Non-anticoagulated Blood Research Articles

Nonionic contrast agents and coagulation.

The chemotoxicity of nonionic and ionic contrast media is discussed with reference to chemical structure. Second-generation nonionic contrast media have the lowest chemotoxicity of any agents because of the uniform distribution of hydrophilic groups around the molecule, which mask the hydrophobic iodine atoms. Previous investigations show that second-generation nonionic agents have the same qualitative hematologic effects as other contrast agents. The less marked quantitative hematologic effects of second-generation contrast media compared with conventional agents are consistent with the low toxicity profile of these agents. A study was conducted in which a nonionic and a conventional ionic contrast medium were observed on microscopic slides after small drops of nonanticoagulated blood were added to each. The disordered aggregations of red cells observed were larger in the nonionic agent but easily dispersed with minimal disturbance, suggesting limited clinical significance. A series of coagulation experiments was conducted in which two types of plastic syringes and glass syringes, containing either iohexol or diatrizoate, were contaminated with blood and inspected for blood clots. An ELISA assay for fibrinopeptide A also was performed. Clot formation was uncommon and appeared to be related to syringe material.

Experimental basis for the use of red cell transfusion in the management of anemic-thrombocytopenic patients.

The Baumgartner perfusion technique was used as an experimental model to study the combined influence of red cell (RBC) and platelet counts on the interaction of platelets with the subendothelium. At normal hematocrit and a platelet count of 100,000 per microliter, platelet adhesion and platelet aggregate (PAG) formation on subendothelium were statistically decreased. At lower platelet counts (50,000/microliter), there was an even more marked reduction in the formation of PAGs. The critical role of RBCs was demonstrated in experiments at low hematocrit; the formation of PAGs was impaired in perfusions at 20 percent hematocrit at any platelet count tested. Platelet deposition on subendothelium was almost absent at 50,000 platelets per microliter, suggesting a negative synergistic effect for the association of low hematocrit (20%) with a low platelet count. Perfusion experiments carried out with nonanticoagulated blood drawn directly from anemic patients with mild thrombocytopenia (43,000-58,000 platelets/microliter) before and after RBC transfusion were in agreement with previous experiments that indicated that normalization of both platelet count and hematocrit is required to achieve optimum hemostasis. Our data give experimental support for the transfusional management of patients with anemia and thrombocytopenia.

Altered in vitro hemostasis, clotting, and thrombolysis after oral nifedipine in normal volunteers.

In this study the authors used the Haemostatometer, a new instrument to monitor the pattern of hemostatic plug formation that occurs in holes in polyethylene tubing through which nonanticoagulated blood is flowing under standard conditions. The pattern and speed of blood coagulation, subsequent to hemostasis, was also monitored. Simultaneously, the time until expulsion of hemostatic plugs formed was measured and considered as spontaneous thrombolysis time (STT). In 10 healthy volunteers, blood samples were drawn and tested before and ninety minutes after administration of 10 mg oral nifedipine. After nifedipine, the initial phase of primary hemostasis was delayed (p less than 0.05), the clotting time lengthened (p less than 0.01), and the STT shortened (p less than 0.01). The authors conclude that the effect of nifedipine on hemostasis and thrombolysis could contribute to its therapeutic efficacy.

Citrate toxicity during massive blood transfusion.

THE USE OF sodium citrate as the blood anticoagulant for transfusion science dates from 1914 to 1915 and the almost simultaneous publication of the work of four independent investigators. Articles from Hustin,l Agote,2 Weil,3 and Lewisohn,4 published between May 1914 and January 1915, each reported the successful use of citrated blood for human transfusion. Because citrate was known to be toxic to animals, Lewisohn carefully titrated the minimum concentration of citrate required to prevent clotting. Despite the demonstration by Weil that citrated blood could be stored for several days and still be effective,3 and the discovery by Rous and TurnerS that citrated blood supplemented by dextrose was capable of more prolonged storage, citrated blood was not quickly accepted by the general medical community. Though used with success in limited trials on the battlefield in World War 1,6 transfusions with citrated blood were often associated with chills and fever which were incorrectly attributed to the citrate. Febrile reactions were so common with citrated blood that during the 1920s most transfusions consisted of the rapid transfers of nonanticoagulated whole blood, and the use of stored citrated blood did not become commonplace until the mid-1930s. 7 While improvements in transfusion technology and the establishment of blood banks made the administration of blood a standard procedure in the operating room, blood usage was generally limited to a few units for any given patient. Advances in knowledge of the biochemistry of citrate and calcium led to an improved understanding of their interaction as well as the relationship of serum ionized Ca + + to total serum calcium. The development of citrate toxicity due to acute hypocalcemia was demonstrated in dogs as early as 1944. After Wodd War II surgical techniques of increas-

