Metabolism and Function of Human Platelets Washed by Albumin Density Gradient Separation

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to plateletrich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [¹⁴C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (ADP) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [¹⁴C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE₁ or PGD₂. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37deg;C compared with 25deg;C did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.     

Albumin Density Gradient Separation and Washing of Platelets and the Study of Platelet Coagulant Activities

Summary: Platelets were centrifuged into a specific gravity gradient made by mixing the interface between platelet rich plasma (PRP) and a 40–45% aqueous solution of bovine albumin. By this method of albumin density gradient separation (ADGS) it was possible to centrifuge platelets without disruptive squashing upon a hard surface. The separated platelets were washed in calcium-free Tyrode's solution without releasing coagulant activity. Platelet yields after multiple washings were high. Morphology and ADP-induced aggregation of platelets washed by ADGS (in contrast to platelets washed by conventional methods) were indistinguishable from those of unwashed platelets tested as PRP.
The activities of factors V and XI remained in close association with platelets after four successive washes by ADGS, whereas factors VIII, IX and XII were entirely removed.     

Platelet-dependent thrombin generation after in vitro fibrinolytic treatment.

BACKGROUND. Fibrinolytic therapy is associated with frequent rethrombosis. There is evidence of both increased coagulation and platelet activation. METHODS AND RESULTS. Platelet-rich plasma (PRP) or washed platelets were incubated with the fibrinolytic agents urokinase, recombinant tissue-type plasminogen activator (rt-PA), or plasmin at concentrations consistent with those in the plasma of patients treated for myocardial infarction. All of the fibrinolytic agents induced a more rapid generation of thrombin and decreased the clotting times of non-contact-activated PRP than in untreated PRP. This effect was not blocked by the inclusion of thrombin inhibitors during the fibrinolytic treatment. Washed platelets derived from rt-PA-treated PRP induced more rapid thrombin generation when resuspended in untreated plasma or treated plasma. Washed platelets were treated with plasmin, rt-PA, and urokinase and added to platelet-poor plasma. Platelets treated with either plasmin or rt-PA increased the ability of washed platelets to support thrombin generation, but urokinase was without significant effect. CONCLUSIONS. These results indicate not only that plasmin can cause increased platelet support of prothrombin activation but also that rt-PA in the absence of plasminogen can have a direct effect on the platelet, which increases thrombin generation.     

Hereditary Giant Platelet Syndrome

The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultrastructurally normal; they contained increased storage pools of adenine nucleotides and heparin-neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet-rich plasma was smaller than normal. Platelet coagulant activities including contact product forming activity, intrinsic factor-Xa forming activity and platelet factor 3 activity were normal or increased, but collagen-induced coagulant activity was absent whether tested in washed platelet suspensions or platelet-rich plasma. Platelet washing experiments showed decreased binding of factors V and VIII to hereditary giant platelets and no detectable factor XI in washed platelet suspensions. It is concluded that (1) the hereditary giant platelets studied lacked a binding mechanism for factors, V, VIII and XI; (2) the normal development of collagen-induced coagulant activity apparently depends upon the binding of factor XI to the platelet membrane; and (3) the defective prothrombin consumption observed in these patients may have resulted from the failure of their platelets to bind factor XI.     

Evidence that ''Activated'' Prothrombin Concentrates Enhance Platelet Coagulant Activity

The infusion of an 'activated' prothrombin concentrate in five haemophilic patients with factor VIII inhibitors and two haemophilic patients without inhibitors resulted in all cases in a shortening of the whole blood clotting time and improvement of the thromboelastographic indices of blood coagnlation. In only three of the patients was a definite shortening of the activated partial thromboplastin time observed. In all seven patients an enhancement of the platelet coagulant activity was found using a recently described method. Similarly, platelets isolated from PRP to which the 'activated' prothrombin concentrate was added at a concentration approaching the maximal concentration achieved following in vivo infusion, showed an increase in platelet coagulant activity. This enhanced platelet coagulant activity could neither be explained by an increase of the factor V level or platelet factor 3 availability on the platelets nor by adsorption of an active clotting enzyme present in 'activated' prothrombin concentrate onto the platelet surface. These findings indicate that 'activated' prothrombin concentrate enhances platelet coagulant activity by increasing the activity of the platelet factor X activator recently described. This mechanism may explain how 'activated' prothrombin concentrates by-pass factor VIII or IX inhibitors.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

