Collagen Suspension Research Articles

Characteristics of the aggregation of platelets and the formation of prostaglandins (PGs) in rat

Unlike human platelets, arachidonic acid (AA) at 1 m m could not induce aggregation of citrated rat platelets. However, preincubation of platelet rich plasma (PRP) with AA (1 m m) enhanced the aggregation induced by collagen suspension or adenosine diphosphate (ADP), and this enhancement was abolished by the preincubation of PRP with indomethacin. Arachidonic acid at a higher concentration (6 m m) induced aggregation, but it was not attenuated by the preincubation of indomethacin or imidazole indicating that this aggregation may not be augmented by PGs or their intermediates. Preincubation of PRP with imidazole neither abolished the enhancement of ADP-induced or collagen-induced aggregation by AA (1 m m), nor attenuated the aggregation induced by ADP or collagen alone; but it inhibited the synthesis of thromboxane (TX) by more than 90%. This suggested that rat platelets are not as sensitive to aggregation by TXA 2 as human platelets. Preincubation of mepacrine hydrochloride (0.5 m m) with PRP prevented the induction of aggregation by ADP or collagen, but it did not inhibit the aggregation by AA (6 m m). Mepacrine also inhibited the formation of PGs and TXA 2. Unlike citrated platelets, arachidonic acid even at 0.25 m m induced aggregation of heparinized rat platelets, implying that the aggregation of rat platelet is more dependent on calcium concentration than human platelets.

Ultrasonic propagation properties of collagen

Ultrasonic absorption measurements in the range 0.5-56 MHz, and measurements of velocity at 8.87 MHz, were made in collagen suspensions in which molecular integrity was preserved, in a range of concentrations 0.07-0.7% by weight, at 10 to 20 degrees C. Absorption and velocity values, per unit concentration, exhibited by collagen are much greater in magnitude than those exhibited by globular proteins.

Aggregation and inhibition of rat platelets, and the formation of endoperoxide metabolites

In citrated rat platelets, arachidonic acid (AA) at 1 mM could not induce aggregation unlike human or rabbit platelets. However, preincubation of platelet rich plasma (PRP) prepared in sodium citrate with AA (1 mM) enhanced the aggregation induced by collagen suspension or adenosine diphosphate (ADP), and this enhancement was abolished by the preincubation of PRP with indomethacin. Arachidonic acid at a higher concentration (6 mM) induced aggregation; but it was not attenuated by preincubation of indomethacin indicating that this aggregation may not be augmented by prostaglandins (PGs) or their intermediates. The major product of endoperoxide metabolites formed during collagen-induced aggregation was thromboxane A 2 (TXA 2) as measured in terms of TXB 2, the stable metabolite. Preincubation of citrated PRP with imidazole neither abolished the enhancement of ADP-induced or collagen-induced aggregation by AA (lmM), nor attenuated the aggregation induced by ADP or collagen alone; but imidazole inhibited the synthesis of TXB 2 by more than 90%. In heparinized platelets, arachidonic acid at 0.25 mM induced the aggregation, and this was inhibited by preincubation of the PRP with indomethacin. Heparinized PRP was much more sensitive to aggregating agents than citrated PRP. This implied that aggregation of rat platelets is more dependent on calcium than human or rabbit platelets. Preincubation of heparinized PRP with imidazole inhibited AA-induced aggregation, and the inhibition was abolished by the preincubation of vitamin E. This result implied that inhibition of AA-induced aggregation by imidazole is due to products of platelet lipoxygenase rather than due to the inhibition of TXA 2 synthesis.