Excessive Deposition of Fibrin, Platelets and Platelet Thrombi on Vascular Subendothelium During Contraceptive Drug Treatment

We investigated the effect of oral contraceptives on thrombogenesis induced by subendothelium of rabbit aorta (SE), exposed to flowing non-anticoagulated blood in an annular flow chamber. Six healthy women on sequential contraceptive drugs (0.05 mg aethinylestradiol/day, 0.125 mg desogestrel/day) were compared with 6 women without hormonal contraception and 6 men. On contraceptive drug treatment, blood values were significantly increased for fibrinogen (2.6 +/- 0.2 vs 1.9 +/- 0.1 g/l) and fibrinopeptide A (3.9 +/- 0.9 vs 0.9 +/- 0.1 ng/ml), whereas antithrombin III was decreased (81 +/- 4 vs 97 +/- 6%). Fibrin deposition on vascular subendothelium was more than four-fold increased when measured morphologically (63.4 +/- 2.5 vs 14.6 +/- 6.8% coverage of SE surface with fibrin) as well as immunologically (29.3 +/- 2.2 vs 4.5 +/- 1.9 micrograms fibrin/cm2 of SE). Thrombus volumes were more than two-fold increased in women with contraceptives (9.0 +/- 1.4 vs 3.7 +/- 1.0 micron 3/micron 2). Our study shows that during contraceptive drug treatment the exposure of flowing blood to vascular subendothelium leads to excessive deposition of fibrin and platelet thrombi. Measurement of blood interactions with subendothelium might be of predictive value in hypercoagulable states such as contraceptive treatment.

Role of shear rate and platelets in promoting fibrin formation on rabbit subendothelium. Studies utilizing patients with quantitative and qualitative platelet defects.

The deposition of platelets on subendothelium of rabbit aortic segments exposed to non-anticoagulated human blood increased progressively with increasing wall shear rates (50-2,600 s-1), whereas fibrin deposition decreased. Studies in normal subjects and patients with platelet disorders suggested that, under the conditions used, platelets were essential for fibrin deposition at intermediate (650 s-1) but not low (50 s-1) shear rates. Fibrin deposition was markedly diminished in a patient with Scott syndrome whose platelets have a diminished capacity to bind Factor Xa and activate Factors IX and II. In glycoprotein IIb-IIIa deficiency, fibrin deposition was normal (or somewhat increased), whereas in glycoprotein Ib deficiency the association of fibrin with platelets, but not subendothelium, was decreased. The findings indicate that platelets, perhaps through surface localization of coagulation proteins, promote fibrin deposition on subendothelium at arterial shear rates and suggest that agents directed against platelet coagulant properties could be antithrombotic.

