets promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions. Activated sickle platelet supernatant (ASPS) promoted high levels of sickle erythrocyte adherence to MEC (55.4 +/- 3.9 erythrocytes/mm2) but only moderate adherence of normal erythrocytes to MEC (14.1 +/- 0.7 erythrocytes/mm2). When MEC were incubated with an antibody (OKM5) against CD36 (a thrombospondin [TSP] receptor), platelet supernatant mediated sickle erythrocyte adherence was inhibited 86%, suggesting that TSP participated in the adherence. To further define the role of TSP in adherence, additional studies using purified TSP were performed. At a concentration of 0.2 micrograms/mL TSP in serum-free media (SFM), sickle erythrocyte adherence to MEC was 33.9 +/- 2.7 erythrocytes/mm2 and sixfold greater than either sickle erythrocyte adherence in the absence of TSP or normal erythrocyte adherence in the presence of TSP. Doubling the concentration of TSP to 0.4 micrograms/mL proportionally increased adherence of sickle erythrocytes. Incubation of MEC with OKM5 or anti-alpha v monoclonal antibodies inhibited TSP-mediated sickle erythrocyte adherence more than 95%. These data suggest that activated platelet release factors, including alpha-granule TSP, which promote receptor-mediated sickle erythrocyte adherence to microvascular endothelium. Such factors released during in vivo platelet activation could contribute to vaso-occlusive complications by promoting erythrocyte adherence and microvascular occlusion.
Heparin-induced thrombocytopenia can be a serious and difficult-to- diagnose complication of heparin therapy. Serum from patients with heparin-induced thrombocytopenia can cause heparin-dependent platelet aggregation, but the low sensitivity and specificity of this test limit its clinical usefulness. In this report we describe an assay for heparin-induced thrombocytopenia that is both sensitive and specific. The improvement in the assay was accomplished by measuring platelet release instead of aggregation and by measuring platelet release at two heparin concentrations. The rationale for the use of two heparin concentrations was that sera from patients with heparin-induced thrombocytopenia caused release at therapeutic but not at high concentrations of heparin. Twenty-eight sera samples from patients suspected of having heparin-induced thrombocytopenia and 573 controls were coded and tested in the assay. The patients with possible heparin- induced thrombocytopenia were ranked according to the likelihood of having this disorder by using prospectively defined criteria. The test had a high specificity (99%); only one of 573 controls showed a positive result. The test was also very sensitive, and the likelihood of a positive test result was directly correlated with the clinical likelihood of the patient having heparin-induced thrombocytopenia. Six of six patients with definitive heparin-induced thrombocytopenia had positive test results, whereas zero of four patients in whom the diagnosis was unlikely had positive test results. The two-point test for heparin-induced thrombocytopenia represents a sensitive and specific test for this disorder. This test may be useful not only in confirming the diagnosis of this disorder but also may provide information about its pathogenesis.
Human plasma contains an inhibitor of tissue factor-initiated coagulation known as the lipoprotein-associated coagulation inhibitor (LACI) or also known as the extrinsic pathway inhibitor (EPI). A competitive fluorescent immunoassay was developed to measure the plasma concentration of LACI in samples from normal individuals and patients with a variety of diseases. The LACI concentration in an adult control population varied from 60% to 160% of the mean with a mean value corresponding to 89 ng/mL or 2.25 nmol/L. Plasma LACI levels were not decreased in patients with severe chronic hepatic failure, warfarin therapy, primary pulmonary hypertension, thrombosis, or the lupus anticoagulant. Plasma LACI antigen was decreased in some, but not all patients with gram-negative bacteremia and evidence for disseminated intravascular coagulation. Plasma LACI levels were elevated in women undergoing the early stages of labor (29%), in patients receiving intravenous tissue-type plasminogen activator (45%), and in patients receiving intravenous heparin (375%). A radioligand blot of the pre- and post-heparin plasma samples shows the increase to be in a 40-Kd form of LACI. Very low levels of plasma LACI antigen were found in patients with homozygous abetalipoproteinemia and hypobetalipoproteinemia, diseases associated with low plasma levels of apolipoprotein B containing lipoproteins. Following the injection of heparin into one patient with homozygous abetalipoproteinemia, the plasma LACI antigen level increased to a level comparable with that in normal individuals after heparin treatment.
Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1- arginine chloromethyl ketone. These studies suggested that the platelet- activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.
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.
