EDTA-induced changes in platelet structure and function: adhesion and spreading

Ethylenediamine tetraacetic acid (EDTA) is an effective anticoagulant, but unfortunately causes structural, biochemical and functional damage to human platelets. Some of the functional injuries, such as adhesion to and spreading on surfaces, are considered irreversible. The present investigation has evaluated that hypothesis. Our findings indicate that platelets from EDTA platelet-rich plasma (PRP) or CCD PRP to which EDTA has been added do not adhere to glass or plastic surfaces. However, when platelets from EDTA PRP or CCD PRP containing added EDTA are washed and resuspended under conditions reported to cause irreversible dissociation ofthe fibrinogen receptor, GPIIb/IIIa, then washed and resuspended in buffer containing Ca 2+ and Mg 2+ ions will adhere and spread in the same manner as platelets not exposed to EDTA. The ability to recover adhesive function may explain why EDTA platelets are able to sustain clot retraction as well as CCD platelets.     

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.     

EDTA-induced changes in platelet structure and function: clot retraction

Ethylenediamine tetracetic acid (EDTA) is known to cause structural, biochemical and functional injury to blood platelets, including irreversible dissociation of the fibrinogen receptor, glycoprotein alpha IIb beta 3 (GPIIb/IIIa). Despite inability to adhere to glass, spread, and to aggregate in response to adenosine diphosphate (ADP) and other agonists, EDTA-treated platelets support clot retraction as well as untreated cells. The present study has used clot retraction under isometric tension and electron microscopy to determine if there are any differences in platelet-fibrin interactions of clots formed from blood collected in EDTA or platelets from blood drawn into citrate (CCD) anticoagulants. No physical differences could be identified. Polymerizing fibrin bound intimately to aggregates developing from EDTA platelets undergoing shape change, internal transformation, adhesion and spreading on fibrin strands oriented in the long axis of contraction. The results suggest that reassociation of irreversibly dissociated GPIIb/IIIa takes place immediately after initiation of clot retraction, or that a significant proportion of GPIIb/IIIa receptors on resting platelets are inaccessible to EDTA and become available after activation by thrombin.     

EDTA-induced changes in platelet structure and function: clot retraction

Ethylenediamine tetracetic acid (EDTA) is known to cause structural, biochemical and functional injury to blood platelets, including irreversible dissociation of the fibrinogen receptor, glycoprotein alpha IIb beta 3 (GPIIb/IIIa). Despite inability to adhere to glass, spread, and to aggregate in response to adenosine diphosphate (ADP) and other agonists, EDTA-treated platelets support clot retraction as well as untreated cells. The present study has used clot retraction under isometric tension and electron microscopy to determine if there are any differences in platelet-fibrin interactions of clots formed from blood collected in EDTA or platelets from blood drawn into citrate (CCD) anticoagulants. No physical differences could be identified. Polymerizing fibrin bound intimately to aggregates developing from EDTA platelets undergoing shape change, internal transformation, adhesion and spreading on fibrin strands oriented in the long axis of contraction. The results suggest that reassociation of irreversibly dissociated GPIIb/IIIa takes place immediately after initiation of clot retraction, or that a significant proportion of GPIIb/IIIa receptors on resting platelets are inaccessible to EDTA and become available after activation by thrombin.     

Vitamin E reduces platelet adhesion to human endothelial cells in vitro

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol. Vitamin E (0.5-1.0 mM) was added to PRP or washed platelets. Endothelial cells in monolayer were incubated with thrombin-activated platelets (1 or 2 U/ml). After 1 hr of incubation, non-adhered platelets were removed and counted. Treating of PRP with alpha-tocopherol inhibited platelet adhesion to endothelial cell monolayer. This effect was dose dependent on concentrations of alpha-tocopherol and thrombin. In our experiments PRP was treated with alpha-tocopherol and endothelial cell monolayer was used as test surface. These findings agree with previous observations on the adhesivity of platelets to synthetic surfaces after dietary vitamin E in healthy volunteers. When washed platelets were incubated with alpha-tocopherol, no significant reduction of adhesion was detectable. As preincubation of washed platelets with alpha-tocopherol does not inhibit platelet adhesion, it may be supposed that the effect of vitamin E does not occur in a directly cellular mechanism. The data suggest that alpha-tocopherol may reduce platelet adhesiveness probably after incorporation by plasma lipoproteins.     

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.     