Effects of Cephalothin and Penicillin G on Platelet Function in Vitro

High concentrations of cephalothin or penicillin G inhibit a number of the functions of human or rabbit platelets in citrated platelet-rich plasma (PRP) and in suspensions of washed platelets. The reactions shown to be inhibited are: ADP-induced shape change and the primary and secondary phases of aggregation and release induced by ADP or adrenaline in human citrated PRP; release and aggregation of washed human platelets exposed to collagen, thrombin, vasopressin, or the ionophore A23,187; aggregation of washed human platelets exposed to phytohaemagglutinin from Phaseolus vulgaris (PHA) or polylysine; release induced by concanavalin A or PHA in suspensions of washed platelets from rabbits; platelet adherence to a collagen-coated surface or to the damaged intimal surface of the rabbit aorta; platelet factor 3 availability; lysis of rabbit platelets by an antiserum directed against them; and clot retraction. Neither antibiotic affected serotonin-induced aggregation; a high concentration of cephalothin slightly inhibited the initial rate of serotonin uptake. Penicilloic acid showed about half the inhibitory effect of penicillin G on ADP-induced aggregation. In citrated human platelet-rich plasma, ampicillin and oxacillin inhibited ADP-induced aggregation to the same extent as similar concentrations of penicillin G; in suspensions of washed platelets, however, ampicillin was less inhibitory than penicillin G or oxacillin. Platelet ultrastructure, assessed by transmission electron microscopy, was not visibly altered. Evidence that the antibiotics become bound to platelets is the finding that platelets incubated with the antibiotics and resuspended in fresh media showed less response to aggregating agents compared with control platelets. Penicillin G and related antibiotics may be inhibitory because they coat the platelet surface. Their effects on platelet functions are probably responsible for excessive bleeding and increased bleeding times observed in patients and volunteers receiving high doses of these antibiotics.     

Activation of human platelets by immune complexes prepared with cationized human IgG

The present study shows that the ability of soluble immune complexes (IC), prepared with human IgG and rabbit IgG antibodies against human IgG, to trigger platelet activation was markedly higher for IC prepared with cationized human IgG (catIC) compared with those prepared with untreated human IgG (cIC). CatIC induced platelet aggregation and adenosine triphosphate release in washed platelets (WP), gel-filtered platelets (GFP), or platelet-rich plasma (PRP) at physiologic concentrations of platelets (3 x 10(8)/mL) and at low concentrations of catIC (1 to 30 micrograms/mL). On the contrary, under similar experimental conditions, cIC did not induce aggregation in PRP, WP, or GFP. Low aggregation responses were only observed using high concentrations of both WP (9 x 10(8)/mL) and cIC (500 micrograms/mL). Interestingly, catIC were also able to induce platelet activation under nonaggregating conditions, as evidenced by P-selectin expression. Cationized human IgG alone did not induce platelet aggregation in PRP but triggered either WP or GFP aggregation. However, the concentration needed to induce these responses, was about eightfold higher than those required for catIC. The responses induced either by catIC or cationized human IgG were completely inhibited by treatment with heparin, dextran sulphate, EDTA, prostaglandin E1, or IV3, a monoclonal antibody against the receptor II for the Fc portion of IgG (Fc gamma RII). The data presented in this study suggest that IgG charge constitutes a critical property that conditions the ability of IC to trigger platelet activation.     