Studies on method of collagen-induced platelet aggregation

Preparation procedures of collagen suspension in testing platelet aggregation by Born's method are presented a great deal, but there are few reports on the concentrations of collagen in detail.Recently, we are able to obtain easily a commercial reagent of collagen suspension (Collagenreagent HormR, Hormon-Chemie Munchen, ETC), and then would like to try to be standardized in its method. ETC contains 1mg per ml of native collagen fibrils from equine tendons suspended in an organic isotonic buffer of pH 2.7. Collagen suspension from bovine achilles tendons (Sigma, BTC) treated with 0.03N HCl was prepared as control, which contains the same concentration of collagen as ETC. ETC was used straight from the vial, not diluted with buffer as diluting medium.1) Each concentration of BTC and ETC requires 20μg, 10μg per ml in platelet rich plasma (PRP) at final concentration respectively, which show about fifty percent of aggregation values after five minutes adding each reagent.2) Normal ranges (mean±S. D.) of BTC —and ETC—induced aggregation were 48.6±15.6%, 52.0±12.5% each other.3) We obtained the good reproducibility of these reagents, but there was no difference between BTC and ETC in reliability. The lag times (mean±S. D.) in aggregating process were 47.8±10.2 seconds in BTC and 16.3±1.3 in ETC.4) ETC was fairly stable against heating or freezing than BTC, and no change in aggregating activity of ETC in storage at 4°C was recognized for at least a half year. In this period, we did not see any coarse fibrils of collagen.5) Examing the sensitivity of reagents to collagenase, aggregating activity of ETC was more decreased than that of BTC, and then it was suggested that ETC might be microfibrillar collagen.6) The effects of sucrose on polymerization of monomer collagen were tested and activities in these reagents were reduced with corresponding the dose of sucrose, but no difference was detectable between them.7) We investigated the sensitivities of these reagents to platelet aggregation in 13 samples from patients with aspirin or other antithrombotic drugs ingested and one patient from Glanzmann's thrombasthenia, but there was no difference in aggregation values between these reagents.These data suggest that ETC is available to standardize the test af collagen—induced platelet aggregation.

Hereditary abnormality of platelet aggregation attributable to nucleotide storage pool deficiency

Abstract An abnormality of platelet aggregation has been detected in six family members with mild bleeding tendencies. In citrated platelet-rich plasma, primary aggregation induced by ADP or epinephrine and agglutination in response to ristocetin were present but second wave aggregation and aggregation in response to collagen suspension were absent or greatly reduced. Sodium arachidonate-induced aggregation was normal although aggregation in response to prostaglandin G2 was reduced and depended entirely on the presence of plasma or ADP. Further tests indicated that the platelets produced prostaglandins but did not release ATP in response to thrombin or sodium arachidonate. Platelets from the patients were found to contain reduced amounts of ADP and 5- hydroxytryptamine and to be unable to retain radioactivity during prolonged incubation at 37 degree C with radiolabeled 5- hydroxytryptamine. Although electron microscopy revealed an absence of very dense bodies, the platelets appeared otherwise normal. The findings are discussed in relation to previous studies of nucleotide storage pool deficiency and the light they shed on platelet physiology in general.

Hereditary abnormality of platelet aggregation attributable to nucleotide storage pool deficiency

An abnormality of platelet aggregation has been detected in six family members with mild bleeding tendencies. In citrated platelet-rich plasma, primary aggregation induced by ADP or epinephrine and agglutination in response to ristocetin were present but second wave aggregation and aggregation in response to collagen suspension were absent or greatly reduced. Sodium arachidonate-induced aggregation was normal although aggregation in response to prostaglandin G2 was reduced and depended entirely on the presence of plasma or ADP. Further tests indicated that the platelets produced prostaglandins but did not release ATP in response to thrombin or sodium arachidonate. Platelets from the patients were found to contain reduced amounts of ADP and 5-hydroxytryptamine and to be unable to retain radioactivity during prolonged incubation at 37 degree C with radiolabeled 5-hydroxytryptamine. Although electron microscopy revealed an absence of very dense bodies, the platelets appeared otherwise normal. The findings are discussed in relation to previous studies of nucleotide storage pool deficiency and the light they shed on platelet physiology in general.

Transcatheter embolization with microfibrillar collagen in swine.

Transcatheter embolization of the gastrosplenic and renal arteries was performed in 7 domestic swine with suspensions of microfibrillar collagen (MFC), a recently developed topical hemostatic agent. Embolized vessels remained occluded 2 days following embolization. Recanalization occurred by 14 days, although some occlusion in distal vessel persisted at 3 months. Pathologically a marked granulomatous arteritis was noted at 14 days which resolved by 3 months. Gastric ulcers or infarcts were present in two animals. The severe vasculitis and gastric pathology observed with MFC in this study would seem to preclude its use as a general embolic agent. It may be useful, however, in the preoperative embolization of neoplasms, especially in pediatric patients since its physical characteristics allow it to be easily injected through small diameter catheters.

