Effects of normal and sickle erythrocytes on prostacyclin release by perfused human umbilical cord veins.

We compared the effects of normal (AA) and sickle (SS) erythrocytes (RBC) on endothelial cell release of prostacyclin by perfused human umbilical cord veins. Two equal-length segments of fresh umbilical cords were perfused first with serum-free Dulbecco's modified Eagle's medium (DMEM) to establish the basal prostacyclin production rate for each segment; then one segment was perfused with SS RBC and/or plasma, while the other segment was simultaneously perfused with AA RBC and/or plasma. Aliquots of perfusate were removed at intervals for measurement of the stable prostacyclin metabolite 6-keto-prostaglandin F1 alpha (6-keto-PGF). Basal prostacyclin production by segments from the same cord was very similar, but it varied considerably among segments from different cords. Therefore, the ratio of prostacyclin release with RBC and/or plasma to basal prostacyclin release for each segment was used to compare prostacyclin release among segments from different cords. Mean prostacyclin release was significantly higher from segments perfused with SS RBC in autologous plasma than from segments perfused with AA RBC in autologous plasma at 15, 30, and 60 min. However, no significant differences in mean prostacyclin production were observed between segments perfused with SS vs. AA RBC in DMEM or between segments perfused with SS vs. AA plasma alone. No significant correlations were observed between prostacyclin production and either the viscosity of SS and AA RBC in autologous plasma or DMEM or the adhesiveness of SS and AA RBC to cultured human umbilical vein endothelial cells. We conclude that SS RBC in autologous plasma cause increased prostacyclin release from perfused human umbilical cord veins. The perfused human umbilical cord vein system may be a useful model for comparing the response of vascular endothelium to SS and AA RBC and plasma under controlled-flow conditions.

Elevated immunoreactive tumor necrosis factor and interleukin-1 in sickle cell disease.

To determine whether tumor necrosis factor (TNF) and interleukin-1 (IL-1) might be involved in the pathogenesis of sickle cell disease and its complications, TNF-alpha and IL-1-alpha were measured using enzyme-linked immunosorbent assay in 59 plasma samples from 34 adult subjects with Hb SS or Hb SC who did not have documented infections. Tumor necrosis factor was elevated on at least one occasion in 27 subjects, including 18 of 21 subjects in the steady state and 13 of 19 subjects during painful crisis. Interleukin-1 was elevated on at least one occasion in 6 subjects, including 3 subjects in the steady state and 3 subjects in crisis. All subjects with elevated IL-1 also had elevated TNF. Tumor necrosis factor and IL-1 were similarly elevated in the steady state and during painful crisis. No correlation was noted between TNF or IL-1 levels and the extent of activation of coagulation, as measured by plasma levels of the fibrin D-dimer fragment, the overall severity of vascular occlusive disease in each subject, or the presence of specific vascular occlusive complications. We conclude that plasma TNF is frequently elevated in subjects with sickle cell disease, and IL-1 is also elevated in some subjects. A direct role for these cytokines in the pathogenesis of vascular occlusion in sickle cell disease was not demonstrated, but an indirect role was not excluded.

Large-vessel occlusion in sickle cell disease: pathogenesis, clinical consequences, and therapeutic implications.

Much of the morbidity and mortality in sickle cell disease (SCD) is caused by tissue ischemia and infarction resulting from vascular occlusion. Research in this area has been dominated by the hypothesis that vascular occlusion in SCD is due primarily to microvascular obstruction by sickle erythrocytes (SS RBC), yet there is no direct evidence that microvascular occlusion is responsible for any of the vasocclusive complications of SCD. In this paper an alternate hypothesis is proposed: that thrombotic occlusion of larger arteries and veins is an important factor in many of the vasocclusive complications of SCD. Large-vessel cerebral arterial disease (intimal hyperplasia with superimposed thrombosis) has clearly been established as the most important cause of stroke in SCD, and considerable evidence suggests that pulmonary arterial thrombosis/embolism is a major cause of pulmonary infarction and hypertension. The involvement of large-vessel thrombosis in painful crisis, aseptic necrosis of bone, priapism, leg ulcers, retinopathy, and miscarriage has not been adequately investigated. Large-vessel occlusion in SCD is probably a consequence of the abnormal adhesive and procoagulant properties of SS RBC, which produce endothelial damage, secondary intimal proliferation, and thrombosis. Techniques currently used to treat large-vessel occlusion in other disorders (antiplatelet and anticoagulant agents, thrombolytic therapy, angioplasty, endarterectomy, and vascular bypass surgery) should be considered in sickle cell subjects with large-vessel occlusion, especially in the cerebral vasculature.