Mechanisms of platelet aggregation by Streptococcus sanguis, a causative organism in infective endocarditis

Summary. The ability of certain strains of Streptococcus sanguis to aggregate human platelets in vitro may be related to their virulence in the pathogenesis of infective endocarditis. We have studied the mechanisms of aggregation of human platelets by S. sanguis strain NCTC 7863. Platelet aggregation follows incubation of S. sanguis cells with platelet-rich plasma from normal, healthy adults, after a lag of 7–19 min. Platelet aggregation was accompanied by 5-hydroxytryptamine release and thromboxane B₂ production. Aggregation was prevented by aspirin and by EDTA. Platelets from two patients with Glanzmann's thrombasthenia did not respond to bacteria. Fixed, washed platelets resuspended in normal plasma were not agglutinated by S. sanguis.
Blocking the glycoprotein Ib receptor with a monoclonal antibody inhibited aggregation of PRP. However, S. sanguis did not induce von Willebrand factor (vWF) binding to platelets; nor did the bacteria prevent ristocetin-induced platelet agglutination or vWF binding. The aggregation response was not related to plasma vWF activity levels in normal subjects or in patients with von Willebrand's disease.
The platelet response to S. sanguis therefore resembles true aggregation, requiring the cyclo-oxygenase pathway and the presence of glycoprotein IIb/IIIa. The mechanism also involves glycoprotein Ib, but not apparently through irreversible binding of vWF.     

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.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

Platelet interaction with subendothelial extracellular matrix: platelet- fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction

Cultured endothelial cells produce an extracellular matrix (ECM) to which platelets adhere and spread, ultimately resulting in platelet aggregation, thromboxane B2 production, and serotonin release. We have investigated the role of fibrinogen binding to the platelet GPIIb/IIIa complex in these reactions by comparing normal platelet-rich plasma (PRP), PRP from patients with Glanzman's thrombasthenia (whose platelets lack the GPIIb/IIIa complex), PRP in the presence of a monoclonal antibody that blocks the binding of fibrinogen to the GPIIb/IIIa complex, platelets washed free of fibrinogen, and washed platelets to which fibrinogen was added. Although platelet aggregation was virtually completely inhibited in the samples in which the normal interaction between fibrinogen and GPIIb/IIIa was impaired, adhesion of platelets to the matrix, spreading, and release of [14C]-serotonin were not affected. All of the platelet preparations released significant amounts of T X B2 with time, but there was a decrease in the amount produced by both the thrombasthenic and antibody-treated platelets. We conclude that the interaction of fibrinogen with platelet GPIIb/IIIa is not required for platelet adhesion to ECM or for adhesion-induced shape change or serotonin release. On the other hand, the platelet-fibrinogen interaction may play some role in augmenting adhesion-induced T X B2 production, and it is absolutely required for adhesion-induced platelet aggregation.     

The role of plasma fibronectin in platelet adhesion to collagen

Human washed platelets were eluted from columns of Sepharose 4B linked to different preparations of collagen in order to evaluate cell adhesion. Collagen preparations characterized by low and high affinity toward platelets were identified. In our experiments, fibronectin purified from human plasma modified platelet adhesiveness, though not dramatically. When washed platelets, resuspended in a buffer containing fibronectin, were filtered on a low-affinity collagen-Sepharose, a significant increase in their adhesion occurred. A similar modification could be observed when platelets were allowed to adhere to the same collagen-Sepharose preconditioned with fibronectin. The effect of fibronectin was otherwise negligible when the high-affinity collagen was used for the experiments.     

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.     

Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium

Close platelet-to-platelet contact induced by weak agonists in a medium with a low concentration of Ca2+ leads to thromboxane A2 (TXA2) formation, release of granule contents, and secondary aggregation. These responses do not occur in a medium containing Ca2+ in the physiological range (1 to 2 mmol/L). Experiments were done to determine whether feedback amplification is required to generate amounts of TXA2 that are sufficient to cause secondary aggregation and the reactions associated with it, or whether close platelet-to-platelet contact alone is sufficient to generate enough TXA2 to produce these responses. Platelets were washed and resuspended in a modified Tyrode solution to which no calcium salt was added that contained 0.35% albumin and apyrase. This medium contains 20 mumol/L Ca2+ and 1 mmol/L Mg2+. Platelets were aggregated with adenosine diphosphate (ADP) in the presence of fibrinogen, agglutinated with polylysine, or after pretreatment with chymotrypsin, aggregated with fibrinogen. In the low- Ca2+ medium, all these agonists caused platelets to adhere to each other, followed by secondary aggregation with TXA2 formation and release of granule contents. When Ca2+ (1 to 2 mmol/L), aspirin, or the thromboxane receptor blocker BM 13.177 was present, the secondary responses did not occur; dazoxiben decreased thromboxane formation, but did not prevent secondary aggregation or release. Aspirin-treated platelets were less responsive to ADP, U46619, or TXA2 in the low-Ca2+ medium, which indicated that the secondary responses of untreated platelets were not caused by a generalized increase in sensitivity. The reactions that result from close platelet-to-platelet contact in a low- Ca2+ medium can be caused by a wide variety of weak agonists; the secondary aggregation response and release of granule contents are dependent on TXA2 formation and on feedback amplification by TXA2 or the prostaglandin endoperoxides. The secondary responses caused by weak agonists in citrated platelet-rich plasma (which has a concentration of Ca2+ similar to the low-Ca2+ medium used in the present studies) do not occur at the concentration of Ca2+ in circulating blood and thus may have little biologic relevance.     

