Incubation Of Normal Plasma Research Articles

Mild hyperhomocysteinemia is associated with increased TAFI levels and reduced plasma fibrinolytic potential.

Mild hyperhomocysteinemia (HHcy) has been shown to be associated with impaired fibrinolysis, but some aspects of this association are unclear. Using an in vitro model of physiological relevance, we investigated whether and how HHcy influences plasma fibrinolytic potential. We studied 176 patients with previous venous thromboembolism, 58 with HHcy, 118 with normal total homocysteine (tHcy) levels (NHcy), at least 3 months after withdrawal of anticoagulant therapy. Plasma fibrinolytic potential was measured as the fibrinolysis time of tissue factor (TF)-induced clots exposed to 15 ng mL(-1) t-PA. Fibrinolysis time was longer in HHcy than in NHcy patients, but this difference disappeared when the assay was performed in the presence of the TAFIa inhibitor, PTCI. Plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI) and factor VIII (FVIII) were higher in HHcy than NHcy, whereas PAI-1, fibrinogen and endogenous thrombin potential were similar. Using multivariate analysis, plasma tHcy was identified as an independent predictor of fibrinolysis time. Experiments in which native fibrinogen was replaced by purified fibrinogen suggested that alterations of fibrinogen structure did not contribute appreciably to the hypofibrinolysis of HHcy plasma samples. The acute increase of tHcy either in vivo (after an oral methionine load) or in vitro (after incubation of normal plasma with 0.5 mm DL-Hcy) had no effects on fibrinolysis or TAFI levels. We describe an enhanced TAFI-related antifibrinolytic activity in mild HHcy, which might account for the reported heightened thrombosis risk; however, it is unknown whether HHcy is causally related to hypofibrinolysis or an associated bystander.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Activation of the Plasma Kinin Forming Cascade along Cell Surfaces

Proteins of the plasma kinin-forming cascade bind to endothelial cells and activation of the cascade can be initiated along the surface. The light chain of high molecular weight kininogen (HK) (domain 5) and factor XII bind to gC1qR, the heavy chain of HK (domain 3) binds to cytokeratin 1 and the interactions are zinc dependent. Prekallikrein binds to domain 6 of HK. Antisera to gC1qR and cytokeratin 1 inhibit binding and activation. Incubation of normal plasma with endothelial cells leads to gradual conversion of prekallikrein to kallikrein, while plasma deficient in factor XII or HK are inactive within a 2-hour time frame. Thus factor XII is critical for activation to proceed. Augmentation of these reactions may occur when C1 inhibitor is functionally deficient or with ACE inhibitors which also inhibit kininases.

Uremic patients have decreased shear-induced platelet aggregation mediated by decreased availability of glycoprotein IIb-IIIa receptors

Bleeding and platelet dysfunction are prominent features of uremia. Shear-induced platelet aggregation (SIPA) involves the interaction of von Willebrand factor (vWF) with platelet membrane glycoproteins (GP) Ib and IIb-IIIa, the same receptor-ligand pair involved in in vivo adhesion and aggregation of platelets in the arterial circulation. We have used a modified rotational cone-plate viscometer to measure SIPA and calcium flux in platelets. Flow cytometric analysis of the surface expression of GP Ib and IIb-IIIa was performed using fluorescein isothiocyanate-conjugated monoclonal antibodies CD42b and CD41a, respectively. Uremic patients showed decreased SIPA (controls, 43% ± 2% [mean ± SEM]; chronic renal failure patients, 36% ± 3%; chronic hemodialysis patients, 26% ± 2%; P < 0.0001) along with a decrease in GP IIb-IIIa (controls, chronic renal failure patients, and chronic hemodialysis patients, 840 ± 25, 649 ± 42, 661 ± 38 mean fluorescence intensity, respectively; P < 0.0001). Glycoprotein Ib in uremic patients was not significantly different from normal. Chronic hemodialysis patients also demonstrated increased platelet-bound fibrinogen ( P < 0.0001) and platelet-bound vWF ( P = 0.01). Calcium flux and thromboxane B 2 generation during SIPA of uremic platelets were normal. However, uremic plasma showed twice the normal concentration of vWF ( P < 0.0001) and sodium dodecyl sulfate agarose gel electrophoresis revealed the presence of fibrinogen fragments. Mixing experiments demonstrated an inhibitory effect of uremic plasma on SIPA of normal platelets (decreased from 39% ± 3% at baseline to 31% ± 3% after incubation in uremic plasma) along with an activation-independent increase in platelet-bound fibrinogen and platelet-bound vWF. When uremic platelets were incubated in normal plasma, their SIPA increased (from 12% ± 5% at baseline to 18% ± 4% after incubation in normal plasma; P = 0.002), although it did not return to normal. These results suggest that the uremic platelet dysfunction results from decreased GP IIb-IIIa availability due to receptor occupancy by fibrinogen fragments (and possibly vWF fragments).