Platelets, coagulation, and fibrinolysis in sickle cell disease: their possible role in vascular occlusion.

Recent evidence suggests that sickle cell disease (SCD) can be considered a hypercoagulable state, in which both platelet activation and thrombin generation are abnormally increased. Although thrombosis is now known to play an important role in at least one of the vasocclusive complications of SCD, namely stroke, the significance of hypercoagulability in the pathogenesis of vascular occlusion in SCD remains unclear. This review summarizes current evidence regarding platelet, coagulation, and fibrinolytic abnormalities in SCD and their possible role in vascular occlusion. Potential implications for therapy are also discussed.

Can storage of thawed cryoprecipitate be extended to more than six hours?

Fresh-frozen plasma (FFP) and cryoprecipitate both contain Factors I and VIII, however thawed FFP may be stored at 1-6 degrees C for 24 hours, but thawed cryoprecipitate may only be stored at 1-6 degrees C for six hours when used for Factor VIII content. To determine whether it would be safe and effective to extend the storage period of thawed cryoprecipitate from 6 to 24 hours, Factor VIII (and fibrinogen) levels were measured in 25 units of cryoprecipitates immediately on thawing and at 6 hours and 24 hours thereafter. The Factor VIII activity level decreased to 86% of the original activity level within 6 hours, but the drop between 6 and 24 hours was relatively small. Eighty percent of the original activity was still present at 24 hours after thawing. The fibrinogen level decreased to 87% of the original level within 6 hours but remained stable between 6 and 24 hours. Additional testing showed that fibrinogen levels remained stable between 6 and 74 hours. These data suggested that the storage of thawed cryoprecipitate might be extended to 24 hours when this blood product is used for Factor VIII content and to 74 hours when it is used for fibrinogen content. Furthermore, the lack of prohibition on the use of cryoprecipitate that has been thawed for more than six hours and stored at 4 degrees C for its fibrinogen content seems reasonable.

Phase I trial of a 5-day course of carbetimer.

Carbetimer (carboxyimamidate) is a low molecular weight derivative of ethylene/maleic anhydride polymer. This compound has demonstrated antitumor activity against several animal models with a daily x 5 schedule appearing most effective. A phase I clinical study of the daily x 5 schedule repeated every 28 days was therefore performed. Forty-one evaluable patients received 66 evaluable cycles of Carbetimer at daily doses ranging from 100-11,000 mg/m2. Hypercalcemia was the dose limiting toxicity with both patients at the 11,000 mg/m2 daily dose level and one patient who received 6 cycles of drug at the 4200 mg/m2 dose level developing severe hypercalcemia not explained by the underlying malignancy. Mild nausea, concentration and rate dependent arm pain at the site of infusion, proteinuria, and coagulopathy were also seen. Calcium balance studies revealed hypercalciuria, suggesting increased mobilization of calcium rather than renal retention. In vitro coagulation studies revealed concentration dependent prolongation of the partial thromboplastin time and thrombin time. No complete or partial responses were seen. However mixed response or biochemical response (reduction in serum lactic dehydrogenase) were seen in 5 patients with melanoma or renal cancer. Due to unacceptable toxicity at the 11,000 mg/m2 daily dose level, Carbetimer 8500 mg/m2 is the recommended dose for a 5-day treatment schedule every 28 days. Special attention should be directed toward possible activity against melanoma and renal cancer.

Human umbilical vein endothelial cells secrete transcobalamin II.

Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.

Increased circulating endothelial cells in sickle cell crisis.