Preparation of washed platelets from non-anti coagulated human blood

Sodium citrate is almost always used to anticoagulate blood for the preparation of washed platelet suspensions. Several adverse effects of citrate on platelet functional responses have been reported. We investigated the extent of activation of platelets and plasmatic coagulation during the preparation of washed platelets from human blood to which no anticoagulant was added. Washed platelets from native blood (PNB) were prepared by passing freshly-drawn human blood rapidly through a mixed Sephadex G-25/G-50 column to remove divalent cations. Gel-filtered blood (GFB), diluted by the elution medium containing 0.35% albumin and 2U/ml apyrase, was obtained within 5 minutes of vene-puncture. Using CaCI 2, the GFB was found to contain a mean of 1.65 x 10 mM calcium. Fibrinopeptide A measurements indicated less activation of plasmatic coagulation in GFB than in citrated blood. Measurements of β-thromboglobulin and platelet factor 4 during the various stages of the preparation of PNB showed no platelet activation. No platelet aggregates, as measured by the platelet aggregate ratio method, were observed in GFB. Transmission electron microscopy showed intact discoid platelets similar to those in platelet-rich plasma. The reduced activation of platelets and of plasmatic coagulation was due to the more effective removal of calcium ions from the blood by gel filtration than chelation by citrate. PNB may thus provide a model for studying the requirements for calcium in platelet function in the absence of any citrate-platelet interactions.

Platelet Adhesion and Thrombus Formation on Subendothelium in Platelets Deficient in Glycoproteins IIb-IIIa, Ib, and Storage Granules

Abstract Patients whose platelets are deficient in glycoprotein (GP) Ib, IIb- IIIa (thrombasthenia), or granule substances (storage pool deficiency, SPD) were studied to define further the properties of platelets that mediate platelet adhesion and thrombus formation on subendothelium. Both nonanticoagulated and citrated blood were exposed to everted, de- endothelialized rabbit vessel segments under controlled flow conditions and shear rates varying from 650 to 3,300 sec-1. Morphometry was used to measure platelet thrombus dimensions and the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets. Adhesion was defined as C + S. The results in SPD demonstrated (1) reduced thrombus dimensions in delta-SPD (pure dense granule deficiency) in proportion to the magnitude of the dense granule defect; (2) an even greater reduction in thrombus dimensions in patients with combined deficiencies of alpha and dense granules (alpha delta-SPD); and (3) impaired platelet adhesion at several conditions in alpha delta-SPD and, in delta-SPD, a hematocrit-dependent impairment of adhesion in citrated blood at 2,600 sec-1. In thrombasthenia, platelets were present as a monolayer on the subendothelial surface in both nonanticoagulated and citrated blood, indicating an absolute requirement for GPIIb-IIIa in promoting platelet-platelet interaction at all shear rates and perfusion times. Two types of abnormalities in platelet-vessel wall interactions were observed. In nonanticoagulated blood, the percentage of platelets in the C phase was consistently increased at all shear rates, but C + S values were normal. These observations indicate that platelets deficient in GPIIb-IIIa do not spread normally on the subendothelial surface exposed to nonanticoagulated blood. With citrated blood, the C + S value in thrombasthenia was reduced at both 800 and 2,600 sec-1, as in von Willebrand's disease, and a similar degree of reduction (about 50%) was observed in normal blood treated with a monoclonal antibody to GPIIb- IIIa. The findings, together with theoretical considerations, are consistent with an hypothesis that GPIIb-IIIa mediates the spreading of platelets on subendothelium following the initial attachment through GPIb and that GPIIb-IIIa may be considered an adhesion site on the platelet membrane. Abnormalities of GPIIb-IIIa may, depending on the conditions of study, result in either increased values of C platelets or decreased values of C + S. The results of the study further suggest that a complex interaction of platelet granule factors and membrane GP mediate platelet adhesion and thrombus formation.

A new, ideal technique to monitor thrombolytic therapy.

In an earlier article we have reported a technique which allows the measurement of haemostasis from whole blood by the "tube bleeding" principle. The technique is now progressed by using non-anticoagulated blood and therefore clotting-time is also measured. We report here that the technique allows a simple thrombolytic assay to perform from the same blood sample used for haemostasis measurement by detecting the length of time until the haemostatic plugs crack or are expelled. Evidence is presented that this "spontaneous" expulsion is mainly due to thrombolysis. Generation of thrombin during haemostasis is indicated by the inhibitory effect of hirudin. 125I-fibrinogen accumulates (28-fold) in the haemostatic plugs. Plasminogen activators (streptokinase and urokinase) reduce while inactivation of plasminogen by specific antibody greatly prolongs the expulsion-time. The effect of cytochalasin B shows that the retraction of the haemostatic plug is not a decisive factor of the expulsion. As the technique allows the simultaneous measurement of haemostasis, thrombolysis and clotting, it seems ideal for monitoring thrombolytic therapy.

Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules

Patients whose platelets are deficient in glycoprotein (GP) Ib, IIb-IIIa (thrombasthenia), or granule substances (storage pool deficiency, SPD) were studied to define further the properties of platelets that mediate platelet adhesion and thrombus formation on subendothelium. Both nonanticoagulated and citrated blood were exposed to everted, de-endothelialized rabbit vessel segments under controlled flow conditions and shear rates varying from 650 to 3,300 sec-1. Morphometry was used to measure platelet thrombus dimensions and the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets. Adhesion was defined as C + S. The results in SPD demonstrated (1) reduced thrombus dimensions in delta-SPD (pure dense granule deficiency) in proportion to the magnitude of the dense granule defect; (2) an even greater reduction in thrombus dimensions in patients with combined deficiencies of alpha and dense granules (alpha delta-SPD); and (3) impaired platelet adhesion at several conditions in alpha delta-SPD and, in delta-SPD, a hematocrit-dependent impairment of adhesion in citrated blood at 2,600 sec-1. In thrombasthenia, platelets were present as a monolayer on the subendothelial surface in both nonanticoagulated and citrated blood, indicating an absolute requirement for GPIIb-IIIa in promoting platelet-platelet interaction at all shear rates and perfusion times. Two types of abnormalities in platelet-vessel wall interactions were observed. In nonanticoagulated blood, the percentage of platelets in the C phase was consistently increased at all shear rates, but C + S values were normal. These observations indicate that platelets deficient in GPIIb-IIIa do not spread normally on the subendothelial surface exposed to nonanticoagulated blood. With citrated blood, the C + S value in thrombasthenia was reduced at both 800 and 2,600 sec-1, as in von Willebrand's disease, and a similar degree of reduction (about 50%) was observed in normal blood treated with a monoclonal antibody to GPIIb-IIIa. The findings, together with theoretical considerations, are consistent with an hypothesis that GPIIb-IIIa mediates the spreading of platelets on subendothelium following the initial attachment through GPIb and that GPIIb-IIIa may be considered an adhesion site on the platelet membrane. Abnormalities of GPIIb-IIIa may, depending on the conditions of study, result in either increased values of C platelets or decreased values of C + S. The results of the study further suggest that a complex interaction of platelet granule factors and membrane GP mediate platelet adhesion and thrombus formation.

Aggregation of Washed Platelets from Non-Anticoagulated Human Blood Is Not Reversible

Most of the knowledge acquired on platelet function and biochemistry has been obtained from platelets prepared from blood anticoagulated with sodium citrate. Using washed platelets from human blood (PNB) to which no anticoagulants were added, we report on responses not observed with platelets prepared from citrate-anticoagulated blood. Native blood was passed rapidly (within 5 min of venepuncture) through a Sephadex G-25/G-50 column to remove divalent ions and thus prevent coagulation. Platelets were separated from the gel-filtered blood by differential centrifugation. Responses of PNB to thrombin, collagen, calcium ionophore, ristocetin, release of 14C-5hydroxytryptamine and beta-thromboglobulin, and generation of thromboxane A2 were similar to those observed for citrated platelets. Comparison of PNB with thrombin-treated platelets, which demonstrate an increase of platelet factor 3 activity, a reduction of the adenylate energy charge and an impairment of clot retraction, indicated the absence of platelet activation. Unlike citrated platelets, however, aggregation of PNB in response to ADP was irreversible in the presence of Ca2+ and fibrinogen, even at concentrations as low as 0.2 microM ADP, with aggregation taking up to 25 times longer to reach the same extent of aggregation as for citrated platelets. PNB did not aggregate to epinephrine even in the presence of Ca2+ and fibrinogen. Sodium citrate impaired ADP-induced aggregation and clot retraction of PNB. Thus citrate affects platelet function and may cause changes resulting in the unphysiological behaviour and responses of platelets.

