Localization of atherosclerotic lesions in the human basilar artery.

The topography of atherosclerotic lesions in the human basilar arteries has been studied quantitatively by digitizing images of the excised vessels and producing contour probability maps. Fifteen basilar arteries were obtained at autopsy (age--61 +/- 2; males--6, females--9; black--6, white--9), fixed in formalin, opened along the ventral aspect and stained grossly with Sudan IV to delineate fat-containing lesions. Photographs of the flattened arteries were analyzed and the presence or absence of sudanophilic lesions was determined at approximately 1000 identical sites on all vessels. The probability of finding a lesion at each site was determined and a contour probability map was constructed. Fifty-two percent of the area of the mean contour map was involved with lesions. The extent of the sudanophilic lesions decreased as one proceeded distally from the origin of the basilar artery at the confluence of the vertebral branches (i.e. proximal 1/3--56%; middle 1/3--49%; distal 1/3--43%; P less than 0.04). Significantly more sudanophilic material was observed on the ventral (outer curvature) as opposed to the dorsal (inner curvature) surfaces (55%, 43% respectively; P less than 0.03). These data suggest that hemodynamic forces associated with confluent flow and curvature may be important in the localization of sudanophilic lesions in the proximal and ventral aspects of the human basilar artery.

Platelet plug formation in an extracorporeal unit.

Platelet plugs were formed in an extracorporeal unit from flowing venous blood and studied by electron microscopy. The unit consisted of a stainless steel needle threaded into a section of silicone rubber tubing that was constricted to form a slit-like stenosis equivalent in cross-sectional area to an arteriole 100 mu in diameter. Blood was allowed to flow at a steady pressure from an antecubital vein through a collection line and the attached unit until bleeding was stopped by the formation of a platelet plug at the stenosis. Electron microscopy of the plugs showed closely packed aggregated platelets. No fibrin was detected. The formation of a stable plug in the absence of fibrin was considered a measure of the capacity of platelets in hemostasis and thrombosis to aggregate and resist the force of the blood current.

Comparative study of thrombolysis by high and low molecular weight urokinase in an in vitro perfusion system.

Two urokinase preparations of different molecular weights were standardized against the International Reference Preparation for Urokinase and compared in an in vitro whole blood perfusion system using thrombi formed with whole blood and 125I-fibrinogen. Plasminogen was added to one group and normal saline to the other. Thrombolysis, as well as plasminogen and plasmin inhibitor levels were monitored over a 60 minute period following the addition of the urokinase to the perfusion mediums. There was a high correlation as well as no significant difference found between the percents lysis caused by the high and low molecular weight urokinase. Added plasminogen resulted in a rapid decrease of plasmin inhibitors in both urokinase groups. In the saline groups this decrease was highly negatively correlated with the percent lysis. It is concluded that both high and low molecular weight urokinase behave similarly in an in vitro whole blood thrombolytic perfusion system over the time period studied.

The effect of hyperlipoproteinemia on thrombolysis: in vitro studies using purified lipoproteins from normolipemic donors.

To determine whether or not lipoproteins affect thrombolysis in vitro using Chandler's loop method, VLDL, LDL, and HDL fractions from healthy male donors were obtained by ultracentrifugation. The lipoproteins were used to enrich whole blood or fibrin thrombi radiolabeled with 125-I-fibrinogen. Lipoprotein-enriched or unenriched thrombi were perfused in rotating Chandler loops with lipoprotein-enriched or unenriched plasma, respectively. Lysis was initiated by adding high molecular weight urokinase or single chain pro-urokinase to the perfusion medium. In some experiments, plasminogen was also added. Variation in the amounts of these activators and plasminogen permitted study of the effect of lipoproteins over a range of thrombolysis. No consistent statistically significant difference in the degree or time course of lysis of lipoprotein-enriched vs. unenriched thrombi or perfusion media was found. These studies, using normal lipoproteins, do not preclude the possibility that lipoproteins from patients with type IIa, IIb, or IV hyperlipoproteinemia may be genetically abnormal or may function pathologically, resulting in an effect on thrombolysis.

Adherent platelets and surface microthrombi of the human aorta and left coronary artery: a scanning electron microscopy feasibility study.