To determine whether increased numbers of circulating endothelial cells, a possible indicator of endothelial injury, are present in subjects with sickle cell disease, we measured circulating endothelial cells in 30 normal subjects and in 23 subjects with sickle cell anemia. Mean circulating endothelial cells were significantly higher (P less than 0.025) in the sickle cell subjects than in the normal subjects. Circulating endothelial cells were significantly higher than normal in 10 sickle cell subjects studied during painful crisis (P less than 0.01) but not in 13 sickle cell subjects studied while in the steady state. To control for the known stimulatory effect of cigarette smoking on circulating endothelial cells, we analyzed the results for smokers and nonsmokers separately. Mean circulating endothelial cells were not significantly higher in sickle cell subjects who smoked (n = 10) than in normal subjects who smoked (n = 8), but were significantly higher (P less than 0.05) in sickle cell nonsmokers (n = 13) than in normal nonsmokers (n = 22). Among nonsmoking sickle cell subjects, mean circulating endothelial cells were significantly higher than normal (P less than 0.01) during painful crisis (n = 7), but not in the steady state (n = 6). We conclude that circulating endothelial cells are significantly increased in sickle cell crisis, and may indicate the occurrence of acute endothelial injury during episodes of microvascular occlusion.

Clinical disorders of fibrinolysis: a critical review.

Much progress has recently been made in understanding the biochemistry and physiology of endogenous fibrinolysis. As a result, a better understanding of the mechanisms and clinical consequences of disordered fibrinolysis has emerged. Increased fibrinolytic activity is an uncommon but important cause of hemorrhagic disease. Congenital disorders of fibrinolysis which cause bleeding include increased plasma plasminogen activator activity and deficiency of alpha-2 antiplasmin. Acquired disorders associated with increased fibrinolytic activity and bleeding include liver cirrhosis, amyloidosis, acute promyelocytic leukemia, some solid tumors, and certain snake envenomation syndromes. Increased fibrinolysis is important to recognize because epsilon-aminocaproic acid (EACA) may be required to prevent or control bleeding. Diminished fibrinolytic activity has been associated with a variety of thrombotic disorders, but a direct cause-and-effect relationship has yet to be established. Congenital abnormalities of fibrinolysis associated with thrombosis include plasminogen deficiency, decreased endothelial generation of plasminogen activator activity, and certain abnormal fibrinogens. Thrombosis in these disorders is effectively managed with warfarin. Diminished fibrinolysis has also been reported in "idiopathic" venous thrombosis, oral contraceptive-induced and post-operative venous thrombosis, coronary artery disease, cerebrovascular disease, systemic lupus erythematosus, and thrombotic thrombocytopenic purpura, but the significance of abnormal fibrinolysis in these disorders is uncertain. Large, prospective studies of fibrinolytic variables as risk factors for vascular and thrombotic disease are needed to determine whether pharmacologic augmentation of impaired fibrinolysis could be useful in the prevention or treatment of these disorders.

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP.

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.

Effect of the lupus anticoagulant on endothelial fibrinolytic activity in vitro.

We investigated the effect of plasma and serum from 10 subjects with the lupus anticoagulant and thrombosis and 9 normal subjects on the secretion of tissue-type plasminogen activator (t-PA) and its rapid inhibitor (type 1 plasminogen activator inhibitor, or PAI-1) by cultured human endothelial cells. Confluent monolayers of human umbilical vein endothelial cells were incubated for 48 hours with plasma or serum diluted ten-fold in serum-free endothelial cell growth medium, and the secretion of t-PA and PAI-1 measured by enzyme-linked immunosorbent assay. No consistent differences in mean t-PA and PAI-1 release were found between cells exposed to normal plasma or serum and plasma or serum from subjects with the lupus anticoagulant and thrombosis. No plasma or serum sample produced consistent inhibition of t-PA release or stimulation of PAI-1 release (defined as t-PA levels less than the mean minus two standard deviations for normal subjects, and PAI-1 levels greater than the mean plus two standard deviations for normal subjects, respectively). These findings do not support a role for altered endothelial fibrinolytic activity in the pathogenesis of thrombosis in subjects with the lupus anticoagulant, and are consistent with previous observations that these subjects have normal fibrinolytic activity in vivo.

Elevated fibrin D-dimer fragment in sickle cell anemia: evidence for activation of coagulation during the steady state as well as in painful crisis.