Protraction of whole blood and plasma clot lysis in patients with high levels of an inhibitor of tissue-type plasminogen activator

The influence of the newly discovered, fast-acting inhibitor of tissue-type plasminogen activator (t-PA) on the lysis time of plasma clots was studied by visual observation of lysis of clotted citrated plasma after addition of purified t-PA. To a series of plasma samples with various concentrations of naturally occurring PA-inhibitor purified t-PA was added to a final concentration, which in pooled normal plasma is sufficient to induce clot lysis within a few hours. In those plasma samples with a high free inhibitor level, determined by measuring the recovery of the activity of added purified t-PA, clot lysis was retarded. Whole blood clots were made by clotting freshly collected non-anticoagulated blood with thrombin after admixture of a trace amount of radiolabeled fibrinogen and a fixed amount of t-PA. Lysis rate, read from the appearance of radioactivity in the serum after centrifugation, was significantly lower in clots obtained from subjects with a high free inhibitor level than in those with a low inhibitor level. It is concluded that the PA-inhibitor protracts clot lysis and may be relevant for physiological fibrinolysis.

Thrombogenetic studies of bioprosthetic heart valve surfaces I. In vitro platelet adhesion in a static system

Contact angle measurements, histological examinations and platelet adhesion tests on native and glutaraldehyde-treated porcine aortic valve leaflets, are reported. Contact angles measured with water or saline on native and treated leaflets do not differ appreciably. Values of less than 10° for both the dry and wet leaflets were observed. Histological examination of the native and fixed tissue surfaces was carried out with both optical and scanning electron microscopy. Native leaflet surfaces appeared to be smoother with longer and straighter fibres beneath the surface than treated leaflets. Platelet adhesion tests were performed with fresh, non-anticoagulated human whole blood as well as with platelet suspension. Static platelet adhesion tests were carried out by depositing droplets of blood on the test surface. The blood droplets were washed off after a contact time of 2 minutes for whole blood and up to 32 minutes for platelet suspension. The adhering platelets were microscopically observed and counted. The platelet density on the native leaflets was found to be significantly higher than on the treated tissues. However, the platelet density did not vary with glutaraldehyde concentration in the range from 0.2 to 1.0% ( v v ).

Destruction of ristocetin cofactor by coagulation at 4°C

Ristocetin cofactor (VIIIR:RCo) and factor VIII-related antigen (VIIIR:Ag) were measured in anticoagulated and non-anticoagulated blood incubated at 4°C, room temperature (RT) or 37°C for 24 hours. A marked decrease in VIIIR:RCo, to almost undetectable levels, and a smaller decrease in VIIIR:Ag occurred when whole blood clotted at 4°C. These changes were slight 8r absent when blood clotted at RT or 37°C. VIIIR:RC8 lost at 4°C was not recoverable by further incubation at 37°C but the less-marked loss of VIIIR:Ag was partially recovered. In blood which had clotted at 4°C there was a change in the electrophoretic profile of VIIIR:Ag on crossed immunoelectrophoresis: there was more anodal migration of the VIIIR:Ag peak, consistent with a decrease in the mean molecular size. Further experiments showed that the decrease in VIIIR:RCo during coagulation at 4°C preceded the decrease in fibrinogen levels. In cell-free plasma VIIIR:RCo also decreased markedly when coagulation occurred at 4°C. The results show that loss of VIIIR:RCo occurs when blood is allowed to clot at 4°C: this is not due to cryoprecipitation and does not require the presence of blood cells. The data suggest that it is probably caused by plasma proteases activated early in the coagulation pathway.

Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion.