Immunocytochemical localization of fibrinogen during thrombin-induced aggregation of washed human platelets

Because thrombin aggregates afibrinogenemic platelets and platelets from patients with the gray platelet syndrome and because antibodies to fibrinogen inhibit thrombin-induced aggregation only at low concentrations of thrombin, the role of fibrinogen in the formation of thrombin-induced aggregates was investigated further with human platelets washed and resuspended in Tyrode-albumin solution containing apyrase, either with or without added Ca2+ (2 mmol/L). Samples for immunocytochemical assessment of fibrinogen distribution were taken at several times (up to five minutes) after aggregation induced by 0.5 U/mL of thrombin. Glutaraldehyde-fixed samples were embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. By 10 seconds, small aggregates formed, and granules were centralized; alpha granules were heavily labeled with immunogold, but the platelet surface was not. As large aggregates formed, granule swelling or fusion occurred, and in some areas granule material seemed to be in contact with the exterior. In these experiments with no added fibrinogen, there were some clusters of gold particles on the platelet surfaces remote from sites of granule discharge, but there were large areas where platelets were in close contact with little or no fibrinogen detectable between them. No fibrin was visible up to five minutes after the addition of thrombin, which indicated that fibrinogen from the granules does not readily become available for fibrin formation in the ambient fluid. Similar results were obtained in media with and without added Ca2+. Thus at least some aggregation in response to thrombin can occur without the participation of released fibrinogen, and much of the granule fibrinogen appears to remain localized at sites where granules fuse with the plasma membrane or the open canalicular system. Incubation of unstirred samples with thrombin for ten minutes resulted in the formation of small aggregates, extensive gold label in regions connected to the exterior of the platelets, but very little gold labeling of the platelet membrane and no visible fibrin formation. When the platelets were aggregated in the presence of external fibrinogen, the morphological changes within the platelets were the same, but fibrinogen rapidly became associated with the entire platelet surface, and visible fibrin formed within 30 seconds in the medium containing 2 mmol/L Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Textured biomaterials as a model for studying formation of focal contacts and rearrangement of the contractile cytoskeleton in platelets

Fibres of textured biomaterials (BM) enable platelets to adhere with formation of focal contacts. The contact structure and the reaction of the contact associated contractile cytoskeleton were studied using fibres of different flexibility/mobility: butyl-S-Sepharose (S), Polysulfone (PS) and Polyurethane (PU). Ultrastructural and immunocytochemical investigations were carried out to obtain information on the influence of tension on (1) the structure of the focal contacts; (2) the constricting cytoskeleton known to retract adherent collagen or fibrin fibres and (3) the cable-like bundles of actomyosin as observed in the clot. Fibre network from S spheres and 0.3 mm thick frozen sections of PS or PU were incubated with citrated PRP or with washed platelets at 37 C for 6 to 30 min while stirring for contact or activation with ADP or thrombin. Flexible fibres of the BM were found in deep invaginations of the plasmalemma associated with the constricting cytoskeleton. Focal contacts (mediated by fibrinogen as shown immunocytochemically) with fibres which were fixed in the texture or inflexible (PU) induce cable-like bundles of micofilaments containing myosin. These bundles pass across the cytoplasm and connect the contacts with the fibres or with other platelets, as demonstrated by computer-assisted 3-dimensional reconstruction. The model used indicates that retraction is possible as long as fibres are mobile and that cable-like bundles occur when the locomotion of platelets is blocked by immobile fibres. The interaction of platelets with textured BM reflects the situation during collagen or fibrin condensation. The findings may contribute to an understanding of platelet reactions on textured surfaces in grafts.     