Treatment of human serum with sulfosalicylic acid structurally alters digoxin and endogenous digoxin-like immunoreactive factor.

Pretreatment of human serum with 5-sulfosalicylic acid (SSA) as used in the Abbott TDx digoxin assay produces deglycosylated congeners of digoxin (DIG) and of endogenous digoxin-like immunoreactive factor (DLIF). Using high-performance liquid chromatography analysis, we observed differences in the degree and pattern of DIG breakdown products among five patients. The aglycone digoxigenin was the major product in several samples. Smaller amounts of the bis- and mono-digitoxosides and unidentified products less polar than DIG were sometimes present. Treatment of DLIF-containing plasma with SSA produced similar patterns of DLIF-breakdown products. Incubation of normal plasma containing DIG with SSA for up to 30 min caused little change in measured DIG by TDx and radioimmunoassay (RIA) but decreased to 50% in the ACS DIG assay. These results are consistent with the near 100% cross-reactivities of deglycosylated DIG congeners in the TDx and RIA assays compared to their lower cross-reactivities in the ACS assay. We conclude that the breakdown of DIG and DLIF during treatment of serum with SSA may compromise the accuracy of TDx DIG assays and may explain discrepancies observed in other studies between digoxin immunoassays. This study underscores the importance of understanding the effects of pretreatment strategies used for analytes measured by immunoassay.

Humoral factor, intraplatelet calcium, and hypertension.

Plasma humoral factors which modulate the transmembrane distribution of sodium and calcium have been identified in hypertensive patients and have been hypothesized to be involved in the etiology of essential hypertension. In cross-incubation experiments, we have found that plasma of hypertensive subjects elevated basal and stimulated intraplatelet calcium levels, while plasma of normal subjects has an opposite effect on platelets from hypertensives. Basal intraplatelet calcium in normal platelets was 108 +/- 5 nmol/L and rose to 142 +/- 3 nmol/L (P less than .001) after incubation in plasma from hypertensive patients. Platelets from hypertensive patients had basal calcium levels of 182 +/- 11 nmol/L which fell to 127 +/- 11 nmol/L (P less than .01) after incubation in normal plasma. Hypertensive plasma potentiated the rise in intraplatelet calcium in response to ADP and PAF. Hypertensive patients treated experimentally with plasmapheresis exhibited a disappearance of the plasma factor responsible for elevating intraplatelet calcium. These results indicate the presence of a plasma humoral factor in hypertensives which elevates intraplatelet calcium and sensitizes platelets to agonist stimuli.

Protein C Degradation in vitro by Neutrophil Elastase

Purified protein C is completely degraded into small peptides by in vitro incubation with purified elastase. Protein C is a rather sensitive substrate as degradation is already accomplished by low elastase concentrations (molar enzyme-to-substrate ratio 1:510) and short incubation periods (5 min-60 min). Protein C in a PPSB coagulation factor concentrate is equally degraded and similar split products are detected by blotting techniques. The protein C activity (measured by a chromogenic substrate) is faster reduced by elastase than the protein C concentration (measured by an ELISA). Incubation of normal plasma with high elastase concentrations (5.7 nmol/ml plasma) results in reduction of the protein C band while no split products are detectable. The pathophysiologic significance of the effects of elastase on protein C remains to be elucidated.

Interaction of Plasma Kallikrein with Protein C Inhibitor in Purified Mixtures and in Plasma

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of plasma kallikrein with protein C inhibitor in purified mixtures and in plasma

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of monoclonal antibodies that inhibit the function of protein C inhibitor. Evidence for heparin-independent inhibition of activated protein C in plasma

Monoclonal antibodies specific for protein C inhibitor (PCI) partially blocked the inactivation of activated protein C (APC) in plasma, whereas in a purified system, the PCI activity could be completely blocked. The inactivation of APC in normal and in PCI-depleted plasma was similar in the absence of heparin. The addition of heparin did not change the rate of inactivation of APC in PCI-depleted plasma, whereas in normal plasma a rapid phase of inhibition of APC was followed by a slower phase of inhibition. The slower phase was identical to the rate of inhibition of APC in the absence of heparin. After incubation of normal plasma with a monoclonal antibody specific for PCI that blocked its activity, there was no difference in heparin-dependent or heparin- independent inhibition of APC. These results indicate that in the absence of heparin PCI is unable to inactivate APC in a plasma environment.