Changes in Platelet Aggregation in Whole Blood,Plasma and Washed Platelets in Streptozotocin-induced Diabetic Rats: Time-dependent Change in the Antiaggregatory Activity of Diabetic Rat Plasma

Time-dependent changes in platelet aggregation in whole blood, platelet-rich plasma (PRP) and washed platelets were studied in streptozotocin-induced diabetic rats. Collagen-induced aggregation of whole blood and PRP from diabetic rats were significantly reduced within 8 weeks after induction of diabetes, although that in washed platelets were increased from 8 weeks. Plasma from diabetic rats within 8 weeks attenuated platelet aggregation, whereas diabetic plasma at 12 weeks showed no inhibitory effect. Insulin treatment normalized aggregation in whole blood and PRP and abolished the antiaggregatory activity of diabetic plasma. These results suggest the plasma antiaggregating activity appears in the early stage of diabetes, which may contribute to the hypoaggregation in whole blood and PRP. The inhibitory activity disappeared in the later stage. Plasma factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet characterized.     

Changes in Platelet Aggregation in Whole Blood, Plasma and Washed Platelets in Streptozotocin-induced Diabetic Rats: Time-dependent Change in the Antiaggregatory Activity of Diabetic Rat Plasma

Time-dependent changes in platelet aggregation in whole blood, platelet-rich plasma (PRP) and washed platelets were studied in streptozotocin-induced diabetic rats. Collagen-induced aggregation of whole blood and PRP from diabetic rats were significantly reduced within 8 weeks after induction of diabetes, although that in washed platelets were increased from 8 weeks. Plasma from diabetic rats within 8 weeks attenuated platelet aggregation, whereas diabetic plasma at 12 weeks showed no inhibitory effect. Insulin treatment normalized aggregation in whole blood and PRP and abolished the antiaggregatory activity of diabetic plasma. These results suggest the plasma antiaggregating activity appears in the early stage of diabetes, which may contribute to the hypoaggregation in whole blood and PRP. The inhibitory activity disappeared in the later stage. Plasma factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet characterized.     

血小板聚集初期形状变化与聚集的关系及其影响因素分析

目的探讨血小板聚集初期形状改变与聚集的关系,并分析其影响因素。方法制备小鼠、大鼠富血小板血浆（PRP）和人洗涤血小板,以生理盐水、贫血小板血浆（PPP）或Tyrode液调整血小板计数来建立不同的血小板体系,二磷酸腺苷（ADP）或胶原诱导聚集,光学法测定血小板形状变化指标（最大负值和达最大负值时间）以及最大聚集率。结果人的最大负值、达最大负值时间比小鼠增加（P均〈0.05）,最大负值比大鼠增加（P〈0.01）;大鼠达最大负值时间比小鼠增加（P〈0.01）。生理盐水稀释人PRP比PPP稀释人PRP最大负值减小,达最大负值时间延长（P均〈0.05）。在人PRP中,胶原所致最大负值比ADP减小（P〈0.05）,达最大负值时间增加（P〈0.01）;在人洗涤血小板中,胶原所致最大负值、达最大负值时间均比ADP增大（P均〈0.01）;ADP和胶原在洗涤血小板中比PRP中的最大负值、达最大负值时间（除应用胶原变大外）减小（P均〈0.01）。多元回归分析显示,聚集率与最大负值和达最大负值时间正相关（r分别为0.49、0.48,P均〈0.01）。结论血小板聚集初期变形促进聚集,人的比大鼠、小鼠增强,生理盐水稀释PRP和洗涤血小板减弱,在PRP中胶原比ADP所致变形减弱,在洗涤血小板中增强。     

Effect of lead and cadmium on platelet aggregation]

Two heavy metals, lead and cadmium, are frequently found as pollutants in many systems. Their effect upon platelet aggregation was investigated, both in human and rat platelet rich plasma and washed platelets. ADP-induced aggregation of human platelets was inhibited by 50%, using concentrations of free lead between 2-4 mM and free cadmium between 0.05 and 0.2 mM. Rat platelets were about ten times more sensitive to the effect of lead than human PRP. 50% inhibition of epinephrine-induced aggregation was attained at lower concentrations of metal, than the concentrations needed for ADP-induced aggregation. The effect was more apparent upon the first phase, which was lengthened, both with PRP and washed platelets. The aggregation of human and rat washed platelets by calcium was inhibited by concentrations of the metals within micromolar ranges. When A 23187 was used as the aggregating agent, the inhibition by the metals was only partial. Cysteine, at approximately tenfold concentrations, reversed the effect of the metals. Cadmium appeared more effective than lead as an inhibitor of platelet aggregation in all systems. Since only high levels of metal inhibit aggregation, more sensitive organs or systems would show alterations, due to these metals at an earlier stage and at lower concentrations.     