Various coagulation abnormalities are associated with pregnancy. Several investigators have suggested that there may be a unique procoagulant associated with amniotic tissue and fluid. We identified a cysteine proteinase from malignant tissue, cancer procoagulant, and had reason to believe a similar proteinase may be present in amniotic tissue. Amniotic fluid and extracts of amnion-chorion were purified by immunoaffinity chromatography with an antibody that was developed to cancer procoagulant antigen. The purified amnion-chorion procoagulant initiated clotting in normal and factor VII-deficient citrated human plasma and directly activated pure human factor X in a two-stage clotting assay. It was inhibited by 1 mmol/L of iodoacetamide and 0.1 mmol/L of HgCl2, and the procoagulant activity was activated by 10 mmol/L of KCN; these are classic properties of cysteine proteinases. The pure amnion-chorion procoagulant had the same mol wt and immunologic determinants as cancer procoagulant from rabbit V2 carcinoma, as determined by crossed immunodiffusion, suggesting that the same or very similar proteins were associated with both tissues. Thus, this procoagulant may be derived from both undifferentiated and dedifferentiated cells.
Coagulation and fibrinolytic activities were studied in 18 subjects with Behçet's disease and compared with results from 14 matched control patients suffering from sero-negative arthritis. Significantly higher plasma concentrations (median and range) were found in Behçet's patients for the following variables: fibrinogen 3.7 (1.7-6.9) vs 3.0 (2.0-5.1) g/1, p <0.05; von Willebrand factor antigen, 115 (72-344) vs 74 (60-119)%, p <0.002; plasminogen activator activity (106/ECLT2) 219 (94-329) vs 137 (78-197) units, p <0.002; tissue plasminogen activator inhibitor (t-PA-I) activity, 9.1 (5.5-19.3) vs 5.1 (1.8-12.0) IU/ml, p <0.002; and PAI-1 antigen, 13.9 (4.5-20.9) vs 6.4 (2.4-11.1) ng/ml, p <0.002. Protein C antigen was significantly lower: 97 (70-183) vs 126 (96-220)%, p <0.02. No differences were observed in antithrombin III activity or antigen, factor VIII coagulant activity, fibrinopeptides A and Bβ15-42, plasminogen, α-2-antiplasmin, functional and immunological tissue-plasminogen activator, thrombin-antithrombin complexes and D-dimer. Levels of tissue plasminogen activator inhibitor (activity and antigen) correlated with disease activity while fibrinogen and von Willebrand factor concentrations did not. Seven of the 18 subjects with Behçet's disease had suffered thrombotic events but it was not possible to distinguish these from the 11 patients without thrombosis using the assays performed. The results suggest the abnormal fibrinolytic activity in Behçet's disease is due to increased inhibition of tissue plasminogen activator. No abnormality of coagulation or fibrinolytic activity specific to Behçet's disease was detected.
The present work was conducted to bring information about the enhanced procoagulant activity of blood platelets in diabetes mellitus, as compared to normal control platelets. The inquiry was performed on platelets from either human patients or hamsters with streptozotocin-induced diabetes. The experiments comprised functional assays of factor Xa and thrombin generation in the presence of normal or diabetic platelets, using saturating amounts of coagulation factors. The prothrombinasic and tenasic complexes activation was measured by using discontinuous amidolytic tests with chromogenic substrates (S-2238 for FXa and S-2222 for thrombin). The results showed that, in contrast to the low prothrombin and factor X converting activity of normal platelets, the thrombin and FXa generation on diabetic platelets was increased by 3-7 times for either humans or hamsters. These findings may be relevant for the pathogenesis of thrombotic complications known to occur in diabetes mellitus.