As part of a feasibility phase of an investigator-initiated multicenter NIH supported study on the Pathobiological Determinants of Atherosclerosis in Youth (PDAY), we report observations on microthrombi and adherent platelets on the intima of the aorta and left anterior descending coronary artery. The long-term objective of this cooperative study is to define more precisely the pathogenesis of atherosclerosis during late childhood and early adulthood and to investigate the influence of selected risk factors known to be associated with clinically manifest disease in later life. Scanning electron microscopy (SEM) was utilized to survey broad areas of arterial intima. Of 109 specimens studied from 52 cases, microthrombi composed of a mixture of aggregated platelets and fibrin and measuring approximately 30-70 micron in size were observed in about 4% [corrected] of the specimens and in about 6% of the cases, while individually adherent platelets were observed in approximately 7% of the specimens and about 10% of the cases. Microthrombi and adherent platelets may be important in atherogenesis by stimulating proliferation of intimal smooth muscle cells through the release of a growth factor from platelets. This feasibility study has shown that SEM is a rapid and effective method for surveying large areas of arterial intima for the study of adherent platelets and microthrombi.

Molecular exchange between blood and in vitro thrombi.

Uptake of radioiodinated Lys-plasminogen (125I-PLG), albumin (125I-ALB), and tritiated water (3H-H2O) by in vitro thrombi and interchange of these molecules with blood in an in vitro perfusion system were investigated. The radioisotopes were taken up by thrombi either by incorporation during formation in radiolabelled blood or by perfusion of preformed nonradioactive thrombi in radiolabelled blood. Release of the radioisotopes into a perfusion medium of nonradiolabelled blood was then monitored over a 120-min period. The small molecules of 3H-H2O were rapidly released from the thrombi and achieved equilibrium with the perfusion medium by 120 min in that the specific radioactivity (cpm/mg) of the thrombi equalled that of the medium. The larger molecules of 125I-PLG and 125I-ALB were more slowly released and did not reach equilibrium with the perfusion medium over the period studied. Release of 125I-ALB was intermediate between that of 3H-H2O and 125I-PLG. The greater retention of 125I-PLG by the thrombi was consistent with the high binding affinity of plasminogen for fibrin. The demonstrated movement of these radioisotopes between medium and thrombus suggests that thrombi exist in blood in a dynamic state of flux, exhibiting a fluid exchange of molecules between the interstitial compartment of the thrombus and blood.

Kinetics of urokinase-induced thrombolysis in a biphasic in vitro system.

The role and effect of added lys-plasminogen (lys-PLG) on urokinase-induced thrombolysis in an in vitro biphasic system were investigated. The kinetics of lysis of whole blood thrombi was followed in perfusion mediums of normal plasma, PLG-deficient plasma and normal saline using a high and a low concentration of urokinase (UK). The lysis of standard whole blood thrombi in whole blood perfusion mediums to which had been added UK alone or UK plus lys-PLG was compared to whole blood thrombi enriched with lys-PLG by incorporation during thrombus formation or by adsorption during perfusion. In addition, the kinetics of lysis of PLG-deficient fibrin thrombi perfused in PLG-deficient plasma or normal saline was studied when lys-PLG had been added to the thrombus, to the perfusion medium or to both thrombus and medium. In PLG-deficient plasma from which plasmin inhibitors had not been removed, thrombolysis was minimal even at a high concentration of UK. This effect could be neutralized, and to some extent, regulated, by lys-PLG enrichment of the medium. Both PLG-incorporated and PLG-adsorbed whole blood thrombi gave initial and sustained acceleration of UK-induced lysis in comparison with standard nontreated thrombi. It is concluded that in a blood-thrombus biphasic thrombolytic system induced by UK, there is interaction between the phases, and that PLG in both phases influences thrombolysis.

Uptake of 125I-Lys-plasminogen by in vitro thrombi.

The specificity, distribution and rate of uptake of radiolabelled 125I-Lys-plasminogen by in vitro thrombi was investigated. 125I-Lys-plasminogen was added to whole blood perfusion mediums containing preformed thrombi and to whole blood prior to thrombus formation. Uptake was assessed by means of radioisotopic analysis and autoradiography. The plasminogen was taken up by thrombi during and after their formation. The largest percentage was in the fibrin component. epsilon-Aminocaproic acid-blocking experiments confirmed the specificity of plasminogen binding to fibrin. Autoradiography of the thrombi revealed plasminogen in the RBC-fibrin part and in platelet-fibrin aggregates. Plasminogen uptake and penetration into preformed thrombi were found to increase as a function of time. However, formation of thrombi from plasminogen-enriched blood was a more effective means for increasing the plasminogen content of thrombi than perfusion of preformed thrombi in a plasminogen-enriched medium, over the time period studied.

Methods for eliminating interferences in digoxin immunoassays caused by digoxin-like factors.