Binding of Factor VIII to Platelets in the Presence of Ristocetin

Ristocetin agglutinates platelets in platelet-rich plasma (PRP) containing factor VIII-related von Willebrand factor (WF). When the clumps were separated from the supernatant and resuspended in buffer, the platelets dispersed if the buffer was free of ristocetin and the PRP had been treated with aspirin or EDTA to prevent the platelet release reaction. Binding to platelets was determined by measuring WF (ristocetin cofactor assay), VIIIR-antigen (AG, by radioimmunoassay), and coagulant activity (C) in the initial plasma, the supernatant remaining after removing the clumps formed by ristocetin, and the eluate produced after resuspending the clumped platelets in buffer. No activity was bound to platelets in the absence of ristocetin. After agglutinating platelets in PRP with ristocetin, WF, AG and C activities in the supernatants were about 65% of the initial plasma values, and in the eluates about 9%. Formalin-fixed washed platelets resuspended in 1 in 3 plasma, and platelets in PRP diluted 1 in 3 also agglutinated when shaken with ristocetin. There was no difference between results with fixed and unfixed platelets. In one series of experiments, the supernatants had about 60% of the initial WF and AG activities; since these activities were the same, they may measure the same substance. In a later series, WF and C were compared; in shaken samples the supernatants had 39% of the initial WF activity and 56% of the initial C activity, and in unshaken samples the supernatants had 3% WF and 29% C. The significantly lower values for C binding suggest that this activity is located on a different molecule from WF-AG and that the molecules are more readily separated in diluted than in undiluted plasma. Activity in the eluates was inversely proportional to that in the supernatants although the initial activity was never completely recovered. WF and C binding increased with platelet number and ristocetin concentration. Platelets exposed to ADP before fixation showed less agglutination than control fixed platelets and bound less WF. Fixed platelets incubated for 15 min with 30 μg trypsin/ml and washed, or platelets from a patient with the Bernard-Soulier syndrome failed to agglutinate and bound virtually no WF, AG or C. We conclude that WF, AG and C reversibly bind to fixed or unfixed platelets in the presence of ristocetin, that binding is similar in undiluted plasma but greater for WF and AG than C in plasma diluted 1 in 3, and that ADP and trypsin alter binding, probably by affecting a platelet receptor.     

Effect of heparin on platelet aggregation

The effect of heparin on platelet aggregation was systematically examined on platelets in plasma (PRP), as well as on gel-filtered, washed, and formaldehyde-fixed platelets. Results indicate that, although heparin causes a mild potentiation of platelet aggregation in the PRP systems, a significant inhibitory activity is observed when heparin is added to isolated platelets. This inhibitory activity appears to be specific and not related to the impurities in the heparin preparations, as heparinase, as well as protamine, effectively neutralizes the heparin-mediated inhibitory activity on platelet aggregation. Although heparin-mediated inhibitory activity can be demonstrated in the presence of a number of different agonists (ADP, arachidonic acid, thrombin, Ionophore A23187, epinephrine, and ristocetin), the most pronounced inhibition is seen in the presence of ristocetin. Further studies show that heparin enhances thromboxane generation in isolated platelets. Platelets pretreated with heparin, however, fail to respond to preformed thromboxane. These findings suggest that, in addition to the potentiation of thromboxane production in platelets, heparin may also attribute some change(s) to the platelet(s)/platelet membrane, which interferes with their ability to respond to the agonists of platelet aggregation. This antiaggregatory activity of heparin was found to be inhibited by a factor(s) present in plasma but not in serum.     