Functional activities and nonenzymatic glycosylation of plasma proteinase inhibitors in diabetes

The functional activity of three of the major plasma protease inhibitors, α 1-proteinase inhibitor, antithrombin III and α 2-antiplasmin, has been measured in a series of persons with diabetes mellitus and compared with healthy controls. The mean specific functional activity of both diabetic plasma antithrombin III (50.8 ± 7.0 U/mg) and α 1-proteinase inhibitor (35.3 ± 5.6 mU/mg) was found to be significantly lower than in healthy controls (57.9 ± 6.0 U/mg) and (42.2 ± 10.7 mU/mg). No difference was detected in α 2-antiplasmin activity or levels. The glycosylated protein fraction of 83% of diabetic plasmas showed the presence of less than 1% of the total α 1-proteinase inhibitor when isolated by phenylboronate affinity chromatography. Incubation of normal plasma with 250 mmol/l glucose (a level approximately 6 × higher than encountered in uncontrolled diabetes) resulted in a 17% and 6% decrease in antithrombin III and α 1-proteinase inhibitor activities. We conclude that the decrease in specific activity of α 1-proteinase inhibitor is not related to nonenzymatic glycosylation, but the decrease in antithrombin III specific activity may be related.

Tangier disease. In vitro conversion of proapo-A-ITangier to mature APO-A-ITangier.

Tangier disease is a disorder characterized by low levels of apo-A-I and high density lipoproteins. The defect in Tangier disease is an abnormal A-I apolipo protein, designated apo-A- ITangier . In normal subjects, apo-A-I is secreted as proapo -A-I with subsequent extracellular conversion to mature apo-A-I. The major form in normal plasma is mature apo-A-I with small amounts of proapo -A-I. In Tangier disease, proapo -A- ITangier is present in roughly equivalent concentrations compared to mature apo-A- ITangier . It has been proposed that the defect in Tangier disease is in the conversion of pro- to mature apo-A- ITangier . To test this, proapo -A-I was isolated from normal and Tangier subjects, and the conversion to the mature form by plasma from normal and Tangier subjects was analyzed. Incubation of radiolabeled normal proapo -A-I in normal plasma anticoagulated with heparin was associated with progressive conversion to mature apo-A-I over 24 h (initially 85% of the radioactivity was in the proapo -A-I isoform; at 24 h 33% radioactivity remained in the pro-isoform). Proapo -A- ITangier was also converted to the mature isoform during 24 h of incubation in normal plasma. Initially, 84% of radioactivity was in proapo -A- ITangier , and by 24 h the radioactivity in this isoprotein had decreased to 36%. A similar pattern of conversion was also observed when proapo -A- ITangier was incubated in Tangier plasma. The proteolytic conversion of both normal proapo -A-I and proapo -A- ITangier was unaffected by the serine protease inhibitors phenylmethylsulfonyl fluoride (1 mM) or aprotinin (200 Kallikrein-inactivating units/ml), but was inhibited by EDTA (0.1%). These results indicate that proapo -A- ITangier can be converted to mature apo-A- ITangier by the converting enzyme in normal plasma. In addition, plasma from a Tangier subject can convert both normal and Tangier proapo -A-I to the mature form. These results establish that proapo -A- ITangier can be rapidly converted to mature apo-A- ITangier , and there is no deficiency of the converting enzyme activity in Tangier disease.

Generation of Inactive Renin in Vitro and in Vivo: Reversible Inactivation of the Active Plasma Enzyme in Rats

1. Active and total (trypsin treatment) plasma renins were measured in normal Wistar rats and in rats in which the renin-angiotensin system was stimulated by ether anaesthesia. 2. After incubation of normal plasma in vitro in the absence of angiotensinase inhibitors, active renin declined. This decline was shown to be due to the conversion of active renin into an inactive form, which could be re-activated by trypsin. 3. In plasma from renin-stimulated rats, the rate of decline of active renin in vitro was accelerated; however, the relative amount of inactive renin generated was decreased. 4. Ligation of the kidneys of the ether-anaesthetized animal resulted in a build-up in vivo of inactive renin concomitant with the decline of active renin. 5. These data demonstrate the conversion of active into inactive renin in vitro and indicate that inactive renin can also be generated in vivo from the active form of the enzyme. 6. Multiple forms of inactive renin may exist; some may be true ‘prorenins’ (renin zymogens) produced in the kidney, and others may result from post-biosynthetic modifications of the active plasma enzyme.

