Platelet Adherence to Collagen: Role of Prostaglandin—Thromboxane Synthesis

The effect of aspirin and indomethacin on adherence of human platelets to collagen was assessed by affinity chromatography on collagen/Sepharose. Studies were done using platelets from normal subjects and subjects ingesting aspirin. Blood was anticoagulated with EDTA, EGTA or citrate, and platelets were suspended in plasma or buffer after separation by centrifugation or gel filtration. The adherence to collagen of platelets from subjects ingesting 1200 mg aspirin per day decreased significantly (P less than 0.05 or below). A significant reduction in adherence also occurred after exposure to aspirin and indomethacin in vitro. The reduction in adherence was associated with inhibition of malondialdehyde (MDA) production and was unaffected by the presence of CP/CPK. Exposure of platelets to aspirin at 24 degrees C caused no impairment of adherence. The addition of 1.0 mM Ca++, 0.5 or 1.0 mM Mg++, or 1 mM arachidonic acid significantly diminished the inhibition of adherence by aspirin. Imidiazole had an effect on adherence opposite to that of aspirin. Changes in release of 14C-serotonin in general paralleled changes in adherence. The data suggest that aspirin and indomethacin impair but do not fully inhibit platelet adherence to collagen. Factors affecting this inhibitory activity in our system are the presence of plasma and availability of divalent cation. Platelet-collagen adhesion appears partly dependent upon prostaglandin-thromboxane synthesis and may be promoted by a different pathway that is stimulated by divalent cations and unaffected by inhibition of prostaglandin synthesis.     

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.     

Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.     

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.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

The Role of Thrombin in ADP-Induced Platelet Aggregation and Release: a Critical Evaluation

S ummary . The role of thrombin in ADP-induced aggregation and release in vitro was critically examined. The addition of heparin or hirudin to citrated platelet rich plasma did not prevent aggregation or release. The addition of citrate to heparinized plasma restored secondary aggregation and release. Hirudin did not prevent irreversible aggregation. These results are incompatible with the hypothesis that thrombin is a primary and necessary mediator of platelet aggregation (Ardlie & Han, 1974; Han & Ardlie, 1974a, b, c). This hypothesis is based in part on the assumption that EDTA enhances the elution of clotting factors from platelets; we found no enhanced elution of factors II, V, X, VIII, IX or XI when platelets were washed in EDTA.     

Platelet secretion defect in patients with the attention deficit disorder and easy bruising

Platelet function was evaluated in 12 patients with the attention deficit disorder and lifelong history of easy bruising. Aggregation and 14C-serotonin secretion studies in platelet-rich plasma in response to adenosine diphosphate (ADP), epinephrine, and arachidonic acid did not reveal striking abnormalities. Secretion of adenosine triphosphate (ATP), ADP, beta-hexosaminidase, and beta-glucuronidase by gel-filtered platelets in response to the divalent cation ionophore A23187 and low concentrations of thrombin (less than or equal to 0.1 U/ml) was impaired in patients as compared to normals. The aggregation response to A23187 (4 microM) was absent in 8 of the 12 patients. The total stores of the secretable constituents, the retention of incorporated 14C-serotonin, and the arachidonate metabolism of the platelets were normal. Our findings suggest a new platelet disorder with impaired secretion mechanism, without storage pool deficiency or impaired arachidonate metabolism. The secretion defect in platelets represents a tissue disorder in a functional psychiatric disease. We refocus attention on the role of platelets as a model for neurons in functional disorders, with emphasis on secretion mechanisms rather than amine uptake, storage, and metabolism.     

Platelet functions in recombinant hirudin-anticoagulated blood

The influence of genetically engineered recombinant hirudin (r-hirudin) on platelet functions was studied. Depending on the concentration, r-hirudin inhibits the thrombin-induced aggregation and 14C-serotonin secretion to the same extent as native hirudin. Comparative studies in blood anticoagulated by r-hirudin, heparin or citrate show a significantly lower spontaneous platelet aggregation in r-hirudinized blood. The extent of the ADP-induced aggregation is nearly the same in r-hirudinized, heparinized or citrated plasma. In r-hirudinized plasma, however, aggregation is reversible. In contrast to heparinized or citrated plasma, adrenaline causes only a very slight aggregation in r-hirudinized plasma. ADP- or adrenaline-induced secretion of 14C-serotonin do not occur in r-hirudinized plasma. The collagen- as well as the PAF-induced aggregation and 14C-serotonin release in hirudinized plasma do not differ significantly from those in heparinized or citrated plasma. r-Hirudin is a suitable anticoagulant for studying platelet functions because it does not produce any alterations in platelet reactions and does not provoke any changes in the ionized calcium concentration in blood.     

ADP and epinephrine-induced release of platelet fibrinogen

Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.     

The Effect of Prostaglandin E1 on Platelet Function in Vitro and in Vivo

S ummary Low concentrations of prostaglandin E₁ (PGE₁) inhibit ADP-induced aggregation in pig and rabbit citrated platelet-rich plasma and in suspensions of washed platelets. Higher concentrations also inhibit the initial change in shape induced by ADP and the release of platelet ATP, ADP and serotonin caused by stimuli such as collagen, thrombin, antigen-antibody complexes and gamma-globulin-coated polystyrene particles. PGE₁ is not taken up by platelets and its effects can be removed by resuspending platelets in fresh medium. Immediately following an intra-arterial injection, ADP-induced platelet aggregation is suppressed, but after 5 min the response returns to normal. PGE₁ inhibits haemostasis in rabbits when given as a continuous infusion. It is concluded that the effect of PGE₁ on haemostasis involves inhibition of the release of ADP from platelets exposed to collagen and thrombin, and inhibition of ADP-induced aggregation.     