Thrombotic events occur frequently in myeloproliferative disorders, namely polycythaemia vera and essential thrombocythaemia. Standard diagnostic criteria are designed quite stringent, so that a number of patients could be underdiagnosed. Spontaneous erythroid colonies formation from bone marrow or peripheral blood in the absence of exogenous erythropoietin is considered a reliable index of myeloproliferative disorder even at early stages. Endogenous erythroid colonies (EECs) formation was assessed in 43 patients having recently suffered from venous thrombosis prior to 45 years and without a previous diagnosis of hematological disease favouring thrombosis. A screening for coagulative abnormalities associated with thrombophilia was also carried out: in 5 patients (11.6%) a plasmatic thrombogenic defect was found (quantitative deficiency of antithrombin III, 1 case, protein C, 2 cases, protein S, 1 case, and plasminogen, 1 case). In 10 patients (2 males and 8 females) (23.2%) EECs assay was positive, allowing diagnosis of myeloproliferative disease even though 7 of them did not fulfill standard diagnostic criteria. In the other 3 patients who met the criteria for diagnosis of overt myeloproliferative disease the thrombotic event was the inaugural manifestation. In all these EECs-positive patients thrombosis involved mesenteric and portal veins (n = 4), hepatic veins (n = 3), portal vein (n = 2), mesenteric vein (n = 1). One of them was simultaneously affected from congenital protein C deficiency. Thus latent or atypical forms of myeloproliferative disease as well as the overt stages were the most frequent recognized cause of splanchnic venous thrombosis, accounting for 55% of the cases of our series. On the contrary no EECs-positive subject was found among the 25 patients with other sites of thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Although numerous studies have provided indirect evidence for enhanced platelet activity in sickle cell anaemia, little attention has been directed to examination of platelet alpha and dense granule release in the sickling disorders. We simultaneously measured by radioimmunoassay plasma levels of the alpha granule constituents β‐thromboglobulin (β‐TG) and platelet factor 4 (PF4) in 43 children with sickle cell anaemia in steady state and 24 patients during severe vaso‐occlusive crisis. β‐TG levels during steady state (50 ± 3.6 ng/ml, mean ± SEM) were greater (P<0.001) than in normal controls (36 ± 1.6), but there was no additional significant rise during crisis (55 ± 5.9). PF4 levels were similar (P= 0.12) in both steady state (10 ± 1.2 ng/ml) and crisis (9.3 ± 2.3) to those of normal controls (6.0 ± 0.8). The similarity of β‐TG/PF4 ratios in normal and sickle cell anaemia patients as well as the positive correlation (P<0.05) between platelet count and β‐TG and PF4 suggested that an artefactual in vitro platelet activation was responsible for some of the observed increased β‐TG and PF4 levels. Further evidence against enhanced platelet activity in these sickle cell patients included normal intraplatelet content of the dense granule constituent 5‐HT and a normal ATP/ADP ratio. From this data we conclude that platelet activation in children with sickle cell anaemia appears minimal.
Meta-analysis of data from several controlled trials has shown that low-dose aspirin reduces the risk of pregnancy-induced hypertension (PIH) and intrauterine growth retardation (IUGR) in women at high risk of these disorders. We have assessed the efficacy of low-dose aspirin in women judged to be at moderate risk.
Women were included on prophylactic criteria— age under 18 or over 40 years, mild or moderate chronic hypertension (diastolic pressure between 90 and 110 mm Hg), nephropathy with normal renal function and blood pressure, history of PIH or IUGR, and twin pregnancy—or therapeutic criteria—PIH or early signs of IUGR in current pregnancy. Eligible women were randomly assigned treatment with 50 mg aspirin daily until delivery (583) or no treatment (523); 18 and 46 women, respectively, were lost to follow-up. The groups were well matched for baseline characteristics. We found no differences between the no-treatment and aspirin groups in numbers of spontaneous (5 vs 2) or therapeutic (1 vs2) abortions, stillbirths (14 vs 13), perinatal mortality (35·7 vs 28·6 per 1000 births), mean birthweight (2858 [SD 729] vs 2874 [795] g), proportion of infants with birthweights below the 10th centile (95 [18·3%] vs 117 [19·0%]), or births before 37 weeks' gestation (184 [35·6%] vs 209 [33·9%]). Nor did the groups differ in the frequency of PIH with or without proteinuria (51 [15·2%] vs 81 [19·3%]). There was no difference in mean birthweight between the treatment groups in separate analyses according to criteria for trial entry and week of gestation at randomisation.
Our study gives little support to the notion that low-dose aspirin is beneficial in women at moderate risk of PIH or IUGR.
The authors have studied 21 patients with Crohn's disease and have looked for signs of platelet and blood coagulation activation, by measuring platelet factor 4, β thromboglobulin and fibrinopeptide A; a fibrinolytic system study with tissue plasminogen activator assessment has also been made. Beta thromboglobulin, platelet factor 4 and fibrinopeptide A were increased in 80 per cent, 100 per cent and 60 per cent of cases respectively. Beta thromboglobulin was significantly correlated with the Van Hees activity index. Plasminogen before venous stasis was significantly decreased and 9 patients had a plasminogen release defect. The relationship between Crohn's disease and thrombosis might partially be explained by a release of inflammation mediators and/or endotoxins; these mediators might induce thrombosis by interfering with the antithrombogenic properties of the endothelial cell. In conclusion these data prove that active Crohn's disease is currently associated with a prethrombotic state, present biologic tests that might predict a venous or arterial thrombosis at short term are not available.
Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.