Blood interaction with the subendothelium of rabbit aorta was investigated in an annular perfusion chamber using patients with von Willebrand's disease, hemophilia, and afibrinogenemia. The vessels were exposed to nonanticoagulated blood for a range of flow conditions (wall shear rates of 650-3,300 s-1) and exposure times (1.5-10 min). The resultant platelet and fibrin interaction was quantified by the use of several morphometric techniques, one of which was developed to measure more precisely the dimensions (height and volume) of platelet thrombi attached to the subendothelium. A major finding was that under flow conditions in which little or no defect in platelet adhesion was observed in von Willebrand's disease, platelet thrombus height and volume in this disorder were significantly reduced as compared with normal controls or patients with hemophilia. Thus, Factor VIII/von Willebrand factor (VIII/VWF) may mediate not only the adhesion of platelets to subendothelium but also platelet-platelet attachments necessary for normal thrombus development. The level of Factor VIII:coagulant activity (VIII: C) was also observed to influence the resultant thrombus height and volume deposited on subendothelium, presumably through the generation of thrombin or some other procoagulant factor preceding fibrin formation, since normal values of thrombus dimensions were always observed in a patient with a fibrinogen deficiency. The influence of VIII:C became greater as shear rate was reduced, whereas as shear rate was increased, VIII/VWF was more dominant in determining the resultant platelet deposition on subendothelium. Thus, the deficiencies of VIII:C and VIII/VWF in hemophilia and von Willebrand's disease can lead to various abnormalities in platelet and fibrin association with subendothelium. The importance of a particular deficiency will depend strongly on the local blood flow conditions.

A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue.

A new method for the covalent binding of heparin to artificial surfaces has been developed. The heparinized surface releases insignificant amounts of heparin and can be regarded as stable. The blood contact properties as studied in vitro revealed that the surface was highly thromboresistant in terms of reduced platelet adhesion, surface catalyzed adsorption and inhibition of thrombin and capacity to prevent clotting of nonanticoagulated blood.

Serum-constituents analyses: effect of duration and temperature of storage of clotted blood.

We studied the effects on 25 analytes of duration of contact of serum with non-anticoagulated blood and of temperature. Serum was separated after blood was allowed to stand, for 0, 2, 4, 6, 8, 24, or 48 h at 4, 23, or 30 degrees C. Results obtained for bilirubin, albumin, zinc sulfate turbidity, thymol turbidity, cholinesterase (EC 3.1.1.8), alkaline phosphatase (EC 3.1.3.1), leucine aminopeptidase (EC 3.4.11.1), amylase (EC 3.2.1.2), total cholesterol, triglycerides, beta-lipoprotein, serum urea nitrogen, creatinine, uric acid, and gamma-glutamyltransferase (EC 2.3.2.2) were not influenced by storage at 4, 24, or 30 degrees C for as long as 48 h. Negligible differences were seen for potassium in sera in contact with cells as long as 24 h at 23 degrees C and for inorganic phosphorus after 48 h at 4 degrees C. However, at 4 degrees C we noted an increase at 8 h, a slight decrease at 30 degrees C. Statistically significant changes were seen for total protein and calcium after 48 h at 30 degrees C; for aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2), between 8 and 24 h at 23 degrees C and as soon as 6 h at 30 degrees C; for lactate dehydrogenase (EC 1.1.1.27) after 8 h at 30 degrees C and between 8 and 24 h at 23 degrees C; for glucose at 24, 4, or 2 h of storage at 4, 23, or 30 degrees C, respectively; for inorganic phosphorus after 48 h at 23 degrees C or 8 h at 30 degrees C; for potassium after 4 h at 4 degrees C or 24 h at 30 degrees C; and for sodium after 48 h at 4 degrees C or 6 h at 23 or 30 degrees C.