Influence of dianhydrogalactitol on some platelet functions in vitro

Platelet-rich plasma was incubated with dianhydrogalactitol (DAG) in vitro. This moderately diminished platelet aggregation induced by adrenaline and to a smaller degree by adenosine diphosphate /ADP) or collagen. The availability of platelet factor 3 induced by ADP, adrenaline or collagen was decreased irrespective of the inducer. The release of platelet factor 4 was unaffected. Reptilase clot retraction induced by ADP or collagen was slightly inhibited. Uptake of 14C-serotonin was slightly decreased. Uptake of 14C-adenine was normal. Platelet adhesion onto glass was not depressed. DAG did not release lactic dehydrogenase from the cytoplasm of the platelets. Coagulation factors were not affected.     

Further Evidence for the Role of Thrombin in the Platelet Release Reaction Caused by Various Agents, and the Nature of Biphasic Platelet Aggregation

Studies were undertaken to resolve conflicting reports about the possible involvement of thrombin in the release reaction and second phase aggregation caused by ADP.
Washed human platelets suspended in a buffered solution responded poorly to ADP, with no second phase aggregation or release of [³H]₅HT. In contrast, washed platelets suspended in dialysed plasma containing all intrinsic pathway clotting factors responded to ADP with second phase aggregation and release of radioactivity. This response depended on an optimum calcium concentration above which aggregation was impaired. Biphasic aggregation and release of platelet contents by ADP, epinephrine and collagen in dialysed plasma was inhibited by hirudin and heparin. The inhibitory effects of hirudin and heparin were not observed when citrate was present.
These observations provide further evidence for the central role of thrombin in the release reaction and biphasic aggregation caused by various agents and indicate that these platelet reactions are not artifacts due to a citrate. Since the effect of citrate on aggregation and the inhibitory actions of hirudin and heparin were produced by another chelating agent, viz EGTA, it is suggested that the citrate effect is due to reduction of cation concentration.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.     

Aggregation of Human Platelets by Bovine Platelet Fibrinogen

Aggregation of human platelets by addition of purified bovine platelet fibrinogen is described. Bovine plasma fibrinogen showed the same but much weaker effect. Human fibrinogen produced no aggregation. No absolute requirement for divalent cations or plasma proteins could be demonstrated. The aggregation of washed platelets appeared monophasic whereas in platelet-rich plasma it was usually biphasic. The use of inhibitors of ADP-induced platelet aggregation, inhibition of intracellular ATP-production, enzyme-catalyzed removal of ADP, and direct determinations of ADP in the medium showed the second phase to be mediated by ADP released from the platelets whereas the first phase was nearly independant of ADP. While the ability of platelet fibrinogen to aggregate platelets and to clot with thrombin were otherwise intimately interconnected, some aggregation activity remained after heat-denaturation of the platelet fibrinogen.     

Fibronectin-mediated inhibition of human platelet activation by crosslinked immunoglobulin G

The effect of purified human plasma fibronectin on stimulation of human platelets with crosslinked immunoglobulin G (IgG) was tested by means of aggregometry, 14C-serotonin release and fibronectin-to-platelet binding experiments. Incremental additions of fibronectin to gel-filtered platelets (8 X 10(8)/ml) followed by 25 micrograms/ml bisdiazoniumbenzidine (BDB)-crosslinked IgG produced a dose-related inhibition of platelet aggregation and suppression of 14C-serotonin release from the platelets. Evidence was obtained that upon stimulation of the platelets with BDB-IgG, fibronectin becomes bound to its receptor and that further platelet activation is inhibited when a sufficient number of receptors is occupied.     

Factors affecting the observation of a synergistic response in platelet aggregation induced by pairs of excitatory agonists

When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 μM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).     

The role of fibronectin in platelet aggregation

A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.     

Influence of cis-platinum on some platelet functions in vitro

Cis-platinum added to citrated platelet rich plasma in vitro did not influence the aggregation of the platelets, their adhesion to glass, their release of platelet factor 4 or availability of platelet factor 3 and acid phosphatase. Neither any effect on the uptake of 14C-serotonin, the reptilase clot retraction or the coagulation system has been observed.     

Heparin, Platelets and Blood Coagulation: Implications for Low-Dose Heparin Prophylactic Regimens in Venous Thrombosis

S ummary . Heparin has been shown to inhibit the second phase of aggregation and the platelet release reaction induced by ADP. These platelet responses to ADP are mediated by thrombin, generated through the coagulation pathways, on the platelet membrane (Ardlie & Han, 1974), and evidence was obtained that the inhibitory action of heparin is due to inactivation of coagulation factor Xa in the presence of XaI. In contrast, it has been shown that thrombin adsorbed to the platelet surface is protected from destruction by its natural inhibitor and heparin. Since trace amounts of heparin were able to augment the inhibitory action of XaI on factor Xa, this phenomenon has relevance to low-dose heparin regimens for prophylaxis of venous thrombosis. It is proposed that postoperative thrombosis is due to abnormal platelet function caused by accelerated coagulation, and that heparin prevents thrombosis through its anticoagulant action on factor Xa. It is also suggested that trace amounts of heparin which may be present normally in the circulation may act as a natural anticoagulant.
One of two commercial preparations of heparin caused washed platelets, prepared by some, but not all, of the methods used, to aggregate and release their constituents. However, it was shown that these effects of heparin on washed platelets are simply a consequence of storage of platelets at 4°C, emphasizing the need to keep platelets at 37°C for in vitro studies. Heparin failed to inhibit the release reaction of platelets in citrated-plasma, and it is suggested that this may be due to interference by citrate. These observations with washed platelets and citrated plasma are considered to be artefacts and thus have no relevance to heparin action in vivo .     

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.