THE OCCURRENCE of thrombosis, both venous and arterial, has been recognized for more than 30 years as a distinct complication of ulcerative colitis. The literature on this subject consists of individual case reports or incidental observations usually in clinical studies concerned with other aspects of ulcerative colitis.¹⁻⁸ As a result, the stated incidence of this complication varies widely. The incidence of thrombosis in the larger series of cases is shown in Table 1.
The occurrence of thrombosis in patients with ulcerative colitis appears predictable since many of the factors characteristic of this disease are known or believed to encourage thrombosis: toxemia, anemia, emaciation, prolonged rest in bed, and frequent surgical procedures. However, as early as 1936, Bargen and Barker,² as a result of their observations on the extensive propagation of such thrombi, considered the possibility that an increased coagulation tendency of the blood occurs in patients with ulcerative
A sensitive and specific test was used to identify a platelet-agglutinating factor in sera from patients with thrombotic thrombocytopenic purpura. Serum from patients plus a preparation rich in large multimers of factor VIII: von Willebrand factor were added to target platelets, and agglutination occurred in 41 of 48 samples. Edetic acid, heparin, or heating, but not aspirin, monomeric IgG, or dansylarginine N-(3-ethyl-1,5-pentanediyl)amide inhibited the platelet-agglutinating factor. In-vitro agglutination requires the presence of a platelet-agglutinating factor and large multimers of von Willebrand factor. High concentrations of either component lowers the amount of the other required for platelet agglutination. Some patients may be more susceptible to the agglutinating factor because of a congenital or acquired abnormality in processing unusually large multimers of von Willebrand factor or because of infections or inflammatory disorders that lead to increased synthesis of large multimers of von Willebrand factor.
Previous work has shown enhanced aggregation and thromboxane synthesis by platelets from diabetic subjects. We have compared thromboxane synthesis by platelets from normal subjects with that of platelets from two groups of insulin-dependent diabetic patients: one group receiving conventional depot insulin therapy and the other continuous subcutaneous insulin infusions. Thromboxane synthesis was significantly higher with platelets from the conventionally-treated diabetic patients than that observed for control subjects. Patients on continuous insulin infusions were similar to control subjects. This group of patients also had better control of glycemia. The effect on thromboxane production might be related to normalization of plasma lipids which occurs with continuous infusion insulin therapy.
. A specific plasminogen activator inhibitor was isolated from the plasma of pregnant women by matrix-bound, cross reacting monoclonal antibodies against placental plasminogen activator inhibitor. The pregnancy plasma plasminogen activator inhibitor (PP-PA-I) was found to be immunologically different from the inhibitor produced by endothelial cells. Its molecular weight was 70 000 daltons. It formed complexes with urokinase (u-PA) and with plasminogen activator of the tissue type (t-PA), similar to those formed by the placental plasminogen activator inhibitor (Pl-PA-I). It did not inhibit plasmin. For measuring PP-PA-I, an enzyme-linked immunosorbent assay (ELISA) was designed using monoclonal and polyclonal antibodies against the placental inhibitor. Concentrations of PP-PA-I increased successively during pregnancy, and fell sharply after delivery. This inhibitor could not be detected in normal non-pregnancy plasma. The results indicate that the inhibitor isolated from pregnancy plasma is responsible for the depressed fibrinolytic activity during pregnancy, and that the placenta is the source of the inhibitor.
. We measured plasma sulphur amino acids in twenty-two patients with chronic renal failure and compared the findings with those obtained in twenty-two normal subjects. In fasting blood (08.00 hours) cysteine-homocysteine mixed disulphide was significantly increased in the renal patients, mean values (± SD) being 8.2 ± 3.4 and 3.1 ± 1.0 μmol/l respectively (P < 0.001). The increase was positively correlated with reduced renal function, as assessed by serum creatinine (r= 0.62; P < 0.01). Homocystine was detected in nineteen patients, the mean concentration (± SD) being 1.7 ± 0.6 μmol/l; it was not found in any normal subject. Methionine levels were not different but there were significant increases in cystine (P < 0.001) and taurine (P < 0.05) in the patients. Similar values for these amino acids were found in a second blood sample drawn at 16.00 hours. Changes in the other neutral and acidic amino acids measured were in agreement with those reported in chronic azotaemia. We concluded that plasma levels of all the principal sulphur amino acids except methionine are elevated in chronic renal failure emphasizing the importance of the kidney in sulphur excretion. Prolonged accumulation of homocysteine and cysteine-homocysteine mixed disulphide may be relevant to the development of accelerated vascular disease in patients with chronic renal failure by producing endothelial damage.
Pediatric Research (1984) 18, 242A–242A; doi:10.1203/00006450-198404001-00894