Effects of α-tocopherol (Vitamin E) on the Ultrastructure of Human Platelets In Vitro

Washed human platelets were incubated with increasing concentrations of α-tocopherol. Spontaneous aggregation was induced by tocopherol (0.5 mM or above). Aggregation was inhibited by ethylenediaminetetraacetate and platelet activation was reduced by prostaglandin E(1). Using electron microscopy, it was confirmed that tocopherol caused platelet disruption to some extent and the released components may have generated aggregation. These effects were not observed in platelet-rich plasma. Spontaneous activation was not observed when the concentration of tocopherol was 0.03 mM or lower. Concentrations of tocopherol between 0.075 mM and 0.0075 mM had inhibiting influences on activation of washed platelets by thrombin. Tocopherol (between 0.1 mM and 0.005 mM) changed activation of washed platelets by cationized ferritin in that it facilitated the first phase of aggregation but reduced the second phase in an indirect proportional manner. The results show that the effects of tocopherol in washed platelet preparations are not comparable to those observed in plasma and that the platelet membrane must be regarded as a crucial target for vitamin E.     

Platelet aggregation and disaggregation in the streptozotocin induced diabetic rat: the effect of sympathetic inhibition

Alterations in platelet function have been observed in a number of diabetic states. Increased responsiveness to platelet-aggregating agents in diabetes associated with increased catecholamine production and/or turnover suggested that heightened sympathetic activity may contribute to this increased platelet aggregation response. To investigate this possibility, we made male Wistar-Furth rats diabetic with streptozotocin and treated them either with adrenergic inhibitors (clonidine, yohimbine, reserpine) or saline. After 2 weeks, arterial blood samples were collected in 3.8% sodium citrate or acid citrate dextrose (ACD). Platelet-rich plasma (PRP) was prepared, and platelet aggregation studies were conducted directly or conducted on washed platelets prepared from PRP collected with ACD. Platelet aggregation in response to ADP by PRP was reduced while the rate of disaggregation was increased in platelets from diabetic animals when compared to controls. However, platelet aggregation in response to ADP in washed platelets was increased in diabetic animals when compared to controls. Clonidine, reserpine and yohimbine significantly decreased the diabetes-induced increase in maximum aggregation. Thrombin-induced aggregation was not altered by diabetes or any of the treatments. The platelet size was increased in the diabetic animals and was decreased toward controls by clonidine, reserpine and yohimbine treatment. These studies suggest that diabetes increases platelet aggregation response in diabetic rats, and that blockage or suppression of adrenergic activity reverses or attenuates the diabetes-induced hypersensitivity to ADP.     

Influence of the Pure Synthetic PAF (Platelet Aggrregating Factor) on Clot Retraction and Platelet Aggregation

Pure synthetic platelet aggregating factor (PAF) (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) induces a dose-dependent platelet aggregation in platelet-rich plasma (PRP) and in gel-filtered platelets. Irreversible platelet aggregation was observed at final concentrations of PAF higher than 2 times 10^-7 mol/l, while reversible or two-wave aggregation was obtained with lower final concentrations. The second wave was inhibited by acetylsalicylic acid, indomethacin, dipyridamole, EDTA, EGTA, theophylline, caffeine, PGE₁ and verapamil. PAF does not induce reptilase clot retraction (RCR); however, it does not inhibit RCR induced by ADP or thrombin. Since all substances known to activate platelets also induce RCR, the lack of this activity by PAF would support the existence of a third pathway in platelets.     