The fibrin D-dimer degradation fragment is produced by plasmin degradation of cross-linked fibrin. Elevated plasma D-dimer levels indicate increased plasmin degradation of cross-linked fibrin, and are therefore an indirect indication of increased thrombin activity and fibrin formation. With an ELISA assay sensitive to plasma D-dimer levels of less than 50 ng/ml, elevated levels of D-dimer were found in 23 of 25 subjects with sickle cell disease tested during the steady state, and in all 21 subjects tested serially while hospitalized for painful crisis (total of 40 samples). Mean D-dimer was 79 +/- (SD) 25 ng/ml in 35 normal subjects, 566 +/- 739 ng/ml in sickle cell disease subjects in the steady state, and 1,038 +/- 1,010 ng/ml in sickle cell subjects during painful crisis; these differences were highly significant (p less than 0.001). No correlation was observed between levels of D-dimer in the steady state and the time since the last painful crisis; D-dimer was elevated in all 7 patients tested more than 6 months after their last painful crisis. Most steady-state patients did not have other microvascular occlusive manifestations, such as active leg ulcers or aseptic necrosis of bone, which could explain the increased D-dimer levels observed. It is concluded that increased thrombin activity and fibrin formation are features of steady-state sickle cell disease, and that they further increase during painful crisis. Possible causes and implications of increased thrombin activity and fibrin formation in subjects with sickle cell disease are discussed.

Endothelial-dependent fibrinolysis in subjects with the lupus anticoagulant and thrombosis.

To investigate the hypothesis that diminished endothelial fibrinolytic activity contributes to the pathogenesis of thrombosis in patients with the lupus anticoagulant (LA), we assessed the ability of endothelium to release tissue-type plasminogen activator (t-PA) in response to standardized venous occlusion (VO) of the arm, and the extent of inhibition of t-PA, in 11 subjects with LA and a history of thrombosis and in 36 healthy normal subjects. The mean rise in plasma t-PA antigen after VO, the mean plasma free t-PA activity after VO, and the mean plasma t-PA inhibitor level prior to VO were not significantly different in subjects with LA and thrombosis and in normal subjects. Four subjects with LA and thrombosis (36%), and five of 36 healthy control subjects (14%) generated no detectable free t-PA activity after VO ("non-responders"); this difference was not statistically significant. All four "non-responders" with LA and thrombosis had normal t-PA antigen release after VO, indicating that the lack of detectable free t-PA activity after VO was due to increased inhibition of released t-PA. We conclude that abnormally reduced endothelial fibrinolytic activity is not present in the majority of subjects with LA and thrombosis. In the subset of subjects with LA and thrombosis who generate no detectable t-PA activity after VO, a stimulatory effect of LA on endothelial production of t-PA inhibitor cannot be excluded.

Protein S deficiency in sickle cell anemia.

To investigate the status of the protein C-protein S anticoagulant pathway in sickle cell disease, we measured protein C, total and free protein S, and C4b-binding protein levels in 20 subjects with sickle cell disease (Hb SS or SC). Mean total and free protein S levels were both significantly lower in subjects with sickle cell disease than in normal individuals, but greater reductions were observed for free S. The free protein S level was below the mean -2 SD for normal subjects in 12 subjects with sickle cell disease; the total protein S level was below this level in three subjects. Mean C4b-binding protein levels were normal in subjects with sickle cell disease, both during painful crisis and in the steady state, and no correlation was observed between the levels of C4b-binding protein and free protein S, suggesting that the low free protein S level was not caused by increased levels of C4b-binding protein. Crossed immunoelectrophoresis of plasma samples from eight subjects with sickle cell disease showed marked reductions in free protein S, with normal levels of protein S bound to C4b-binding protein. In contrast to the protein S level, mean protein C activity was normal in subjects with sickle cell disease, both during painful crisis and in the steady state. However, the protein C level was below the mean -2 SD for normal subjects on at least one occasion in four subjects with sickle cell disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue type plasminogen activator antigen and activity in sickle cell disease.

To investigate the hypothesis that diminished endothelial fibrinolysis is present in sickle cell (SS) disease plasma, tissue type plasminogen activator (t-PA) antigen titres were measured before and after a standard stimulus of endothelial t-PA release (venous occlusion of the arm), and plasma t-PA activities after venous occlusion in 33 subjects with SS disease and in 32 healthy subjects. Mean plasma t-PA antigen titres before and after venous occlusion, and mean plasma t-PA activities after venous occlusion did not differ significantly between SS patients and normal subjects. No significant differences in mean t-PA antigen and activity were observed between samples taken from inpatients being treated for acute pain crisis (18 subjects, 30 samples) and samples taken from subjects in the steady state (23 subjects, 26 samples). No consistent differences were seen between painful crisis and steady state samples in eight SS patients studied while in crisis and in the steady state. No correlation was observed between any fibrinolytic variable in SS patients and the overall severity of microvascular occlusive disease as measured by a standard scoring system. It is concluded that the capacity of endothelium to synthesise and release t-PA is not impaired in SS disease, and that excessive inhibition of released t-PA, leading to reduced t-PA activity in plasma is also not a feature of SS disease, either in the steady state or during painful crisis.