Effects of corticosteroids on eosinophil chemotaxis and adherence.

Therapeutic doses of corticosteroids frequently induce eosinopenia; however, the mechanism(s) involved remain obscure. To investigate this question, we studied the effects of corticosteroids on eosinophil adherence and migration. Eosinophils from normal donors were prepared by dextran sedimentation and Hypaque gradient centrifugation to 45-96% purity. Adherence was measured by filtration of whole blood and isolated eosinophils through nylon wool columns. Before prednisone administration, adherence was 83.8+/-3.2% for eosinophils in heparinized blood and 82.1+/-3.2% for isolated eosinophils. 4 h after oral prednisone administration whole blood eosinophil adherence was reduced to 53.9+/-10.7%; at 24 and 48 h adherence was normal. In contrast, isolated eosinophils showed no decrease in adherence 4, 24, or 48 h after corticosteroid administration. Similarly, in vitro addition of hydrocortisone to isolated eosinophils at 0.01 and 2.0 mg/ml did not reduce adherence. Eosinophil migration was tested in modified Boyden chambers by "lower-surface" and "leading-front" methods, using zymosan-activated serum and buffered saline to assess chemotactic and random migration, respectively. In vitro incubation of eosinophils with hydrocortisone or methylprednisolone produced a dose-dependent inhibition of chemotaxis. Using lower-surface methods the minimal concentration effecting substantial inhibition was 0.01 mg/ml for both drugs. At 2.0 mg/ml hydrocortisone and methylprednisolone inhibited eosinophil chemotaxis 82.6+/-4.4% and 85.0+/-3.5%, respectively. Using leading-front chemotaxis techniques significant inhibition was detected at 0.001 mg/ml hydrocortisone. Eosinophils incubated and washed free of corticosteroids responded normally to chemoattractants, indicating that the inhibitory effect of these drugs was reversible. Hydrocortisone at 2 mg/ml inhibited random eosinophil migration, although this effect was not apparent at lower concentrations. Corticosteroids did not act as chemotactic factor inactivators and were not toxic as measured by trypan blue exclusion. Eosinophils obtained from donors who had received 40 mg of prednisone orally for four days showed normal chemotactic responses, probably reflecting the fact that the cells were washed free of plasma before testing. In contrast, incubation of eosinophils in plasma from donors who had received a 300-mg bolus of hydrocortisone induced 46.1+/-4.5% more inhibition of chemotaxis than did incubation in normal plasma. These results indicate that: (a) eosinophil adherence is transiently reduced following in vivo corticosteroid administration, (b) eosinophil chemotaxis is inhibited by both in vitro and in vivo administration of corticosteroids, and (c) the chemotaxis inhibiting effect is nontoxic, cell-directed, dose-dependent and reversible. Inhibition of eosinophil adherence and chemotaxis may in part explain how corticosteroids produce eosinopenia and decrease the local accumulation of eosinophils.

Chemotaxls inhibition by plasma from patients with atopic dermatitis

We previously reported depressed polymorphonuclear leukocyte and monocyte chemotaxis in patients with severe atopic dermatitis. The degree of impairment roughly correlated with the disease severity and chemotaxis was noted to improve rapidly with clinical remissions. This rapid improvement suggested the presence of a short-lived plasma inhibitor of leukocyte function. We used a radio?labeled PMN chemotaxis assay to assess plasma effect on migration. PMN?s were incubated in plasmas from patients with varying degrees of atopic dermatitis then washed and assessed for migration toward endotoxin-stimulated serum attractant. lnhibitory effect varied widely and correlated directly with clinical extent and severity of dermatitis. Serial studies on individual patients showed lessening of plasma inhibitory activity during remissions. PMN?s from patients with atopic dermatitis showed improved migration after incubation in normal plasma. There was no evidence for circulating chemoattractants nor chemotactic factor inactivators. There appears to be a circulating inhibitor of chemotaxis present in plasma during acute flares of atopic dermatitis; the molecular nature of the inhibitor remains to be elucidated.