A study of the relationship between the platelet shape and it's function

It is widely accepted that the platelet which is originally discoid is deformed into spiny spheres by ADP, thrombin or collagen (viscous metamorphosis) and forms thrombus by its function-adhesiveness and aggregation. But we have hardly achieved the quantative analysis of the morphological changes and disclosed the relationship between the deformed platelets and their functions.In order to clarify this relationship, we examined the influences of the following procedures on human platelets.1) preservation (non-anticoagulated whole blood, citrated whole blood), 2) stirring (the same blood samples), 3) preservation after stirring (citrated whole blood) and 4) surgical operation with laparotomy on 10 patients with various diseases.After fixing the platelets by 1% glutaraldehyde, the platelets were classified into disc, hemisphere, sphere and the other form, and checked for pseudopod formation under a light microscope. As a platelet function, platelet retention rate was measured by a modified Hellem-II method.Platelet shape was easily changed and pseudopods issued by preservation and stirring. These morphological changes were brought about more strikingly by stirring than by preservation. The retension rate decreased when spherification and pseudopod formation had prominently advanced. However, it increased when the incidences of non-discoids and the pseudopods increased to some degree, e. g. when citrated blood was preserved up to 60min. at room temperature and when it was stirred for 1min. These phenomena were similarly observed in vivo condition (during operation).These results suggested that some relationship existed between the platelet shape and its function and that halfly deformed platelets were in an activated phase and prominently deformed platelets were rather in a refractory phase. This relationship was probably derived fromnot only extra-platelet factors such as activation of plasma coagulation factors (contact factor and prothrombin) but also from internal factors (or character) of platelets. The latter factors were considered to be more important because both shape change and the increase of retention rate were demonstrated in some experiments to occur before the silicon PTT became shortened.

Platelet Adhesion from Nonanticoagulated Rat Arterial Blood*

SummaryPlatelet adhesion to glass bead is measured using nonanticoagulated arterial blood from adult male rats. Parameters studied include: 1) surface area of glass exposed to platelets, 2) time of contact and rate of platelet flow over the beads, 3) differing blood-gas interface, 4) adhesion to siliconized polyvinyl surfaces, and 5) heparinization of donors. Parameters kept constant include: 1) diameter of glass beads, 2) diameter of glass bead chamber, and 3) body temperature of the donor animals. No air was mixed with the blood. Concentration of hemoglobin released into the plasma supernatant was measured.Platelet adhesion from each aliquot of each sample increases as glass surface area increases, and the time of contact between the platelets and surface increases. Adhesion did not change when nitrogen was substituted for room air at blood-gas interface. Heparinization of donor animal did not alter adhesion. Platelets adhere to siliconized polyvinyl at greater rate than to nonsiliconized polyvinyl. Hemoglobin concentration in the plasma supernate did not correlate directly with percent of platelet adhesion. These results are similar to those reported for native and anti-coagulated venous and anticoagulated arterial blood.This technique does not require in vitro handling of platelets. It can be applied to other animals and to humans.

Intravascular Hemagglutination Studied with the Living Extracorporeal Eye

Abstract Direct and filmed observations of events during intravascular hemagglutination have been made using our Living Extracorporeal Eye preparation. Agglutinated erythrocyte masses in the arterioles are separated by plasma gaps but tied to one another in short chains by fibrin threads and have a surface deposition of fibrin. This fibrin forms under circumstances that make autocatalytic coagulation unlikely. This seems to indicate that if thrombin evolution occurs, it must be localized to the areas of exposed erythrocyte stroma and adjacent endothelial cell surfaces altered by deoxygenation. These masses plug some of the microcirculatory channels and venous many-tailed casts build from multicentric sites at the exits of venules with stilled flow. The light and electron microscopic structure of such casts shows a core of aggregated erythrocytes with surface fibrin and platelet deposition. The above events are not prevented by prothrombinopenic, leukopenic or thrombocytopenic reacting bloods but are abolished by resuspending from each of the reacting bloods, the autologous erythrocytes, leukocytes, and platelets in the supernate of its centrifuged heat-defibrinated plasma. Hydrocortisone, benadryl and 6 per cent clinical dextran do not alter the reaction. When calcium ion binding agents are also present, heparin abolishes the fibrin formation and sharply reduces the magnitude of erythrocyte aggregates so that vigorous perfusion of the microcirculation continues. Heparin alone is ineffective in suppressing interspecies hemagglutination and erythothrombus formation when non-anticoagulated bovine blood is intermixed with heparinized diluted human blood. The obstructing arteriolar masses and the many-tailed venous cast induced by hemagglutination, are promptly and completely disaggregated by plasma plus streptokinase or by urokinase or by 5M urea solution perfusion. Osmotically lysed erythrocytes lead to similar erythrothrombosis only when stasis is induced by interrupting arterial perfusion for a minute or more.