Hereditary Disorders of Platelet Function

The most common hereditary disorders of platelet function are those in which there is decreased platelet aggregation in response to more than one of the commonly used aggregating agents, collagen, ADP, adrenaline and arachidonic acid. When measured, there is usually also a reduction in the extent of the platelet release reaction and often also thromboxane production. In a proportion of these cases it is possible to demonstrate that there is a decrease in or absence of the contents of the dense storage granules which accounts for the decrease in platelet responsiveness. In most other cases the primary cause of the decreased responsiveness has not been determined, although in some cases deficiency of cyclooxygenase or thromboxane synthetase has been demonstrated. Investigation of patients with these disorders is often difficult because the tests involved are difficult to subject to adequate quality control, their sensitivity and specificity has not been adequately defined, and lack of reproducibility renders it difficult in less severe cases to be certain of abnormality even after repeat tests. Much less common but of great interest are the disorders in which the primary abnormality is in one of the glycoproteins on the platelet surface. In Glanzmann's thrombasthenia glycoprotein IIb.IIIa is absent or greatly decreased leading to failure of activated platelets to bind fibrinogen to their surface. In contrast to the decrease in aggregation seen in the above disorders, the platelets do not aggregate at all in response to the usual aggregating agents. In Bernard-Soulier syndrome there are severe deficiencies of three glycoproteins, particularly lb, leading to inability to bind von Willebrand's factor and consequent inability of the platelets to aggregate in response to ristocetin. Study of the disorders of platelet function will continue to contribute to our ability to detect and treat these disorders and to our knowledge of platelet physiology and biochemistry.     

Platelet aggregation in whole blood from patients with Glanzmann's thrombasthenia

We examined platelet aggregation in platelet-rich plasma (PRP) and in whole blood from two patients with Glanzmann's thrombasthenia. In PRP, aggregation was measured by monitoring the changes in light absorbance that occurred in response to aggregating agents; to measure platelet aggregation in whole blood, we used a platelet counting technique. In PRP, the patients' platelets showed defective aggregation in response to ADP, adrenaline, arachidonic acid (AA), and collagen, but normal agglutination occurred in response to ristocetin. In whole blood, however, platelet aggregation in response to the aggregating agents appeared to be either very similar to that which occurred in blood from normal subjects or only slightly reduced. There was a reduced response to all concentrations of ADP and to low concentrations of collagen but a normal response to all concentrations of adrenaline, AA, and higher concentrations of collagen. Conversely, there seemed to be an increased agglutination response to ristocetin. The abnormality in our two patients with Glanzmann's thrombasthenia probably lies in the inability of their platelets to form large, macroscopic aggregates rather than in platelet aggregation per se.     

Platelet thromboxane synthesis and release reactions in myeloproliferative disorders

A group of patients with myeloproliferative disorders was studies with respect to platelet aggregation responses, release of beta-thromboglobulin and incorporated 5-hydroxy-tryptamine, and synthesis of thromboxane b 2. In all patients the resting plasma beta-thrombo-globulin was elevated. Aggregation responses were frequently impaired to adrenaline, arachidonic acid, A23187 and the prostaglandin endoperoxide analogue, U44069. Both 5-hydroxy-tryptamine and beta-thromboglobulin release were greater with patients' platelets than with those of controls in response to adrenaline, ADP and U44069. The patients' platelets produced more thromboxane B2 than did controls, irrespective of the agonist used, yet those aggregating agents which are thought to act by generating thromboxane A2 were relatively ineffective in causing aggregation. This might reflect resistance to thromboxane A2 action in these patients, which is met by increased thromboxane formation.     

Further Studies on a Specific Platelet Antibody Found in Bernard-Soulier Syndrome and its Effects on Normal Platelet Function

An IgG antiplatelet antibody found in a multitransfused patient with Bernard-Soulier syndrome (BSS), reacted with a normal platelet surface antigen of 150 000 daltons which was similar to the glycoprotein missing from BSS platelets. The BSS platelet antibody (BSS-Pab) aggregated all control platelets which then released ADP and 5-HT and synthesized thromboxane. When mixed with the antibody, BSS platelets did not aggregate, did not release ADP and 5-HT and failed to synthesize thromboxane. The BSS-Pab was not inactivated by incubation with BSS platelet stroma. While the antibody did not aggregate thrombasthenic platelets, its aggregating activity was lost after incubation with their stroma. The BSS-Pab did not provoke ADP or 5-HT release or thromboxane synthesis in thrombasthenic platelets or in the platelets of a patient with platelet cyclooxygenase deficiency or in normal platelets treated with indomethacin. The aggregating, release and synthetic responses of platelets after binding of BSS-Pab to its membrane antigen (probably glycoprotein I) requires the presence of glycoprotein IIb and/or IIa and the normal metabolism of arachidonic acid.     

Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation

The possible participation of proteases in human platelet aggregation was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of protease. Substrates used were synthetic substrates for the trypsin type as well as for the chymotrypsin type of protease. All these inhibitors and substrates inhibited platelet aggregation and serotonin release induced by ADP, collagen, epinephrine, or thrombin. In ADP- and epinephrine-induced platelet aggregation the second phase of aggregation was most efficiently inhibited. The inhibitors suppressed the formation of malondialdehyde during platelet aggregation. Release by aggregating agents of arachidonate and its metabolites from indomethacin-treated platelets as well as nontreated platelets was also inhibited. The inhibitors apperar to interact with stimulated platelets but not with unstimulated platelets. These observations suggest that the interaction of an aggregating agent with its platelet receptor activates a unique precursor serine protease that in turn activates platelet phospholipase to liberate arachidonic acid (the precursor of the potent platelet aggregating agent thromboxane A2) from platelet phospholipids.     

Altered platelet function in cirrhosis of the liver: impairment of inositol lipid and arachidonic acid metabolism in response to agonists

Hemorrhagic disorders are common in patients with liver cirrhosis and result from several factors including impaired platelet function. We evaluated platelet aggregation and arachidonic acid metabolism in response to standard agonists in platelet-rich plasma from 12 cirrhotic patients with mild impairment of liver function (Child A), 12 patients with severe liver dysfunction (Child B and C) and 12 healthy subjects. Platelet aggregation and thromboxane A2 production were consistently reduced in patients with severe liver impairment. To determine whether the platelet dysfunction is due to an intrinsic platelet defect or a circulating inhibitor, we measured platelet aggregation and thromboxane A2 synthesis on washed platelets in healthy subjects and in Child B and C patients. The aggregating response of washed platelets in response to thrombin, collagen and arachidonic acid was markedly reduced, suggesting an intrinsic platelet defect. The biochemical events underlying platelet aggregation were investigated by prelabeling platelets with [1-14C]arachidonic acid. Thrombin-induced activation of phospholipase C (measured as the release of [1-14C]phosphatidic acid) and phospholipase A2 (measured as the release of [1-14C]arachidonic acid and its metabolites) was greatly impaired in platelets from patients with severe liver impairment. We conclude that in advanced cirrhosis there is a severe reduction in platelet aggregatory response to physiologic agonists due to an intrinsic platelet defect which is related to an impairment of the platelet transmembrane signaling mechanism induced by receptor stimulation.     

Impairment of Platelet Thromboxane A2 Generation and of the Platelet Release Reaction in Two Patients with Congenital Deficiency of Platelet Cyclo-oxygenase

Two cases of thrombocytopathia with congenital deficiency of platelet cyclo-oxygenase were investigated. The platelet release reaction was impaired. There was a marked decrease of aggregation with collagen and with adrenalin and a total absence of aggregation with sodium arachidonate. The platelet response to labile aggregation stimulating substance (LASS, mostly thromboxane A2) was normal. There was no biosynthesis of prostaglandin cyclic endoperoxides or of thromboxane A2 from arachidonic acid. Basal levels of platelet PGE1 were lowered although plasma levels were normal. Thrombin decreased the cyclic AMP content of patients' platelets and also that of control platelets pretreated with aspirin. The patients platelets showed no ultrastructural difference when compared with control platelets, except for a slight decrease of granule volume, but, in contrast to control platelets, thrombin (0.02 U/ml) did not provoke contraction of the patients' platelets.     