Streptokinase-induced platelet aggregation. Prevalence and mechanism

BACKGROUND. Streptokinase (SK) is a bacteria-derived protein and one of the plasminogen activators that is currently available for therapeutic use. Exposure to SK induces synthesis of specific antibodies that may initiate platelet aggregation and paradoxical clot propagation during treatment. METHODS AND RESULTS. Using platelet-rich plasma (PRP), we found that SK (5,000 units/ml) but not urokinase (2,500 units/ml) or recombinant tissue-type plasminogen activator (2,500 units/ml) caused platelet aggregation in PRP from 14 of 100 normal volunteers. In 13 consecutive patients treated with SK for acute myocardial infarction, SK-mediated platelet aggregation was induced in five patients within 1 week after treatment. SK-mediated platelet aggregation was associated with significantly increased titers of both anti-SK antibodies and SK-neutralizing activity in plasma; it was partially inhibited by aspirin (1 mM) and by aprotinin (500 kallikrein inhibitor units/ml) and completely inhibited by tranexamic acid (1 mM) and by prostaglandin E1 (9 microM). Addition of SK (1,000 or 5,000 units/ml) induce a statistically significant dose-dependent thromboxane B2 release in mixtures of PRP with plasma from subjects with SK-induced aggregation but not in samples of PRP mixed with plasma from nonresponders; addition of recombinant tissue-type plasminogen activator (1 or 50 micrograms/ml) did not induce thromboxane B2 release. Mixing experiments with PRP and immunoglobulin G from reactive and nonreactive donors revealed that SK-induced aggregation requires the presence of anti-SK antibodies. When 125I-SK (50 nM) was used, platelets preincubated with plasminogen (0.5 microM) bound 9,500 +/- 600 (mean +/- SEM, n = 6) molecules SK/platelet, which increased to 25,000 +/- 3,100 molecules/platelet after thrombin stimulation. Tranexamic acid (1 mM) blocked specific binding of SK to resting platelets. CONCLUSIONS. These data demonstrate that SK-induced platelet aggregation is initiated by the binding of anti-SK antibodies to the SK-plasminogen complex located on the platelet surface. SK-induced platelet activation may limit the therapeutic effectiveness of the drug, and in view of the high prevalence of aggregation in a normal population, prospective evaluation of the effects of platelet aggregation during treatment with SK is warranted.     

Platelet aggregation occurs in congenital afibrinogenaemia despite the absence of fibrinogen or its fragments in plasma and platelets, as demonstrated by immunoenzymology

Platelet aggregation of an afibrinogenaemic patient's platelet rich plasma (PRP) was greatly decreased when ADP was used for stimulation. In the presence of collagen or arachidonic acid the changes in light transmission recorded during platelet aggregation of patient's PRP were similar to those observed with normal PRP but the size of aggregates appeared to be smaller in comparison with those observed with normal platelets. In addition, thrombin-induced aggregation of washed platelets was similar to normal platelets. The interpretation was made possible because the fibrinogen level in plasma and in platelets was found to be almost nil as demonstrated by both an Elisa procedure described here and the determination of fibrinopeptide A (fpA). Furthermore, fibrinogen fragments, which could result from abnormal synthesis and therefore replace fibrinogen in platelet aggregation, were undetectable by immunological analysis using specific antibodies against A alpha, B beta and gamma chains and 10 different monoclonal antibodies against fibrin degradation products.     

Contribution of the Platelet Factor V Content to Platelet Factor 3 Activity

S ummary . The procoagulant activity obtained from bovine thrombocytes has been compared to that of lipids isolated from platelets, with and without the addition of purified bovine factor V. A one-stage assay, which consisted of delipidated bovine plasma containing RVV-activated factor X, was used to assess the activity. At low lipid concentrations no difference in coagulant activity was found between sonicated vesicles of extracted platelet lipid and lysed platelets. At higher lipid concentrations, however, the extracted lipids were found to be less active than lysed platelets. Determination of factor V in suspensions of gel-filtered platelets demonstrated that suspensions containing 2 × 10⁹ platelets per ml possessed about 1% of the factor V activity present in a normal bovine plasma pool. Platelet lysis by sonication produced a five-fold increase in factor V activity. Addition of factor V to sonicated vesicles of extracted platelet lipid, so as to produce an identical factor V activity per amount of lipid as found in lysed platelets, decreased the clotting time only in the higher lipid concentration range. A further three-fold increase in the amount of factor V added to the lipid vesicles made the coagulant properties of the lipid vesicles indistinguishable from those of lysed platelets over the whole range of phospholipid concentrations tested. When the conditions of the test were changed by diminishing the concentration of factor Xa in the substrate plasma, the difference between lysed platelets and extracted platelet lipid disappeared completely. It is concluded that the higher coagulant activity of lysed platelets, as compared to that of extracted platelet lipid, can be ascribed to platelet factor V activity. Therefore there is no compelling necessity to postulate the existence of a specific procoagulant factor in the platelet other than factor V or phospholipids.