Impaired fibrinolysis in coronary artery disease.

We assessed endogenous endothelial-dependent fibrinolysis in 99 subjects with coronary artery disease (CAD) documented by angiography and in 28 control subjects with normal coronary arteries on angiography. We used specific, sensitive assays for plasma tissue-type plasminogen activator (t-PA) antigen, t-PA activity, plasminogen activator inhibitor (PAI) activity, plasminogen, and alpha-2 plasmin inhibitor (alpha-2 PI). Mean PAI activity was significantly higher, and mean t-PA activity after venous occlusion of the upper arm (a standard test of the capacity of vascular endothelium to release t-PA) was significantly lower in subjects with CAD than in subjects with normal coronary arteries. The mean increment in t-PA antigen after venous occlusion was significantly lower than normal in subjects with CAD with onset of symptoms before age 45 years. Subjects with CAD had a significantly increased mean plasma fibrinogen level compared with control subjects, and a significant positive correlation was observed between PAI activity and plasma fibrinogen in subjects with CAD. No significant abnormalities of plasminogen or alpha-2 PI were observed in any subset of subjects with CAD. These data support an association between impaired fibrinolysis and CAD, with contributions from both increased PAI activity and in younger subjects from reduced endothelial t-PA release.

Protein C deficiency resulting from possible double heterozygosity and its response to danazol.

A unique family with protein C (PC) deficiency is described. The proband had a history of renal vein thrombosis as a newborn and iliofemoral thrombosis at the age of 6 years. After 6 months of heparin treatment, discontinuation of anticoagulation therapy was accompanied by persistent hypofibrinogenemia with increased fibrinogen consumption. With continuous infusion of heparin, fibrinogen turnover normalized, and the child has remained free of thrombosis. Both the immunologic level of PC and the functional activity measured by amidolytic assay were moderately reduced (47% and 34%, respectively). Functional activity of PC measured by its anticoagulant activity was disproportionately lower (14%). A 3-year-old asymptomatic sibling had a similar disproportionate reduction of PC anticoagulant activity compared with the amidolytic activity or immunologic level. The mother demonstrated type I PC deficiency with a proportionate reduction in immunologic protein levels (59%), anticoagulant activity (52%), and amidolytic activity (46%), whereas the father had type II PC deficiency with normal immunologic protein levels (102%), normal amidolytic function (98%), but a low anticoagulant function (50%). An abnormal PC molecule was detected by two-dimensional immunoelectrophoresis in the father and two children. These data are consistent with the hypothesis that the children are doubly heterozygous for two different types of PC deficiency inherited from each of the parents. A 14-day trial of danazol in the proband resulted in a rise in the PC antigen concentration from 66% to 98% but no change in PC anticoagulant function.

Tissue plasminogen activator antigen and activity in disseminated intravascular coagulation: clinicopathologic correlations.

The fibrinolytic response to disseminated intravascular coagulation (DIC) has been considered important both in preventing thrombosis and in contributing to hemorrhage. Detailed studies of fibrinolysis in DIC are lacking, however. We measured tissue plasminogen activator (t-PA) antigen and activity levels in 74 patients with DIC, 53 hospitalized patients with similar illnesses without DIC, and 36 healthy normal subjects, using sensitive, specific assays. Mean t-PA antigen levels were significantly higher in patients with DIC than in either hospitalized control subjects or normal individuals in most disease categories studied. Highest t-PA antigen levels were seen in patients with liver disease, but patients with DIC without liver disease also had significantly elevated t-PA antigen. Detectable free t-PA activity was infrequently seen in patients with DIC, however. No correlation was found with either thrombotic or hemorrhagic complications and levels of t-PA antigen or activity in DIC, with the possible exception of increased t-PA activity in two patients with intracranial hemorrhage. Bleeding complications were seen almost exclusively in patients with underlying anatomic lesions, and thrombotic complications were usually associated with other known predisposing factors. We conclude that t-PA antigen is usually elevated in DIC, but free t-PA activity is infrequently seen, most likely because of increased levels of t-PA inhibitor. The presence or absence of free t-PA activity does not appear to predict which patients with DIC will develop hemorrhage or thrombosis.