Effects of streptokinase, urokinase, and recombinant tissue plasminogen activator on platelet aggregability and stability of platelet aggregates

STUDY OBJECTIVE--The aim of the study was to evaluate the effects of streptokinase, urokinase and recombinant tissue plasminogen activator (TPA) on platelet aggregability and metabolism and the stability of preformed platelet aggregates. DESIGN--The experiments (n = 15 for each condition) were performed on citrated plasma or on platelet suspensions in phosphate buffered saline, both with a standardised platelet count of 250 x 10(9).litre-1. SUBJECTS--were healthy volunteers. MEASUREMENTS AND MAIN RESULTS--With both ADP (1 mumol.litre-1) and collagen (1 mg.litre-1) as aggregating agents, streptokinase at greater than or equal to 10(5) units.litre-1 led to reduction in the rate of platelet aggregation. With collagen and in most instances with ADP, this was associated with a decreased extent of aggregation, though in five out of 30 cases with ADP as aggregating agent, a conversion from reversible to irreversible aggregation occurred with streptokinase. Urokinase inhibited platelet aggregation at greater than or equal to 3 x 10(5) units.litre-1 with both aggregating agents. TPA inhibited aggregation at greater than or equal to 1 mg.litre-1 with ADP and at greater than or equal to 3.3 mg.litre-1 with collagen as aggregating agent. The inhibitory effect was still present when the platelets were suspended in saline. Platelet synthesis of thromboxane on stimulation with collagen, and of c-AMP on stimulation with prostaglandin E1, was markedly reduced by either agent. The stability of platelet aggregates, as assessed photometrically during a 90 min exposure to stirring stress, increased when streptokinase or urokinase was added to platelet rich plasma, but remained uninfluenced with TPA. CONCLUSIONS--Urokinase and TPA inhibited platelet aggregability uniformly and in a dose dependent manner. Streptokinase inhibited platelet aggregation in most instances, but led to a stimulation of aggregation in a minority of cases. These effects of the thrombolytic agents on platelets might have an influence on the occurrence of bleeding and of reocclusion after thrombolytic therapy.     

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia

We found a novel platelet aggregating factor in a patient with steroid- responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus- response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.     

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.     

Platelet responsiveness and biosynthesis of thromboxane and prostacyclin in response to in vitro cocaine treatment

Preincubation of rabbit platelet-rich plasma with cocaine hydrochloride, at low and high concentrations, increased the platelet responsiveness to arachidonic acid, in terms of the aggregating response and the thromboxane production. The thromboxane levels released by collagen-stimulated platelets were increased after incubation with low concentrations of cocaine, while marked decreases were observed after incubation with high doses of cocaine. No effects on platelet aggregation induced by collagen and ADP were observed when low concentrations of cocaine were added; on the other hand, high doses of the anaesthetic were found to block the aggregating effects of these two agents. Specific studies showed cocaine to have an inhibitory activity on prostacyclin release when the aortic tissue was mechanically and thermically stimulated. By contrast, the prostacyclin synthesis by 'exhausted' aortic rings incubated with arachidonic acid appeared to be enhanced after addition of cocaine. These results lead us to believe that cocaine modifies both the Ca++ membrane binding and the extent of Ca++ influx, thereby increasing the permeability to arachidonic acid and altering the affinity of the membrane binding sites for the aggregating agents.     

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.     

Platelet dysfunction and alteration of prostaglandin metabolism after chronic alcohol consumption

To study the effects of chronic alcohol consumption on platelet functions, the rate of arachidonate-induced platelet aggregation, the production of malondialdehyde in platelets, and plasma levels of prostaglandin endoperoxide metabolites were examined in 88 chronic alcoholics and 24 healthy controls. The rate of platelet aggregation and the production of malondialdehyde in platelets were greater in chronic alcoholics both on admission and 1 week after. However, these alterations returned to the level of healthy controls within 4 weeks of abstinence from alcohol and were independent of the number of circulating platelets. Furthermore, on admission, plasma levels of thromboxane B2 were significantly increased in chronic alcoholics when compared with those of healthy controls (400.8 +/- 36.5 versus 241.7 +/- 28.9 pg/ml plasma; p less than 0.025) and were also significantly correlated with malondialdehyde production in washed platelet debris (r = 0.6049; p less than 0.001). In contrast, plasma levels of 6-keto prostaglandin F1 alpha and prostaglandin E were not altered after chronic alcohol consumption. As a result, the ratio of 6-keto prostaglandin F1 alpha to thromboxane B2 was markedly decreased in chronic alcoholics (0.31 +/- 0.03 versus 0.62 +/- 0.13; p less than 0.001). These results strongly suggest that the imbalance in prostaglandin endoperoxide metabolites is produced by chronic alcohol ingestion. Moreover, a significant correlation was observed between platelet aggregation rate and malondialdehyde production during platelet aggregation (r = 0.559; p less than 0.005). Thus, we conclude that chronic alcohol consumption alters platelet thromboxane metabolism, which is likely associated with the increased ability of platelets to aggregate.     