Platelet aggregability in chronic arterial occlusive diseases

It is generally accepted that platelets play an important and primary role in arterial thrombosis. Present study was carried out to clarify the platelet aggregability in patients with Buerger's disease (TAO) and Arteriosclerosis obliterans (ASO).Sixty male cases with TAO aged 25-65 (average and SD: 43.2±9.7), 60 male cases with ASO aged 46-75 (62.5±8.1), and 50 healthy males aged 28-70 (48.5±12.0) were studied with optical density method.According to the clinical course of the disease, the patients of each group were further classified into 4 stages: stationary, improving, chronically aggravating, and acute occlusive stages in ASO or acute stage of thrombophlebitis migrans (TPm) in TAO.Four concentrations of ADP and three concentrations of collagen suspension were used to induce aggregation. The maximal aggregation rate (MAR, described as percentage of maximal transmittance change of PRP in 5 minutes after adding the aggregating agents to that of PPP) and the appearance rate of the second aggregation at each ADP concentration were employed as the parameters of aggregability.In patients with TAO in the aggravating and stationary stages, reduced aggregability was found at the lower concentrations of aggregating agents. In the improving stage the aggregability was almost normal and significantly enhanced in the TPm stage.In patients with ASO in the stationary and improving stages, enhanced aggregability was found, and it was the most marked in the acute occlusive stage. In the aggravating stage, the aggregability was normal.In the chronically aggravating stage, chronic narrowing of arterial lumen is suspeited to derelope probably due to repeated thrombus formation and its partial dissolution and partial organization. In the improving stage such a process does not take place and the development of the collateral circulations or recanalization of the thrombosed arteries lead to the improv ement of the peripheral circulation. This difference between the two stages may account for the difference of the platelet aggregability, reduced in the aggravating stage and maintained normal in the improving stage. This relative difference of the platelet aggregability between the two stages were found not only in patients with TAO but also with ASO.It was of interest that in patients with TAO the platelet aggregability in the stationary stage was the same as in the aggravating stage. This result lead to the speculation that in patients with TAO there exists even in the stationary stage a certain change similar to that in the aggravating stage.In patients with ASO enhanced platelet aggregability was noticed in the stationary stage. This result cannot be explained only by the effect of aging and the contribution of some plasma factors is probable.In the acute occlusive stage in ASO and the TPm stage in TAO, markedly enhanced platelet aggregability was observed. This may be explained partially by the relatively increased number of young platelets in the circulating blood secondary to the rapid platelet consumption by the acute thrombus formation. Considering that the patients in such an acute stage are in a stressed condition, many other unknown factors might contribute to the enhanced platelet aggregability.

Platelet adherence to collagen: A simple, reproducible, quantitative method for its measurement

A simple, reproducible method for quantitating platelet adherence to collagen is described. Platelet rich plasma is stirred with a stable collagen suspension, non-adherent platelets counted by Coulter counter, and percent adherence calculated. Platelet aggregation is prevented by EDTA. Platelet adherence was found to reach a maximum value within 5 minutes and also reached a maximum value with increasing stirring speed and collagen concentration. It fell exponentially with increasing platelet count although the number of adherent platelets increased to an apparent maximum suggesting saturation of collagen binding sites. Percent platelet adherence fell linearly when pH was increased. At physiologic pH a non-adherent platelet population of approximately 25% was present. Platelets of different sizes adhered to a similar extent suggesting that platelet age may not influence adherence. A relatively narrow range for normal percent adherence was established and serial determinations in a single individual showed only limited variability with time. By changing either the platelet count or collagen concentration adherence can be varied from < 20% to 70%. The technique is sufficiently versatile that it should be useful for studying many aspects of the platelet/collagen adhesion reaction.

The investigation of collagen induced platelet aggregation by the screen filtration pressure technique.

A modification of the screen filtration pressure technique has been developed to allow platelet response to collagen to be measured. Using a commercial collagen suspension this method has been found to be both simple to perform and to give highly reproducible results. Changes in platelet response to collagen after administration of acetylsalicyclic acid were clearly detected and this suggests that collagen screen filtration pressure may provide a sensitive method for evaluating the effect of platelet release inhibiting drugs.

Some physical properties of the pulmonary arterial bed deduced from pulsatile arterial flow and pressure.

This study aimed to quantify changes of vascular compliance and resistance of the proximal and the peripheral pulmonary arterial vessels when vascular smooth muscle was stimulated. These above vascular characteristics were derived from registrations of pulsatile pressure and flow in the pulmonary artery (PA). An in situ cat lung preparation was used, with the right heart by-passed by a pulsatile blood pump. Vascular input impedance was derived from PA pulsatile pressure and flow recordings, and impedance characteristics were used for calculation of the variables of a simple lumped analog representation of the arterial bed. PA smooth muscle was stimulated by infusions of collagen suspension, by general hypoxia and by noradrenaline injections. Collagen caused 40% reduction of vascular compliance (C), no changes in proximal arterial resistance (R1) and 180% increase in peripheral vascular resistance (R2). Hypoxia caused 50% reduced C, 20% increased R1 and 7u% increased R2. Noradrenaline caused 20% reduced C and 30% increased R1 and R2. These results, together with results derived from simulation of the observed impedance changes in a computer model of the lung arterial bed, indicated that collagen infusion elicited contraction of small and medium-sized arteries, with increased arterial volume as result of increased distending pressure. Hypoxia and noradrenaline, seemed both to cause contraction of the total arterial bed. This effect being most pronounced during hypoxia.