Studies on the degradation of lipoprotein‐X

Abstract. Three patients suffering from prolonged obstructive jaundice were studied after surgical removal of biliary obstruction. Four days after surgery the initial cholesterol esterification rate in the plasma rose from very low activities to values three to four times higher than those found in normal subjects. During this time lipoprotein‐X decreased in concentration. The initial cholesterol esterification rate returned to normal values when plasma lipids and lipoproteins reached normal levels.During incubation of normal plasma with lipoprotein‐X, changes in the lipid composition of the incubation mixture were observed which appeared to be catalysed by lecithin:cholesterol acyltransferase. The formation of esterified cholesterol and lysophosphatidylcholine exceeded that of control experiments when lipoprotein‐X was replaced by buffer solution in the incubation mixture. Lipoprotein‐X disappeared as monitored by electrophoresis on agar and measured after zonal ultracentrifugation, and lipoproteins with flotation behaviour of high density lipoprotein2, enriched in polar lipids, appeared.When plasma was incubated separately with lipoprotein‐X1 and lipoprotein‐X2in vitro, the T1/2 of these lipoproteins were 24 h and 9 1/2 h, respectively. Addition of lipoprotein‐X2 to normal plasma resulted in an increased initial cholesterol esterification rate with a sigmoidal response curve. Lipoprotein‐X1 increased the initial cholesterol esterification rate also but to a lesser extent.Our findings indicate that incubation of normal plasma with lipoprotein‐X results in the disappearance of this abnormal lipoprotein and the appearance of a high density lipoprotein2‐like particle mediated by enzymic cholesterol acyltransfer.

Activity of inhibitors on plasmatic erythropoietin and on the renal and splenic erythropoietic factors in the rabbit.

The author has studied the action of Cleland's reagent and Koshland's reagent on the erythropoietic factors present in the kidneys and the spleen of normal rabbits, in the plasma of bled rabbits and in the mixtures obtained through incubation of normal plasma with kidney and spleen homogenates. The results show that Cleland's reagent inhibits the activity of the renal and splenic erythro-stimulant factors, whereas it has no effect upon the erythropoietin in anaemic plasma and in plasma-kidney and plasma-spleen incubation mixtures. The activity of erythropoietin, on the other hand, is inhibited by Koshland's reagent, which has no influence on the renal and splenic erythro-stimulant factors.

Preliminary evidence for a human prolactin releasing factor.

Plasma from normal human subjects was subjected to a 3-phase methanol extraction. An increase in prolactin release from rat anterior pituitary tissue in vitro obtained with increasing doses of the plasma extract, while no increase in TSH release occurred. Control studies with an extract of simulated plasma using incubation medium with human serum albumin resulted in less release than a comparable plasma extract dose. The plasma extract did not lose activity with heating to 100 degrees C for 10 min, and incubation of normal plasma at 37 degrees C for 4 h prior to methanol extraction resulted in no loss of prolactin releasing activity. The plasma extract was subjected to cascade ultra-filtration through UM-10, UM-2, and UM-0.5 filters with no significant loss of prolactin releasing activity. Finally, the extract contained no TRH immunoreactivity. These results support the existence of a heat stable, ultra-filterable circulating PRF in human plasma. The origin of this material is unknown.

Platelet Adsorptive Properties and Platelet Extracts in Thromboplastin Generation

Abstract (1) Simple incubation of normal platelets in saline for 10 minutes at 37 C. markedly diminishes their activity in the thromboplastin generation test. This loss of activity is due to the removel of a factor(s) from the platelet and is present in the saline extract. (2) These "attenuated" platelets retain their effect on the recalcification time, prothrombin consumption, and clotting time of whole blood. (3) Attenuated platelets, as well as platelets thought to be qualitatively defective, regain normal activity in the TGT after incubation in normal plasma or in plasmas from patients with qualitative platelet defects. (4) Attenuated platelets, in contrast to qualitatively defective platelets, are restored to normal, as measured by the TGT, after treatment with saline extracts of normal platelets. Attenuated platelets, however, do not function normally after incubation with saline extracts of qualitatively defective platelets. (5) The possible mechanisms involved are discussed, and it is concluded that the phenomenon involves the adsorption of plasma factor(s) by the platelet. The nature of the plasma factor(s) is not known.