Endogenous, circulating digoxin-like immunoreactive factors (DLIF) are known to cross react with many antisera used in digoxin assays, complicating the quantification of serum digoxin. We have explored ways to decrease or remove the interference from DLIF in such assays. Prolonging the assay incubation from 30 to 60 min decreased apparent digoxin concentrations (attributable to DLIF) by an average of 68% in the digoxin radioimmunoassays studied. The serum protein-binding of DLIF was also exploited to remove them from serum. Ultrafiltration, performed as part of a simple centrifugation step, removed approximately 90% of the DLIF. Analytical-recovery studies with true digoxin also demonstrated ultrafiltration to be an adequate method (greater than 95% of digoxin was recovered) for routine clinical use. Heat- or acid-precipitation of protein removed DLIF less effectively. Appropriate incubation times and ultrafiltration can dramatically minimize or eliminate DLIF interference in digoxin immunoassays.

Localization of ferritin-conjugated anti-fibrin-fibrinogen in platelet aggregates produced in vitro.

A ferritin-conjugated anti-fibrin/fibrinogen was localized by means of light and electron microscopy in artificial in vitro thrombi formed in the presence of the labeled antibody, and in preformed ADP-induced platelet aggregates. The ferritin was distributed throughout the central and peripheral regions of the columns of aggregated platelets in the thrombi. In the preformed ADP aggregates, ferritin was deposited only by infiltration from surrounding plasma and was confined to the periphery of the columns. The even distribution of ferritin in the central zone of the platelet columns of the thrombi indicated a specific reaction had occurred before or during thrombus formation unrelated to infiltration of plasma. In the artificial thrombi, the ferritin-labeled antibody was localized on the surface layer of platelets and on the bridging structures composed of the combined surface layers in the narrow spaces between cohesive platelets. Vesicles and alpha granules within the platelets also were tagged. The absence of obvious fibrin between narrow interspaces and within the platelets indicated that the antibody had reacted with fibrinogen or partly polymerized fibrin at these sites. Many invaginations of the platelet membrane containing dense fibrillar material were interpreted to be alpha granules discharging their contents during the "release reaction" at the time of aggregation. This material, which was tagged by the ferritin-conjugated antibody, merged with the interplatelet bridges to suggest that released fibrinogen from within the platelet contributed to the structural bond and strengthened it. A layer of dense fibrin and altered platelets in the periphery of the columns of aggregated platelets in the artificial thrombi contained the platelets and limited further growth of the aggregates. The fibrin was thought to be derived from infiltrated plasma as well as from released intraplatelet fibrinogen. Platelet fibrinogen thus appeared to take part both in the cohesion of aggregated platelets and in the stabilization of the aggregates formed.

A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.

This report is the continuation of two earlier reports that defined human arterial intima and precursors of advanced atherosclerotic lesions in humans. This report describes the characteristic components and pathogenic mechanisms of the various advanced atherosclerotic lesions. These, with the earlier definitions of precursor lesions, led to the histological classification of human atherosclerotic lesions found in the second part of this report. The Committee on Vascular Lesions also attempted to correlate the appearance of lesions noted in clinical imaging studies with histological lesion types and corresponding clinical syndromes. In the histological classification, lesions are designated by Roman numerals, which indicate the usual sequence of lesion progression. The initial (type 1) lesion contains enough atherogenic lipoprotein to elicit an increase in macrophages and formation of scattered macrophage foam cells. As in subsequent lesion types, the changes are more marked in locations of arteries with adaptive intimal thickening. (Adaptive thickenings, which are present at constant locations in everyone from birth, do not obstruct the lumen and represent adaptations to local mechanical forces). Type II lesions consist primarily of layers of macrophage foam cells and lipid-laden smooth muscle cells and include lesions grossly designated as fatty streaks. Type III is the intermediate stage between type II and type IV (atheroma, a lesion that is potentially symptom-producing). In addition to the lipid-laden cells of type II, type III lesions contain scattered collections of extracellular lipid droplets and particles that disrupt the coherence of some intimal smooth muscle cells. This extracellular lipid is the immediate precursor of the larger, confluent, and more disruptive core of extracellular lipid that characterizes type IV lesions. Beginning around the fourth decade of life, lesions that usually have a lipid core may also contain thick layers of fibrous connective tissue (type V lesion) and/or fissure, hematoma, and thrombus (type VI lesion). Some type V lesions are largely calcified (type Vb), and some consist mainly of fibrous connective tissue and little or no accumulated lipid or calcium (type Vc).