Collagen suspension for platelet aggregation test

Collagen-induced pletelet aggregation in Born's method is very important in hemostatic tests, and many reports of this test have been presented. There are only few reports which were described about the concentration of collagen. But very high dose of collagen were used in these reports. The reasons are suspected it is difficult to make small particle of collagen fiber and collagen suspension is not so stable.In this report, the method how to make stable collagen suspension containing small sized collagen fiber is presented, and minor changes in collagen-induced platelet aggregation of hemorrhagic patients are detected by using this collagen suspension.One hundred mg of bovine Achilles tendon collagen (Sigma Chem. Co., St. Louis, Mo. U. S. A.) (lot No., 92C-8060, 53C-8140) in immersed in 50ml of 0.03N. HCl and homogenized with “Virtis Homogenizer 45” (Virtis Co., U. S. A., flask; 16-207, blade; 16-108), at 10, 000rpm, for 40 minutes, in ice bath. The homogenized collagen suspension had milky colour and high viscosity. This suspension was stored in plastic tubes and in refrigerator until use. This suspension was diluted in plastic tube with 0.003N. HCl in normal saline, pH was about 3.3. The concentration of collagen was decided by absorbance at O. D. 280nm in spectrophotometer. Scince the diluted collagen suspension which had same value of 100μg/ml of standard bovine serum albumin solution in Lowry's method, showed 0.1 at O. D. 280mμ, so we called the concentration of collagen was 100ug/ml.The final concentration of 2μg/ml this collagen suspension was able to induced platelet aggregation completly. But the final concentration of 3μg/ml of collagen suspension was seemed to be safety range, and the final concentration of this collagen suspension was enough dose to induce platelet aggregation.Two female hemorrhagic patients, only purpura of legs, had normal values in hemostatic tests except slightly prolonged bleeding time, decreased platelet adhesiveness and slight decrease in ADP-induced platelet aggregation, showed remarkable decrease of collagen-induced platelet aggregation in the final concentration of 3μg/ml. However one of these patients was normal in 10μg/ml collagen-induced platelet aggregation.We think this stable and small sized collagen fiber suspension is useful for routine collagen-induced platelet aggregation test in Born's method, because this collagen suspension is homogenious and has high reproducibility, then low concentration of collagen can detect the minor change of platelet aggregability.

A technique for the evaluation of platelet-collagen adhesiveness in vitro.

A method for the evaluation in vitro of platelet-collagen adhesiveness has been developed. This method is based on counting platelets both before and after their contact with an insoluble collagen (IC) suspension. The results are expressed as adhesiveness index (AI). A number of variables which could influence the test has been evaluated and standardized in a way suitable for the routinary use of the technique described.

Newborn Platelet Dysfunction: a Storage Pool and Release Defect

Per cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to highdose ADP (20 muM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 muM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns' PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns' platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.

The effect of dextran on collagen-induced platelet aggregation in vitro

Dextran was shown to potentiate platelet aggregation induced by acid soluble collagen in vitro . Both the extent of aggregation and the lag time were influenced by dextran. In contrast, platelet aggregation induced by suspensions of insoluble collagen was not affected by dextran. Further experiments using a turbidimetric technique demonstrated that the rate of precipitation of collagen fibres from tropocollagen was enhanced in the presence of dextran. This effect could be demonstrated both in salt solutions and in plasma. It is proposed that the potentiation by dextran on platelet aggregation induced by acid soluble collagen can be explained by the increased rate of collagen fibre formation in the presence of dextran.

Essential Athrombia

A constitutional platelet function disorder in a twelve year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported. Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1-4 MUM) although a small wave of primary aggregation was obtained by very large ADP concentrations (25-50 muM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Release of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal. The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of "essential athrombia."