Nonreversible loss of platelet aggregability induced by calcium deprivation

Platelets lose their ability to aggregate when deprived of divalent cations. This usually was studied by incubating human citrated platelet- rich plasma with EDTA or EGTA and then adding enough CaCl2 to combine with the chelating agent. Incubation for 5-7 min at 37 degrees C caused irreversible loss of the platelets' ability to adhere to glass and to aggregate with ADP, epinephrine, A23187, vasopressin, or serotonin or upon rewarming after chilling and markedly reduced aggregation with collagen or thrombin. Control samples incubated with saline, CaEDTA, or CaEGTA were not inhibited. Untreated platelets washed and incubated in solutions treated with resins that remove divalent cations lost their ability to aggregate in 30 min. More than about 0.26 mM Mg2+ partially protected the platelets. Unlike aggregation, ADP-induced shape change, clot retraction caused by thrombin or ADP plus reptilase, and thrombin- induced 14C-serotonin release were not inhibited after incubation. Aggregability was not restored by prolonged incubation with CaCl2, adding normal plasma, or washing the platelets. Its loss was temperature and pH dependent, occurring in 2 min at 43 degrees C but not in 7 min at 30 degrees C, and at pH 7.8 but much less at pH 7.2. The defect was not associated with an increase in platelet cyclic AMP, a decrease in metabolic ATP, or the presence of free ADP.     

High-speed platelet adhesion under conditions of rapid flow.

The recognition of exposed collagen by circulating platelets is an initial step in the formation of the hemostatic plug or a thrombus after vascular injury. Theoretical calculations of the speed of platelet function required for effective hemostasis have suggested very short reaction times. However, it is not known how fast platelets can adhere to collagen under arterial flow conditions or which membrane proteins are involved. We have used a continuous-flow, microaffinity column linked to a resistive-particle counter to detect platelet adhesion. Adhesion of human platelets to native type I collagen was extremely rapid, with exponential half-times as short as 240 ms, and was nearly complete by 2 s. This RGD-independent process was not associated with platelet aggregation or secretion. The monoclonal antibody 6F1 directed against the glycoprotein Ia/IIa complex inhibited adhesion, suggesting that this complex plays an important role in the initial phases of platelet-collagen interaction under flow conditions. In addition, divalent cations were required for adhesion, as indicated by inhibition with EDTA in plasma and the dependence on Mg2+ for washed platelets.     

Evidence that Endotoxins Enhance the Factor X Activator Activity of Washed Human Platelets

The in vitro effect of several bacterial endotoxins on human platelets was determined. Nine different endotoxins failed to induce aggregation in platelet-rich plasma (PRP) or of platelets washed by two different methods; four of them which we studied further failed to induce [14C]serotonin release in PRP. In contrast, using recently described test systems for platelet coagulant activity, all the endotoxins shortened the latent period occurring before aggregation of a mixture of washed platelets, normal serum, and CaCl2, and the clotting time of this mixture upon addition of fibrinogen. Washed platelets obtained from PRP preincubated with endotoxin had a higher platelet coagulant activity than platelets obtained from PRP preincubated with buffer. Washed platelets contribute to thrombin generation by providing factor V, a factor X activator and possibly phospholipid. Since the endotoxins did not influence the factor V activity of platelets or the platelet factor 3 activity, either in PRP or using platelets washed by albumin density gradient centrifugation, it is suggested that they enhance the factor-X activator activity of human platelets.     

Direct Assessment of Platelet Adhesion to Glass: A Study of the Forces of Interaction and the Effects of Plasma and Serum Factors, Platelet Function, and Modification of the Glass Surface

Platelet adhesion to glass has beendirectly determined with a cover slipchamber. These studies have separated the functions of platelet adhesionto a foreign surface from platelet cohesion (aggregation). The forces ofthe platelet-glass interaction have beenstudied, and the qualitative variablesaffecting this interaction have beendefined. Fibrinogen and calcium ormagnesium are required for adhesionof either unwashed or washed platelets in plasma. Platelet adhesion infresh serum requires thrombin andprobably the fibrinogen of the plateletsurface, since washed platelets arenot adherent in fresh serum. Plateletsincubated at 37°C for 48 hr can neitherspread nor adhere to glass, a defectthat may reflect decreased plateletsurface area and membrane deformability. Platelets are equally asadherent to siliconized glass as to untreated glass surfaces. However, platelets cannot adhere to glass treatedwith the hydrogen-bonding polymer,poly(ethylene oxide). In contrast tosilicone, poly(ethylene oxide)-treatedglass surfaces do not impede bloodcoagulation. These surface propertiesfurther distinguish the two major factors involved in thrombogenesis: platelet adhesion and plasma coagulation.

Submitted on May 29, 1972 Revised on July 7, 1972 Accepted on July 10, 1972