Epinephrine induces a late thromboxane-dependent platelet shape change and enhances synergistically the shape change induced by other platelet agonists

Epinephrine is the only physiological platelet activator which induces platelet aggregation without a preceding change in platelet shape. The reason why epinephrine cannot induce this shape change is not known. Electron microscopically, we could show that during the first phase of epinephrine-induced platelet aggregation, the platelet aggregate is composed of discoid platelets, lying in rather loose contact with neighbouring platelets. During the second wave of epinephrine-induced aggregation (this is when thromboxane (TX)A(2) production has taken place), platelets have completely lost their discoid shape and are very tightly bound. In EDTA-platelet rich plasma (PRP), we could demonstrate a clear synergistic action of epinephrine 10-20 μM on the first phase of shape change (disc-to-sphere transformation), induced by low concentrations of arachidonic acid (AA), collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). In combination with moderate concentrations of AA or collagen, epinephrine induced a clear aggregation-independent secretion of platelet granules, which in the absence of epinephrine, only takes place with higher inducer concentrations. All these synergistic actions could be demonstrated in the aggregometer and electron microscopically. To explain these findings, we hypothesize that the inability of epinephrine to induce a shape change that precedes aggregation is due to slow generation of TXA(2) which is only formed as a positive feedback mechanism of aggregation. This TXA(2) will bind to its own receptor and produce a shape change coinciding with the second wave of epinephrine-induced aggregation. Collagen, in contrast, induces very rapid TXA(2) generation, causing Ca(2+) mobilization and myosin light chain-phosphorylation, leading to shape change, clearly before aggregation starts.     

Evidence against a platelet cyclooxygenase defect in uraemic subjects on chronic haemodialysis

Defective platelet thromboxane synthesis has been described in uraemia and attributed to a 'functional cyclooxygenase defect'. We have studied platelet aggregation and generation of immunoreactive thromboxane B2 (TXB2) in 11 subjects on a chronic haemodialysis programme. The platelet function abnormality of uraemia was confirmed, maximal aggregation in response to collagen (2 and 4 micrograms/ml) and sodium arachidonate (1.5 and 3.0 mM) being significantly depressed. However, increased platelet aggregation in response to sodium arachidonate 0.75 mM was noted. Due to the reduced haematocrit, the platelet concentration in platelet-rich plasma (PRP) of uraemic subjects was significantly lower than that of controls; when TXB2 generation in PRP adjusted to 200 X 10(9) platelets/l was assessed, no evidence for a defect of cyclooxygenase was found, although reduced synthesis of TXB2 in response to thrombin was noted. Furthermore, increased thromboxane generation by uraemic PRP in response to sodium arachidonate 0.75 mM was detected. We conclude that the mild platelet abnormality in uraemic subjects treated by haemodialysis is not explained by a 'functional cyclooxygenase defect', although an abnormality of thrombin-induced thromboxane synthesis may be present. Furthermore, the tendency to increased aggregation and thromboxane synthesis in response to a low concentration of arachidonic acid may contribute to the thrombotic tendency which is also described in such subjects.     