CARDIOPULMONARY DYSFUNCTIONS CAUSED BY INTRAVENOUS COLLAGEN-INDUCED RELEASE OF ADP AND ATP FROM PLATELETS

Bovine tendon collagen suspension (4.5 mg/kg) was injected rapidly into the femoral vein of 14 normal (untreated) and 8 busulfan treated rats. Trasient effects included decreased platelet counts and arterial PO2, increased central venous pressure, apnea, bradycardia and variable A-V block. These findings were most prominent within 1 minute after injection and subsided or disappeared by 10 minutes. During this period, ADP and ATP in platelet-free plasma from carotid arterial blood were measured in a liquid scientillation counter using the firefly luciferase assay. In normal (untreated) rats, collagen injection was followed by increases in plasma ADP and ATP, a rise in plasma hemoglobin and minimal changes of fibrinogen and hematcrit. Pathological observations indicated the platelet emboli in pulmonary vessels. In contrast, rats made thrombocytopenic by intraperitoneally infected busulfan prior to collagen injection had minimal or no change in platelet count, plasma ADP, ATP, hemoglobin, fibrinogen, or cardiopulmonary functions following collagen injection. These findings suggest that collagen injection causes release of ADP and ATP from platelets; released ADP induces platelets to form aggregates which lodge in the coronary and pulmonary microcirculations and elsewhere, resulting in thrombocytopenia and the cardiopulmonary dysfunction, in the presence of shear, of red cells with vessel surfaces altered by platelet aggregates.

Evaluation of a quantitative platelet-collagen adhesiveness test system

A method is described for the quantitative measurement of the blood platelet-collagen reaction, in which the adhesiveness of blood platelets is measured with a standard collagen suspension. Though glass has been used in many blood platelet adhesiveness tests, collagen fibers are a more biological substrate. The test, as developed, involves the mixing of a pre-determined volume of a standard collagen suspension (that which produces ∼ 50% adhesion in normal PRP) to an aliquot of platelet-rich plasma (platelet count adjusted to 400,000/mm 3), shaking with a vortex shaker for 5 seconds, and fixing immediately with glutaraldehyde fixative. The mixture is then centrifuged at 100 × g and a platelet count determined on the supernatant. Percent decrease in platelet count over blank is used as the platelet adhesiveness index. If the PRP-collagen mixture is shaken for 5 seconds but not fixed with glutaraldehyde for 30 seconds, platelet aggregation also occurs. This decreases the platelet count even further; such a measurement can be used to distinguish conditions or agents that affect aggregation but not adhesion. The method is illustrated by determinations of the effects of chlorpromazine and EDTA in vitro and aspirin in vivo , and the effect of dialysis on the adhesiveness index and platelet aggregation in uremic patients. The results indicated that chlorpromzine inhibited adhesion while EDTA did not, but EDTA inhibited the subsequent aggregation. Aspirin did not inhibit adhesion, but inhibited aggregation. The uremic patients had a very low adhesiveness index before dialysis. Dialysis improved the adhesiveness index, but it still was lower than normal. This method could be valuable both clinically and preclinically to test compounds for pharmacologic activity.

Effect of temperature on platelet interaction with subendothelium exposed to flowing blood.

Platelet interaction with subendothelium exposed to whole blood in an annular perfusion chamber was investigated at 37, 20 and 4 °C. Decrease in temperature caused a significant decrease in the coverage of the subendothelial surface by platelets as well as in the formation of platelet microthrombi. In contrast, the total percentage of platelets in their initial state of attachment (contact) was increased. The corresponding decrease in platelets spread onto the surface (adhesion) suggested that the transition reaction from platelet contact to adhesion was significantly diminished by the reduction in temperature. Results of aggregation experiments in platelet-rich plasma with adenosine diphosphate and collagen suspensions were consistent with a decreased adhesion reaction to collagen.Mass transfer theory was used to compare platelet deposition results at 37 and 20 °C and suggested on of the following: (1) a platelet diffusivity in whole blood which was significantly more sensitive to temperature than the diffusibility of molecular solutes or (2) a significant decrease in the rate of the platelet attachment reaction at 20 °C.

Blood Coagulation and Platelet-function Parameters before and during Parenteral Administration of Medroxyprogesterone Acetate as a Contraceptive Agent

SummaryThe effects of medroxyprogesterone acetate on blood coagulation and platelet collagen aggregation were studied in nineteen women. After parenteral administration of the compound for 4 and 7 months, the activated partial thromboplastin time showed a significant decrease. The percentage of the prothrombin time test rose after 4 months, but not significantly after 7 months ; factor II decreased after 7 months of treatment. The reaction time during which no aggregation occurred after addition of a collagen suspension to a stirred platelet-rich plasma, was shorter after both 4 and 7 months of treatment. Other coagulation and platelet-function parameters were not altered. Although a trend toward hypercoagulability was detected, the changes were not the same as those reported for combined oral contraceptives.