Snake Venom Constituents that Affect Platelet Function

Snake venoms contain complex mixtures of proteins with biological activities. These venom proteins affect blood coagulation and platelet function in various ways. Many inducers and inhibitors of platelet aggregation have been isolated from snake venoms, especially from the Crotalidae and Viperidae families. According to their biochemical properties and modes of action, they can be classified into ten groups: (1) thrombin-like enzymes which show higher activity towards platelets than towards fibrinogen; (2) procoagulant enzymes which generate thrombin and then activate platelets indirectly; (3) noncoagulant glycoproteins which activate platelets independently of ADP release or thromboxane formation; (4) lectin-like peptides which cause agglutination of platelets through binding to a sugar moiety; (5) coagglutinins whose activities are dependent on the presence of von Willebrand factor (vWF); (6) membrane-active polypeptides which potentiate the aggregating action of other inducers by activation of platelet endogenous phospholipase A(2); (7) phospholipase A(2) enzymes which show biphasic aggregating and inhibitory effects on platelets; (8) α-fibrinogenases which degrade the α (A) chain of fibrinogen; (9) 5'-nucleotidase or ADPase which act on the ADP released from platelet dense bodies; and (10) fibrinogen-receptor antagonists which interfere with the interaction of fibrinogen and glycoprotein IIb/IIIa on activated platelets. These venom proteins are unique research tools for study of the haemostatic process and some of them are potential antithrombotic agents.     

The Platelet Defect Associated with Albinism

S ummary . Investigations on the blood of two albino women with a haemostatic defect have revealed a specific abnormality of their platelets. This consists of an inability of the platelets to store adenine nucleotides in a metabolically inert form, and consequently of an impairment of the release reaction whereby normal platelets are induced to aggregate by agents, including collagen and thrombin, that act indirectly through the release of adenosine diphosphate ( ADP ). These patients' platelets also have a greatly impaired ability to take up and store 5-hydroxytryptamine (5HT) and adrenaline. It is suggested that the haemostatic failure previously observed in association with albinism is a result of such a defect of storage and release of adenine nucleotides by the platelets. Investigations on seven other albinos without haemorrhagic symptoms have shown no abnormality of platelet aggregation or nucleotide storage.     

Comparison of fibrinogen association with normal and thrombasthenic platelets on exposure to ADP or chymotrypsin.

Although 125I-fibrinogen becomes associated with washed platelets from normal human subjects during ADP-induced shape change and aggregation, 125I-fibrinogen did not become associated with washed plateletes from a thrombasthenic subject during ADP-induced shape change and the platelets did not aggregate. Platelets from control and thrombasthenic subjects were treated with chymotrypsin, which is known to degrade platelet membrane glycoproteins. More 125I-fibrinogen became associated with chymotrypsin-pretreated platelets from normal subejcts than with untreated platelets, and fibrinogen caused the enzyme-treated platelets to aggregate. 125I-fibrinogen did not become associated with chymotrypsin-pretreated thromobasthenic platelets, and fibrinogen did not aggregate them. Thus, there appears to be a defect in thrombasthenic platelets that prevents the association of fibrinogen with them.     

Characterization of a platelet membrane protein of low molecular weight associated with platelet activation following binding by monoclonal antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.     

Myocardial ischemia: platelet and thromboxane concentrations in cardiac lymph and the effects of ibuprofen and prostacyclin

Blood platelets have been implicated in several mechanisms leading to and/or modifying myocardial ischemia. Cardiac lymph examination allows insight into the extracellular fluid that is in equilibrium with the capillary blood. In order to obtain an index of platelet activation during coronary artery events in the awake chronic animal, we wished to ascertain whether evaluation of cardiac lymph would detect changes in platelet activation resulting from a vascular occlusion. The study used conscious dogs in which cardiac lymph vessels had been previously cannulated by open-chest surgical protocol. The concentrations of immunoreactive thromboxane B2 and platelet counts were assessed in the cardiac lymph during the control period, the 10-60 minute occlusions, and the reperfusion periods. The same protocols were effected on another series of dogs after infusion of ibuprofen or prostacyclin. Initially, immunoreactive thromboxane B2 concentrations in the systemic blood and cardiac lymph were identical. A three-fold increase in immunoreactive thromboxane B2 concentrations occurred in untreated animals and was accompanied by a fall in platelet count in the lymph. The infusion of ibuprofen or prostacyclin, which inhibit platelet aggregation by different mechanisms, prevented both the decrease in platelets and the increase in immunoreactive thromboxane B2. In this study, intravascular events resulting from coronary occlusion invoke a rapid rise of immunoreactive thromboxane B2 in the extravascular fluid. A decrease in platelet escape into the extravascular compartment is interpreted as a result of intravascular aggregation promoting decreased platelet numbers. Thus, examination of continuously flowing cardiac lymph allows rapid detection of intravascular activation of platelets in the awake animal in the absence of surgical trauma.